Table of contents :

• cell types of the adaptive immune system : lymphopoiesis

Even the ontogeny of the adaptive immune system recapitulates its phylogeny. All the maturation stages are Ag-independent : the bone marrow produces 10⁶ cells / day. Homo sapiens body contains ~ 2 ^. 10¹² lymphocytes.
• pluripotent lymphoid stem cell (PLSC) / multipotent lymphoid progenitor (MLP) (CD34⁺) in bone marrow
‡
+ SCF
• bipotent lymphoid progenitor (CD34⁺)

BCL11A / EVI9 functions as a myeloid or B cell proto-oncogene in mice and humans, respectively. Bcl11a is essential for postnatal development and normal lymphopoiesis. Bcl11a mutant embryos lack B cells and have alterations in several types of T cells. Phenotypic and expression studies show that Bcl11a functions upstream of the transcription factors Early B cell factor (EBF) and Pax5 / BSAP in the B cell pathway. Transplantation studies show that these defects in Bcl11a mutant mice are intrinsic to fetal liver precursor cells. Mice transplanted with Bcl11a-deficient cells died from T cell leukemia derived from the host. Thus, Bcl11a may also function as a non-autonomous T cell tumor suppressor gene. The early B cell–specific mb-1 promoter is hypermethylated at CpG dinucleotides in HSCs but becomes progressively unmethylated as B cell development proceeded. EBF, a transcription factor required for B lymphopoiesis, potentiated activation of mb-1 by the transcription factor Pax5 (which alone cannot activate endogenous mb-1 transcription in a plasmacytoma cell line). EBF and the basic helix-loop-helix transcription factor E47 each contributed to epigenetic modifications of the mb-1 promoter, including CpG demethylation and nucleosomal remodeling. EBF function is enhanced by interaction with the transcription factor Runx1^ref.
‡
+ IL-2, IL-3, IL-4, IL-5, IL-6, IL-7 (=> up-regulation of antiapoptotic Bcl-2 and MCL-1^ref (which selectively inhibits the proapoptotic protein BIM), is essential both early in lymphoid development and later on in the maintenance of mature lymphocytes), IFN-g, EBF (TF for CD79a / Iga):
• early B progenitor (CD10 / CALLA⁺19⁺34⁺38⁺45 / B220 / LCA⁺79a / Iga⁺79b / Igb⁺, MHC II⁺, TdT⁺). In bony fishes B cells develop since day 4 in pacreas and since week 3 in pronephros (head kidney), which remains the main source organ.

‡
+ ? (inhibited by IFN-a => TYK2 => DAXX), ZAP70
• early pro-B cell (CD10 / CALLA⁺19⁺24 / HSA⁺34⁺38⁺40⁺43⁺45 / B220 / LCA⁺79a / Iga⁺79b / Igb⁺, MHC II⁺ IgM^- BP1⁺) : D_HJ_H rearrangement.


‡
+ Pax-5 (TF for CD19 and transcriptional repressor for Notch1), cyclin D3^ref, BLNK / SLP65, ZAP70
• late pro-B (a.k.a. pre-BI or stage B/C) cell (CD10 / CALLA⁺19⁺20⁺25^-38⁺40⁺43⁺45 / B220 / LCA^int): V_HD_HJ_H rearrangement. To survive and proliferate, pro-B cells must be able to interact with intramarrow stromal cells and to react to soluble and membrane factors that these cells produce. Moreover, to differentiate into pre-B cells, pro-B cells must initiate and successfully complete the V(D)J recombination program at the IgH locus, and to express on cell membrane a m heavy chain (mmH) capable of pairing with the invariant (i.e. non rearranged) surrogate light (SL / yLC) chains (SLC) (a heterodimer formed by CD179a / VpreB and CD179b / l5 / l-like 1). Such surrogate sIgM joins to CD79a / Iga and CD79b / Igb to form the pre-B cell receptor (pre-BcR / preBcR / pBcR) complex, allowing B cells to proliferate in a ligand-independent manner : the nonimmunoglobulin portion of l5 mediates cell-autonomous pre-BcR signalling via a crosslinking that involves a linking factor produced by the pre-B cell itself or non-covalent interactions between 2 l5 non-immunoglobulin regions. Based on the observations that the surrogate L chains also assemble on the surface of mm^- pro-B cells, it was proposed that membrane protein complexes analogous to pre-BcR and BcR may regulate some of the survival and differentiation processes in early pro-B cells. More recently, Iga and Igb have also been on the surface of mm^- pro-B cells in protein complexes that do not contain surrogate L chains, but 4 other proteins, one of which has been identified as calnexin. m gene recombination is not affected in pro-B cells lacking Iga, Igb, or both molecules, and the size and phenotype of the pro-B cell population is also not affected in vivo.

Until recently, it was generally assumed that all H chain proteins were capable of bringing about these changes; however dysfunctional H chains neither mediate heavy chain allelic exclusion nor drive clonal maturation, due to the inability to pair with SLC and form a pBcR. Significantly, failure to pair with SLC is also indicative of the inability of the H chain protein to pair with conventional L chain. Analysis of the Ig rearrangements from early B cells has revealed that as many as 50% of all H chain proteins are dysfunctional. Since V_H genes have been found that can encode for either a functional or a dysfunctional H chain the inability to pair with SLC must arise as a result of differences within the CDR3 sequence : the CDR3, formed by DNA recombination between V_H, D, and J_H genes, is the only sequence that varies between H chains using the same V_H gene.
‡
+ p110^d subunit of PI3K, CD21 / CR2 / C3dR / EBVR, Syk, ZAP70, myeloid-cell leukemia 1 (MCL1)^ref (a component of the signalling pathway downstream of IL-7, which binds BIM and prevents BIM-mediated activation of the pro-apoptotic proteins BAK1 and BAX)
• large pre-B cell (CD7⁺9⁺10 / CALLA^+?19⁺20⁺21 / CR2 / C3dR / EBVR⁺24⁺38⁺40⁺43^-45 / B220 / LCA⁺79a / Iga⁺79b / Igb⁺179a / VpreB⁺179b / l5⁺, mmH⁺, MHC II⁺).

‡
• small pre-B cell or early immature cell (CD7⁺9⁺10 / CALLA^+?19⁺20⁺21 / CR2 / C3dR / EBVR^-24⁺38⁺40⁺43^-79a / Iga⁺79b / Igb⁺) : V_LJ_L rearrangement. 76% of antibodies from early immature B cells are self-reactive and polyreactive.

‡
• immature B cell

(CD19⁺20⁺21 / CR2 / C3dR / EBVR^-23^-24 / HSA^hi40⁺79a / Iga⁺79b / Igb⁺ IgM⁺IgD^lo/-, E₂R⁺). 43.1% of antibodies from immature B cells are self-reactive and a few are polyreactive.
‡
+ Syk
• newly formed or new emigrant or transitional (TR) type 1 (T1) B cell

(CD1^lo19⁺20⁺21 / CR2 / C3dR / EBVR^lo/-22^lo23^lo/-24 / HSA^hi40⁺45 / B220 / LCA^lo79a / Iga⁺79b / Igb⁺ IgM^hi/+IgD^lo/-)
Located in bone marrow, peripheral blood, B-cell zones of spleen PALS. 20% of antibodies from T1 B cells are self-reactive, with the potential to contribute to autoimmune diseases.
‡
+ Rac1, Rac2^ref
• transitional (TR) type 2 (T2) B cell

(CD1^hi19⁺20⁺21 / CR2 / C3dR / EBVR^hi22⁺23^hi24^int40⁺45 / B220 / LCA^lo79a / Iga⁺79b / Igb⁺, MHC II⁺, IgM^hiIgD^hi)
Located in B-cell zones of spleen PALS
‡
+ BOB.1 (which downregulates CD22), BLNK / SLP65
• resting (naive) mature B or B0 cell (CD13^-15^-19⁺20⁺21 / CR2 / C3dR / EBVR⁺22⁺23⁺24⁺25⁺32 / FcgRII⁺38⁺39⁺40⁺45 / B220 / LCA^hi72⁺75⁺79a / Iga⁺79b / Igb⁺, MHC II⁺IgM⁺IgD^-).

Approximately 65% of B cells generated in human bone marrow are potentially harmful autoreactive B cells. Most of these cells are clonally deleted in the bone marrow (negative selection by BcR editing as in the thymus for T lymphocytes), while those autoreactive B cells that escape to the periphery are anergized or undergo clonal deletion by AICD before becoming mature B cells. Escape of self-reactive B cells from tolerance permits production of pathogenic auto-antibodies; recent studies suggest that extended B lymphocyte survival is a cause of autoimmune disease in mice and humans. Spontaneous death of resting B cells is regulated by nuclear localization of PKCd that contributes to phosphorylation of histone H2B at Ser¹⁴ (S14-H2B) : treatment of B cells with BAFF prevents nuclear accumulation of PKCd^ref. Those occasionally found in the thymus were once called T_h5 cells in mice. Bone marrow produces 10⁹ B cells/day. Mature B lymphocytes represent 10% of bone marrow lymphocytes, 10-15% of peripheral blood lymphocytes (0.3 ^. 10⁹ B cells/L = 1.5 ^. 10⁹ B cells), and 50% of spleen lymphocytes. Once terminated the Ag-independent development in primary lymphoid organs, mature B cells undergo Ag-dependent maturation in secondary lymphoid organs.
‡
+ interaction between BcR and self-Ag => differentiation, expression and maintenance
• B-1 cell (CD9⁺11b / a_M integrin⁺14^lo15^-19⁺20⁺21 / CR2 / C3dR / EBVR^lo23^lo/-40⁺43⁺45 / B220 / LCA^lo72⁺w125 / IL-5Ra⁺ IgM^hisIgD^lo) : CXCL13 / BLC-mediated homing from fetal liver and omentum to peritoneal and pleural cavities up to adolescence : not recirculating, they are absent from lymph nodes, spleen and peripheral blood. They produce IL-10 and IL-10R (autocrine signalling). They enter S phase in response to phorbol ester alone. B-1 B cells are not usually associated with autoimmune diseases (being able to undergo class switch recombination and somatic hypermutation) : anyway typical B-1 antibodies do not seem to undergo extensive hypermutation, perhaps because of a lack of interaction with T cells. They represent the cell of origin for B cells chronic lymphocytic leukemia (B-CLL).  On Ag encounter they might give rise to plasma cells that produce a V_H selected nonimmune repertoire of "natural" serum IgM (natural antibodies (NAb)) and substantial amounts of IgA in the gut and mesenteric lymph nodes. They are considered to be innate lymphocytes as among the known BcR specificities typical of B-1 cells are autoantigens (=> natural autoantibodies (nAAbs)), such as plasma membrane phospholipids (eg V_H11-V_k9 or V_H12-V_k4 Ig genes - expressed by 5-15% of the peritoneal B-1 cells in normal mice - react with phosphatidylcholine (PtC)), and conserved epitopes present on common pathogens, such as polysaccharide moieties : this set of specificic VDJ rearrangements encoded in the germline with short or absent N sequences is evolutionarily retained and provides at low affinity a degree of serological protection against a range of microorganisms prior to the immunization that accompanies microbial pathogenesis. There is mounting indirect evidence that self-Ags drive the development of naive B cells, but Hardy's murine model, dealing with a monoreactive autoantibody directed against a membrane-bound Ag, is, to date, the only direct proof of such a positive selection of B cells. However, as mentioned above, nAAbs are mostly polyreactive, and mainly react with conserved soluble self-Ags such as ssDNA, IgG, thyroglobulin, or cytokines. Precisely because of the polyreactivity profile of these nAAbs, demonstrating the role of a given autoantigen by its elimination is difficult.
• B-1S cell (precursor in adult bone marrow => in spleen) CD11b / a_M integrin^-
• B-1P cell (precursor in fetal liver => in peritoneal cavity) CD11b / a_M integrin⁺
• B-1a B cells (CD5 / Ly-1⁺11b / a_M integrin^-23^-24 / HSA^lo43⁺45 / B220 / LCA^int IgM^hisIgD^lo). Their number is reversibly increased in mice deficient for serum "natural" IgM (negative feedback ?).
• B-1b B cells (CD5 / Ly-1^-11b / a_M integrin⁺23^-24 / HSA^lo43⁺45 / B220 / LCA^int IgM^hisIgD^lo) : CD5 / Ly-1 binds membrane lipids and modulates BcR signalling through SHP-1 / PTPN6 recruitment, causing an altered responsiveness to BcR ligation compared with their B-2 cohorts (marked diminished ability for intracellular Ca²⁺ mobilization, aberrant proliferation, and increased apoptosis after BcR cross-linking)
... or ...

without interaction between BcR and self-Ag

• B-2 cell (conventional B cell) (CD1^lo5 / Ly-1^-19⁺21⁺23⁺24 / HSA⁺72⁺sIgM⁺sIgD^-) :

enter S phase in response to anti-Ig stimulation; located in :
• bone marrow
• peripheral blood : 16 to 28% (of these, 4-12.7% have sIgG, 1-4.3 have IgA, 6.7-13% have IgM, and 5.2-8.2 have IgD).
• spleen :
+ p110^d subunit of PI3K
• marginal zone (MZ) B cells(CD1D^hi9⁺11a / a_L integrin⁺18 / b₂ integrin⁺21^hi22^hi23^lo/-24 / HSA^int29 / b₁ integrin⁺49d / a₄ integrin⁺ IgM^hiIgD^lo/-S1P₁⁺S1P₃⁺) : lower threshold for activation, mainly involved in responses against TI-Ags ; express Gab1 and Id2. MZ B cell localization to the marginal zone depends on responsiveness to the blood lysophospholipid S1P, with S1P₁ signaling overcoming the recruiting activity of CXCL13. In mice lacking the chemokine CXCL13, S1P₁-deficient marginal zone B cells reacquired a marginal zone distribution. Exposure to LPS or antigen caused marginal zone B cells to downregulate S1P₁ and S1P₃ and to migrate into the splenic white pulp^ref. CXCL13-independent homing; non-recirculating thanks to a₄b₁integrin---CD106 / VCAM1 and a_Lb₂ integrin---CD54 / ICAM-1 interactions (both ligands are upregulated by LT-b); upon stimulation with bacterial products they undergo CXCL13-dependent migration to the B-cell follicles at the T-B junction of the spleen where they divide and generate large clones of IgM-secreting plasma cells within 3 days. They are considered to be innate lymphocytes. Their number is reversibly increased in mice deficient for "natural" serum IgM (natural antibodies (NAb)) (negative feedback ?).
• follicular (FO) B cells (CD1D^int9^-21^int22^hi23^hi IgM^int/loIgD^hi) (CXCL13-dependent homing from adult bone marrow into the follicles; recirculating)
• lymph nodes (recirculating)
They enter S phase in response to anti-Ig stimulation.
... or ...

+ Notch1 (vs. mNumb)

• common T-cell and NK progenitor (C-TNKP) (CD3deg⁺19^-34⁺117 / c-kit / SCFR⁺)
‡
• NK progenitor or pro-T1 (CD2^-3deg⁺56⁺161⁺)

‡
+ IL-2, IL-15
• natural killer (NK) cell / large granular lymphocyte (LGL) / null cell (CD2⁺3^-4^-8^-(rarely 8ab⁺)14^-15^-16a / FcgRIIIA⁺19^-46⁺55 / DAF⁺56 / NKH1 / NCAM / Leu-19⁺57 / HNK1 / Leu-7^{+ (absent in cord blood NK cells !)}85⁺94⁺178 / Apo1L / FasL⁺226 / DNAX accessory molecule 1 (DNAM1)⁺244 / 2B4⁺, MHC II^-, TcR^-, asyalo-GM₁⁺) : small eosinophilic granules in the cytoplasm; 5÷15% of blood lymphocytes (more abundant in TAP^-/- individuals), localized mainly in spleen but not in thymus and lymph nodes. CD94.1-expressing blood cells in the urochordate Botryllus schlosseri might be ancestral NK cells : they mediate cytotoxic lesion at the point of contact between colonies


• NK gene complex (NKC) is on human chromosome 12p13
• leukocyte receptor cluster (LRC) on chromosome 19q13.4 (evolved by gene duplication, together with FcaR gene; 15 haplotypes => > 100 genotypes) : diversity at the KIR locus has been generated by an evolutionary history of expansion and contraction, presumably due to combinations of duplication and unequal crossing over within the locus. Up to 12 KIR genes appear to be expressed, and KIR haplotypes consist of various combinations of KIR genes. The number of putatively expressed KIR genes present on a single haplotype ranges from 7 to 11, depending primarily on the presence or absence of activating KIR loci. Estimates of the extent of KIR genotype diversity within the population suggest that far < 0.24% of unrelated individuals can expect to have identical genotypes. The most common Caucasian haplotype, the "A" haplotype (frequency of ~ 47-59%), contains only 1 activating KIR gene (KIR2DS4) and 6 inhibitory KIR loci (KIR3DL3, -2DL3, -2DL1, -2DL4, -3DL1, and -3DL2). The remaining "B" haplotypes are very diverse and contain 2 to 5 activating KIR loci (including KIR2DS1, -2DS2, -2DS3, and-2DS5).
NK cell receptors don't cover the whole set of class I HLA alleles and are not expressed on 100% of NK cells.
Diversity in KIR gene content, KIR specificity for particular HLA allotypes, and the unlinked physical location of the KIR loci relative to the HLA loci give rise to the possibility that, for some KIR loci, any given individual may encode receptor only, ligand only, both receptor and ligand, or neither one. NK cell receptors (NKR) are all type II transmembrane proteins.
• inhibitory receptors (also expressed on memory CD8⁺ >> CD4⁺ ab and gd T cells) ...
• ... have cytoplasmic ITIM(s) that, upon binding to MHC I, become phosphorylated by c-Src and allow recruitment of SHP1 / PTPN6 and/or SHP2, LCK and talin :
• killer cell Ig-like (or inhibitory) receptors (KIR) / CD158 family : they undergo Zn²⁺- (and Co²⁺- ?) induced multimerization.
• KIR2DLs (long intracytoplasmic tail with 2 ITIMs)
• KIR2DL1 / CD158a / p58.1 (D1 and D2 Ig domains) --- group 2 HLA-C allotypes (HLA-C2)
• KIR2DL2 / CD158b / p58.2 (D1 and D2 Ig domains) --- group 1 HLA-C molecules (HLA-C1)
• KIR2DL3 / CD158b / p58.3 (D1 and D2 Ig domains) --- group 1 HLA-C molecules (HLA-C1)
• KIR2DL4 (D0 and D4 Ig domains) --- HLA-G
Anyway it has activating function !!!
• KIR3DLs (3 Ig domains (D0, D1 and D2), long intracytoplasmic tail with 2 ITIMs)
• KIR3DL1 / NKB1 / NKR p70 --- HLA-B serotypes that have the Bw4 >> Bw6 epitope (determined by amino acids positions 79-83 in a₁ domain of the molecule), e.g. HLA-B27 : HLA Bw4 molecules with Ile⁸⁰ (Bw4-80I) may be better ligands than those containing Thr⁸⁰.
• KIR3DL2 / NKR p140 --- HLA-A3 and HLA-A11
• killer cell lectin-like receptors (KLR) (belonging to C-type (Ca²⁺-dependent) lectin SF).
• CD94 / KLRD1
• KLRC1-derived NKG2A isoform (both ITIMs are required for mediating the maximal inhibitory signal, but opposite to KIR, the membrane distal ITIM is of primary importance rather than the membrane-proximal ITIM : this probably reflects the opposite orientation of the ITIMs in type II vs type I proteins).
• KLRC1-derived NKG2B isoform
The receptor may occur either as a CD94 homodimer or as a disulfide bound CD94 heterodimer with NKG2A or NKG2B) --- HLA-E charged with HLA-A, HLA-B, HLA-C, or HLA-G leader peptides (so cells that don't express MHC Ia have no ligand on their surfaces : such charging is TAP-independent as leader peptides are already available in RER lumen) or with a peptide from the chaperonin HSP60, up-regulated in stressed cels (in this latter case the complex cannot be recognized by CD94/NKG2A, allowing killing of stressed cells). Expression of both CD94 and NKG2A is induced by IL-12. CD94/NKG2A is continuously recycled between the cell surface and cytoplasm, and that this recycling is independent of ligation and the transmission of inhibitory signals. Because CD94/NKG2A receptors are in the constant presence of ligand expressed by normal cells, the detachment of recycling from ligation/inhibitory processes likely facilitates the maintenance of a pool of CD94/NKG2A receptors on the cell surface available for interaction with HLA-E
• Ig-like transcripts (ILTs) / leukocyte inhibitory receptors (LIRs) / monocyte-macrophage inhibitory receptors (MIRs) family
• CD85 / LIR-1 / MIR7 / ILT-2 --- HLA class Ia and HLA-G1
• LIR-2 / MIR10 /  ILT-4 --- HLA class Ia and HLA-G1
• ... have a different unknown STP :
• tetraspanin family
• CD81 / TAPA-1 --- protein E2 on HCV envelope
• CD161 / KLRB1 / NKR-P1A / NKR-P1B, NKR-P1D, and NKRP1F^ref (CD161b/d/f) (contains an extracellular domain with several motifs characteristic of C-type lectins, a transmembrane domain, and a cytoplasmic domain. The KLRB1 protein is classified as a type II membrane protein because it has an external C terminus.) --- C-type lectin superfamily 2, member D (CLEC2D) / osteoclast inhibitory lectin (Ocil) / Clr-b / CLAX / LLT-1, a member of a previously cloned group of C-type lectin-related (Clr) proteins linked to the NKR-P1 receptors in the mouse NK gene complex (NKC). Expression of Ocil/Clr-b on mouse tumor cell lines inhibits NK cell-mediated killing. Inhibition is blocked with a new mAb (4A6) specific for Ocil/Clr-b. Ocil/Clr-b is displayed at high levels on nearly all hematopoietic cells, with the exception of erythrocytes, in a pattern that is similar to that of class I MHC molecules. Remarkably, Ocil/Clr-b is frequently down-regulated on mouse tumor cell lines, indicating a role for this receptor-ligand system in a new form of "missing self-recognition" of tumor cells^ref.
• tyrosine phosphorylation of the cytoplasmic tail of SH2D1B / EAT-2 and ELF3 / ERT adaptors, which associate with the SLAM-related receptor CD244 / 2B4, represses natural cytotoxicity and IFN-g secretion^ref
• activating receptors are linked to intracellular adapters :
• adapter FceRI g chain or CD247 / z chain (ITAM-containing) => ZAP70 and Syk => PI(3)K STP
• CD16a / FcgRIIIA --- IgG (mediates ADCC)
• NKp30 / 1C7 / B144 / LST1 / D6S49E --- ? (also used for DCs killing).

[pp65, the main tegument protein of HHV-5 / HCMV, inhibits NK cell cytotoxicity by a direct and specific interaction with the activating receptor NKp30, leading to dissociation of the linked CD3z from NKp30^ref]
• NKp46 --- HA_{Influenza virus}, ?
• Killer cell Ig-like (or inhibitory) receptors (KIR) / CD158 family : they undergo Zn²⁺- (and Co²⁺- ?) induced multimerization.
• p50^KIR2DSs (2 Ig domains (D1 and D2), short intracytoplasmic tail with no ITIM) --- HLA-C, non-MHC molecules (foreign or microbial antigens expressed on infected cells, normal cell surface proteins that are aberrantly expressed, or complexes of pathogen-derived peptides bound to MHC class I molecules), ?
• KIR2DS1 / CD158a / p50.1 ---
• KIR2DS2 / CD158b / p50.2 --- group 1 HLA-C molecules (HLA-C1)
• KIR2DS3 ---
• KIR2DS4 / PAX / p50.3 ---
• KIR2DS5 ---
• KIR3DS1 (3 Ig domains (D0, D1 and D2), short intracytoplasmic tail) --- at least some of the HLA-Bw4 allotypes in HIV-1⁺ individuals.
• adapter p14^{DNAX-activating protein of 12 kDa (DAP12) / KARAP} homodimer (recruited by electrostatic interaction between transmembrane domains; ITAM-containing) => ZAP70 and Syk => PI(3)K STP. This STP is required to trigger NKG2D-dependent cytokine release by NK cells.
• killer cell lectin-like receptors (KLR) (belonging to C-type (Ca²⁺-dependent) lectin SF).
• CD94 / KLRD1
• NKG2C / KLRC2
• NKG2E / KLRC3
• NKG2I / KLRE1 (40% homology with CD94 and NKG2D, but lacks any signalling motifs or positively charged residues in its putative transmembrane region, which are characteristics of the NKG2 family, and shows little similarity to other family members in its putative ligand-binding domain, indcating that it is likely to interact with unique ligands; homodimer or heterodimer ? The earlier report indicated that it can dimerize with an unidentified partner containing an ITIM motif and form an inhiitory receptor complex^ref, while a later report suggests it is an activating NK-cell receptor involved in initiating pathways that lead to NK-cell-mediated allograft rejection^ref, just like CD94)
The receptor may occur either as a CD94 homodimer or as a disulfide-bound heterodimer --- HLA-E charged with HLA-A, HLA-B, HLA-C, or HLA-G leader peptides (so cells that don't express MHC I molecules have no HLA-E on their surfaces ! Such charging is TAP-independent as leader peptides are already in RER lumen)
• NKp44 --- HA_{Influenza virus}, ?
• adapter DNAX-activating protein of 10 kDa (DAP10) / phosphoinositide-3-kinase adaptor protein (PIK3AP) (no ITAM motif => PI(3)K and AKT STP !)
• killer cell lectin-like receptors (KLR) (belonging to C-type (Ca²⁺-dependent) lectin SF)
• NKG2D / NKG2F / KLRC4-L (homodimer) ---
• MICA and MICB (K_d = 5 ^. 10^-7 M)
• ULBPs
The binding of NKG2D to its various ligands is generally of higher affinity than many immunoreceptor-ligand interactions, with a K_dranging from 10^-6 M to 4 ^. 10^-9 M. Despite the marked differences in their amino-acid sequences, the different ligands interact with NKG2D similarly (diagonally across the a-helical surface of each of these ligands, similar to the mode of binding of a TcR to an MHC molecule), and the receptor does not seem to undergo marked conformational changes (induced fit) to accommodate different ligands. Notably, most of the receptor amino-acid residues that dominate binding to the different ligands are the same, and several of the contact residues on the ligands are conserved, expecially those that are thought to contribute most of the binding energy. Another interesting feature of the structures is that different residues in the 2 NKG2D monomers that make up the homodimer dominate binding to the asymmetric a₁ and a₂ domains of the ligands. The NKG2D signal is strong enough to overcome inhibitory signalling by MHC-specific receptors in some cases.
The DNA damage pathway regulates innate immune system ligands of the NKG2D receptor^ref
• adapter SAP / SH2D1A
• CD244 / 2B4 --- CD48 / Blast-1 / OX-45 / Hu Lym3
• adapter ? => ERK STP
• CRACC --- ?
• DNAX accessory molecule 1 (DNAM1) / CD226 --- poliovirus receptor (PVR) and the d and a isoforms of CD115 / poliovirus receptor-related 2 (PRR2) / herpesvirus entry mediator B (HVEB) / nectin-2 (at adherens junctions of myelo-monocytic and megakaryocytic hematopoietic, neuronal, endothelial and epithelial cells)
The susceptibility of the target cell to killing by NK cells depends on the balance of the ligands for activating and inhibitory receptors. This is reminiscent of the balance between proapoptotic and antiapoptotic members of the Bcl-2 family proteins that control intrinsic apoptosis and presumably reflects the fact that "fuzzy" decision-making mechanisms are more resistant to errors than discrete, all-or-none processes. Peroxiredoxin (PRDX1) is required in both NK cells and non-immune cells for optimal NK function.
See also activation of NK cells.
• KLRH1
Comprising the third largest lymphocyte population, NK cells recognize and kill cellular targets and produce pro-inflammatory cytokines. These potentially self-destructive effector functions can be controlled by inhibitory receptors for the polymorphic MHC class I molecules that are ubiquitously expressed on target cells. However, inhibitory receptors are not uniformly expressed on NK cells, and are germline-encoded by a set of polymorphic genes that segregate independently from MHC genes. Therefore, how NK-cell self-tolerance arises in vivo is poorly understood. NK cells acquire functional competence through 'licensing' by self-MHC molecules. Licensing involves a positive role for MHC-specific inhibitory receptors and requires the cytoplasmic inhibitory motif originally identified in effector responses. This process results in 2 types of self-tolerant NK cells—licensed or unlicensed—and may provide new insights for exploiting NK cells in immunotherapy. This self-tolerance mechanism may be more broadly applicable within the vertebrate immune system because related germline-encoded inhibitory receptors are widely expressed on other immune cells^ref.
Redirected killing is an experimental system for determining the capacity of a NK-cell receptor to induce cytotoxicity. NK cells coated with antibody specific for a candidate receptor are assessed for their ability to kill target cells that express an Fc receptor to which the antibody binds.
Bibliography : Carrington, Mary and Norman, Paul : The KIR gene cluster, Bethesda (MD):National Library of Medicine (US), NCBI; 2003.
Web resources : NKcells.info by Volker Huppert

or ...

+ ? to become a :

• primary prothymocyte / haematopoietic precursor / bone-marrow derived progenitor / pro-T2 (CD7⁺34^- or CD7⁺34⁺) : needs ..
• Ikaros (which associates with nucleosome-remodelling histone (NuRD) complex, a multisubunit complex containing nucleosome remodeling and HDAC activities, including methyl-CpG binding domain protein 3 (MBD3), metastasis-associated gene family, member 2 (MTA2), p66a, ...)
• PU.1 (=> up-regulation of M-CSFR, G-CSF-R and IL-7Ra : whereas the transcription factor PU.1 regulates IL-7R expression in mouse pro–B cells via a GGAA motif, GA binding protein (GABP) ab₂ binds to this site and is essential in the regulation of IL-7R expression in T cells, where PU.1 is not expressed^ref)
• Notch1
• Pax3
• GATA3
• IL-15Ra
... to undergo T-cell commitment (see also T-cell development). CCR7 is crucial for migration of early thymic immigrants from the corticomedullary region to the subcapsular region (more important in the adult structured thymus) and migration of positively selected thymocytes from the cortex to the medulla (more important in newborns than in adult mice)^{ref1, ref2}. The only progenitors in blood with efficient T lineage potential are Lin^-SCA1^hic-kit^hi (LSK cells). The blood LSK population, like its counterpart in the bone marrow, contains HSCs and nonrenewing, multipotent progenitors, including early lymphoid progenitors and CD62L⁺ cells previously described as efficient T lineage progenitors. CLPs can not be identified in the circulation, suggesting they are not physiological T lineage precursors. Blood LSK cells are the principal circulating progenitors with T lineage potential^ref.
‡
+ ?
• secondary prothymocyte / early T lineage progenitor (ETP)  (CD7⁺, cCD3deg) : 10⁶ primary prothymocytes enter the thymus daily through ...
• the capsule and the septa (in embryo)
• blood vessels (after thymus became vascularized) at corticomedullary junction
... then placing in cortical subcapsular portion. Developing thymocytes within the thymic microenvironment are subject to an osmotic stress that is effectively countered by NFAT5-dependent responses. ETPs can also develop via a common lymphoid progenitors (CLP)-independent pathway.
See also in vitro techniques to study the development of thymocytes.
‡
+ IL-1b, IL-2, IL-6, IL-7, + Notch1 on T cells (vs. mNumb) --- Notch ligands (on TECs), ...
• thymic lymphoid double-negative (DN1) progenitor (CD1a^-4^-8^-25^-34⁺44⁺117 / c-kit / SCFR⁺CCR7⁺) : it is located in the deep cortex and needs CD117 / c-kit / SCFR/ CD132 / g_c, preTcRa/ CD132 / g_c, BCL11B, and IL-7Ra to maturate.

‡
• tertiary prothymocyte / pro-T DN2 cell (CD1a⁺4^-5⁺7⁺8^-19^-25⁺34⁺44⁺CCR7⁺, cCD3deg). It is located in the midcortex.

‡
+ BMP2, BMP4, myeloid-cell leukemia 1 (MCL1)^ref (a component of the signalling pathway downstream of IL-7, which binds BIM and prevents BIM-mediated activation of the pro-apoptotic proteins BAK1 and BAX)
• early pre-T DN3 cell (CD1a⁺2 / LFA-2⁺19^-25⁺44^-CCR7⁺) : it is located in the outer third of the cortex and needs ...

‡
+ RAG1, RAG2, Ku80, DNA-PK_CS / XRCC7, TcRbd, cCD3egx, p56^LCK, Jak3, Csk, 45 / B220 / LCA, ZAP70, Syk, Fyn, CD117 / c-kit / SCFR, IL-2Rg, IL-7Ra, TCF1a, GATA3^ref, Rho, LAT, SLP76, Vav1, c-Myb^ref, survivin^{ref1, ref2}, and Runx2
induction of preTCR signaling resulted in rapid elevation of c-Myc protein levels, only required for preTCR induced proliferation but dispensable for developmental progression from the DN to the DP stage^ref.
... to undergo b-selection / pre-TcR checkpoint : b-chain rearrangements occur and successfully rearranged b-chain associates with preTcRa and CD3 g-, d-, e, and z-chains to form the preTcR complex. Signaling from the complex leads to survival, differentiation from DN3 to DN4, proliferation, and allelic exclusion : it channels cells into the ab-lineage because it reduces the number of gd T cells with productive TcRb rearrangements. The absence of signaling due to the lack of functionally rearranged b-chain leads to cell death and a developmental block at the DN3 stage. There exists an obvious similarity of proximal signaling events between the constitutively signaling pre-TcR and the ligated TcRab : in a TcRa^-/- background, the pre-TcR promotes a biased positive selection of CD8⁺T cells. The pre-TCR complex can deliver its signal autonomously through oligomerization of the pre-TCR a-chain mediated by charged residues in the extracellular domain^ref
• late pre-T DN4 or triple-negative (TN) cell (CD1a⁺3^-4^-8^-19^-25^-44^-)

‡
It is controversial whether cells commit to the T-cell lineage prethymically or intrathymically^{ref1, ref2}. Aided by modern cell-sorting techniques, many studies in the mouse have recently readdressed these issues. Adult murine BM has been demonstrated to contain precursors with a restricted T-/B-lymphoid potential, so-called common lymphoid progenitors (CLPs)^{ref1, ref2}. However, the earliest thymic immigrants were shown to differ from CLPs in several aspects^ref. In peripheral blood, T-lineage potential appeared to be restricted to Lin^–Sca-1⁺c-Kit⁺ (LSK) progenitor populations, rather than to CLPs^ref. Recently, 2 detailed analyses of the earliest subpopulations in the murine thymus showed variable lineage potential of different subsets^{ref1, ref2}. Together, these studies point toward a model in which a range of BM-derived progenitors colonize the thymus, probably including multipotent progenitors and more lineage-restricted precursor cells^ref. In both humans and mice, the most immature cells in the thymus do not express CD4 and CD8 and therefore are called double negative (DN). In humans, the DN stage can be further subdivided by staining for CD34 and CD1^{ref1, ref2}. Studies in the murine thymus have shown that the DN1 subset (the earliest DN subset in the mouse) contains multilineage progenitors^{ref1, ref2, ref3, ref4, ref5}. CD34 levels decline as the cells mature^{ref1, ref2}, concomitantly with expression of CD1^ref. This is usually detected by staining for CD1a, but CD1b, CD1c, CD1d, and CD1e are also expressed. The CD34⁺CD1a^–cells start to rearrange their TCRD genes, but mostly have their TCRG and TCRB loci still in germ line position, whereas expression of CD1a is accompanied by rearrangements of TCRD (V to DJ), TCRG, and TCRB (D to J) loci^ref. Gene expression, lineage potential, and self-renewal capacity of the 2 most immature subsets in the human thymus, namely CD34⁺CD1a^- and CD34⁺CD1a⁺ thymocytes, were examined. DNA microarrays revealed the presence of several myeloid and erythroid transcripts in CD34⁺CD1a^– thymocytes but not in CD34⁺CD1a⁺ thymocytes. Lineage potential of both subpopulations was assessed using in vitro colony assays, bone marrow stroma cultures, and in vivo transplantation into NOD/SCID mice.
• CD34⁺CD1a^– cells (0.4% of all thymocytes^ref) contained progenitors with
• lymphoid (both T and B), myeloid, and erythroid lineage potential
• B-cell precursor potential of CD34⁺CD1a^– thymocytes has never been reported, despite significant numbers of B cells in the human thymus^{ref1, ref2, ref3}. Furthermore, in the thymus we could detect the presence of all BM B-cell progenitor stages, indicating that B cells develop in the thymus, albeit at much lower frequencies than in the BM^ref.
• the capability of human CD34⁺ thymocytes to develop into NK cells and DCs has been demonstrated by several studies, in both fetal^{ref1, ref2} and adult^{ref1, ref2} thymus.
• myeloid potential of CD34⁺ thymocytes was investigated by a number of studies several years ago, yielding contradictory results. Although one study could generate myeloid colonies from CD34⁺ as well as from CD34^– thymocytes^ref, others could not confirm any myeloid potential in CD34⁺ thymocytes^ref. De Yebenes et al^ref demonstrated that myeloid DC precursors can be generated from CD34⁺ thymocytes in cultures containing M-CSF.
• megakaryocytic potential was suggested by the expression of NF-E2 in CD34⁺CD1a^– thymocytes and was confirmed by culturing thymocytes in suspension in the presence of TPO and SCF. Although CD34⁺CD1a⁺ thymocytes did not survive, CD34⁺CD1a^– thymocytes could be cultured in this condition for at least 14 days and developed into large cells resembling megakaryocytes, although definitive flow cytometric characterization (eg, by staining for CD41) was hindered by high autofluorescence^ref
Remarkably, development of CD34⁺CD1a^– thymocytes toward the T-cell lineage, as shown by TcR gene rearrangements, could be reversed into a myeloid-cell fate. A close lineage relation between myeloid cells and T cells has also been suggested by experiments in the fetal mouse, by Kawamoto et al.^ref. These cells resemble hematopoietic stem cells but, by analogy with murine thymocytes, apparently lack sufficient self-renewal capacity. Both CD34⁺CD1a^– and CD34⁺CD1a⁺ thymocytes failed to repopulate NOD/SCID mice.  In the mouse it has been shown that proliferation of a precursor that enters the thymus is limited to several weeks and that no permanent endogenous stem cell exists^ref. This makes it likely that, similar to the corresponding cells in the mouse, CD34⁺CD1a^- cells have very limited self-renewal. Thus, it is possible that the thymus is seeded by a HSC that rapidly loses self-renewal capacity and reduces non–T-cell potential after entering the thymus and interacting with molecules on the thymic epithelium, for instance, Notch ligands. Another possibility is that only a minute fraction of the cells that seed the thymus retains sufficient self-renewal and that this population is too rare to be detected with currently used assays. In line with this, it may be that a similar situation exists in the mouse, but that very stringent lineage depletion or insufficient cell numbers used in colony assays have excluded detection of, for instance, erythroid potential. An alternative explanation for the fact that CD34⁺CD1a^– thymocytes could not repopulate NOD/SCID mice is that they lack BM homing capability. One of the adhesion molecules necessary for BM homing is CXCR4^{ref1, ref2}, but using flow cytometry no significant differences in CXCR4 expression was detected between CD34⁺ UCB cells, which efficiently repopulate NOD/SCID BM, and CD34⁺ thymocytes; absence of other homing signals in CD34⁺ thymocytes cannot be excluded. Poor homing capability can be considered as an additional argument against the existence of a thymic HSC, because murine HSCs were shown to have high BM-seeding efficiency^ref. Frequencies of myeloid and erythroid potential, as determined by in vitro colony assays, were markedly lower in the CD34⁺CD1a^– thymocytes than in CD34⁺ UCB cells, probably because UCB contains higher numbers of more mature progenitors. This is visible, for instance, in the higher expression of CD13/33 and MPO on CD34⁺ cells from UCB. Also in the OP9 cocultures we found low frequencies of myeloid and B-lineage progenitors in the CD34⁺CD1a^– subset^ref.
• CD34⁺CD1a⁺ cells (0.6% of all thymocytes^ref) yielded only T-cell progenitors (a very limited NK and no DC precursor activity of CD34⁺CD1a⁺ cells^ref), demonstrating their irreversible commitment to the T-cell lineage. In contrast to the immature characteristics of the CD34⁺CD1a^-– cells, the CD34⁺CD1a⁺ thymocytes showed a T-cell–specific gene-expression pattern and did not yield any non–T-lineage cells in in vitro cultures. Together these findings suggest that human CD34⁺CD1a⁺ thymocytes are the equivalents of murine DN3 cells, in which final commitment to the T-cell lineage occurs^ref. This confirms a recently proposed scheme in which the stages of human T-cell development are highly similar to those in the mouse^ref.
These precursor cells exit the thymus before TcR rearrangements and permanently colonize the precursor compartment of lymphoid organs such as the thymus and the gut for the generation of 'euthymic like' CD8aa⁺ intraepithelial lymphocytes^ref.
+ Ets2
• intermediate single-positive (ISP) T-cell / intrathymic T progenitor (ITTP) (CD1a⁺2 / LFA-2⁺3^-4⁺8ab^-19^-25^-34^-44^-TcRab^- or CD1a⁺3^-4^-8ab⁺25^-34^-44^-TcRab^-) : they express IL-4, but attenuate GATA3 expression, recruit DNA methyltransferases (Dnmts) to the Il4-Il13 locus and downregulate IL-4 expression as they mature into T cells. They contain mature TCRB (V to DJ) rearrangements^ref

‡
+ IL-7, IL-15 (which induces local chromatin modifications specific for the variable gd-region gene segment and enhanced accessibility conducive to subsequent targeted gene rearrangement. This cytokine-directed tissue-specific TCR repertoire formation probably reflects distinct TCR repertoire selection criteria for gd and ab T cell lineages adopted for different antigen-recognition strategies^ref)
• gd T lymphocytes / (1÷5%): in the inner cortex. (CD1abcde⁺2 / LFA-2⁺3deg⁺4^-7⁺8ab^+/-10^-15^-19^-45 / B220 / LCARA^-RO⁺NKG2D⁺, z⁺h⁺TcRgd^hi)
• CD2 / LFA-2⁺3deg⁺4^-5⁺7⁺8ab^+/-10^-13^-15^-19^-, TcRgd^hi : 3÷4% blood lymphocytes ; 50% intraepithelial T lymphocytes (IELs) in skin, intestinal and pulmonary epithelium. They act as a first line of defense against infections and cancers on the basis of their ability to respond directly to soluble protein and non-protein antigens of endogenous origins (e.g. hsp). In many animal species, gd T cells are the first lymphocyte lineage to be generated during fetal development : although theoretically ~ 10²⁰ different TcR could be realized, an enormous selective pressure is exerted on the development of gd T cells throughout life to produce populations of cells that express TcR encoded by specific gene segments. The human gd T cell repertoire has the greatest diversity during the prenatal stage of development : after birth and up to 6-10 years age there is a progression to oligoclonality (human Vg gene nomenclature is that of Lefranc). Intrinsic molecular constraints - rather than antigen selection - limit the generation of functional receptors in these populations :
• Vg3 gd T cells can be generated by fetal liver hematopoietic stem cells (FL HSC), but not by adult long-term reconstituting HSC (LT-HSC).
• Vg4 gd T cells can be generated by fetal liver hematopoietic stem cells (FL HSC), but not by adult long-term reconstituting HSC (LT-HSC).
• VgX-Vd1gd T cells use CD31 / PECAM1a for transendothelial migration (TEM). In bowels (intestinal IELs (iIELs / i-IELs) : 25-60% all gut T lymphocytes are partially activated CD8aa⁺44^lo/int45 / B220 / LCARB^hi103 / a_E integrin⁺134 / OX40^lo178 / Apo1L / FasL^loLy-6C^lo : they express high levels of FasL gene whose surface expression is controlled at posttranscriptional level) their TcR binds to MICA and MICB expressed on enterocytes (binding poorly occurs also through the costimulatory receptor NKG2D - as for NK cells - which anyway doesn't deliver any costimulatory signal 2). Other clones bind to lipohexapeptides (during Lyme arthritis), HLA-A2, HLA-A24 and CD1C. Most have naive phenotype and no canonical sequence motif. Vd1-expressing T lymphocytes are often clonally expanded in human transplant patients that are infected with HHV-5 / HCMV, a virus that is known to cause the upregulation of MIC expression by infected cells.
• Vg9-Vd2gd T cells (> 97% all human gd T cells !)use CD161 / NKRP1a for transendothelial migration (TEM). They have unique reactivity toward :
• organic phospholigands / monoalkyl phosphates from Mycobacterium spp., Escherichia coli, Plasmodium falciparum, and Francisella tularensis.
• isopentenyl pyrophosphate (IPP)
• prenyl phosphates
• nonphosphorylated alkylamines
• nucleotide conjugates
• bisphosphonates (zoledronate and pamidronate) (during tuberculoid leprosy)
• SEA
• mycobacterial Hsp65_180-188 / tetanus toxoid_1229-1252 + HLA-DRw53 (during rheumatoid arthritis)
• GroEL homolog on Burkitt's lymphoma cells
As cell activation requires cell-to-cell contact, the phosphoantigens might be presented through some conserved, as yet unidentified, surface molecule. They produce T_h1-type cytokines : TNF-a, IFN-g and MIF (the latter indirectly increases TNF-a and IL-1b secretion, counteracting the inhibitory effect of glucocorticoids). They represent 100% lymphocytes in fetal liver and 5% total adult peripheral blood lymphocytes. They are considered to be innate lymphocytes, although in the perinatal period sequences express N-diversification and cells have a naive phenotype (CD45 / B220 / LCARA⁺). They express NKG2D for reception of costimulatory signals from MICA and MICB, which are not sufficient to activate these cells. Some recognize CD1C.
They don't undergo thymic education : so they are not MHC-restricted and inherently self-reactive. Anyway normal gdlymphocyte development is dependent on the development of conventional ab T lymphocytes. Self-reactivity is regulated by the transient expression of antigens after stress, injury or infection, restricted tissue distribution of antigens and any co-stimulatory signals, high-threshold antigen density for activation and short half-life. Recognition of antigen is not always dependent on direct binding to the gd TcR. They may act as either helper (with a T_h1 > T_h2 cytokine release profile) or killer cells (even on activated macrophages). They have pro-inflammatory functions early in disease, but anti-inflammatory during the late stage of disease.
... or ...

+ the ATP-dependent chromatin remodeller chromodomain helicase DNA binding protein 4 (CHD4) / Mi-2b — which was previously thought to be a negative regulator of gene expression — is a positive regulator of CD4 expression associating directly with the Cd4 proximal enhancer through its interactions with the E-box-binding protein TCF12 / HEB^ref

• ab prethymocyte / cortical or common double-positive (DP) thymocyte ( > 95%) :

(CD1abcde⁺2 / LFA-2⁺4⁺5⁺7⁺8ab⁺10⁺15^-19^-45 / B220 / LCARA^-RO⁺, TcRab^low + cCD3deg)
They represent 80% of all thymic cells and are located in the inner cortex, where a first, partial negative selection occurs : those DP thymocytes that bind with high avidity to the most abundant self peptides expressed by cortical epithelial cells are deleted. DP cells regulate the differentiation of early thymocyte progenitors and gd T lymphocytes, by a mechanism dependent on the transcription factor RORgt, and the lymphotoxin (LT)  receptor (LTR)^ref.
‡
+ Egr1, c-Myb^ref
• mature or medullary thymocyte

(CD1^-2 / LFA-2⁺3deg⁺4⁺5⁺7⁺8ab⁺10^-11a^-13^-15^-18 / LFA-1⁺19^-28⁺45 / B220 / LCARA⁺RO^-49d/VLA-4^-152/CTLA-4^-154/40L/gp39^-, TcRab^hi). SLAP links the E3 ligase activity of c-Cbl to the TCRz, allowing for stage-specific regulation of TCR expression via ubiquitination and degradation, preventing the accumulation of fully assembled recycling TCR complexes^ref.
In the cortex they undergo positive selection / self-MHC restriction : according to the "strenght of signal" paradigm, T cells with TcR capable of weakly binding self-MHC - a.k.a. restriction elements -  expressed on cortical thymic epithelial cells (cTECs) continue to proliferate and differentiate, while the others - i.e. those with no or strong TcR signals - undergo programmed cell death (PCD) in the thymus within 3 or 4 days of their last division^{ref1, ref2, ref3, ref4, ref5, ref6}. T cells with high-affinity class II reactive ab TCRs are functional, but there is an affinity threshold above which an increase in affinity does not result in significant enhancement of ab T cell activation^ref. Calcineurin regulatory subunit (Cnb1) is essential^ref.
The specificity can undergo several changes as the a-chain genes continue to rearrange. The ability of a single developing thymocyte to express several different rearranged a-chain genes during the time that it is susceptible to positive selection must increase the yield of useful T cells significantly; without this mechanism many more thymocytes would fail positive selection and die. However, this continued rearrangement of a-chain genes also makes it likely that a significant percentage of T cells will express 2 receptors, sharing a b chain but differing in their a chains. Indeed, one can predict that if the frequency of positive selection is sufficiently low, roughly 1 in 3 mature T cells will have 2 a chains at the cell surface. This was confirmed recently for both human and mouse T cells. T cells with dual specificity might be expected to give rise to inappropriate immune responses if the cell is activated through one receptor yet can act upon target cells recognized by the second receptor. However, only 1 of the 2 receptors is likely to be able to recognize peptide presented by a self MHC molecule as, once the cell has been positively selected, a-chain gene rearrangement ceases. Thus, the existence of cells with 2 a-chain genes productively rearranged and 2 a chains expressed at the cell surface does not seem to challenge the importance of clonal selection, which depends on a single functional specificity being expressed by each cell.
If the binding specificity of the unselected repertoire were completely random, only a very small proportion of thymocytes would be expected to recognize an MHC molecule. However, it seems that the variable CDR1 and CDR2 loops of both chains of the TcR, which are encoded within the germline V gene segments, give the TcR an intrinsic specificity for MHC molecules. This is evident from the way these 2 regions contact MHC molecules in crystal structures. An inherent specificity for MHC molecules has also been shown by examining mature T cells that represent an unselected repertoire of receptors. Such T cells can be generated in fetal thymic organ cultures, using thymuses that do not express either MHC class I or MHC class II molecules, by substituting binding of anti-b-chain antibodies and anti-CD4 antibodies for the receptor engagement responsible for normal positive selection. When the reactivity of these antibody-selected CD4 T cells is tested, roughly 5% are able to respond to any one MHC class II genotype and, because they developed without selection by MHC molecules, this must reflect a specificity inherent in the germline V gene segments. This germline-encoded specificity for MHC should significantly increase the proportion of receptors that can be positively selected in any one individual
Naive thymocytes are highly motile at low intracellular calcium concentrations, but during positive selection cells become immobile and show sustained calcium concentration oscillations. Increased intracellular calcium is necessary and sufficient to arrest thymocyte motility. The calcium dependence of motility acts to prolong thymocyte interactions with antigen-bearing stromal cells, promoting sustained signaling that may enhance the expression of genes underlying positive selection^ref.
T cells that survive positive selection traverse the corticomedullary junction and interact with APCs (mainly with bone marrow-derived DCs and macrophages and to a lesser extent with medullary thymic epithelial cells (mTECs)) : T cells with a high-affinity TcR for self-MHC (alone or combined with a thymus-derived self-peptide) are (negative selection)^{ref1, ref2, ref3} ... :
• deleted by activation-induced cell death (AICD) (in a Bim-, Bak1-, Bax- and LIGHT / TL5-dependent manner)
• or undergo a variety of nondeletional mechanisms, including :
• down-modulating the levels of coreceptor molecules
• anergy (blockade of the NFAT-AP1 interaction)
• raising the activation thresholds in T cells.
Ectopic gene expression in mTECs is triggered by LT-b or TNF-b / LT-a signaling via LTbR^ref and activation of theautoimmune regulator (AIRE), whose loss-of-function mutations cause APECED and Omenn syndrome). AIRE contains 2 plant homeodomains (PHDs) which have recently been shown in other proteins to facilitate polyubiquitylation of target proteins : PHD1 pof AIRE has E3 ligate activity, which is abolished by common disease-causing mutations. By contrast, PHD2 is responsible for transcriptional repression. It remains to be determined whether the E3 ligase activity modifies proteins in the AIRE-containing transcriptional complex or has an independent effect^ref
Anyway it is likely that thymus doesn't represent all body self Ags => some self-reactive clones survive, requiring peripheral tolerance mechanisms.
The whole of the 2 selections is a.k.a. ab TcR checkpoint / thymic education / thymic selection, and it kills between 95% and 99% of all thymocyte progeny. Once the cell is committed to CD4⁺ or CD8⁺ lineage, the remaining locus remains silent even if the silencer element is removed. Finally they exit the thymus and travel to the periphery. Following export from the thymus, naive T cells require continued engagement by MHC molecules to survive in the periphery and for homeostatic expansion. Notably, T cells with different TcRs display major discrepancies in their ability to undergo homeostatic expansion. Thus, the T cell repertoire shaped in the thymus is subject to further molding in the peripheral lymphoid organs. The nature of the specific peptides involved in peripheral survival/expansion remains controversial. Peptides involved in peripheral expansion may be identical with those that support intrathymic positive selection, or be structurally different but sharing the same affinity or avidity. However the postselection T cell repertoire is anatomically secluded from the site in which it has been positively selected as, because of the hematothymic barrier, mature T lymphocytes never reenter the thymus cortex.
Mature T lymphocytes (10¹² in an adult human being) represent 70-80% of peripheral blood lymphocytes (but peripheral blood contains only 2% of total body lymphocytes), 90% of lymphocytes in thoracic duct, 30-40% of lymphocytes in lymph nodes (in paracortex), and 20-30% of lymphocytes in spleen  (in TCZ of PALS).
‡ + b-catenin (a target of TCR-CD3 signals in thymocytes and mature T cells)
They need ... :
... IL-2Rb, IL-15, IL-15Ra, Fyn, Ets1, Itk, IRF1, GM-CSFR, CD132 / g_c CD1D, sTNF-b and LT-b to become a :
(CD44^lo62L^-69⁺161 / NKR-P1A^-)
‡
(CD44^hi62L^-69⁺161 / NKR-P1A^-)
‡^ref
• natural killer T (NKT) cell (CD3deg⁺10^-13^-14^-15^-19^-44^hi62L^-/lo69⁺) express CD3, at least one NK marker, and a semi-invariant and self-reactive TcR. These cells constitute < 0.1% of the mononuclear cells and < 1% of the CD4⁺ T cells in the peripheral blood^ref
• category I (CD49B / integrin a₂^-161 / KLRB1 / NKR-P1A^+/-) : TcR rearrangement occurs randomly and the cells that bear the Va24Ja15 rearrangement (forming an invariant CDR3a, but having very variable CDR3b regions and preferential co-expression with Vb11) undergo positive selection by CD1D⁺CD4⁺8⁺ (DP) thymocytes (differential feature from positive selection of conventional T cells !). In fact they recognize :
• GD₃, atumour-associated disialoganglioside
• isoglobotrihexosylceramide (iGb3), a lysosomal glycosphingolipid of previously unknown function. Impaired generation of lysosomal iGb3 in mice lacking b-hexosaminidase b results in severe NKT cell deficiency, suggesting that this lipid also mediates development of NKT cells in the mouse. Expression of iGb3 in peripheral tissues may be involved in controlling NKT cell responses to infections and malignancy and in autoimmunity^{ref1, ref2}
• glycosphingolipids (glycosylceramides) from Gram-negative, LPS-negative a-Proteobacteria such as Ehrlichia muris and Sphingomonas capsulata^ref. In contrast, Gram-negative, LPS-positive Salmonella typhimurium activates NKT cells through the recognition of an endogenous lysosomal glycosphingolipid, iGb3, presented by LPS-activated dendritic cells. Glycosylceramides are an alternative to LPS for innate recognition of the Gram-negative, LPS-negative bacterial cell wall^ref
• other undetermined autologous glycolipid Ag(s) (mimicked by the unusual a-anomeric - but no the b-anomer ! - glycosphingolipid a-galactosylceramide (a-Gal-Cer) / KRN7000, originally discovered in the marine sponge Agelas mauritiana, and its synthetic analogues : < 1 nM is required to fully activate the Va24i population in vivo !)
• phosphatidyl-choline and phosphatidyl-ethanolamine extracted from cypress grains^ref
... presented by APCs in the context of the antigen-presenting molecule CD1D : the CD1D specificity relies on the TcRa-chain, which is normally acquired by developing thymocytes at the DP stage. Inhibitory NK receptors probably prevent unchecked autorectivity for CD1D. As the expression of CD161 / KLRB1 / NKR-P1A is acquired on maturity after activation in extrathymic tissues, they are referred just as Va24⁺ invariant T cells (Va24i oriNKT or NKT^inv). Expression of Aire in thymic medullary epithelial cells (MECs) is required for development of iNKT cells, suggesting a role for iNKT cells in APS1^ref. They are less frequent in humans than are the Va14i T cells in mice. They locate in thymus (where they are unable to respond due to interaction with the thymic stroma), peripheral blood (4% all ab T-cells), spleen (3%), liver (30÷50%; LFA-1-dependent migration), bone marrow, lungs (CCL2-dependent migration) and peripheral lymph nodes (0.5%). They all express CD122 / IL-2Rb : they are considered to be innate lymphocytes and killer cells. 4 subsets exist :
• CD4^-8^- (DN)
• CD4⁺ produce more IL-4 than DN ones, but are less able to induce expression of perforin after exposure to cytokines.
• CD8aa⁺ don't produce IL-4. CD8 acts as a coreceptor for CD1D.
• CD8ab⁺ have never been described, and are thought to be deleted (negative selection) during ontogeny due to their high binding avidity to CD1D : although this is an attractive hypothesis, the affinity of CD1D for CD8ab misured by biacore is several orders of magnitude lower than the affinity of classical MHC class I molecules for CD8ab : further it is about the same as for CD8aa.
Their number decreases in the peripheral blood in various autoimmune diseases (with the possible exception of psoriasis and the hyperplastic thymus in myasthenia gravis). They can be expanded very rapidly in vitro in presence of a-Gal-Cer, APCs+IL-2 or IL-7+IL-15. Only about 50% of the iNKT cells in the peripheral blood are CD4⁺ cells^ref : they preferentially produce IL-4 and IL-13^{ref1, ref2, ref3, ref4}. They can be identified with CD1d tetramers loaded with a-Gal-Cer, monoclonal antibody 6B11 (which specifically recognizes the CDR3 region of the V24-J18 T-cell receptor of human iNKT cells^ref).
• category II (CD49B / integrin a₂^?161 / KLRB1 / NKR-P1A⁺) has more diverse TcRs that bind undetermined ligands presented in the context of the antigen-presenting molecule CD1D. These cells are mainly located in thymus (?), liver, spleen and bone marrow (?). 2 subsets exist :
• CD4^-8^- (DN)
• CD4⁺
• category III (CD49B / integrin a₂^+/-161 / KLRB1 / NKR-P1A⁺) has more diverse TcRs that bind undetermined self ligands presented in the context of MHC class I or other molecules. These cells are mainly located in the spleen and bone marrow, compared with the thymus and liver.
• CD4^-8^- (DN)
• CD4⁺
• CD8aa⁺ don't produce IL-4. CD8 acts as a coreceptor for CD1D.
• category IV (CD49B / integrin a₂⁺161 / NKR-P1A^+/-) has more diverse TcRs that bind undetermined ligands presented in the context of MHC class I or II molecules. These cells are mainly located in thymus (?), liver, spleen and bone marrow.
• CD4⁺
• CD8aa⁺ don't produce IL-4.
• Va2/Vb21 T-cell line is specific for small sulphated polyaromatic structures, and the magnitude of the T-cell response induced by these structures varies depending on their pattern of hydroxylation and methylation. Although these antigens are not physiological, they have similar chemical features to several commonly used antibiotics and anti-inflammatory drugs known to induce strong hypersensitivity reactions in humans, suggestiong that these drugs might function as NKT-cell antigens^ref.
See also activation of NKT lymphocytes.
• mucosal-associated invariant T (MAIT) cells : hVa7.2-Ja33 or mVa19-Ja33 TCR rearrangement are preferentially located in the gut lamina propria of humans and mice. Selection and/or expansion of this population requires B lymphocytes, as MAIT cells are absent in b₂-microglobulin or B-cell-deficient patients and mice. In addition, we show that MAIT cells are selected and/or restricted by MR1, a monomorphic MHC class I-related molecule that is markedly conserved in diverse mammalian species and can stimulate cytokine release from MAIT cells. MAIT cells are not present in germ-free mice, indicating that commensal flora is required for their expansion in the gut lamina propria, while are more abundant in mice that lack TAP or Ii, in which the number of classical T lymphocytes is low because of the reduced expression of MHC class I and II molecules. This indicates that MAIT cells are probably involved in the host response at the site of pathogen entry, and may regulate intestinal B-cell activity^ref.
... or TcRa, CD4, CD3d, MHC II, invariant chain, b2m, 45 / B220 / LCA, CD86, ZAP70, RasGRP, Vav1, Csk, Itk, GADS, Id3, GATA3^ref and ERK1 to become a :
• precursor of T-helper or T-inducer cells (T_hp) or single-positive (SP4) resting (naive) lymphocyte (CD2 / LFA-2⁺3deg⁺4 / Lyt1⁺5⁺8^-10^-13^-14^-15^-19^-24 / HSA^lo25^-29⁺45 / B220 / LCARA⁺69⁺NKG2D^-, TcRab^hi)

‡ c-Myb^ref
• CD4⁺ T-helper (T_h0) or T-inducer cell (CD2 / LFA-2⁺3deg⁺4⁺5⁺8^-10^-13^-14^-15^-19^-45 / B220 / LCARO⁺62L / MEL-14⁺69⁺90 / Thy-1⁺, x⁺h⁺, TLR3⁺9^+ref     TcRab^hi). They produce low levels of all the cytokine kinds produced by T_h1, T_h2 and T_Reg, but expecially IL-2, IL-4 and IFN-g. See also activation of naive CD4⁺ T_h0 lymphocytes.

... or FOXP3 ... to become a ....
• naturally occurring suppressor / (negatively) regulatory T cells (T_reg), continuously produced in thymus :
• CD4⁺19^-25^hi45 / B220 / LCARB^lo103 / a_E integrin⁺/ 152 / CTLA-4⁺ CCR4⁺CCR8⁺TNFRSF18 / GITR⁺T_R1 lymphocyte  (10% of total CD4⁺ T lymphocytes). Differential regulatory capacities of the CD62L / L-selectin^hi and CD62L / L-selectin^lo subsets in vivo reflect differences in homing properties, rather than suppressor potential per se. They secrete :
• large amounts of IL-10 (=> control of inflammatory reactions)
• CD38^- subset  produce TGF-b₁ and/or IL-4
• CD38⁺ subset : they don't produce neither TGF-b₁ nor IL-4
Found even in Peyer's patches.
The differentiation, acquisition of functional capacity and formation of a sizeable pool of suppressor T cells in the thymus (including induction of Foxp3 expression in thymocytes) is independent of IL-2 signaling, but that interleukin 2 was essential for the survival of mature Foxp3⁺ T_reg cells in vivo^{ref1, ref2}. In vitro, they fail to produce IL-2 after TcR stimulation, suppress both CD4⁺ and CD8⁺ T cell responses (even to an unrelated Ag !), but require TcR stimulation for induction of their suppressor function via mechanism depending on the location and context of the inflammatory response that they are called upon to suppress :
• direct suppression :
• IL-10- and TGF-b₁-dependent mechanism
• T-cell-T-cell contact-dependent, IL-4-, IL-10- and TGF-b₁-independent mechanism that suppress IL-2 and IFN-g production in the responder population.
• indirect suppression :
• inhibit the upregulation of expression of co-stimulatory molecules on APCs required for the activation of CD25^- T cells and, thereby, indirectly inhibit the induction of IL-2 production and the proliferation of the CD25^- responder T cells
• regulation of immunosuppressive tryptophan catabolism in DCs
• CTLA-4-dependent mechanism requiring B7 expression and cytokine production by the DCs. CTLA-4 upregulation leads to enhanced IFN-g production, which in turns upregulates transcription of the gene encoding indoleamine 2,3-dioxygenase (IDO) in DCs, the enzyme that catalyses the initial step of tryptophan degradation, linked with tolerance induction during pregnancy, transplantation and autoimmunity^ref. Ttryptophan metabolites in the kynurenine pathway, such as 3-hydroxyanthranilic and quinolinic acids, will induce the selective Fas-independent apoptosis in vitro and in vitro of murine thymocytes and T_h1 but not T_h2 cells, in a fashion qualitatively similar to dexamethasone^ref
• T_Reg cells cultured in the presence of bacterial LPS induce tryptophan catabolism by DCs in a CTLA-4-independent but cytokine-dependent way.
Probably they arise in thymus as T cells that recognize self-antigens with an intermediate affinity and escape an altered negative selection receiving a signal that renders them anergic and suppressive. They require a crucial IL-2 signal in the thymus for development (and also in vitro for expansion), but can be maintained for long times in the periphery by other cytokines, such as IL-4, the production of which is dependent on co-stimulatory signals. They are resistant in vivo and in vitro to Fas-induced activation-induced cell death (AICD), including clonal deletion following viral SAg-induced activation : even if sensitivity to apoptosis can be increased by contact with IL-2-producing CD4⁺25^- T cells in vitro, this resistance may be important in maintaining a permanent population of regulatory T cells that can control and eventually suppress autoreactive T cells or harmful T cells activated during normal immune responses. By allowing the persistence of parasites, IL-10 secreting T_reg cells might benefit the host by maintaining protective immunity.
• CD4⁺25^-45 / B220 / LCARC^-T lymphocyte. Although they are less potent on a cell-for-cell basis, they contribute to at least an equal extent because of their greater numerical representation (90%) in the CD4⁺ T-cell population.
CD27 expression has been proposed as a new surface marker^ref : it binds to N-terminal part of SIVA / CD27BP, a pro-apoptotic protein^ref.
Unseparated CD4⁺ T cells from tolerant mice are more potent at suppression than either CD4⁺CD25⁺ or CD4⁺CD25^– populations alone, which indicates a synergistic interaction between the 2 types of CD4⁺ T_Reg cell. It is also important to note that characterization of the potency of such T_Reg-cell populations has not been carried out in T-cell-replete animals, and so effects of homeostasis have not been excluded. Moreover, the demonstration that CD4⁺CD25⁺ T_Reg cells are antigen specific requires evidence of specific regulation in a 'criss-cross' experiment : CD4⁺CD25⁺ T_Reg cells tolerant of type-A grafts should allow rejection of type-B grafts, and vice-versa. Until this type of experiment is carried out, specificity cannot be claimed from one-way studies. Furthermore :
• naive (donor antigen-inexperienced) CD4⁺CD25⁺ T_Reg cells can suppress rejection in the adoptive-transfer systems
• CD4⁺CD25⁺ T_Reg cells from mice that are tolerant of allografts are not markedly more potent than CD4⁺CD25⁺ T_Reg cells from naive mice
Alternative hypotheses should be considered :
• antigen-specific regulation is an exclusive property of CD25^– T_Reg cells, whereas CD4⁺CD25⁺ T_Reg cells are not important for antigen-specific regulation but rather act in a non-specific manner that is irrelevant to tolerance
• CD4⁺CD25⁺ T_Reg cells can act on both effector T cells and antigen-specific CD25^– T_Reg cells, modulating their function in an antigen-nonspecific manner. Specificity would come from CD25^– T_Reg cells. In this way, the CD4⁺CD25⁺ T_Reg cells could act in concert with the CD25^– T_Reg cells to give an overall antigen specificity when the system is taken as a whole.
Apart from naturally occurring T_Reg cells that are continuously produced from thymus, other sets of regulatory T-cells can be obtained in vitro pulsing BMDC with Ag under certain conditions (whose in vivo counterpart is usually unknown) :
• 1) several days with ...
• IL-10(=> down-regulates CD80and CD86)
• IFN-a
• CD2 --- CD58 / LFA-3 interaction
• CD3deg
• CD46 / MCP
=> CD4⁺10^-13^-14^-15^-19^-25⁺27⁺45 / B220 / LCARA⁺RB^lowRO^-152 / CTLA-4⁺LIGHT⁺ that produces :
• high levels of IL-5
• high levels of IL-10 (autocrine stimulation)
• high levels of IFN-g
• high levels of TGF-b₁, which inhibits T_h1 differentiation, stimulates T_h2 differentiation and suppresses in vitro proliferation and IFN-g secretion by CD8⁺ T cells
• low levels of IL-2
• no IL-4.
They can both enhance or suppress immune responses. Activation is Ag-dependent.
• 2) CD40^-BMDC_imm (so IL-10 independent) :
• naturally occurring DC_imm
• BMDC treated with NFkB translocation inhibitors (e.g. BAY)
• RelB^-/- BMDC
• CD40^-/- BMDC
=> CD4⁺25^- T lymphocytes
‡
+ TGF-b₁
CD4⁺25⁺152 / CTLA-4⁺ cells : non-proliferating, IL-10-producing. Suppressive activity requires cell-cell contact and is APC-independent and Ag-independent. When transferred into naive wild-type mice they can suppress previously primed immune response
In experimental systems that are used to test for regulatory functions, regulation could not be in fact a special job, but rather a competition between normal T lymphocytes for...
• IL-2 in in vitro co-culture assay of naive responder cells and suppressor cells. It does not show any involvement of inhibitory cytokines such as IL-10 or TGF-ß.
• self-peptide-MHC molecules and IL-7 in in vivo transfer of small numbers of naive T cells to severely lymphopenic hosts, which then generate exaggerated immune responses resulting in immune pathology. Lymphopenia is invariably associated not only with homeostatic expansion of the remaining (or adoptively transferred) lymphocyte populations in response to endogenous peptide–MHC ligands and IL-7, but also with immune reactivity to both self and foreign determinants that would not normally be apparent in T-cell-replete hosts. Those T lymphocytes with higher avidity for self-peptide-MHC molecules (CD5⁺) have increased competitive (and hence suppressive) potential. Anyway IL-7^-/- mice have a robust regulatory FOXP3⁺ CD4⁺ T cell compartment that prevents T-cell mediated disease. Regulatory FOXP3⁺ CD4⁺CD25^- T cell population has the potential to restore a functional CD4⁺CD25⁺ T cell compartment through an IL-7 independent pathway^ref.
Antigen-dependent killing of B lymphocytes : activated CD4⁺CD25⁺ T cells could inhibit proliferation and Ig secretion of LPS-activated B cells, but the mechanisms of action remained unclear. Activated T cells are able to inhibit B-cell proliferation by directly inducing apoptosis of the proliferating B cells themselves. This mechanism is quite different from how T_regs control T-cell proliferation, in which T_regs prevent IL-2 production by proliferating T cells. CD4⁺CD25⁺ T cells do not induce B-cell apoptosis through the pathways that would normally be associated with lymphocyte-lymphocyte interactions (ie, Fas/FasL, TNF/TNFR, or TRAIL/TRAILR). Instead, the apoptotic event requires a combination of granzyme B and perforin activities. The requirement for these 2 pathways is reminiscent of the manner in which CD8 CTLs kill their targets through MHC class I–dependent antigen recognition. In fact, activated CD4⁺CD25⁺ T cells produce and release granzyme B similarly to CD8⁺ T cells, an activity not found in CD4⁺CD25^– T cells. B-cell apoptosis mediated by CD4⁺CD25⁺ T cells can be antigen-dependent. CD4⁺CD25⁺ T cells from mice bearing a transgenic TCR that recognizes an ovalbumin peptide were used for these experiments. Proliferating B cells were divided into 2 separate groups, one pulsed with ovalbumin and one unpulsed. Ovalbumin-specific CD4⁺CD25⁺ T cells were dramatically more efficient at killing antigen-pulsed B cells, demonstrating that this T_reg activity is indeed antigen dependent^ref.
... or TcRa, CD8ab, b₂m, CD3d, TAP1, 45 / B220 / LCA, IRF1, Id3, ERK1, ZAP70, RasGRP, Vav1, Itk and GADS to become a :
• single-positive (SP8) resting (naive) cytotoxic T-lymphocyte (CTL or T_c)  or T-killer (T_K)(CD2 / LFA-2⁺3deg⁺4^-5⁺8a / Lyt2⁺b / Lyt3⁺10^-11b^-13^-14^-15^-19^-45 / B220 / LCARA⁺57⁺62L / MEL-14⁺69⁺, x⁺h⁺, BLT₁ / P2Y₇^lo, NKG2D-S^-, NKG2D-L^-, TcRab^hi). See also activation of naive CTLs.

Prevailing knowledge dictates that naive ab T cells require activation in lymphoid tissues before differentiating into effector or memory T cells capable of trafficking to nonlymphoid tissues. CD8⁺ recent thymic emigrants (RTEs) migrated directly into the small intestine. CCR9, CCL25 and a₄b₇ integrin were required for gut entry of CD8⁺ RTEs. After T cell receptor stimulation, intestinal CD8⁺ RTEs proliferated and acquired a surface phenotype resembling that of intraepithelial lymphocytes. CD8⁺ RTEs efficiently populated the gut of LT-a-deficient mice, which lack lymphoid organs. These studies challenge the present understanding of naive ab T cell trafficking and suggest that RTEs may be involved in maintaining a diverse immune repertoire at mucosal surfaces^ref.
... or several days of stimulation with Ag and IL-10 (with or without synergizing by IFN-a) to become a ...
• veto cell (CD8ab⁺10^-14^-15^-) : a subset of suppressor cells that are passively recognized by autoreactive CTLs that recognize MHC on the veto cells; this one-way recognition results in the elimination of the autoreactive CTLs. They inactivate every CTL that attempts to kill them after being activated by self Ags => they protect from autoimmune reactions suppressing T_h1 immunity. Veto activity was defined in 1980 by Miller^ref as the capacity to specifically suppress cytotoxic T-cell precursors (CTL-p) directed against antigens of the veto cells themselves but not against third-party antigens. Interestingly, it has been shown that some of the most potent veto cells are of T-cell origin and, in particular, a very strong veto activity was documented for CD8⁺ CTL lines or clones^{ref1, ref2, ref3, ref4, ref5, ref6, ref7}. The specificity of the veto effect mediated by CTL clones was shown by several studies to be unrelated to their TcR specificity^{ref1, ref2, ref3}. The suppression of effector CTL-p directed against the veto cells is both antigen specific and MHC restricted, resulting from the unidirectional recognition of the veto cell by the responding CTLs, but not vice versa^ref. Furthermore, it has been shown that this suppression is mediated by apoptosis^{ref1, ref2} and that coexpression of both CD8 and Fas ligand (FasL) is a prerequisite for the veto reactivity of the CTLs^ref. Veto CTLs induce apoptosis in the effector T cells by a Fas-FasL–mediated mechanism. Upon engagement between the TCR of the effector cell and class I (MHC I) of the veto cell, the effector cell is activated and Fas is up-regulated, allowing for the FasL on the veto CTL to induce apoptosis. However, inhibitors such as FLIP protect the activated effector T cell. The high affinity afforded by the interaction between CD8 on the veto cell and class I (MHCIa3) on the effector cell enables prolonged association until FLIP is down-regulated and apoptosis can take place.
Normal blood contains 1000÷2500 T cells / mL = 5÷12.5 ^. 10⁹ T cells
• CD4⁺ T cells are 500÷1600 / mL = 2.5÷8.4 ^. 10⁹ CD4⁺ T cells
• normal T_h-to-T_s ratio = 2:1
• CD8⁺ T cells are 300÷900 / mL = 1.5÷4.5 ^. 10⁹ CD8⁺ T cells
So normal CD4⁺/CD8⁺ T-cells ratio in blood is 1.5.

Ins(1,4,5)P₃ 3-kinase isoform B (ITPKB) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P₄) define an essential signaling pathway for T cell precursor responsiveness and development.
Several different lines of evidence demonstrate that thymus T cell production does not determine the number of peripheral T cells. First, in the young adult mouse, T cell production in the thymus largely exceeds the number of cells required to replenish the peripheral T cell pools. Mice manipulated to have reduced rates of thymus T cell production can attain normal peripheral T cell numbers. Secondly, in mice grafted with multiple thymuses, the increased thymus mass and T lymphocyte production does not lead to the proportional increase of the peripheral T cell production capacity. Peripheral T cells in absence of the thymus in thymectomized hosts or when transferred into T cell-deficient hosts are capable of considerable expansion. In a normal mouse, there are mechanisms that control both T cell survival and division in the peripheral pools and keep T cell numbers constant. It has been proposed that competition for resources or complex cell interactions play a role in lymphocyte homeostasis. However, the mechanisms involved remain elusive. In mice, at least, there is evidence of extrathymic T cell development. The model for this are the nude mouse, which is congenitally athymic, or athymic radiation chimeras reconstituted with normal hemopoietic precursors. These mice initially have no T cells (and are often used as recipients of grafts of human cells and tissues for experiments), but as they age, one can find fully functional TcRgd⁺ T cells. No one yet knows where these cells arise. Bone marrow and the appendix (the only 2 other sites where RAG is only known to be expressed) may be the sites of extrathymic T cell development. It is likely that a fourth (or more) site is where extrathymic T cell development occurs. There is no evidence of T cell development in the blood, spleen or lymph nodes.
The survival of naive T lymphocytes requires continuous ...
• ... contact with self peptides bound to MHC molecules
• ... exposure to IL-7, which maintains the activity of anti-apoptotic (anti-death) pathways. However, the limiting amounts of IL-7 that are available relative to the large number of T cells suggests that a mechanism must exist that enables cells to compete successfully for this resource without risking the loss of antigenic diversity as represented by the whole T cell population. IL-7 ligation induces down-regulation of IL-7Ra via activation of the transcriptional repressor growth factor independent 1 (GFI1)^ref
In consort, these 2 ligands are presumed to induce a form of low-level signaling that is sufficient to keep the cells alive but does not induce them to enter the cell cycle.
The size of the naive peripheral lymphocyte pool is the result of constant and dynamic modulation reflecting the interplay between lymphogenesis in primary lymphoid organs, selection of newly emergent cells into the long-lived pool, survival in the periphery, and depletion following antigenic encounter. Despite these conflicting forces, naive lymphocyte populations are maintained at remarkably constant numbers by poorly understood homeostatic mechanisms :
• lymphocyte survival, which is currently considered the dominant mechanism, is an active process requiring signals delivered through cell surface receptors that control the expression of antiapoptotic molecules
• in B cells : BcR and BAFF-R, requires Btk, c-Rel, and p50^NFkB1
• in T cells : TcR and IL-7R
It has been proposed that competition between lymphocytes for ligands that promote survival is a major regulatory control on lymphocyte numbers.
• Ag-independent homeostatic proliferation in lymphopenic hosts
• in B cells : T cell independent, requires Btk
• in T cells : requires peptide-MHC complexes, IL-7, and p56^LCK
 
• Ag-specific receptors in the adaptive immune system are all type I transmembrane proteins. The major part of involved proteins arise from duplication and divergence of a Ig precursor gene, coding for a 110 amino acids-long p12^{Ig domain} : so the actual molecules contain a natural multiple of such domain. The response induced upon antigen binding doesn't depend on antigen structure.See also Database of Sequences of proteins of Immunological Interest.
• receptors binding Ag intracellularly : the human leukocyte antigens (HLAs) of the major histocompatibility complex (MHC). MHC diversity is a balance between evolutionary pressure to increase diversity for recognition of a greater number of potential pathogens and evolutionary presure to decrease the number of T lymphocytes that are deleted during negative selection. Receptor variability is high in a species but low in the organism, so that some Ags may not to contain peptides that can bind any receptor and so the host is not protected. On the other hand, individuals that have some MHC alleles can be induced to have an adaptive artificial immune response protective against some Ags (e.g. melanoma-associated MAGE Ag is recognizable by HLA-A2 individuals). Previous studies looking at the structure of MHC molecules have used recombinant forms consisting of only the extracellular domains. However, these structures do not take into account interactions with specific lipid microdomains of the plasma membrane. When electron microscopy is used to analyse the extracellular domains of the MHC class I molecule H–2K^b tethered to a lipid bilayer by a histidine tag, the MHC molecule is shown to lie on its side parallel to the membrane, rather than standing up perpendicular to the membrane as is usually depicted. This orientation is maintained by ionic interactions between lipid head groups in the membrane and the length of the MHC protein. As the supine orientation has been shown to be optimal for binding of the co-receptor CD8, changes in MHC orientation might be another way to regulate T-cell-receptor signalling^ref
• classical MHC class I (a.k.a. MHC class Ia)molecules : invariant p12^{b2-microglobulin} (b₂m : chromosome 15; it pairs with the a₃ domain of p45) + p45^{a chain / heavy chain} (cytoplasmic tail, TM, a₃, a₂ and a₁ domains : the latter 2 domains pair each other to form the platform domain) with extraordinary polymorphism within the Ag recognition site (ARS) :
• HLA-A : 100 alleles
• HLA-B : 200 alleles
• HLA-C : 50 alleles, tissue expression level are far lower than those of HLA-A and HLA-B
• group 1 HLA-C molecules (HLA-C1 : containing Ser⁷⁷Asn⁸⁰ : e.g. HLA-Cw1, HLA-Cw5, and HLA-Cw7)
• group 2 HLA-C allotypes (HLA-C2 containing Asn⁷⁷Lys⁸⁰ : e.g. HLA-Cw2, HLA-Cw4, and HLA-Cw6)
In general, MHC class I genes seem to be more divergent and more rapidly evolving than class II genes. HLA class I lineages are only recognized in great apes and thus maintained up to 6 million years, while certain HLA class II lineages were recognized in prosimians that diverged from human ~ 85 million years.
They are expressed on all cells except RBCs, but are at lower density on neurons, hepatocytes, renal cells, thymocytes, syncitiumtrophoblast, germinal cells and aggressive cancer cells; on the contrary they are found at higher density on leukocytes. Expression is up-regulated by IFN-g via IRF1. They consist of 6 pockets termed A to F : B, C, D, and E pockets are placed at the a-helix-b-fold hunction, while A and F pockets are placed between a-helices. In Golgi apparatus the N-terminal oligosaccharide is further processed.
The cleft formed by a₂ and a₁ domains binds 8÷11 amino acids-long peptides (in a niche of 30 Å x 12 Å, slightly longer in HLA-C) primarily derived from endogenously synthetised proteins from ...
• self (=> self-tolerance or autoimmunity (including tumor immunity)), including epitopes created by post-translational protein splicing (e.g. FGF5 peptide consisting of 5 amino-terminal amino acids and 4 carboxy-terminal amino acids^ref)
• nonself (=> defense from cytosolic pathogens or rejection to minor histocompatibility antigens)
... and processed by specific cytosolic proteases :
• cytosolic 26S proteasome : b₁, b₂ and b₅ subunits are amino-terminal nucleophile (NTN) hydrolases with Thr in active sites. During de novo assembly induced by IFN-g they are totally or partially replaced by the immunosubunits:b₉ / large multifunctional protease 2 (LMP2), is incorporated into newly synthetised proteasomes as b₁i (in place of d), b₇ / LMP7 as b₅i (in place of X), and b₁₀ / MECL-1 as b₂i (in place of Z) to produce so-called immunoproteasomes (their incorporation is cooperative, e g. b₁i before b₂i). Even TNF-a can up-regulate the LMP2 gene, either alone or synergistically with IFN-g. The immunosubunits alter whole proteasome stucture and allow higher efficiency in substrate channelling within the proteasome and cleavage specificity, thereby changing both the quality and the quantity of peptide products. IFN-g-induced coincident biosynthesis of proteasome maturation protein (POMP) and LMP7 and their direct interaction essentially accelerate immunoproteasome biogenesis compared with constitutive 20S proteasome assembly. The dynamics of this process is determined by rapid LMP7 activation and the immediate LMP7-dependent degradation of POMP. Silencing of POMP expression impairs recruitment of both b₅ subunits into the proteasome complex, resulting in decreased proteasome activity, reduced MHC class I surface expression, and induction of apoptosis. Furthermore, immunoproteasomes exhibit a considerably shortened half-life, compared with constitutive proteasomes, allowing cells to return rapidly to a normal situation once immunoproteasome function is no longer required^ref.

[immunoproteasome formation is delayed by the endogenous 31 kDa proteasome inhibitor (PI31), up-regulated by Ad5 to affect presentation of the immunoproteasome-dependent epitope E1B].
IFN-g induces the synthesis of a 11S regulator / PA28 (made up of PA28a and PA28b in a a₃b₄-arranged complex plus PA28g), enhancing peptide presentation in a immunosubunits-independent manner. In fact in vivo the rate-limiting step in protein degradation is not the width of the gate but substrate binding to the 19S regulator, as well as its channelling into the 20S core. Anyway because product exit from the proteasome occurs by diffusion, the width of the gate will determine the diffusion rate : in a situation in which a gate is not fully open, the retention time of a processing intermediate in the catalytic chamber will be increased and the exit of larger peptide products will be hindered (just like in a food-processor, the length of time that the ingredients are processed affects the quality of the product !). The proteasome generates peptides ranging from 3 to 22 amino acids in length, but a relatively larger percentage of antigenic peptides are generated by immunoproteasomes as precursor peptides of 10-12 residues. Because some processed peptides have residues close to their amino termini (Pro, for example) that impair their TAP-mediated transport into the ER, by creating products with an amino-terminal extension the proteasome allows these epitopes to pass unhindered into the ER, where they can be trimmed to the optimum size for binding to MHC class I groove by the IFN-g-inducible ER aminopeptidase involved in antigen processing (ERAAP) (in some rare cases, cytosolic trimming might also take place). Loss of ERAAP disrupts the generation of naturally processed peptides in the ER, decreases the stability of peptide–MHC class I complexes and diminishes CD8⁺ T cell responses. Thus, trimming of antigenic peptides by ERAAP in the ER is essential for the generation of the normal repertoire of processed peptides^ref.
Local variations in proteasome composition might regulate organ-specific autoimmunity, representing an important mechanism to control reactivity of autoreactive CD8⁺ T cells that escape thymic delection.
• other proteases^{ref1, ref2, ref3} : due to their broad cleavage specificity, these proteases can generate only a limited subset of peptides that are appropriate for MHC class Ia binding, and therefore cannot be important for the production of antigenic peptides or substitute for proteasome function, even when expressed together. The same is true for their role in cytosolic trimming of proteasome-derived peptides. The main proteases involved are :
• puromycin-sensitive aminopeptidase (PSA)
• bleomycin hydrolase
• leucyl aminopeptidase (LAP)
• tripeptidyl aminopeptidase II (TPP II) (endoproteolytic and exoproteolytic activities)^ref1,ref2
• furin (found predominantly in the TGN from where it cycles via the cell surface)
• thimet oligopeptidase 1
Peptidases can also destroy epitopes by trimming peptides to below the size needed for presentation. In the cytosol, endopeptidases, especially thimet oligopeptidase, and aminopeptidases degrade many proteasomal products, thereby limiting the supply of many antigenic peptides. Thus, the extent of antigen presentation depends on the balance between several proteolytic processes that may generate or destroy epitopes^ref.
Functional translation from a non-AUG initiation codon, and MHC class I presentation of a peptide encoded by the 3' UTR could markedly increase the number of peptides surveyed by the immune system, making the prospect of defining the peptides resulting in CD8⁺ T-cell mediated immunity and autoimmunity that much harder.
Once produced, peptides are transported in the rough endoplasmic reticulum (RER) lumen by the transporter of antigen peptides (TAP) complex located on RER membrane : TAP complex is an ATP-binding cassette consisting of 4 tapasin / TAPBP / / TAPA glycoproteins assembled with each TAP1 / TAP2 heterodimer (all genes are up-regulated by IFN-g).
[TAP is inhibited by :
• US6 from CMV / HHV-5, a transmembrane protein which binds to the lumenal face;
• ICP47 from HSV-1 / HHV-1]
TAP complex has highest affinity for peptide fragments whose lenght is 8-11 residues.
The class I MHC a chain is stabilized by a lectin chaperonin that binds monoglucosylated N-linked glycans on a chain :
• mobilferrin / calreticulin in the ER membrane. Calreticulin binding depends on the glucose residue in the N-linked glycan and is independent of MHC class I molecule conformation (free MHC class I heavy chains, empty MHC class I-b₂-microglobulin heterodimers and peptide-loaded MHC class I complexes) providing support for the hypothesis that, by binding monoglucosylated N-linked glycans, calreticulin cannot determine the conformation of the protein^ref
• calnexin in the ER lumen.
Once b₂-microglobulin binds to the a chain, the chaperone is released and the complete (heterodimeric) class I MHC molecule associates with ... :
• BiP / hsp70 5 / MIF2 / GRP78
• ERp57 / GRP58 (a thiol-oxidoreductase) : in ERp57^-/- mice, death in utero caused by ubiquitous ERp57 deletion was prevented by specific deletion in the B cell compartment. ERp57 is central for recruitment of MHC class I molecules into the loading complex. In ERp57^-/- cells, interaction of MHC class I molecules with the loading complex is short-lived. Thus, in the steady state, very few MHC class I molecules are present in the loading complex. Surface H-2K^b–peptide expression and stability are reduced, and presentation of a model antigen is decreased. ERp57 does not influence the redox state of MHC class I molecules but is an essential structural component required for stable assembly of the peptide-loading complex^ref
Glucose regulated protein, 94 kDa (GRP94) / gp96 / tumor rejection antigen 1 (TRA1) / adenotin is an Hsp90 that acts as an ER marker. Tapasin brings the class I MHC molecule close to the remaining components of the TAP complex, allowing the fomer to acquire the antigenic peptide from the latter. Assembly of this compex is associated with formation of a disulfide bond between tapasin and ERp57. ERp57 and BiP then dissociate from the now stable class I MHC molecule. The capacity of MHC class I /b₂m/peptide complexes to form dimers may be important during Ag loading.
[some molecules from pathogenic microorganisms inhibit the presentation pathway :
• CMV / HHV-5
• US2 causes the degradation of HLA-DR-a and HLA-DM-a
• US3 retains MHC class I complexes in the ER
• US11 transports MHC class I H chains back into the cytosol, where they are deglycosylated by host N-glycanase and then degraded by the proteasome
• E3-gp19k in Ad downregulates MHC class I translocation
• UL18 in CMV / HHV-5 impairs b₂m binding to MHC class I.
• measles virus
• retroviridae
• canarypox virus
• vaccinia virus
• LCMV
There are amino acid positions in the peptide (termed anchor residues) in which a certain type of amino acid side chain (hydrophobic or basic) is needed to interact with amino acids in the MHC's groove : one of these is always located at the carboxyl-terminus of the peptide and is generated with high fidelity by the proteasome, while the other 1 or 2 are usually located near the amino-terminus (generated by post-proteasomal trimming by amino-exopeptidases). The peptide may be slightly longer than 9 amino acids, with the additional length bulging from the middle of the cleft. N- and C-terminal segments are anchored to A and F clefts, respectively.
Unusually long MHC class I–restricted epitopes are important in immunity, but their 'bulged' conformation represents a potential obstacle to ab TcR–MHC class I docking. The TCR in complex with HLA-B*3508 presenting a peptide 13 amino acids in length was atypical of TCR–peptide–MHC class I interactions, being dominated at the interface by peptide-mediated interactions. The TCR assumed 2 distinct orientations, swiveling on top of the centrally bulged, rigid peptide such that only limited contacts were made with MHC class I. Although the TCR-peptide recognition resembled an antibody-antigen interaction, the TCR–MHC class I contacts defined a minimal 'generic footprint' of MHC-restriction. Thus our findings simultaneously demonstrate the considerable adaptability of the TCR and the 'shape' of MHC restriction^ref

Peptides that bind MHC class I molecules are the best known odortypes.
Recently, MHC class I molecules expressed by neurons that undergo activity-dependent, long-term structural and synaptic modifications have been shown to be important for the retraction of synaptic connections that normally occurs between retina and central targets during development^ref. In the adult CNS, a classical response of neurons to axon lesion is the detachment of synapses from the cell body and dendrites. Lesioned sciatic motoneurons in b₂m or TAP1-null mutant mice show more extensive synaptic detachments than those in wild-type animals. This surplus removal of synapses is entirely directed toward inhibitory synapses assembled in clusters. In parallel, a significantly smaller population of motoneurons reinnervates the distal stump of the transected sciatic nerve in mutants. MHC I molecules, which traditionally have been linked with immunological mechanisms, are thus crucial for a selective maintenance of synapses during the synaptic removal process in neurons after lesion, and the lack of MHC I expression may impede the ability of neurons to regenerate axons^ref.
Class I /b₂m/peptide complexes are recognized by TcRab on the surface of CD8⁺ T lymphocytes. MHC class I /b₂m/peptide complexes dimers arose from transient interaction may be stabilized by coreceptors at the immunological synapse.
Neither the a chain alone nor the a chain-calnexin complex can't reach the plasma membrane. Anyway a smaller fraction of the MHC molecules on the cell surface exist as functionally inactive b₂m-free H chains believed to be formed by the dissociation of b₂m and peptide from the previously cell surface-expressed heterodimers. This form does not bind peptides and is recognized by mAbs that recognize denatured H chains. The biological role of these denatured b₂m-free class I H chains is not clear. It has been shown that b₂m-free class I H chains are spontaneously released from the surface of activated cells and that cross-linking of b₂m-free class I H chains with specific mAbs results in the rapid down-regulation and internalization of these molecules.
The single amino-acid change at position 116 (present in the F pocket of the peptide-binding groove) from aspartic acid in HLA-B*4402 to tyrosine in HLA-B*4405, results in a preference for phenylalanine at position 9 of the peptide. This is due to decreased electronegativity and increased hydrophobicity of the F pocket of HLA-B*4405. HLA-B*4402 cell-surface expression is highly dependent on tapasin, which recruits HLA class I molecules into the peptide-loading complex (PLC). By contrast, HLA-B*4405 has  been shown to be expressed at high levels on the surface of tapasin-deficient cells and without association with the PLC, so being resistant to TAP inhibitors. Furthermore, compared with HLA-B*4402, HLA-B*4405 is more rapidly transported to the surface of both tapasin-deficient and tapasin-sufficient cells by circumventing PLC-mediated retention and peptide optimization in the ER. So, although sacrificing the optimization of peptide cargo by the PLC can come at a cost to the host, tapasin-independent alleles such as HLA-B*4405 might have been selected to provide an advantage during infection with pathogens that interfere with conventional antigen-presentation pathways^ref.

Cross-presentation : antigens synthetised by antigen-donor cells (ADC) can  ...
• ... be processed elsewhere and directly extracellularly bind uncharged MHC class I molecules on DC surface
• ... enter the DC
• directly from the extracellular fluid to the cytosol (unknown "cytosolic diversion" pathway / cytosolic route) => classical MHC class I processing. APC cross-present Ag with fast kinetics since cross-presentation is detectable within 1 h of ADC-pAPC encounter, pearks between 2 and 5 h, and decreases rapidly to almost nil within the next 10-15 h. This kind of cross-presentation does not require apoptosis or necrosis of the ADC, since live-infected cells are acapable ofd supplying Ags to pAPC within few hours of becoming infected.
• by endocytosis (RME, phagocytosis or macropinocytosis) =>
• => endosomal processing by cathepsin D =>
• => regurgitation of peptide antigen from the endosomal compartment onto the cell surface => extracellular binding to discharged MHC class I molecule on DC surface
• if endoplasmic reticulum (ER)-mediated phagocytosis occurred => direct endosomal loading of still available MHC class I molecules. Latex beads labelled with fluorescent ovalbumin were phagocytosed and fluorescence could be detected in the cytoplasm, indicating that exogenous proteins are retro-translocated into the cytoplasm for degradation by proteasomes. Indeed, proteasomes and polyubiquitylated proteins are associated with the cytoplasmic side of the phagosome. The cross-presentation of peptide-MHC class I complexes is partially inhibited by treatment with brefeldin-A (which blocks the secretory pathway), whereas conventional presentation is totally inhibited, supporting the idea that phagosomes are the source of the MHC class I complexes in the brefeldin-A-treated cells^{ref1, ref2}. Various ER-resident proteins (e.g. glucose-6-phosphatase, TAP, tapasin, and calreticulin) are detectable on early phagosomes from immature DCs : functional assays showed that pohagosomes are a site of TAP-dependent peptide loading onto MHC class I molecules, leading to T-cell stimulation^ref. Exogenously added soluble US6 inhibits cross-presentation, showing that the TAP inhibition occurred in a location where internalized proteins have access to ER components^ref. The fusion of endoplasmic reticulum membrane with nascent phagosomes allows intersection of the endosomal system with the endoplasmic reticulum, the classical site of MHC class I peptide loading, and may reconcile the seemingly conflicting evidence indicating both of these sites are crucial in cross-presentation^ref
• => direct injection of pathogen-derived antigenic material into the cytosol (cytosolic diversion) via fusogenic peptides => classical MHC class I processing
• many viruses
• LCMV induces cross-priming by a mechanism dependent on type I interferons, independent of CD4⁺ T cell help or CD40/CD40L interaction, and involves direct stimulation of DCs)
• VP22 (an important coat protein of gallid herpesvirus 2 / Marek's disease virus)
• some Bacteria
• Listeria monocytogenes
• Pseudomonas exotoxin translocation subunit (PET)
• other exogenous proteins unknown pathway
• => backward transport through the secretory pathway => Golgi complex => ER lumen. The endocytised material comes from :
• cellular destruction
• apoptotic cells : DCs phagocytose apoptotic cells and re-present the acquired cellular Ags on MHC class I and MHC class II. Receptors that mediate uptake of apoptotic cells by DCs are CD36 / GPIIIb / GPIV (expressed only in CD8a⁺ DCs), CD51-? / a_Vb₅ integrin, and CD51-61 /  a_Vb₃ integrin.
• necrotic cells
• DCs phagocytose necrotic cells through other receptors
• heat shock proteins (HSPs) : anyway cellular proteins, rather than peptides or heat shock protein/peptide complexes, are the major source of antigen that is transferred from antigen-bearing cells and cross-presented in vivo^ref.
• HSP70 family members partially activate APCs, but subsequent CD40 ligation is required for full activation^ref
• constitutive HSC54 / HSC70 / HSC71 / heat shock 70kDa protein 8 (HSPA8)
• heat-inducible HSP70
• BiP / hsp70 5 / MIF2 / GRP78
• glucose-regulated protein (GRP)170
Only a small number of human DCs pulsed with influenza-A-derived peptides bound to mycobacterial HSP70 are necessary to induce an efficient antigen-specific CTL response (T-cell:DC ratio of 5000:1). Pulsing DCs with the same peptides in the absence of HSP70, even in the presence of a known DC activator such as LPS, is not effective, and CD8⁺ T cells can not be stimulated by peptide-HSP70 complexes alone. 120 pM HSP70-bound peptide can generate a CTL response in vitro, whereas a 2000-fold higher concentration of free peptide is unable to do so. The low concentration of mycobacterial HSP-bound peptide and the small number of stimulated DCs required for a CTL response without additional adjuvnats in this human system requires further investigation as a potential vaccination method. Truncated HSP70 proteins containing the known carboxy-terminal peptide-binding domain only are as efficient as full-length HSP70 at generating CTLs, indicating that they are able to both stimulate and deliver peptide to DCs. By contrast, an HSP70 mutant with markedly decreased peptide-binding affinity, owing to a point mutation in the peptide-binding domain, can still induce the production of pro-inflammatory cytokines by DCs but do not lead to CTL generation, and therefore the delivery of antigen  can be separated from DC stimulation. This mutant HSP70 was unable to generate CTLs even in the presence of excess free peptide, which shows that to be crosspresented, the peptide must be bound to HSP. However, when the concentration of wild-type peptide-HSP70 was not limiting, addition of excess mutant HSP70 that cannot bind peptide enhances the CTL response. So the 2 components of the response can be delivered by separate HSP molecules, indicating that other HSPs might be able to enhance the response to a low concentration of peptide-bound HSP in physiological situations^ref. LOX-1 is a DC receptor for Hsp70^ref. MyD88 is required for cross-priming in vivo^ref
• HSP90
• glucose regulated protein, 94 kDa (GRP94) / gp96 / tumor rejection antigen 1 (TRA1) / adenotin
• HSP110 / APG-1
• mobilferrin / calreticulin
... from necrotic cells (e.g. cancer cells) but not from normal cells carry donor Ags for MHC class I presentation by APC. One of the APC receptors involved in receptor-mediated endocytosis is CD91 / LRP1 / a₂-macroglobulin receptor, which binds to gp96, HSP70, and HSP90. HSP70 is also able to translocate across cellular membranes and gains cytoplasmic entry, which allows the protein Ags to be processed by cytoplasmic proteasomes, and subsequently, to enter the MHC class I pathway.
• capture of antigen in a cell-associated form through the nibbling of small vesicles from the donor cells by the DC, without causing damage
• exosomes
Once reached the ER lumen the antigen may undergo :
• endoplasmic reticulum-associated destruction (ERAD) pathway =>  => classical MHC class I processing. E.g. :
• CT : in the ER lumen the A1 chain is released from the rest of the toxin, so that it can be recognized as a misfolded protein.
• SLT-1
• processing by signal peptidase-like enzyme that cleaves eventual specific cleavate site(s) to create the epitope within ER lumen itself => immediate charging of MHC class I molecule. E.g. :
• cleavage site in the surface exposed N-terminal segment of A subunit of RT (RTA).
Under normal circumstances, cross-presentation is probably less efficient than direct presentation, since cross-presentation requires the additional step of transfer from the ADC to the pAPC : so the level of expression by the donor cell seemes to be very important for successful cross-presentation, with high dose antigens inducing cross-presentation and low-dose antigens being ignored. This makes sense because otherwise B cells or DCs picking up soluble antigens may generally become over-susceptible to elimination by CTLs, a process that would prematurely stop immune responses.
The bias of MHC class I presentation pathway for endogenously synthetised Ags has presumably evolved to ensure that the CD8⁺ CTL activity remains focused on directly infected targets : bystander cells that simply endocytose viral debris from infected neighbouring cells will not process this antigen into the MHC class I pathway and will therefore not to be targeted by CTL. But as naive CTLs have a limited recirculation pattern, they cannot scan the entire body for the presence of antigen : naive CTLs rather scan for the presence of antigen on DCs, that recirculate throughout the entire body. So, if absolute, the inability to cross-present exogenous Ags on MHC class I molecules of limited subsets of normal (i.e. uninfected, self, and non neoplastic) DCs would limit :
• CTL immune responses (cross-priming) against
• extralymphatic tumors
• allografts
• intracellular pathogens
• that do not infect DCs
• that enter DCs but that functionally compromise their ...
• ... MHC class I presentation [although virus-mediated down-regulation or inhibition of target-cell expression of MHC class I will also impair the effector phase of responses induced by cross-priming (see below), this is unlikely to completely block recognition by effector CTLs]
• ... migration to secondary lymphoid compartment
An epitope present in the functional signal sequence (the amino-terminal part of a protein that targets it to the endoplasmic reticulum and is then cleaved) of a GFP fusion protein could not be cross-presented to CTLs at any significant level, whereas the same epitope or a different epitope close to the carboxyl terminus of the mature GFP protein could be efficiently cross-presented due to the efficiency of uptake by pAPCs^ref. Cross-priming is biased towards unprocessed antigens and against sequences that are degraded rapidly after synthesis, such as signal peptides. Cross-priming favours proteasome substrates rather than pre-processed peptides^ref : in their in vivo set-up, cross-priming is based on the transfer of proteins rather than peptides, which casts doubt on the previously suggested role of peptide-binding molecular chaperones in this process. For efficient cross-presentation, vaccine antigens should be engineered for maximum half-life
The cross-priming APC is the immature (i.e. capable of phagocytosis) resting CD8⁺ DC in the T-cell zone (TCZ) of spleen PALS. If primed by LPS, also immature resting CD4^-8^- DCs (migrated from marginal zone to T-cell area of the spleen) become cross-presenting, although with far poorer capacity. In vitro they are more effective cross-presenters than mature DCs (that have lost phagocytic ability), Mf and B cells.
However, the assumption that only DCs can induce CD8⁺ T cells have been overstated because antigenic fibroblasts^ref or extralymphatic tumor cells^ref that lack conventional second signals efficiently induce CD8⁺ T cells if they reach draining secondary lymphoid organs and if helper T cells are available.
• constitutive maintenance of both central and peripheral CD8⁺ CTL tolerance (cross-tolerance). The cross-tolerizing APC is still unknown. Such cross-presentation induces proliferation of naive CD8⁺ CTLs, but ultimately leads to their deletion
• nonclassical MHC class I (a.k.a. MHC class Ib) genes are considerably less polymorphic, of essentially unknown function, and variable in amount and tissue distribution. To date, nonclassical class I Z lineage genes have only been identified in the teleost (bony fish) species, ginbuna crucian carp (Carassius auratus langsdorfii) and common carp (Cyprinus carpio), and in the cyprinid species zebrafish (Danio rerio) and barbus (Barbus intermedius).
• coded in the MHC locus
• HLA-E (associated with class I 6.2-kB HindIII fragment, 5 alleles) is expressed in all cells but found on cell surface only in some but not all activated T cells^{ref1, ref2, ref3, ref4}, and on the surface of extravillous trophoblast that has invaded the maternal decidua^ref (syncytiotrophoblast, villous mesenchymal cells, extravillous trophoblast and several decidual cell types, but staining for HLA-E appeared to be confined primarily to the cytoplasm^ref). HLA-E is structurally homologous to classical MHC class I proteins^{ref1, ref2}, also requiring association with peptide for cell surface expression. In contrast to MHC class I, however, HLA-E only binds a restricted set of nonamer peptides that are primarily derived from the leader sequences of class Ia (HLA-A, -B, -C) and -G molecules^{ref1, ref2, ref3, ref4}, though peptides derived from other cellular^ref and viral^{ref1, ref2} sources have been identified. More recently, HLA-E has also been implicated in the presentation of self-peptides and bacterial antigens to T cells, although the role of these T cells in immune responses remains to be established^{ref1, ref2, ref3, ref4} (16–19). The generation of the MHC signal peptide-derived epitopes binding to HLA-E does not follow the "conventional" antigen-processing pathway described above. During biosynthesis of the MHC class I pre-protein and its translocation into the ER lumen, the signal sequence is cleaved by signal peptidase and subsequently processed by signal peptide peptidase (SPP) / HM13^{ref1, ref2} (20, 21). This second cut occurs in the hydrophobic membrane-spanning region, allowing the release of an 14 residue-long N-terminal signal peptide fragment into the cytosol. Proteasomal activity is then required to trim the extended peptide fragment liberated into the cytosol^ref, consistent with the finding that C-terminal trimming increases the efficiency of TAP transport and is required for peptide binding to HLA-E^ref (20). Peptide entry into the cytosol is consistent with the TAP dependency of HLA-E expression. The epitope-containing fragment is then transported into the ER lumen for loading onto newly synthesized HLA-E^ref (20). 2 nonsynonymous alleles of HLA-E are retained in the human population, maintained through strong balancing selection, distinguished by a single sequence dimorphism at position 107: glycine (E*0101; HLA-E^G) or arginine (E*0103; HLA-E^R)^ref. Biophysical studies have shown that both allelic and peptide sequence differences have significant effects on HLA-E solution stability, correlating with differential cell surface expression levels^ref. E*0101 allele is significantly higher in patients with recurrent spontaneous abortion^ref.
• The expression of the Qa1–self-peptide complex on antigen-activated T cells is a function of the intermediate-avidity interaction between TcRs on T cells and MHC–antigen-peptide complexes presented by conventional APCs during the T-cell activation^ref. The Qa1–self-peptide complexes, as surrogate structures expressed on target T cells, are recognized by the a/b T-cell receptors on regulatory CD8⁺ T cellsand trigger the CD8⁺ T cells to differentiate into effector cells. These effector cells in turn down-regulate any activated intermediate-avidity T cells expressing the same Qa1–self-peptide complexes during the secondary immune response^ref. It binds to CD94 included in either an inhibitory (associated with NKG2A or NKG2B) or activatory (associated to NKG2C or NKG2D) receptor on NK cells and CD8⁺ T lymphocytes (although <5% of CD8 T cells express the receptor in a naive mouse, its expression is upregulated upon specific recognition of antigen). Similar to NK cells, most CD8 T cells that express high levels of CD94 co-express NKG2A, the inhibitory isoform. The engagement of this receptor can lead to a blocking of cytotoxicity. However, these receptors have also been implicated in the cell survival of both NK and CD8 T cells. The level of CD94 expression is inversely correlated with the level of apoptosis in culture^ref. The concomitant presence of the appropriate HLA class I alleles with leader sequence-derived peptides and HLA-E heavy chain may not be sufficient to allow HLA-E surface expression in tumor cell lines (e.g. only 23% in osteosarcomas) as opposed to lymphoid cells^ref.
• HLA-F (associated with class I 5.4-kB Hind III fragment) is not expressed on cell surface other than extravillous trophoblast that has invaded the maternal decidua^refand has unknown function
• HLA-G (associated with class I 6.0-kB Hind III fragment; 8 alleles; 6 isoforms from alternative RNA splicing) has a limited distribution on the cell surface on extraembryonic tissues (i.e., on extravillous trophoblast that has invaded the maternal decidua^ref) and is thought to prevent killing of fetal cells : it is also expressed in many glioblastomas, preventing efficient priming of T_c-cells. In addition, soluble HLA-G isoforms have been detected in the serum of pregnant and nonpregnant women as well as men (produced by Sertoli cells, spermatocytes and spermatids). HLA-G is recognized by the inhibitory receptors KIR2DL4, LIR1 and LIR2 on NK cells.
• y_HLA-J (very similar to HLA-A)
• MHC-class-I-chain related (MIC) molecules
• MICA(383 amino acids, homology < 30%)
• MICB
• MICC
In adults they are expressed at only minimal levels on normal cells (except enterocytes, perhaps as a consequence of stimulation by the neighbouring bacterial flora), while they are abundantly present on distressed cells (e.g. abnormally proliferating synoviocytes in rheumatoid arthritis), virally infected cells (MICA expression is also upregulated as a result of the binding of Escherichia coli adhesin AfaE to CD55 and by cells infected with Mycobacterium tuberculosis) and a wide variety of carcinomas : elements in their promoters are similar to hsp genes and permit their up-regulation of them in stressed and transformed cells. Expression of MICA and MICB by DCs can also be induced by IFN-a. Despite the presence of an MHC a₃-like domain, they do not bind b₂-microglobulin (not required for folding, stability, or cell surface expression) so their expression would not be affected by the loss of b₂-microglobulin, which occurs in certain tumors. They have and have an occluded peptide-binding groove, too small to allow for the binding of anything larger than the equivalent of a 3- or 4-residue peptide. This, plus the polar character of the lining of the pocket and the lack of any electron density not accounted for by protein atoms in the structure, led to the conclusion that they don't bind any peptide or other small-molecule ligand. The platform and a₃ domains are joined through a flexible linker, allowing considerable interdomain flexibility, a feature unique among MHC class I proteins and homologs. They are highly polymorphic (150 MICA alleles and 13 MICB alleles known) and are broadly recognized by ...
• g?d1 TcR on Vd1 gd iIELs
• activating receptor NKG2D on ...
• both resting and activated NK cells
• Vd1 gd iIELs
• activated CD8⁺ab CTLs
• activated macrophages
The existence of MHC class I molecules that are minimally expressed on normal tissues, but induced by transformation or adverse environmental influences, provides an ideal early danger signal for the immune system. A 100-kb deletion including the MICA gene has been reported in the HLA-B48 (B*4801)-associated haplotype in East Asians (Japanese, Koreans) and Angaite Amerindian community in Paraguay (with the novel recombination haplotype between this MIC null haplotype and HLA-B15, HLA-B15-MICA-del-MICB*0107 N). Interestingly, this MICA deletion is accompanied by a MICB null allele (a premature stop codon), MICB0107N. Maybe the expression of both MICA and MICB molecules is indispensable to viability through a yet-to-be understood mutual interaction in immune surveillance.
• HFE / HLA-H (very similar to HLA-A) is expressed in all tissues except brain. It does not bind peptides but regulates the Tf-CD71 / TfR binding : at least 11 isoforms exist. Deficiency leads to hereditary hemochromatosis.
• coded in other loci :
• (HHV-5 / CMV-encoded) UL16 binding proteins (ULBPs) / human retinoic acid early transcript 1 (RAET1) proteins (coded on chromosome 6q25, outside the MHC region). They bind to the activating receptor NKG2D on NK cells and Vd1 gd iIELs.
• ULBP1 / RAET1I
• ULBP2 / RAET1H
• ULBP3 / RAET1N
• ULBP4 / RAET1E
• RAET1L
Pseudogenes :
• RAET1G
• RAET1J
• RAET1K
• RAET1M
Most of the RAET1 proteins, similar to the mouse Rae1 proteins, are attached to the plasma membrane with a lipid linkage (despite RAET1E and RAET1G have transmembrane domains) and have considerable sequence diversity, ranging as low as 35% amino acid identity in pairwise comparison, but all have a similar and unique domain structure, consisting of 2 ectodomains that are distantly related to the a₁ and a₂ domains of MHC class I molecules, except that the site corresponding to the MHC peptide-binding groove is closed off, apparently preventing the binding of peptides and other small molecules. Some of RAET1 proteins are expressed at marked levels by various normal cells at the mRNA level, but cell-surface expression by normal cells is low or has not been documented : they are induced in distressed or HCMV-infected cells.
• CD1 family
• endothelial protein C receptor (EPCR)
• FcRn
• Zn-a₂-glycoprotein I does not bind peptides and promotes triglycerids catabolism. It does not bind b₂-microglobulin.
• thymic leukemia (TL) antigen is a nonclassical class I molecule expressed abundantly on intestinal epithelial cells, monocytes, and dendritic cells, that, in contrast to other MHC class I molecules that bind CD8ab, preferentially binds CD8aa : this interaction modifies responses mediated by TCR recognition of antigen presented by distinct MHC molecules^ref
[UL16, a CMV / HHV-5 glycoprotein, binds activating receptor NKG2D ligands ULBPs and MICB, retaining them in the cell and effectively masking NK-cell recognition]
Other class Ib genes may be transcribed but not translated, or the proteins are not detected on the cell surface.
• MHC class II : p34^{a chain} (TM, a₂and a₁ domains: it is poorly polymorphic) and p28^{b chain} (TM, b₂ and b₁ domains : it is polymorphic). It binds 10÷34 amino acids-long peptides derived from proteins that enter the cell through ...
• endocytosis
• autophagy (e.g. EBNA1, the dominant CD4⁺ T cell antigen of latent HHV-4 / EBV infection. Lysosomal processing after autophagy may thus contribute to MHC class II restricted surveillance of long-lived endogenous antigens including nuclear proteins relevant to disease^ref)
... and are processed by aspecific lysosomal proteases in phagolysosomes. Unlike peptides bound to MHC I molecules, peptides bound to class II MHC molecule are neither anchored at their ends nor severely limited to a precise length thanks to the open ends of the class II MHC grove and it is more difficult to identify "required" amino acids (motifs) in the peptides. Although endosomes contain a variety of proteases, few have been directly implicated in the generation of class II-associated peptides :
• endogenous lysosomal enzymes :
• cathepsin B
• cathepsin D
• asparaginyl endopeptidase (AEP) / legumain (LGMN1) / cysteine protease 1 (PRSC1) is a cysteine protease that specifically cleaves after asparagine residues. Enzyme activation is triggered by acidic pH and appears to be autocatalytic, requires sequential removal of C- and N-terminal pro-peptides at different pH thresholds, and is bimolecular. Removal of the N-terminal propeptide requires cleavage after aspartic acid rather than asparagine^ref. A fully mature, active enzyme is produced following LPS expression in mDCs. Overexpression of this gene may be associated with the majority of solid tumor types. This gene has a pseudogene on chromosome 13. Alternative splicing results in multiple transcript variants encoding different isoforms. AEP is not expresed by B lymphocytes^ref
• internalized exogenous enzymes :
• cathepsin (Cat) G, a serine protease that is not endogenously synthesized by B lymphocytes, is internalized from the plasma membrane and present in lysosomes from human B cells where it represents a major functional constituent of the proteolytic machinery^ref
At least 2 processing events exist : an initial generation of precursor fragment from the intact protein and secondary processing to an optimal size for MHC binding and/or T cell recognition (some processing of MHC-associated peptides occurs subsequent to binding). Many of these class II-associated peptides comprise nested sets that contain the same core sequence but vary in length at the amino or C-terminal ends. C-terminal residues reflect the action of peptidases that cleave at preferred amino acids, while amino termini appear to be determined more by proximity to the class II MHC binding site.
In order to avoid binding to cytoplasmic peptides introduced in the rough endoplasmic reticulum (RER) lumen by TAP, here MHC class II binds with higher affinity the invariant chain (IC) / Ii / p33÷41^CD74. Due to the presence of trimerization region in the Ii, 3 molecules of abIi trimers form a supramolecular complex of a nonamer, (abIi)₃. Aided by the presence of targeting sequence in the Ii, this complex is then transported to the endocytic vesicles, where selective Ii degradation by cathepsin S occurs leaving only the Ii fragment class II-associated invariant chain peptide (CLIP) attached to the peptide-binding groove of the MHC class II molecule. pH lowering  in the late endosome or in the phagolysosome (a.k.a. MHC class II containing compartment (MIIC) / MHC II vesicles (CIIV) compartment) removes HLA-DO heterodimer (a / DN ; b) inhibition on HLA-DM heterodimer (a ; b), which now can detach CLIP allowing the MHC class II molecule to eventually bind a peptide from phagocytated material. HLA-DO is expressed in B cells but not in epithelial cells. MHC class II-bound peptides adopt an extended type II polyproline conformation with a common periodicity, such that residues at P1, P4, P6 and P9 point down into MHC pockets, whereas P-1, P2, P5, P8, and P11 are solvent exposed and point toward the TcR. This is in part due to the extensive, and often conserved, interaction with the peptide backbone. Naturally processed MHC class II-bound peptides possess ragged amino and carboxy termini referred to as peptide-flanking residues (PFRs) because they are outside the MHC anchor residues (P1, P4, P6, and P9) but within the MHC binding groove (e.g., P-2, P-1, P10, P11). P1 appears  to be important for peptide stability. In contrast, residues at P10 and P11 are frequently removed by Ag processing. PFR recognition is a common event characteristic of TcR recognition of MHC class II:peptide complexes : invariably, the PFRs at P-1 and P11 of all of the MHC class II:peptide complexes solved to date are solvent exposed and accessible to the TcR. Interestingly, PFRs are a unique and exclusive feature of peptides bound to MHC class II molecules, in that MHC class I-bound peptides are predominantly of fixed length.


HLA-DR and HLA-DP genes are constitutively expressed in B lymphocytes, spermatozoa and most myeloid cells. Based on studies in streptococcal and leprosydisease systems, it has been suggested that DQ molecules may play a role in immunosuppression, as opposed to the helper function of DR molecules. However, in many other systems, Ag-specific DQ-restricted T cells can be found.
HLA-DP, HLA-DQ, and HLA-DR expression can be induced on activated T lymphocytes and most epithelial (including endothelial) cell types : TNF-a, IL-4 and IFN-g co-up-regulate MHC class II, Ii and cathepsin S expression.
Class II /b₂m/peptide complexes are recognized by TcRab on the surface of CD4⁺ T lymphocytes.
ab T lymphocytes are able to detect even a single peptide–MHC on the surface of an antigen-presenting cell. This is despite clear evidence, at least with CD4⁺ T cells, that monomeric ligands are not stimulatory. Some specific combinations of  soluble peptide–MHC heterodimers (in which one peptide is an agonist and the other is one of the large number of endogenous peptide–MHCs displayed by presenting cells) can stimulate specific T cells in a CD4-dependent manner. This activation is severely impaired if the CD4-binding site on the agonist ligand is ablated, but the same mutation on an endogenous ligand has no effect. These data correlate well with analyses of lipid bilayers and cells presenting these ligands, and indicate that the basic unit of helper T cell activation is a heterodimer of agonist peptide– and endogenous peptide–MHC complexes, stabilized by CD4^ref.
Certain bacterial capsular zwitterionic polysaccharide (ZPS) (e.g. polysaccharide A (PS-A) from Bacteroides fragilis), when catabolized after overnight culture (involving polysaccharide oxidation) and conventionally processed by APCs, can associate with MHC class II molecules and induce CD4⁺ T-cell responses^ref. These observations might explain why some polysaccharide vaccines work so well and might provide new approaches to the design of vaccines for infectious diseases. PS-A can direct normal immune system development in the mouse. Reconstitution of germ-free mice with the bacterial commensal Bacteroides fragilis expanded T cell numbers and restored lymphoid structures that would otherwise have developed abnormally. Expression of PSA was sufficient and necessary for this activity and also reestablished balance in T_h1 and T_h2 cell cytokine responses, through presentation of PSA by dendritic cells. The finding that a bacterial product can implement such direct governance over the mammalian immune system may explain how our microflora help maintain pathogen immunity while preventing unwanted inflammation and allergy^ref.
[UL37 / vMIA from CMV / HHV-5 is a Bcl-2 ortholog that disrupts constitutive MHC class II expression by inducing retention of MHC class II positive vesicles in an abnormal perinuclear distribution.]
Both MHC class Ia and II genes are inherited on chromosome 6p21.3 as an extended (3.2 cM = 4 Mb) or restricted (e.g. DR+DQ) haplotype arranged as follows :
• ribosomal protein S18 (RPS18)
• RXRB
• collagen type XI a₂ (COL11A2)
• MHC class II or D region belongs to isocore L / H1 (low GC content = 40-45%), with high gene density = 1 protein every 30 kb.
• HLA-DP y_b2 (DP stands for D-priming)
• HLA-DP y_a2
• HLA-DP b₁ (82 alleles)
• HLA-DP a₁ (10 alleles)
• HLA-DO a / HLA-DN a
• HLA-DM a (4 alleles; not expressed on cell surface !)
• HLA-DM b (5 alleles; not expressed on cell surface !)
• genes coding for products in the MHC class I presentation pathway
• large multifunctional protease 2 (LMP2) / RING12
• TAP1 (5 alleles)
• LMP7 (4 alleles)
• TAP2
• HLA-DO b
• HLA-DQ y_b2
• HLA-DQ y_b3
• HLA-DQ y_a2
• HLA-DQ b₁ (> 40 alleles)
• HLA-DQ a₁ (20 alleles)
• HLA-DR b₁ (> 200 alleles; DR stands for D-related)
• HLA-DR y_b2
• HLA-DR b₃ (14 alleles; only in some haplotypes !)
• HLA-DR b₄ (9 alleles; only in some haplotypes !)
• HLA-DR b₅ (12 alleles; only in some haplotypes !)
• HLA-DR y_b6 (3 alleles)
• HLA-DR y_b7 (2 alleles)
• HLA-DR y_b8
• HLA-DR y_b9
• HLA-DR a (2 alleles)
• MHC class III region belongs to isocore H3 (the one with the highest GC content = 53%), with low gene density = 1 protein every 15 kb.
• receptor for advanced glycation end-products (AGE) (RAGE)
• LPA acyltransferase d (LPAAT)
• steroid 21-hydroxylase / cyp21A2 / cyp21B
• y_{cyp21A2 / cyp21B}
• C4B
• C4A
• B factor (Bf)
• C2
• MHC class IV region : in the telomeric end of the Class III region, involved in both global and specific inflammatory responses^ref
• hsp70 1A
• hsp70 1B
• hsp70 1-like
• casein kinase 2, b subunit (CKIIb)
• LT-b
• TNF-a
• TNF-b / LT-a
• allograft inflammatory factor 1 (AIF1) gene is induced by cytokines and interferon. Its protein product is thought to be involved in negative regulation of growth of vascular smooth muscle cells, which contributes to the anti-inflammatory response to vessel wall trauma. The gene expresses 3 transcripts
• MHC class I belongs to isocore H3 (the one with the highest GC content = 53%), with low gene density = 1 protein every 15 kb.
• MICB
• MICA
• HLA-B (a)
• HLA-C (a)
• POU5F1 TF
• TCF19 TF
• tubulin b (TUBB)
• y_HLA-X
• HLA-E
• MICC
• y_HLA-J
• HLA-A (a)
• HLA-H / HFE
• y_HLA-H
• HLA-G
• HLA-F
• myelin oligodendrocyte glycoprotein (MOG)
The extended major histocompatibility complex (xMHC) comprises 421 loci, of which 252 are classified as expressed genes, 139 as pseudogenes and 30 as transcripts (based on EST evidence, but without open reading frames). This represents an increase of 345 annotated loci since the first MHC poster in 1991^ref and juxtaposes the classical MHC with several gene clusters, including some of the largest in the human genome^ref. This is the most polymorphic locus (expecially in the peptide binding sites coding regions) in Homo sapiens genome : most polymorphisms occur at specific sites in MHC I a₁ and a₂ domains, and the b₁ domain of MHC II. MHC-polymorphism-driven diversification of the TcR repertoire allows high-avidity CTLs to be generated more readily, and this might be the link between MHC polymorphism and resistance to infection.
DNA methylation profiling of the human MHC : a pilot study for the Human Epigenome Project (HEP)^ref
HLA nomenclature
The most common human HLA class II specificity is HLA-DP4 (which involves at least 2 molecules : HLA-DPA1*0103/DPB1*0401 (DP401) > HLA-DPA1*0103/DPB1*0402 (DP402), which differ from each other by only 3 residues), expressed in 50% in Europe, 76% in Caucasians, 60% in South America, 80% in North America, 60% in India, 2% in Africa, 15% in Japan : in comparison, approximately 6 molecules of HLA-DR are required to cover the same percentage of people. Therefore, we could expect that peptides that efficiently bind to DP401 and DP402 (e.g. NY-ESO-1_158-180, IL3R_127-146, MAGE3_245-268, HSV_283-302, NSP2, and TT_947-963) will have a large impact as epitope-based vaccines at least as HLA-DR-restricted peptides (e.g. TT_830-843, PADRE peptide)..
Gene expressions of MHC class Ia, b₂m, MHC class II (HLA-DP, HLA-DQ, and HLA-DR), invariant chain, HLA-DOA, HLA-DOB, and HLA-DM are all under the control of MHC class II transactivator (CIITA) : it does not directly bind to the promoter region of the genes it regulates; rather, it coordinates the action of numerous transcription factors such as members of the regulatory factor X (RFX), NF-Y and X2BP/CREB families to a compact series of conserved cis-acting elements termed the S/W/Z, X1, X2, and Y boxes found 100-200 bp upstream of all MHC class II genes. CIITA ubiquitination enhances its association of CIITA with both MHC class II transcription factors and the MHC class II promoter. Additional promoter-binding factors such as Oct-2 modulate cell- and stage-specific expression of MHC class II. Transcriptional activation of the CIITA gene occurs through the action of 3 promoters in mice (pI, pIII, and pIV). Immediately downstream of each promoter exists a unique exon 1 that is spliced with the remaining shared exons to form 3 different types of CIITA (I, III, and IV).

pDCs rely strictly on the B cell promoter pIII, whereas macrophages and all other DCs depend on pI^ref
There are only up to 6 different class I (2 HLA-As, 2 HLA-Bs, and 2 HLA-Cs) and up to 22 different class II (2 HLA-DPs, 2 or up to 16 - HLA-DRs, and up to 4 HLA-DQs (2 in cis and 2 in trans)) heterodimers (up to 28 codominant heterodimers !) on the surface of a cell that expresses both class I and class II MHC molecules : but in practice...
• for class I MHC molecules, certain molecules compete with each other for cell surface expression, and the resulting low cell surface expression of specific alleles can lead to a severe reduction in the ability to generate a CTL response
• for class II MHC molecules, a from mom may pair with the b from dad, creating even more different MHC II molecules on the cells (please note some aand b pairings are better fits than others, so "mixed-and-matched" MHC II molecules are not always equally expressed).
Different MHC molecules or different allelic products of one gene bind a different profile of peptides : there may be some or even substantial overlap in the range of peptides bound, but there are clearly differences. MHC associated peptide sequences can be obtained by using Edman degradation (limited by the complexity of the peptide mixture even after HPLC fractionantion) or mass spectrometry to correlate masses in individual HPLC fractions with Edman data : anyway only by using tandem mass spectrometry (MS/MS) it is possible to select and individual peptide ion and subject it to collision-activated dissociation to generate an unambiguous sequence.
Web resources :
• dBMHC at NCBI
• HLA Database at ASHI
• HLA locus at Sanger Center
• HLA Informatics Group at Anthony Nolan Research Institute (ANRI)
• ImMunoGeneTics (IMGT) Database at the European Bioinformatics Institute, UK :
• HLA Database
• MHC Database
• MHCBN : a comprehensive data base of MHC binding and non-binding peptides peptides.
• MHCPEP : a database of > 13,000 MHC-binding peptides
• Official list of human HLA and MIC Ags
• HLA class 1 project at University of Washington
• proteasome processing prediction :
• Prediction Algorithm for Proteasomal Cleavages (PAProC) is a prediction tool for cleavages by human and yeast proteasomes, based on experimental cleavage data^ref
• NetChop
• Histo
• HGMP
• JenPep - Peptide binding database, 8000 entries
• MHCBN: Comprehensive Database of MHC Binding and Non-Binding Peptides
• Functional Immunology (FIMM) is an integrated database focusing on MHC, antigens, T- and B-cell epitopes, and diseases. FIMM was developed at the Knowledge Discovery Department of the Institute for Infocomm Research^{ref1, ref2}
• HLA Ligand/Motif Database
• Biomolecular Interaction Network Database (BIND)
• MHC-Peptide Interaction Database (MPID) (PDB entries)
• MHC-binding epitope prediction
• SVMHC uses support vector machines and currently contains prediction for 26 MHC class I types from the MHCPEP database or alternatively 6 MHC class I types from the higher quality SYFPEITHI database^ref
• PREDICT : prediction of MHC (HLA-DRB1*0401) binding peptides using artificial neural network (ANN), motifs, and Hidden Markov's Model by V.Brusic, Judice L.Y.Koh, Zhang GuangLan and T.Kandasamy
• EpiMatrix by EpiVax, Inc. (on payment)
• HLA peptide binding prediction (human and murine) at BioInformatics & Molecular Analysis Section (BIMAS)
• SYFPEITHI : epitope prediction by BioMedical Informatics (BMI) - Heidelberg contains a collection of MHC class I and class II ligands and peptide motifs of humans and other species accessible as individual entries. Searches for MHC alleles, MHC motifs, natural ligands, T-cell epitopes, source proteins/organisms and references are possible. Hyperlinks to the EMBL and PubMed databases are included. In addition, ligand predictions are available for a number of MHC allelic products. The database content is restricted to published data only^ref. The first natural MHC ligand to be sequenced directly was the nonapeptide SYFPEITHI eluted from H-2 K^d molecules of a mouse tumour line, P815. A GenBank search indicated high homology to a nonapeptide contained within the human tyrosine kinase JAK1: SFFPEITHI, residues 355-363. So SYFPEITHI is a dominant H2-K^d ligand derived from the JAK1 tyrosine kinase^{ref1, ref2}
• Epitope Identification System (EIS) by Epimmune
• MHCPred : predicting the binding affinity for human MHC I and II molecules using additive methods
• RANKPEP : prediction of binding peptides to human and murine class I and II MHC molecules at the Molecular Immunology Foundation
• class I MHC-binding prediction :
• LpPep (for HLA-A2) : various methods have been proposed to predict the binding affinities of peptides to MHC-I molecules based on experimental binding data. They can be classified into 2 groups:
• AIB methods that assume independent contributions of all peptide positions to the binding to MHC-I molecule (e.g. scoring matrices)
• general methods which can take into account interactions between different positions (e.g. artificial neural networks).
The best AIB methods gave significantly better predictions than three previously published general methods, possibly due to the lack of a sufficient training data for the general methods. The best results, however, were achieved with our newly developed general method, which combined a matrix describing independent binding with pair coefficients describing pair-wise interactions between peptide positions. The pair coefficients consistently but only slightly improved prediction accuracy, and were much smaller than the matrix entries. This explains why neglecting them-as is done in AIB methods-can still lead to good predictions^ref.
• NetMHC 2.0 server predicts binding of peptides to a number of different HLA alleles using artificial neural networks (ANNs) and hidden markov models (HMMs). Predictions can be obtained for 12 supertypes, and 120 individual alleles using matrices (ungapped HMMs) generated with data from public databases. Furthermore ANNs have been trained for 10 different alleles representing 7 supertypes^{ref1, ref2, ref3}
• ProPred-I : the promiscuous human and murine MHC class-I binding peptide prediction server
• MAPPP : human and murine MHC-I binding prediction at Max-Planck-Institute for Infection Biology using BIMAS or SYFPEITHI matrices
• class II MHC-binding prediction :
• EpiPredict : HLA class II-restricted T cell epitopes and ligands
• MHC-Thread: HLA class II predictions
• ProPred: MHC class II binding peptide prediction server^ref
• Vaccinome's TEPITOPE is a software package for the prediction of promiscuous HLA-class II binding peptides and human T-cell epitopes.
• receptors binding Ag extracellularly. Their genes have some unique properties : allelic exclusion, gene exclusion and gene rearrangement. The chosen strategy is to produce a clonal distribution of Ag-binding specificities created through random combination of a limited pool of inherited genes, rather than from an inherited gene for each receptor (the latter strategy would need a wider genome !). Each molecule has a N-terminal variable (V) region and a C-terminal constant (C) region. The clonal selection theory, evolved by Burnett (1960), presupposes that the information requisite to the synthesis of the antibody is part of the genetics. While the body develops a wide range of clones of cells necessary to cover all antigenic determinants by random mutation during early embryonic life, those clones which are capable of reacting with antigens of the body ("self’) are destroyed, leaving only those cells which are not oriented to self ("non-self’). The evolution of adaptive immunity appears to have been made possible by the invasion of a putative Ig-like gene by a retrotransposon encoding a gene able to catalyze gene rearrangement, and thus generate diversity. While the invasive ends of the retrotransposon remained into the right place near the Ig-like gene, the retrotransposon-encoded enzyme jumped to another chromosome. Such enzyme corresponds to nowadays looping out recombination-activating genesRAG1 and RAG2, coding for p118 and p58 endonucleases, respectively, expressed also in neurons and still able to catalyze a transposition event that can potentially lead to oncogenic chromosomal translocations as seen in some human T-cell lymphomas. Such dangerous transpositions are limited by the amino-terminal region of RAG1 and the carboxy-terminal region of RAG2 (containing a GTP-binding domain), which inhibit the capture of the target DNA after DNA cleavage. RAG-2 expression is under the control of LEF-1, c-Myb and Pax-5. A Runx-dependent intergenic silencer and an antisilencing element that interact at a distance of 85 kilobases regulate expression of RAG1 and RAG2 in developing CD4⁺8⁺ T lymphocytes^ref.

(reproduced with permission from Nature Reviews Immunology (Vol 3, No. 11, pp 890-899(2003)) copyright Macmillan Magazines Ltd)

(reproduced with permission from Nature Reviews Immunology (Vol 3, No. 11, pp 890-899(2003)) copyright Macmillan Magazines Ltd)

(reproduced with permission from Nature Reviews Immunology (Vol 3, No. 11, pp 890-899(2003)) copyright Macmillan Magazines Ltd)

Gene rearrangements, somatic hypermutations and isotype switches all depend on introduction of double-strand breaks (DSB) in DNA by recombination-activating gene (RAG1) and RAG2, then repaired by the general non-homologous end-joining (NHEJ) machinery (Ku70, Ku80, DNA-PK_CS / XRCC7, DNA ligase IV and XRCC4). Every germ-line V, D and J gene segment has upstream and downstream recombination signal sequences (RSSs) consisting of a conserved heptamer andnonamer motif separated by a spacer of 12 or 23 bp. A pair of RSS with unlike spacers serves to direct a single recombination reaction and to ensure assembly of a biologically useful product. The first phase of V(D)J recombination consists of recognition of a pair of RSSs, synapsis of the 12- and 23-RSS, and cleavage at the signal-coding borders. Nicks mediated by RAG proteins were detectable only in gene segments associated with RSSs containing 12–base pair spacers but not in those containing 23–base pair spacers. These data support a model of capture rather than synapsis for pairwise RSS cleavage during V(D)J recombination^ref. Both recognition and cleavage steps are mediated by RAG1 and RAG2. The cleavage step produces 2 types of broken molecules corresponding to V(D)J recombination intermediates : those with coding ends and those with signal ends. As a consequence of the cleavage mechanism, coding ends are covalently sealed to form a DNA hairpin. In contrast, signal ends are almost always blunt with no sequence loss or addition. The second phase involves the processing and joining of DNA ends, and includes hairpin opening, insertions and deletions at the open coding ends, and formation of a circular signal-joint (sj) (ie the excised interposed sequence) and a coding-joint (cj). One feature unique to the processing of coding ends, and observed at junctional sequences of joined coding segments, is the addition of palindromic (P) nucleotides. Such additions correspond to the gain of several nucleotide complementary to the last few nucleotides of a full-length segment. Addition of P nucleotides is thought to reflect the mechanism whereby hairpin coding ends are opened. Nicking by a single-strand specific endonuclease at a site either 3' or 5' of the hairpin tip would produce P nucleotide overhangs. The second phase of V(D)J recombination also depends upon the function of ubiquitous factors that partecipate in DNA NHEJ. Lymphocytes typically express only one functional antigen receptor, a restriction contributed to by allelic exclusion. In the offspring of a mouse generated by nuclear transfer using a single donor B lymphocyte, all immunoglobulin alleles are inherited as found in the donor lymphocyte. This donor cell had 2 rearranged Ig L chain alleles, both directing the synthesis of light chains that could form functional antigen receptors, one of which was autoreactive. Progeny mice carrying this immunoglobulin light chain allele produced mature B cells, some of which continued to express the autoreactive receptor but required another rearrangement to rescue them from negative selection. Such receptor editing failed to destroy expression of one original light chain allele, thereby recreating dual receptor expression on these surviving B cells. Autoreactive antibodies in serum of mice and humans are due in part to such 'passenger' receptors^ref.
• T cell receptor (TcR) (10^4-5 per T-cell) :p49^a (TM, a₁ and a₂ domains) and p43^b chains (TM, b₁and b₂ domains) or g and d chains.
• a chain : V regions result from rearrangement of ~ 100 different V segments and ~ 100 J segments on chromosome 14q11.2. T early-a promoter is important in targeting primary Tcra rearrangements. T early-a and a previously unknown promoter associated with J_a49 targeted primary recombination to discrete sets of constant  region (C_a)-distal J_a segments and together directed nearly all normal primary recombination events. Furthermore, deletion of the T early-a promoter activated previously suppressed downstream promoters and stimulated primary rearrangement to centrally located J_a segments. Central promoter derepression also occurred after primary rearrangement, thereby providing a mechanism to target secondary recombination events^ref.
• b chain : V regions result from rearrangement of ~ 75-100 different V segments and 2 complexes made up each of 1 D segment, 7-8 J segments and 1 C segment on chromosome 7q34
• g chain : V regions result from rearrangement of 3 different V segments on chromosome 7p15-p14
• d chain : V regions result from rearrangement of 3+1 different V segments, 2 D segments and 3 J segments on chromosome 14q11.2, in the centre of TcRa locus.
The TcR d enhancer (Ed) function depends critically on transcriptional factors core binding factor (CBF) A / Runxx 1, 2, and 3 and CBFB / polyoma enhancer-binding protein 2 (PEBP2), which induces a conformational change in the enhanceosome that is associated with augmented binding of c-Myb.
During TcR rearrangement, excised DNA fragments are mantained in cells episomally as T-cell receptor excision circles (TREC). 2 TREC species, signal-joint TREC (sjTREC) and coding-joint TREC (cjTREC), which are products of the deletion of the TcR d locus from the TcR a locus during the first TcR a gene rearrangement, are produced sequentially by all ab T cells. In most of them, these TREC are generated without prior d rearrangement and are hence identical and can be detected in ~ 70% of abT cells. A maximum of 2 sjTREC and 2cjTREC can be present within one abT cell if the corresponding rearrangement event occurs in both alleles. TREC are exported from the thymus to the periphery within recent thymic emigrants (RTE). Thus, TREC levels in the periphery could reflect RTE numbers. TREC are stable and are not duplicated during mitosis : therefore, TREC concentration is diluited out with each cell division. Whether T cell maturation is complete at the time of thymic exit has been a subject of debate. Using mice transgenic for GFP driven by the RAG2 promoter to identify RTEs, it has been shown that T cell differentiation continues post-thymically, with progressive maturation of both surface phenotype and immune function. In addition, the relative contribution of CD4⁺ and CD8⁺ RTEs is modulated as they enter the peripheral T cell pool^ref.
Allelic exclusion occurs only for b chain, while it is inefficient at the a locus, allowing a sizeable portion of T cells to carry 2 functional TcRs. In fact, rearrangement at the TcRa locus continues until signals for positive selection terminate RAG-1 and RAG-2 expression. Residual levels of RAG-1 and RAG-2 from the first wave of expression are sufficient to initiate recombination at the TcRa locus. However, in the absence of additional RAG expression, rearrangement is limited to the 5' portion of the Ja cluster of one locus. Thus, a secondary wave of RAG expression appears to be initiated with the purpose of extending recombination to the 3' portion of the Ja cluster in the rearranged TcRa locus and to the second TcRa allele. Dual TcRa expression at the cell surface is relatively common in immature thymocytes, but only positively selected TcRa is expressed in mature thymocytes. The primary (nonselectable) TcRa is subject to internalization and intracellular retention. Thus, a decision on TcRa cell surface expression is made based on TcRa engagement during positive selection.
Allelic inclusion of the TcRa locus carries the danger of potential reassembly of an autoreactive receptor that may lead to autoimmune reactions. A special case of allelic inclusion is termed TcR editing (equivalent to BcR editing in mature B lymphocytes), in which interaction of the primary autoreactive receptor with Ag triggers secondary rearrangement. Although safer mechanism of elimination/silencing of autoreactive lymphocytes exists and could be used, the immune system takes the risk to let these cells alive as it allows expansion of the TcR repertoire and multiple checkpoints (regulatory T cells, tissue-specific expression of activating and inhibitory costimulatory molecules, secretion of immunomodulatory cytokines, Fas-FasL interaction, ...) can eventually styill prevent autoreactivity from developing into autoimmunity.
Gene exclusion occurs between d and b chains (both present on chromosome 7), with the former tried before the latter. Only then the rearrangement of the sister chain gene on chromosome 14 occurs : if b chain has been chosen, then a chain is rearranged, while if d chain has been chosen, then g chain is rearranged.
TcR genes do not undergo somatic mutation in regions coding for the CDRs (a safety measure to prevent the development of autoreactive T cells ?). there is no splicing out of the transmembrane domain and thus TcR are not secreted. More, unlike antibodies, TcRs bind not to free Ag but only to Ag fragments presented to them bound to MHC molecules on the surface of APCs. The value of this MHC restriction is to focus the T cell on other cells; the T cell's function involves acting on other cells, and therefore it would not be efficient or safe for T cells to act unless they are already attached to the appropriate cell.
There are 2 Ig domains per TcR chain : a constant domain proximal to cell membrane and a variable domain distal to cell membrane. The variable domain has 3 hypervariable regions (HV) or complementarity-determining regions (CDR). V gene segments generate CDR1 and CDR2 while junctional flexibility and nucleotide addition in V-J and V-D-J generates CDR3.
A T-cell repertoire of ~ 10¹⁵ TcR (~ 25 million specificities) is created.
They recognize only MHC-co-presented, membrane-anchored, 8÷16 amino acids-long, usually hydrophobic peptides. Every TcR is specific for a ligand made up of the combination of a self MHC allele and a non self protein epitope : TcR interaction with peptide-MHC complexes occur by a 2-step process. First the CDR1 and CDR2 loops (histotope) of the TcR chains dock on the MHC helices, then and induced-fit mechanism allows CDR3 (paratope) to interact specifically with the peptide in the MHC groove. Crystallographic analysis of several class-I-restricted and one class-II-restricted TcRs has shown that TcRa has more contacts with peptide than TcRb and is presumably of greater importance in peptide recgnition. Besides, TcRa is also important in making contacts with the a-helices of the MHC. The kinetic proof-reading model indicates that the TcR must be engaged by peptide-MHC for long enough to initiate the complete set of intracellular biochemical signalling events that are required for T-cell activation. The 2 most well-defined parameters of TcR engagement are :
• ligand density
• TcR affinity
Although the ability of a peptide to activate a T cell often correlates with the half-life of the TcR-peptide-MHC complex interaction, not all peptides conform to this rule. K2 and K3 peptides, analogues of a moth cytochrome c (MCC) peptide, were more stimulatory than predicted. Thermodynamic analysis of the TcR peptide-MHC interaction indicated greater changes in heat capacity for K2 and K3, compared with MCC. These changes were associated with structural rearrangement and flexibility after TcR binding. By considering both the half-life of TcR binding and the change in heat capacity, the ability to activate a T cell can be predicted, indicating that conformational change and flexibility contribute to T-cell activation^ref.
The transmembrane regions of both TcR chains are positively charged and can interact with the negatively charged transmembrane regions of a complex of 6 invariant polypeptide chains (d, e, g, g_{FcgRI, FcgRIIIA, and FceRI}, h and CD247 / z : the latters 2 arise from alternative splicing of a same gene), termed the CD3 complex. These chains associate to produce 3 different kinds of dimers on T cell surface :
• ge heterodimer
• de heterodimer
• zz homodimer (majority) or zh heterodimer (minority) or zg_{CD16, CD64 and FceRI} (rare) or zCD16a / FcgRIIIA (rare) or zCD16b / FcgRIIIB (rare)
The z chain is associated to the short intracellular domains of TcR chains. The g, d, and e chains each contain a single ITAM while the z and h chains each contain 3 ITAMs. Upon ligand binding 2 events occur :
• a conformational change in CD3e exposes its Pro-rich sequence (PRS) for binding to the SH3 domain of Nck1.
• TcR-CD3 complex clustering results in cross-phosphorylation of CD3 ITAMs by p56^Lck and then the phosphorylated moieties recruit the SH2 domains of ZAP70 and/or FYN. Further LCK directly phosphorylates and activates Itk, which in turns activates PL-Cg₁.
Assembly of the TcR begins in the ER with the formation of z-containing dimers and dimers of TcR, and is completed in the trans-Golgi compartment when a TcR:CD3 complex associates with a z-containing to form the octameric holoreceptor. Extensive editing in the form of pre-Golgi retention ensures that only corrected folded and assembled TcR complexes are allowed to reach the cell surface. Stable assembly and expression of surface TcR is limited by the amount of available z. The majority (85-95%) of newly synthetized ab and CD3gde are degraded within 4 hours of synthesis, whereas 80-100% of z is long.lived and has a half-life of 10-20 hours. In the case of isolated subunits or partial complexes lacking any chain other than z, retention and degradation in the pre-Golgi compartments also occur. Hexameric complexes lacking z are reported to be transported from the Golgi complex to lysosomes where they are degraded. However, a small proporion of z-deficient TcR is believed to escape to the surface, accounting for the low expression of TcR in T cell hybridomas and mice lacking the z-chain. In contrast, fully assembled octameric complexes are transported to the surface where they are long-lived. Like many other cell surface receptor, TcR-CD3 complexes are constitutively internalized and recycled back to cell surface. The functional consequence of rapid cycling is that surface receptor levels, which regulate the ability of the T cell to be activated, respond rapidly to small changes in the rate constants for internalization and surface transport. Changes in surface TcR also occur physiologically, TcR engagement induces down-regulation of surface TcR levels, activation of protein kinases, and targeting of activated receptors to lysosomes for degradation. Down-regulation of unengaged receptors is also reported to occur during activation. In addition, TcR levels are dinamically regulated during thymic devlopment, with immature DP CD4⁺CD8⁺ cells having 10-fold lower cell surface TcR than more mature T cells that have undergone positive selection. The function of z in maintaining cell surface TcR levels is to mask internalization sequences inherent in other TcR components. Experimentally, reductions in surface TcR can be achieved with reagents that induce PKC activity, while elevations can be obtained with glycolipids such as ceramide, which can activate serine/threonine phosphatases. The z chain can be expressed at the surface independently of the other subunits and cycles at a different rate from the other components : functional dissociation of TcRab from CD3gde occurs after activation. Recent data indicate the presence of 2 TcRab dimers in a single TcR complex.
Although the a- and b-chains of the TcR are clonotypic, the remaining structural and costimulatory interactions (a.k.a. signal 2) are invariant, thereby allowing diversity in Ag recognition while conserving mechanisms of signal transduction upon binding between T-cell and APC (MHC I⁺II⁺) or target cell (MHC I⁺II^-).
Outside the thymus, the TcR genes can undergo affinity maturation by point mutations.
T cells are constantly attaching to, crawling on, and detaching from APCs, with the duration of contact averaging 6-12' when the cells are observed interacting on a collagen matrix. Although collagen may make T cells behave this way, the cells are unlikely to encounter collagen when interacting with APC in the T-cell area of the lymph node and are unlikely to behave that way at that primary site of naïve T-cell activation. By contrast, T cells in the dermis and other solid tissues would always be exposed to abundant collagen, and thus would interact in a serial encounter mode that might be ideal for T-cell effector functions.
For many years work on lymphocyte activation had described the aggregation of antigen receptor clusters as "caps" at the T-cell-APC interface. The breakthrough came with high-resolution 3-D fluorescence microscopy and antibody staining as well as video microscopy using T cells and artificial bilayers^{ref1, ref2}, which revealed a complex organization. Stimulation through both the TcR and other T-cell surface molecules (CD28 and LFA-1) triggers the active transport of TcRs from all around the cell into the interface^ref, forming the central cluster of receptors. This TcR movement sweeps across the adjacent B-cell area and gathers a selection of MHC molecules opposite it. One function of the synapse seems to be directed secretion, akin to its neuronal counterpart. The synapse likely serves to avoid the delivery of secreted factors to the wrong cells, leading to cell death or, perhaps more detrimentally, the inadvertent activation of self-reactive lymphocytes, resulting in autoimmunity. Also at least one report suggests that synapse formation may have a dampening effect, at least at high peptide concentrations^ref.
Immunological synapse (IS) / supramolecular activation cluster (SMAC)^{ref1, ref2, ref3} is divided into :
• central zone (C-SMAC / c-SMAC)
• peripheral zone (P-SMAC / p-SMAC)
Real time evaluation of events in the immunological synapse can be achieved with :
• live-cell fluorescent imaging of T cells in contact with supported planar bilayers : this is a reconstitution method. The molecules in the bilayer can be made laterally mobile and fluorescently labeled, which allows direct observation of interactions of cell surface receptors with molecules in the bilayer. This is the only system where accumulation of fluorescence is sure to have a 1:1 relationship with receptor-ligand interaction. The caveat is that cytoskeletal mechanisms and membrane organization in a live APC are lost. The rigid nature of the planar bilayer is probably not a major drawback because T cell-APC interfaces are generally flat and the change in degres of ffreedom before and after synapse formation is the same in the cell-planar bilayer and cell-cell situation.
• real-time confocal imaging in the T cell-APC interface : it is the more physiological of the two, but interpretation is difficult becayse the lateral and cytoplasmic interactions of each surface receptor cannot easily be controlled
Stages :
• adhesion via LFA-1/ICAM-1 interactions which slows the T cell
• immature immunological synapse formation occurs within 30 seconds
• C-SMAC contains mainly LFA-1/ICAM-1 and very few TcR/MHC:peptide complexes
• P-SMAC contains the TcR which samples the MHC-peptide, LCK and PKCq. Live-cell video microscopy imaging studies show that Ag recognition occurs within 30" of T cell-APC contact, as shown by a sharp increase in [Ca²⁺]_cytosol  (one of the prime responses to signaling) which signal the T cell to stop. A single MHC-peptide is enough for the T cell to trigger a calcium response and to stop. Click here to view signal tranduction pathway from TcR complex (a.k.a. signal 1) (see also canonical and schematicT-cell STP [free registration required]). Within 1', small MHC:peptide clusters form in the contact zone. It takes about 10 peptides to make a synapse. The suppressor ot TcR signalling 1 (Sts1) and 2 (Sts2) are redundant negative regulators of TcR signalling^ref.


T cell activation is initiated and sustained in TCR-containing microclusters generated at the initial contact sites and the periphery of the mature immunological synapse. Microclusters containing TCRs, the tyrosine kinase Zap70 and the adaptor molecule SLP-76 are continuously generated at the periphery. TCR microclusters migrated toward the C-SMAC, whereas Zap70 and SLP-76 dissociated from these microclusters before the microclusters coalesced with the TCR-rich C-SMAC. Tyrosine phosphorylation and calcium influx are induced as microclusters form at the initial contact sites. Inhibition of signaling prevents recruitment of Zap70 into the microclusters. These results indicated that TCR-rich microclusters initiate and sustain TCR signaling^ref.
The earliest step in synapse formation can be directly correlated with topologically driven receptor segregation based on the size of the different receptor-ligand pairs. For example, the 15-nm CD2:CD58 pair segregates from the 40-nm LFA-1:ICAM-1 pair. Receptor topology appears to be very important, because the similarity in size between the CD2:CD48 pair and the TcR: MHCp pair at around 15 nm is critical for sensitive T cell activation on thebasis of experiments where CD48 with additional domains added to lengthen it inhibited T cell activation by APC. The driving force for this segregation might be the thermodynamic advantage of aligning the membrane surfaces with nm precision, which serves to enhance interactions. An energetic penalty associated with membrane bending may define the minimum size of the segregated domains.
• mature immunological synapse formation occurs within 1-5' and can be stable for hours
• C-SMAC contains :
• TcR/MHC:peptide complexes : over 3-20' the TcRs engaged in P-SMAC move from synaptic periphery toward the center. The binding of a T-cell antigen receptor (TCR) to peptide antigen presented by major histocompatibility antigens (pMHC) on antigen-presenting cells (APCs) is a central event in adaptive immune responses. The mechanism by which TCR-pMHC ligation initiates signalling, a process termed TCR triggering, remains controversial It has been proposed that TCR triggering is promoted by segregation at the T cell-APC interface of cell-surface molecules with small ectodomains (such as TCR-pMHC and accessory receptors) from molecules with large ectodomains (such as the receptor protein tyrosine phosphatases CD45 and CD148). Increasing the dimensions of the TCR-pMHC interaction by elongating the pMHC ectodomain greatly reduces TCR triggering without affecting TCR-pMHC ligation. A similar dependence on receptor-ligand complex dimensions was observed with artificial TCR-ligand systems that span the same dimensions as the TCR-pMHC complex. Interfaces between T cells and APCs expressing elongated pMHC showed an increased intermembrane separation distance and less depletion of CD45. These results show the importance of the small size of the TCR-pMHC complex and support a role for size-based segregation of cell-surface molecules in TCR triggering^ref.
• lipid rafts : functional and imaging experiments indicate that in the plasma membrane of T lymphocytes (whose formation is dependent on CD28 costimulation and regulated by WASp) may play an important role in helping to form the structure of the IS. Lipid raft proteins have a random distribution during localized activation of the T-cell receptor^ref. By expressing GPI-linked FRET donor and acceptor proteins in Jurkat T cells, it has been showed that distribution of lipid rafts (identified by the GPI-linked proteins) throughout the cell membrane is not altered by activation with CD3-specific antibody^ref
• CD80 / B7-1 / B7.1 --- CD28
• coreceptors that activate p56^Lck :
• CD8 dimers (usually a disulfide linked ab heterodimer, rarely an aa homodimer: a is required for surface expression of b) (a domain) --- MHC I (non polymorphic a₃ domain (also a₂ domain and b₂-microglobulin ?)
or
• p55^CD4 (containing 4 extracellular Ig-like domains termed D1-D4) homodimers (via K318 and Q344 in the D4 domain) --- MHC II (b₂ domain) : localization in lipid rafts (see below) has a fundamental importance for CD4 (but not for CD8) signalling (and also for HIV-1 entry). Several of the CD4 ligands have been described as multimers, including MHC II as a dimer of ab heterodimers, the chemoattractant cytokine IL-16 as a homotetramer, and HIV-1 gp160 as a trimer.
The aminoterminal region of LCKforms a heterodimeric complex with CD4 or CD8a only in the presence of Zn²⁺, with both CD4 and CD8a forming similar "zinc clasp" cores despite having little sequence similarity in their cytoplasmic regions^ref
• PKCq
• LCK
• FYN
• LAT
• Vav1
c-SMAC increases both TcR activation and TcR degradation : at low levels of stimulation, TcR clustering in the c-SMAC enhances T-cell activation, whereas at high levels of stimulation, the increased phosphorylataion of TcRs in the c-SMAC leads to increased degradation that protects against T-cell death due to overstimulation. This could be a mechanism to ensure that the immune system can respond appropriately to antigenic stimulation over a wide range of magnitudes. The paucity of signalling molecules in the c-SMAC over long periods of time after stimulation is the result of intense signalling, owing to the clustering of TcRs and increased TcR phosphorylation. TcR internalization occurs at a fixed rate and, on the basis of existing data, nonphosphorylated or single-phosphorylated TcRs are returned to the surface of the T cell, whereas double-phosphorylated TcRs are degraded^ref
• P-SMAC contains :
• CD2 --- CD48
• CD11a-18 / LFA-1 / a_Lb₂ integrin --- CD54 / ICAM-1
• CD43 / sialophorin / gpL115 / leukosialin
• CD81 / TAPA-1
• talin 1
The mechanis of SMAC organization is less clear. One leading hypothesis is that the process of cSMAC formation is very similar to antibody-mediated capping, involving an actin-myosin transport process. This transport process may be activated by early signaling that peaks during the time when the synapse is in the process of being formed. There is direct evidence that actin-myosin transport toward the synapse is active during synapse formation. However, the definitive evidence that cSMAC formation takes place through directed transport is lacking. Alternatively when the thermally driven membrane oscillations harmonize with the kinetics of the receptor-ligand interactions, patterns like the mature immunological synapse can be generated in a computer simulator. This synapse assembly model can also reproduce the correlation between the kinetics of TcR:MHCp interaction and mature synapse formation for T_H cells. Another interesting prediction of the synapse assembly model is that some self-MHCps may synergize with agonist MHCp to lower the threshold for mature synapse formation. Recent experiment demonstrate that model self-MHCps do in fact amplify TcR responses to low levels of agonist MHCp and promote mature synapse formation. The major difference between  the "actin-myosin transport" and "synapse assembly" models is that single TcR are expected to follow straight trajectories to the cSMAC in the formed and a "jagged" path to the cSMAC in the latter. Therefore, the 2 models can be distinguished with fluorescence single molecule tracking. The pSMAC acts as a gasket to contain secretion of soluble agents (cytokine and cytotoxic agents) that are released near the cSMAC and held in the synaptic cleft. In this way, the only cell that is acted upon is the cell with which the T cell forms the synapse, and this function is clearly analogous to that of the neural synapse. In the immunological synapse, the gasket is integrin dependent, whereas at the neural synapse cadherins and other molecules form the gasket.
As well as being enriched in certain lipids, a number of protein are also concentrated in rafts largely as a result of modification with GPI anchors or acylation, which pack well into the ordered lipid environment (e.g. p56^LCK and LAT). Src and Syk family kinases are activated early in immunological synapse formation, but this signaling process returns to the basal level after 30'. When T cells conjugated to APC move across the APC surface, they appear to frag the synapse with them. The IS is involved in TcR downregulation and endocytosis, perhaps providing a platform for attenuating the TcR signaling by means of other receptors. An alternative view is that TcR signaling does occur in the cSMAC, but that the receptors are internalized and degraded too quickly to be noticed. The immunological synapse may be terminated by negative signaling receptors like CTLA-4 interacting with CD80 and CD86 and by changes in cellular interactions during the mitosis : both CTLA-4 induction and the first mitosis occur around 36 hours after first contact for a naive T_h cell.
PDZ-domain-containing protein Discs large 1 (Dlg1) is involve in immunological synapse formation. PDZ domains can support heterotypic and homotypic interactions, making proteins that contain them ideal scaffolding molecules. Indeed, Dlg1 is already known to be expressed at synapses between neurons in the central nervous system. In T cells, Dlg1 is recruited with actin to the contact zone with an antigen-presenting cell, and it also associates with T-cell signalling molecules such as LCK. Dlg1 seems to inhibit signalling from the synapse: overexpression of the protein attenuated VAV1-induced transcription activity,whereas Dlg1 suppression potentiated the response to stimulation through CD3^ref.
Web resources : Immunological Synapse Fact Book at Washington University.
One clear characteristic of these surface molecules is that they have strikingly weaker affinities (1-100 mM) than receptors that snatch ligands from 3D environments (in the low nM to pM range)^ref. And yet these surface molecules make no apparent sacrifice in specificity, being just as sensitive to minor ligand changes as much higher-affinity molecules are. This illustrates the unique environment in which surface molecules operate, where diffusion is largely confined to 2D and massive polyvalency and confined space offset low affinity. It also seems important for transient cellular interactions to employ molecular bonds that can easily be broken : multiple cell-surface interactions stronger than about 10 mM could not be disrupted without damaging vesicle membranes^ref. It has long been asserted that T cells are very sensitive to antigen, but published reports estimate that anywhere from 1 to 400 peptides per APC are necessary to fully activate at least some cells. Recently a laboratory developed a procedure to visualize individually labeled peptides bound to MHC molecules on cell surfaces and thus determine exactly how many ligands a T cell was in contact with, and then using video microscopy, follow the response^{ref1, ref2}. Surprisingly, 4 different T-cell lines, representing both helper and killer cells, were all able to detect even 1 molecule of antigen-MHC, though it took about 10 peptides to elicit maximal calcium release and form a stable synapse. This finding could not have been made using population-based assays. Large numbers of cells, even in a clonal population, won't respond to the same stimulus in exactly the same way at the same time. Up until now, we have had to hope that they did, but I predict that single-cell imaging will set the new standards. After all, what good is careful quantitation if it's off by 400-fold? Just as rod cells in the eye can detect even a single photon, CTLs can kill on the advice of only 3 peptide-MHC ligands^ref. This redundancy could be a way to eliminate cytocidal "accidents" due to peptide "noise."
Dephosphorylation and inactivation of Ezrin-Radixin-Moesin (ERM) proteins (general crosslinkers between the actin network near the cell surface and the plasma membrane, inactivated after antigen recognition by the TcR) through a GEF Vav1-small GTPase Rac1 pathway leads to relaxation of the T-cell cytoskeleton and favour conjugate formation with APCs^ref.
Human CTL clones require only cognate peptide-MHC complexes and the adhesion molecule CD54 / ICAM-1 to forma a mature immunological synapse. Interestingly, in the presence of a threshold density of ICAM1 alone, both CTL clones and blood-derived CD8⁺ effector T cells form a transient pSMAC-like structure known as ring junction that lacks a cSMAC and fails to initiate granule polarization. At lower ICAM1 densitites, the NKG2D ligand MICA facilitated their formation. As ICAM1 expression is upregulated during inflammation and MICA expression after cellular stress, the ring-junction formation is enhanced under these conditions to augment immune surveillance by CTLs^ref.
Both the nervous system and the immune system utilize specific molecular recognition events between discrete cells, cell:cell adhesion, positional stability, and directed secretion for communication to fulfill their respective functions. Both systems have evolved highly sophisticated forms of information storage. In the immune system, the synapse functions to provide specificity to the action of otherwise nonspecific soluble agents through confinement to the synaptic cleft and to coordinate cell migration and antigen recognition during induction of the immune response. A critical difference in the functional context of the neural and immunological synapses is in the basic wiring of the systems. The CNS is to a great extent hard-wired and retains precise connectivity patterns throughout adult life, with neurons projecting long axonal processes that form synapses on complex dendritic trees of other neurons that may be quite distant from the cell nucleus. Whereas CNS synapses may be formed and pruned back in the adult, the long dendritic and axonal processes anchor the cell bodies and prevent cell migration. Thus, the CNS synapse is an "action at a distance" junction, in relation to the nucleus where transcription takes place. Therefore, most functions of CNS synapses may be considered "postnuclear" in that they are carried out without a requirement for immediate transcriptional regulation, although synaptic stimulation can lead to transcriptional changes in the long term. CNS synapses can alter their efficacy by processes such as receptor clustering by scaffold proteins. Synapse-forming neurons are terminally differentiated. In contrast, the immune system operates through rapidly migrating T cells and their partners, the dendritic cells (DCs), that congregate in tissues like lymph nodes. This is essential to the operation of the immune system because each T cell expresses a differente antigen specificity and the point at which an antigen will enter the body to become associated with a DC is not predictable. So it is essential that T cells and DCs congregate and make many random contacts to possibly find a match and form a synapse. Migrating T cells assume morphologies similar to the growth cones of neurons but move at least 50 times faster. Thus, the T cell and DC cover greater distances than neurons in search of foreign antigens, but when the synapse is formed it is immediately proximal to the transcriptional machinery in the nucleus. Whereas adhesion in the immune system is integrin mediated, cadherins are believed to function in an analogous capacity at the CNS synapse.
• in the cytotoxic T synapse the CTL is the presynaptic cell and the APC the postsynaptic cell.
• in the helper T synapse the APC is the presynaptic cell directing the exocytosis of MHCp into the nascent synapse. The newly secreted MHCp can then be bound by the TcR on the T_h cell, which represents the postsynaptic side. Cosecreted with the MHC are accessory molecules which promote T cell activation and differentiation
CARMA1 / CARD11 and the Cbl family of ubiquitin ligases scaffolding proteins may determine the polarity of response to antigen by promoting assembly around antigen receptors of competing multiprotein signal complexes : immunosomes versus tolerosomes. Each of the factors that influence immunogenicity or tolerogenicity—stage of lymphocyte differentiation, concurrent engagement of inhibitory or costimulatory receptors, extent of receptor crosslinking, and prior antigen experience—may be integrated in lymphocytes through their capacity to influence the probability of assembling immunosomes versus tolerosomes
TCR revision results in cellular rescue upon expression of an alternate TcR that no longer recognizes the tolerogen. It requires the rearrangement of novel TCR b-chain genes and is limited to recent thymic emigrants (RTE) that have maintained RAG expression and TcR loci in a recombination-permissive configuration
• B cell receptor (BcR) / membrane immunoglobulin (mIg) / surface immunoglobulin (sIg)(10^4÷5 per B cell) : H(eavy)₂L(ight)₂ chains.
• L chain may be k or l.
• k chains result from rearrangement on chromosome 2p12 of ...
• ~ 100 V segments
• 5 J segments
• 1 C segment (IGKC)
• l chains result from rearrangement on chromosome 22q11 of ...
• ~ 100 V segments (several are pseudogenes)
• 6 J-C segments
• IGLC1 (Mcg marker)
• IGLC2 (Kern^-Oz^- marker)
• IGLC3 (Kern^-Oz⁺ marker)
• IGLC4 (pseudogene)
• IGLC5 (pseudogene)
• IGLC6 (Kern⁺Oz^- marker)
... creating l_1-4 chains. The general coding for the p22.5^{L chain} is as follows : V (variable) exon codes for amino acids 1 through 95, the J (joining) exon codes for amino acids 96 through 110, and the C (constant) exon codes for amino acids 111 through 214.
• H chains result from rearrangement on chromosome 14q32 of
• V_H region
• ~ 100-300 V segments
• 5 D segments
• 9 J segments
• 9 C segments (mdI S-box g₃I S-box g₁I S-box y_eI S-box a₁I S-box y_g I S-box g₂I S-box g₄I S-box eI S-box a₂). Another y_e exist on chromosome 9p24.
The IgH locus is controlled by cis-acting elements, including Emu and the 3' IgH regulatory region which flank the C region genes within the well-studied 3' part of the locus. The 5' region of the V_H gene cluster contains a single cluster of DNase I hypersensitive sites within the 5' flanking region, and a candidate Igh regulatory region defined by pro-B cell-specific hypersensitivity (HS1) interacts with factors implicated in regulating VDJ recombination^ref.
The capital Latin letter indicates the chain kind : the Greek letter indicates the chain isotype / class, while the Arab number at the pedex indicates the subisotype / subclass (whose antigenic differences are termed isotypic variation)
For some heavy chains even allotypes / allotypic markers exist (whose antigenic differences are termed allotypic variation) for constant regions :
• Gm (> 30 : G₁m in g₁chain, G₂m in g₂ chain, G₃m in g₃ chain, G₄m in g₄ chain)
• A₂m in a₂ chain (2)
Allelic exclusion involves the H chain locus and requires a preBcR signal : it doesn't allow gene rearrangement (or at least a functional gene rearrangement, i.e. the resulting mRNA in untranslatable (abortive rearrangement)) in the homologous chromosome. This doesn't occur at the L chain locus (allelic inclusion).
Gene exclusion occurs between the k and the l chains, with the former tried before the latter and so present in ~ 60% of Ig molecules.
Gene rearrangement depends more on 3' enhancer than on Ig gene nucleotide sequence : it occurs in germinal centers. The orientation of the RSS, combining a one-turn with a two-turn element, ensures that a V_L segment only joins with a J_L segment and not with another V_L segment; and that V_H, D and J_H join to each other in the proper sequence. Apart from endonucleases and NHEJ machinery, gene rearrangment in BcR also requires terminal deoxynucleotidyl transferase (TdT), STP molecules (gp40÷45^{CD79A / Iga}, gp33÷40^{CD79B / Igb}, 45 / B220 / LCA, Btk) and TFs (Oct-2, EBF, PAX5 / BSAP, GATA2). If no productive rearrangements occur, the B cell dies by apoptosis. Every V gene has its own promoter, leading sequence and intron : the intron is then eliminated in RNA splicing. Immunoglobulin heavy chain rearrangement (V_H-to-DJ_H) occurs only in B cells, as methylation of lysine 9 on histone H3 (H3-K9) inhibits it in other lineages : Pax5 / BSAP, a transcription factor required for B cell commitment, is necessary and sufficient for the removal of H3-K9 methylation in the V_H locus^ref. The D genes may be translated in every of the 3 reading frames (although 2 are preferred) and up to lenghts depending on the resulting CDR3. TdT may add up to 15 random N nucleotides in N regions, i.e. between  between V and D and/or D and J in the CDR3 of the H chain, which bind up to 50% of amino acids in the epitope. P-nucleotide addition consists in cleaving the hairpin loop at the DNA ends to result in new sequences in the junction region.
Transcription, rearrangement, and methylation of the IgH loci depend on activating elements in the J_H-C_m intronic LCR : 5' - MAR - core enhancer Em - MAR - switch region.
Generation of diversity (GOD) :
• VDJ combinations for H chain (A) : 300 V segments ^. 5 D segments ^. 9 J segments = 13,500.
• VJ combinations for average L chain (A) : 80 V segments ^. 5 J segments = 400.
• number of splice sites (s) :  2 for the H chain and 1 for the L chain.
• 5 base variation at s splice sites (B = A ^. 5^s) : 3 ^. 10⁵ for the H chain; 2000 for the L chain.
• insertion of k nucleotides at each splice site can be calculated as DR_n,k + DR_n,k-1 + ... + DR_n,1, where n = 4 (A,T, C and G) and 0 < k < 15. As on average 6 bases are added at each splice site => DR_4,6 + DR_4,5 + DR_4,4 + DR_4,3 + DR_4,2 + DR_4,1 + DR_4,0 = 4⁶ + 4⁵ + 4⁴ + 4³ + 4² + 4¹ + 4⁰ = 5461. This can give 9 ^. 10¹³ different H chains and 2 ^. 10³ different L chains (C = B ^. 5461ⁿ).
• L and H chain combinations :1.8 ^. 10¹⁶. Because sequences (in the splice sites) have to be read in frame, this theoretical number is high. The actual number of antibody possibilities is ~ 1.9 ^. 10⁷.
Anyway the B cell repertoire in mice and humans is shaped by nonrandom use of individual V genes during V(D)J recombination, resulting in a few genes being greatly overrepresented, while many other genes undergo gene rearrangement at much lower frequency. There is much heterogeneity in the recombination signal sequence (RSS) flanking the individual V genes, with some RSSs being closer to the consensus sequence than others. Although these natural variations in the RSS clearly contribute to nonrandom V gene usage, there are certainly many other factors that influence V gene rerrangement frequency. One such factor could be differential accessibility to recombination due to localized changes in histone acetylation, DNA methylation, or chromatin remodeling at the level of gene families or even individual genes. The transcription factors themelves may be involved in targeting chromatin remodeling or gene-specific accessibility, either through effects on germline transcription, or by recruiting chromatin-modifying complexes : although V gene coding region sequences are highly conserved among members of the same family and to a lesser degree between families, core promoter regions between families show little conservation, and the remaining flanking regions of these genes exhibit quite extensive sequence variability. Only the octamer and some TAT box-like motifs are conserved among V gene promoters, while other TF binding sites may only be shared among members of the same family : the majority of V gene promoters therefore bind combinations of TFs unique to each individual gene family. Besides TF binding sites, Ig gene promoter regions contain other regulatory elements (e.g. matrix attachment regions (MARs)).
The resulting protein sequence of the variable (V) regions (the N-terminal portion, composing one homology region, of an immunoglobulin heavy (V_H) or light (V_L) chain that varies in amino acid sequence among chains of a single type : 108 amino acids in V_H and 109 amino acids in V_L(1-97 from V segment, 98-109 from J segment)) is as follows :
• framework (FW) region 1
• complementarity-determining region (CDR) / hypervariable (HV) region 1 : regions a few amino acids in length within immunoglobulin heavy and light chain variable regions at which the amino acid sequence is extremely variable; there are 3 in light chains and 4 in heavy chains. They contain most of the amino acid residues forming the antigen binding site
• FW2
• CDR2 / HV2
• FW3
• CDR3 / HV3
• FW4
Although variable regions vary among antibodies of different specificity (idiotypic variation : the antigenic differences that characterize the different amino acid sequences and structures of immunoglobulin variable regions (idiotypes) and corresponding differences in antigen specificity), all of the immunoglobulins produced by a single clone of plasma cells (a clonotype) have the same variable regions but may have different constant regions.
V segment codes for both the CDR1 and CDR2, while DJ segments code for the CDR3. Binding of an Ab to an Ag involves only a small number of residues within the CDRs designated asspecificity-determining residues (SDRs). The final sequence can be represented according to several numbering schemes :
• Kabat-Wu's numbering scheme is the most widely used but has the following problems
• the position of amino acid insertions in CDR-L1 (27) and CDR-H1 (35) is mistaken.
• the number of (letter) positions available for describing amino acid insertions is often lower than necessary.
The numbering throughout the chains is as follows:
• light chain

       0     1     2     3     4     5     6     7     8     9
      10    11    12    13    14    15    16    17    18    19

      20    21    22    23    24    25    26    27
      27A   27B   27C   27D   27E   27F               28    29
      30    31    32    33    34    35    36    37    38    39
      40    41    42    43    44    45    46    47    48    49

      50    51    52    53    54    55    56    57    58    59
      60    61    62    63    64    65    66    67    68    69
      70    71    72    73    74    75    76    77    78    79
      80    81    82    83    84    85    86    87    88    89
      90    91    92    93    94    95
      95A   95B   95C   95D   95E   95F   96    97    98    99
     100   101   102   103   104   105   106
     106A                                      107   108   109

• heavy chain

       0     1     2     3     4     5     6     7     8     9
      10    11    12    13    14    15    16    17    18    19
      20    21    22    23    24    25    26    27    28    29
      30    31    32    33    34    35
      35A  35B                            36    37    38    39
      40    41    42    43    44    45    46    47    48    49
      50    51    52
      52A   52B   52C   53    54    55    56    57    58    59
      60    61    62    63    64    65    66    67    68    69
      70    71    72    73    74    75    76    77    78    79
      80    81    82
      82A   82B   82C   83    84    85    86    87    88    89
      90    91    92    93    94    95    96    97    98    99
     100
     100A  100B  100C  100D  100E  100F  100G  100H  100I  100J



     100K  101   102   103   104   105   106   107   108   109
     110   111   112   113

• Chothia's numbering scheme places insertions in CDR-L1 and CDR-H1 at the structurally correct positions (30 and 31, respectively).
• light chain

       0     1     2     3     4     5     6     7     8     9
      10    11    12    13    14    15    16    17    18    19
      20    21    22    23    24    25    26    27    28    29

      30    
      30A   30B   30C   30D   30E   30F
            31    32    33    34    35    36    37    38    39
      40    41    42    43    44    45    46    47    48    49
      50    51    52    53    54    55    56    57    58    59
      60    61    62    63    64    65    66    67    68    69
      70    71    72    73    74    75    76    77    78    79
      80    81    82    83    84    85    86    87    88    89
      90    91    92    93    94    95
      95A   95B   95C   95D   95E   95F   96    97    98    99
     100   101   102   103   104   105   106
     106A                                      107   108   109

• heavy chain

       0     1     2     3     4     5     6     7     8     9
      10    11    12    13    14    15    16    17    18    19
      20    21    22    23    24    25    26    27    28    29
      30    31 
      31A   31B
                  32    33    34    35    36    37    38    39
      40    41    42    43    44    45    46    47    48    49
      50    51    52
      52A   52B   52C   53    54    55    56    57    58    59
      60    61    62    63    64    65    66    67    68    69

      70    71    72    73    74    75    76    77    78    79
      80    81    82
      82A   82B   82C   83    84    85    86    87    88    89
      90    91    92    93    94    95    96    97    98    99
     100
     100A  100B  100C  100D  100E  100F  100G  100H  100I  100J
     100K  101   102   103   104   105   106   107   108   109
     110   111   112   113

CDRs can be placed on the numbering scheme according to 4 different definitions :
 

CDR	Kabat	AbM	Chothia	Contact
L1	L24--L34	L24--L34	L24--L34	L30--L36
L2	L50--L56	L50--L56	L50--L56	L46--L55
L3	L89--L97	L89--L97	L89--L97	L89--L96
H1	H31--H35B (on Kabat's  numbering scheme)	H26--H35B	H26--H32..34	H30--H35B
H1	H31--H35 (on Chothia's  numbering scheme)	H26--H35	H26--H32	H30--H35
H2	H50--H65	H50--H58	H52--H56	H47--H58
H3	H95--H102	H95--H102	H95--H102	H93--H101

Note that as the Kabat's numbering scheme places the insertions at H35A and H35B, when applying Chothia's definition on Kabat's numbering scheme, the end of the CDR-H1 varies between H32 and H34 depending on the length of the loop :
• if neither H35A nor H35B is present, the loop ends at Chothia's H32
• if only H35A is present, the loop ends at H33
• if both H35A and H35B are present, the loop ends at H34

Metaanalysis allows recognition of the following common features in a given CDR :

CDR	start	residue before	residue after	length
CDR-L1	approx residue 24	almost always a Cys	almost always a Trp. Typically Trp-Tyr-Gln, but also, Trp-Leu-Gln, Trp-Phe-Gln, Trp-Tyr-Leu	10 to 17 residues
CDR-L2	almost always 16 residues after the end of L1	generally Ile-Tyr, but also, Val-Tyr, Ile-Lys, Ile-Phe	?	almost always 7 residues (except NEW (7FAB) which has a deletion in this region)
CDR-L3	almost always 33 residues after end of L2 (except NEW (7FAB) which has the deletion at the end of CDR-L2)	almost always Cys	almost always Phe-Gly-Xaa-Gly	7 to 11 residues
CDR-H1	approx residue 26 (almost always 4 after a Cys) [Chothia / AbM defintion];  Kabat definition starts 5 residues later	almost always Cys-Xaa-Xaa-Xaa	almost always a Trp. Typically Trp-Val, but also, Trp-Ile, Trp-Ala	10 to 12 residues [AbM definition];  Chothia definition excludes the last 4 residues
CDR-H2	almost always 15 residues after the end of Kabat / AbM definition of CDR-H1	typically Leu-Glu-Trp-  -Ile-Gly, but a number of variations	Lys/Arg-  -Leu/Ile/Val/Phe/ Thr/Ala- -Thr/Ser/Ile/Ala	Kabat definition 16 to 19 residues;  AbM (and recent Chothia) definition ends 7 residues earlier
CDR-H3	almost always 33 residues after end of CDR-H2 (almost always 2 after a Cys)	almost always Cys-Xaa-Xaa (typically Cys-Ala-Arg)	almost always Trp-Gly-Xaa-Gly	3 to 25(!) residues

Each V region is followed by a constant (C) region made up of 3/4 (C_H1-3/4) or 1 (C_L) domains. Papain (a protease from Carica papaya) proteolysis of the Ig creates 2 Fab (fragment antigen binding) fragments and 1 Fc (fragment cell-binding or crystallizable, made up of C_H1 and C_H2 on both H chains), while pepsin proteolysis creates 1 F(ab')₂ fragment and many smaller fragments arising from Fc digestion. In both digestions cleavage occurs in the hinge region, a short flexible Cys- and Pro-rich region between the C_H1 and C_H2 domains of immunoglobulin heavy chains (including interchain disulfide bonds) which allows each of the Fab regions (the “arms” of the Y -shaped immunoglobulin molecule) to move independently (form an angle up to 180°) as necessary to bind to antigens. The C_H2 domains have glycosylation (Fuc-Man-GlcNAc) sites and bind to C1q. C_H3 domains mediate binding to FcRs.
Immunoglobulin carries N-glycosylated oligosaccharides located mainly in the C_H regions. These act as spacers for the immunoglobulin molecule that are important for maintaining effector functions^ref. Because of locations of the sites in the interstitial region between the C_H2 domains, the oligosaccharide chains are incompletely galactosylated and sialylated^ref. Human antibodies do not generally contain O-linked oligosaccharides, with the exception of IgA, which can be O-glycosylated in the hinge region^ref. Glycosylation of the variable region is less common, but it has been found in some antibody molecules. Approximately 18% of the V^ref sequences in the Kabat database have been reported to contain a potential N-glycosylation site^ref, but this includes tumor and normal sequences. The motif for N-glycosylation is Asn-X-Ser/Thr, where X is any amino acid except Pro, Asp or Glu. A few germline V^ref genes have a naturally occurring N-glycosylation site, including V_H1-08, V_H4-34, and V_H5a. Studies of monoclonal immunoglobulins with antibody activity have shown that binding to antigen can be increased^{ref1, ref2} or decreased^ref by the presence of carbohydrate in the V region. There is also evidence that V-region glycosylation can alter physical properties^ref and other characteristics normally attributed to the Fc region^ref
The CDRs recognize soluble (i.e. hydrophilic) epitopes made up of 5-7 amino acids (non-linear !) or 6-7 monosaccharides. The epitope-binding portion (usually the 3 CDRs) is called paratope. When the antigen is known, the immunoglobulin is termed antibody. It is estimated that for each epitope from 50 to 300 antibodies of different affinity will be produced. An antibody molecule is usually divalent (i.e. able to bind 2 different epitopes) when alone. Generally antibodies cross-link the same epitope on 2 different antigen molecules (polygamy or polygamous binding) : occasionally, if the antigen has 2 identical epitopes that are close to each other, the 2 reactive sites of the antibody may react with the same antigen molecule (monogamy or monogamous binding). Look here for theoretical model of interaction between a multivalent Ab and a univalent Ag.
Antibody multispecificity can be mediated by conformational diversity of preexisting isomers (independent of antigen) that can adopt multiple functions and can increase the effective size of the antibody repertoire
 

ABG : directory of 3D structures of antibodies  Antibodies – structure and sequence by Andrew C.R.Martin  BLAST for antibody sequences at NCBI  Calculation of Antigen Selection Pressure on Immunoglobulin Genes Canonical structures and conformations of CDR3 in the VH domain How lymphocytes produce antibody from Cells Alive  Immune Recognition Group ImMunoGeneTics (IMGT) : LIGM Database Mats Ohlin Home Page Mike Clark's Immunoglobulin Structure/Function Home Page The Antibody Cross Reactivity Resource The Antibody Resource Page The Antibody Web V Base : the directory of human V gene sequences  Web Antibody Modelling (WAM)

Click here to view signal transduction pathway from the BcR (see also B-cell Ag receptor pathway [free registration required]) :
• BcR complex consists of a mIg noncovalently associated with a disulfide linked heterodimer CD79a / Iga + CD79b / Igb: both have long cytoplasmic tails containing an ITAM. Ag engaging and cross-linking mIg causes tyrosine kinases to phyosphorylate Y residues of the ITAMs. 45 / B220 / LCA, which dephosphorylates tyrosine residues, is also activated in a negative feedback.
• B cell coreceptor complex is a complex of 3 proteins which intensifies the activating signal resulting from cross-linking of the BcR, reducing by 2 orders of magnitude the number of mIg molecules which must be engaged by Ag for B cell activation :
• CD19 interacts with CD79a / Iga + CD79b / Igb, becomes phosphorylated, and activates the tyrosine kinase Lyn. This amplifies the activating signal transmitted through the BcR. CD19 expression is regulated by Pax5 / BSAP (whose neoplastic activation in t(8;21) in AML induces aberrant expression).
• CD81 / TAPA-1 --- protein E2 on HCV envelope
• CD21 / CR2 / C3dR / EBVR binds to :
• CD23 / FceRII
• complement-coated antigen bound to mIg, cross-linking the coreceptor complex to the BcR. Direct coupling of C3d multimers to protein Ag can increase the immunogenicity by greater than 1000-fold.
Although CR21 has a short cytoplasmic tail that does not have signaling capabilities, it nevertheless exerts potent B cell signaling effects through its noncovalent association with CD19 and CD81 / TAPA-1.
BcR inducibly associates lipid rafts together with CD20 (a 33- to 36-kDa transmembrane phosphoprotein involved in the activation, proliferation, and differentiation of B lymphocytes encoded by a gene on 11q12-p13.1, belonging to the TM4 span tetrafamily together with FceRIB and Htm4; not to be confounded with the tetraspanin family, which include CD37, CD53, CD81 and CD82). The predicted amino acid sequence of the CD20 suggests 4 transmembrane-spanning regions with both N- and C-termini located in the cytoplasm. It works as a putative store-operated Ca²⁺ channel (SOCC) activated by the depletion of intracellular calcium following BcR ligation. The SOCC could be created by CD20 multimers, or be a separate protein activated sterically or via second messengers. CD20 is expressed since pre-B cells to m⁺ B cells to m⁺d⁺ B cells to activated B cell to B blast, while is absent on pre-B and plasma cells. Expression of CD34 and CD20 are mutually exclusive. CD20 expression is higher in GC B cells than in mantle zone B-cell than in marginal zone B-cells. CD20 expression can be upregulated at the transcriptional level by bryostatin^ref via ERK and PKC, but not p38 nor dexamethasone, making B cells more sensitive to apoptosis induced by anti-CD20 monoclonal antibodies : this cause relocation into lipid rafts. The lack of sIg expression in CD20⁺ B cells is a surrogate of neoplasia. CD20^-/- mice display a normal phenotype.
The B-cell specific major raft protein raft-linking protein (RAFTLIN) localizes in rafts by means of an acylated motif and is constitutively associated with the BcR, although it does not bind the BcR directly, being important for BcR signalling and recruitment of Lyn and ganglioside GM₁.
Each B lymphocyte carries around 50,000 sIg on its surface. The BCR is a monomer on the surface of resting cells. Binding of multivalent antigen clusters the BCR, resulting in the simultaneous phosphorylation of and a conformational change in the BCR cytoplasmic domains from a closed to an open form. Notably, the open conformation required ITAM and continuous Src family kinase activity but not binding of the kinase Syk. Thus, the initiation of BCR signaling is a very dynamic process accompanied by reversible conformational changes induced by Src family kinase activity^ref.
Except for TI Ags, a B lymphocyte undergo blastization only if at least one of the peptides derived from the endosomal degradation of the BcR-recognized Ag is able, once MHC II co-presented on cell-surface, to bind the TcR on a CD4⁺ T-cell, otherwise the B lymphocyte undergoes clonal anergy (i.e. the antigen acts as an hapten !). This occurs because the B cell needs cytokines released from activated T cells to produce antibodies. Anyway if such a hapten is associated with an adjuvant capable, once degraded and MHC II co-presented, to be bound from a host TcR, the B lymphocyte can be primed. When a B lymphocyte acts as a APC, the epitope MHC II co-presented to the CD4⁺ T-cell and recognized by the TcR is not necessarily the same that is recognized by its own BcR.
IgM and IgD with the same antigenic specificity are produced through RNA alternative splicing. According to a phylogenetic analysis, the Cd gene appears to have been duplicated from the Cm gene > 300 million year ago. In humans 7÷8S p175^IgDare produced only as membrane-bound, but anyway they are found in plasma as result of B lymphocyte death (9% of carbohydrates ; [ ]_pl : 0÷0.4 mg/mL : because of a highly exposed hinge region, it is highly susceptible to proteolytic cleavage by blood clotting proteases that normally become active when blood is exposed to air; 0.3-1% of all plasma Igs; secretion rate : 0.4 mg/Kg/die ; t_1/2 : 2-3 dd., due to susceptibility to proteolysis). They have no activity against Gram -ve Bacteria.
On the contrary of TcR and IgD, other BcR isotypes are massively secreted : plasma cells secrete 2^.10³ soluble immunoglobulins (sIg) / _s(100 times more than a resting B cell) thanks to alternative polyadenylation site usage that cuts off the 2 membrane exons.
Crystallographers have noted that an antibody bound to an antigen has a different conformation from the same antibody in an unbound form. To explain this, they often invoke an instructive mechanism called "induced fit" - after antigen binds to antibody, interactions take place that convert the initial antibody-antigen complex to a more stable conformation. An alternative selective explanation termed "preexisting equilibrium" posits that 2 antibody conformations - one abundant, one rare - exist in equilibrium when not bound to antigen. Antigen binds selectively to the rare conformation, whose free concentration is maintained by reequilibration from the pool of unbound antibody. When an antibody-antigen complex undergoes crystallization, the rare antibody isomer is preferentially observed; when uncomplexed antibody is crystallized, the abundant form is preferentially observed (the same structural data are obtained with both mechanisms). The induced fit and preexisting equilibrium mechanisms can be distinguished kinetically. Both reactions give fast and slow phases with a distinctly different dependence on concentration. On the basis of this kinetic test, both mechanisms have been shown to operate. Although induced fit may improve antigen binding, the initial structure of the antibody binding site must still be complementary to the antigen, otherwise binding cannot take place. By contrast, the preexisting equilibrium mechanism constrains only one structure to be compatible with an antigen; a second, distinct, antibody conformation could potentially bind to a structurally unrelated antigen. The preexisting equilibrium mechanism can explain - or create - violations of the "one antibody, one specificy" prediction. Eg the crystal structures of 2 unbound isomers of SPE7 mAb have very different binding sites. One form (Ab1) has a relatively flat binding site typical of antibodies that bind to peptides and proteins (TrxShear3), whereas the other isomer (Ab2) has a deep hole typical of antibodies that bind to aromatic haptens (2,4-DNP, ...)^ref. Some additional (induced fit type) rearrangements occur to accommodate each antigen, but does not alter the basic picture that the antigen "selects" one of the 2 available antibody isomers. The binding reactions have 3 kinetic phases : a prebinding conformational isomerization, the initial binding reaction, and a final slow isomerization of the antibody-hapten complex. Antibody conformational isomerism itself is not rare : its frequency is 10 to 20% of antibodies raised to the hapten 2-phenyl-5-oxazolone (Ox)^ref. Conformational isomerism can only be confirmed kinetically, but that is not possible unless pre-steady-state kinetics can be measured, and then only if the binding and isomerization steps can be resolved. A natural concern is whether isomerization-based cross-reactivity occurs in vivo : this model could offer an attractive explanation for observations of aberrant cross-reactivity leading to autoimmune disease and allergy^ref. Diversion of an antibody population into unreactive isomers thermodynamically and kinetically disfavors antigen binding. Multiple conformational states would increase the antibody repertoire and immune diversity without the need for additional lymphocytes expressing distinct antibodies. However, this possible advantage is challenged by the fact that gene-level diversity already confers an over-capacity for antigen recognition. For example, transgenic mice with just 4 V_H and 4 V_L genes mount an effective antibody response to a test antigen^ref. Antibody conformational isomerism may provide an advantage when both conformations recognize an epitope on the same antigen molecule, particularly if the antigen is monomeric. Ligation of the second arm of IgG by antigen, even with a low affinity constant, may improve the functional affinity of an antibody. Monoclonal antibodies cannot aggregate a monomeric antigen because once ligation occlusdes the only epitope, the antigen can form no further cross-linking bonds. But if a second epitope could react with a second antibody conformation, cross-links could form. The importance of cross-linking and immune complex aggregation to antigen clearance, complement activation, endocytosis, and signal transduction has been well established. Finally, a recent finding regarding antibody neutralization of toxins suggests the potential value of antibody isomers that recognize 2 epitopes on the same antigenic toxin molecule. Binding of one antibody to monomeric BoNT/A greatly enhanced binding of a second and third antibody specific for other epitopes : although no single mAb significantly neutralized toxin, a combination of 3 mAbs (oligoclonal Ab) neutralized 450,000 LD₅₀ of BoNT/A, a potency 90 times greater than human hyperimmune globulin. The potency of oligoclonal Ab was primarily due to a large increase in functional Ab binding affinity. The results indicate that the potency of the polyclonal humoral immune response can be deconvoluted to a few mAbs binding nonoverlapping epitopes, providing a route to drugs for preventing and treating botulism and diseases caused by other pathogens and biologic threat agents. Hence, binding enhancement was not epitope specific, nor was it due entirely to a chelate effect. The mechanism of high-potency toxin neutralization by oligoclonal antibody preparations is unknown. Whatever the mechanism, monospecific mAbs on their own can undergo only the initial, low-potency binding step; effectively bispecific isomeric antibodies could achieve high-potency neutralization through a high-affinity interaction with a second epitope^ref.
The main antibody classes are :
• IgM (10% of carbohydrates; [ ]_pl : 0.4÷1.7 mg/mL = 5-10% of all plasma Igs ; secretion rate : 7 mg/Kg/die ; t_1/2 : 5 dd.) are arranged in a 19S p900 pentamer (macroglobulin) or p1150 hexamer in which each IgM molecule has an additional C_m4 domain. Because of sterical obstacles their valence is 5 or 6 instead of 10 or 12, respectively. An additional p15^{J chain} links 2 Fcm in the pentamer. Cm3 triggers classical activation pathway for complement cascade. A tetrameric homolog of IgM is the predominant teleost serum Ig.
• class-switch recombination (CSR) is induced by ...
• CD40 (homodimer on B cells)--- CD154 / 40L / gp39 interaction. The major CD40 isoform lacks exon 6 (which codes for the membrane-associated endodomain) and act as a decoy receptor. CD23 / FceRII expressed on lymphocytes, macrophages, and many other cell types has been reported to also play a role in this process.
• cytokines that induce particular isotype switching :
• switching to IgG₄ requires ...
• IL-13
• IL-4 => E47 / TCF3 =>2 highly conserved intronic E-boxes => activation-induced C deaminase (AID). According to different hypotheses AID may act on ...
• mRNA (current hypothesis : editing enzyme homologous to APOBECs) : deamination of C results in an activating C=>U transition in mRNA coding for the involved endonuclease. Because phosphorylated histone H2AX (g-H2AX, which forms next to double-stranded DNA breaks (DSBs)) focus formation at the IgH loci does not depend on uracil–DNA glycosylase (UNG), but CSR is reduced in the absence of UNG, this indicates that UNG has a role in CSR downstream of DNA cleavage, which contradicts the DNA deamination model. In support of this, no difference in the IgH mutation rate between cells expressing or not expressing the UNG inhibitor Ugi; because mutation results from DNA cleavage during CSR, it seems probable that UNG is not required for DNA cleavage^ref. UNG mutants that lack UNG catalytic activity but retain DNA-binding activity were shown to recover wild-type levels of CSR in UNG-deficient B cells. The importance of UNG for CSR in the absence of its catalytic activity indicates that it might have a structural role, functions as a scaffold to recruit repair proteins to the DNA after DNA cleavage caused by an AID processed endonuclease. The role of such an endonuclease is also supported by showing that de novo protein synthesis is required for DNA cleavage in CSR^ref.
• ssDNA (past hypothesis) : CSR involves transcription through switch regions, which generates ssDNA within R loops. Deamination of dC to U results in a U-G mismatch lesion that could :
• remain uncorrected : C=>T transition (and G=>A transition)
• be wrongly corrected by BER, leading to :
• transversions
• MMR with error-prone DNA polymerases (h, i,z and m)
• template-mediated repair on an upstream V pseudogene
• if the lesion occur in a switch site, repair that involves another switch region would lead to CSR
• undergo uracil removal by the DNA base-excision repair enzyme uracil–DNA glycosylase (UNG) (mutations in UNG cause hyper-IgM (HIGM) syndrome), resulting in strand break^ref
A CSR-specific cofactor binds to the carboxyl terminus of AID : C-terminus mutations impair CSR but not SHM^ref.
AID, a mature B-cell–restricted cytoplasmic antigen, is relocated into the nucleus in 2.5% of CDKN1B^–, CCNB1^– GC cells. The GC dark zone and the outer zone (OZ), but not the light zone, contain nuclear and cytoplasmic AID⁺ blasts. AID⁺ cells in the OZ are in contact with T cells and CD23^– follicular dendritic cells. In addition, AID is expressed in extrafollicular large proliferating B cells, 14% of which have nuclear AID. GC and extrafollicular AID⁺ cells express E47 but not the inhibiting BHLH protein Id2. Outside the GC, AID⁺ B cells are in contact with T cells and show partial evidence of CD40 + bcr stimulation-dependent signature (CCL22, JunB, cMYC, CD30) but lack early and late plasma cell markers. The distribution of nuclear AID is consistent with the topography of SHM and CSR inside the GC and in extrafollicular activated B cells^ref.
They activate the I_H promoter, inducing the production of untranslatable germline I_H-C_H transcripts, which, in turn, facilitate recombination of the switch (S)m region with the targeted downstream S region. The RNA-DNA hybrid structure that forms in vitro is an R-loop with a displaced guanine (G)-rich strand that is single-stranded and whose length can exceed 1 kb. This yields to CSR through triplex formation and looping-out deletion of the genomic DNA between the recombined S regions and generates an extrachromosomal reciprocal switch DNA recombination product, a.k.a. the switch circle (SC), which includes the I_H promoter upstream the targeted S region, and Cm. Under the influence of the I_H promoter, the SC transcribes a chimeric I-Cm product, referred to as the circle transcript (CT). Since SCs are rapidly degraded by nucleases, both SCs and CTs constitute specific molecular markers of ongoing CSR.
AID deaminates 5-methylcytosine in DNA and is expressed in pluripotent tissues: implications for epigenetic reprogramming.
DNA deaminases of the Aid/Apobec family convert cytosine into uracil and play key roles in acquired and innate immunity. The epigenetic modification by methylation of cytosine in CpG dinucleotides is also mutagenic, but this is thought to occur by spontaneous deamination. Aid and Apobec1 are 5-methylcytosine deaminases resulting in a thymine base opposite a guanine. Their action can thus lead to C => T transition mutations in methylated DNA, or in conjunction with repair of the T:G mismatch, to demethylation. The Aid and Apobec1 genes are located in a cluster of pluripotency genes including Nanog and Stella and are co-expressed with these genes in oocytes, embryonic germ cells, and embryonic stem cells. These results suggest that Aid and perhaps some of its family members may have roles in epigenetic reprogramming and cell plasticity. Transition in CpG dinucleotides is the most frequent mutation in human genetic diseases, and sequence context analysis of CpG transitions in the APC tumor suppressor gene suggests that DNA deaminases may play a significant role in tumor etiology^ref.
• switching to IgE requires ...
• low TGF-b₁ : it up-regultes Id2, which acts as a negative regulator of E2A, whose activity is required for IgE class switching.
• IL-4
• IL-13



Both signals are offered by T_h2 cells, or by activated mast cells, basophils and eosinophils. IL-2, IL-5, IL-6, and TNF-a are known to act in concert.
• switching to IgG₁ and IgG₃ requires ...
• IL-10
• switching to IgG₂ requires ...
• ? (a yet unidentified T cell factor)
• switching to IgA₁ and IgA₂ requires ...
• VIP
• IL-10
• TGF-b
CD30 binding by TNFSF8 / CD153 / 30L negatively regulates CSR. Isotype switching is cell division linked : as human B cell switching cytokines (such as IL-4,IL-10, and IL-13) can also promote B cell proliferation they could not be true switching factors.
Anyway sometimes IgE production occurs without DNA deletion and IgM production is reacquired by IgG-producing B-cells. CSR is generally restricted to germinal centres in lymph nodes but may occur also in the lamina propria of gut villuses (IgM => IgA transition).
In plasma protein electrophoresis Ig show a wide range of electrophoretic mobility, from slow g to the a₂ globulins : all Igs form 20% of plasma proteins. Under physiological conditions plasma contains :
• 7S p150^IgGs (3% of carbohydrates; 6÷14 mg / mL = 75÷85%; secretion rate : 33 mg/Kg/die; persist in the skin for up to 2 days; not inactivated if heated at 56° C for 4 hours) : 4 subclasses numbered according to serum concentrations.

Human IgG subclasses book by Research Diagnostics
• 59÷70% IgG₁ (t_1/2 = 23 days; 2 inter H chain S-S bonds; C_g12 activate complement; cross placenta)
• 20÷30% IgG₂ (t_1/2 = 20÷23 days; 4 inter H chain S-S bonds; C_g22relatively inefficient at activating complement; does not cross the placenta)
• 7÷9% IgG₃ (t_1/2 = 7-8 days; 13 inter H chain S-S bonds; C_g32activate complement very effectively, ; cross the placenta)
• 2÷4% IgG₄ (t_1/2 = 21-23 days; 4 inter H chain S-S bonds; C_g42cannot activate complement; cross the placenta)
C_H3 domains of soluble IgG₁, IgG₂, Gm3 s⁺t⁺ IgG₃ and IgG₄ are bound by protein A from Staphylococcus aureus.
• IgA (7% of carbohydrates ; 0.6÷4 mg / mL = 7÷20%; 65 mg/Kg/die ; t_1/2 : 6 dd.). They occur as p160 7S monomers (80%) or polymers (20% : dimers, trimers, tetramers, pentamers linked by disulfide bonds and p14^{J chain}). C_a2 doesn't activate the classical activation pathway of complement, but may activate the alternative activation pathway. They cannot cross the placenta. IgA dimers (pIgA) are formed in which Fcas are bound together by p14^{J chain}, which is expressed also in IgM- and most IgG-secreting plasma cells. 75% of the plasma cells in the body make IgAs, and most of this IgA is released continuously into mucosal secretions.
• IgA₁ (93%): Cys¹⁴⁵ and Cys²⁰⁴ in C_H1, Cys²⁶⁶ and Cys³²³ in C_H2, and Cys³⁶⁹ and Cys⁴³² in C_H3 form the intradomain disulfide bonds characteristic of the Ig fold. Cys¹⁹⁶  and Cys²⁰⁰ in C_H1and Cys²⁴² and Cys²⁹⁹ in C_H2 form additional intradomain disulfide bonds with tremaining Cys residues forming covalent bonds with other chains : 133 to the L chain, 241 and 301 to the H chain, 311 to the secretory component, and 471 to the J chain.

[IgA₁ are cleaved in the hinge region by proteases produced by :
• Streptococcus pneumoniae
• Streptococcus sanguinis
• Clostridium ramosum (which cleaves also IgA₂m(1) allotype)
• Gemella haemolysans
• Proteus spp.
• Prevotella spp.
• Capnocytophaga spp.
• Haemophilus influenzae
• Neisseria gonorrhoeae
• Neisseria meningitidis
• Pseudomonas aeruginosa]
• IgA₂ (7%) : a similar structure has been proposed for IgA₂m(1) allotype, except there is no disulfide bond with the L chain since Cys¹³³ is now Asp (although recent studies have shown that a small amount of HL disulfide-linked IgA₂m(1) is made as an assembly intermediate, only small quantities of HL, H₂L₂, and H₄L₄J are present in the secretions). Although IgA₂m(2) and IgA₂(n) allotypes also lack Cys¹³³, they form covalent bonds with the L chain via undefined Cys residues. IgA₂m(2) differs from IgA₂m(2) and IgA₂(n) at positions 212 and 221 in C_H1 : IgA₂m(1) has Pro²¹² and Pro²²¹ whereas IgA₂m(2) and IgA²(n) have Ser²¹² and Arg²²¹. In IgA₂m(1) the L chain is disulfide bonded to the hinge-proximal region of C_H2 with assembly taking place through an HL intermediate.
Fcas are bound by some isoforms of M protein expressed by Streptococcus agalactiae and by ~ 50% of all Streptococcus pyogenes, but attempts to use these proteins as IgA-purifying reagents have been hampered by unsuitable binding properties and/or affinity for human plasma proteins other than IgA. Anyway the Sap peptide (corresponding to aa 35-83 of protein Sir22 / M22) contains a 29 aa sequence that is sufficient for specific IgA binding.
• 8S p190^{IgE / reagin} (13% of carbohydrates; 17÷450 ng / mL =  0÷200 IU / mL (as 1 U = 2.4 ng) since age 5-7; 0.019 % ; 0.016 mg/kg/die; t_1/2 : 2 dd.). They have an additional C_e4 domain. C_e2 does not activate complement; they cannot cross the placenta. Persist in human skin for up to 6 weeks, inactivated if heated at 56° C for 4 hours. A number of e transcripts produced by alternative splicing between constant region (C_H3 and C_H4) and membrane (M1 and M2) exons and by differential cleavage-polyadenylation at poly(A) sites downstream of the C_H4 and M2 exons have been detected in IgE-producing B cells :
• secreted isoforms^ref : in the long IgE isoform (mLIgE), this domain contains a stretch of 52 amino acids which are absent in the short variant (mSIgE). The 2 membrane isoforms of human IgE assemble into functionally distinct B cell antigen receptors^ref.
• eS1
• eS2 : generated by splicing between a donor splice site immediately upstream of the stop codon in the H-chain constant region exon 4 (C_H4) and an acceptor site located in the 3' part of the second membrane exon. The eS2 H chain is only 6 amino acids longer than the classical secreted Ig H chain (eS1) and contains a C-terminal cysteine, which is a characteristic sequence feature of mu and alpha H chains. However, unlike IgM and IgA, the eS2 C-terminal cysteine (Cys⁵⁵⁴) does not induce polymerization of H₂L₂ molecules (where L is light chain), but rather creates a disulfide bond between the 2 H chains that increases the rate of association into covalently bound H₂L₂ monomers. This C-terminal cysteine also does not function as an intracellular retention element because the eS2 isoform was secreted in amounts equal to that of the epsilon S1, both in B lymphocytes and in plasma cells. The eS2 H chains secreted by B lymphocytes differed from the eS1 H chains in the extent of glycosylation. Interestingly, a difference in glycosylation between B-lymphocytes and plasma cells was also noted for both isoforms. The presence of the Cys⁵⁵⁴ also allowed the identification of a distinctive asymmetric pathway of IgE assembly, common to both types of e H chains^ref.
• intracellularly retained and degraded isoforms because of their inability to assemble into complete IgE molecules. At the protein level, the 5 alternatively spliced e transcripts (eC_H4*, eC_H4-M2', eC_H4'-1, eC_H4'-1-M2, and eC_H3-13-C_H4) corresponded to 4 e heavy chain isoforms^ref

[microbial Ig binding proteins (IBPs) are produced by protozoa^ref, viruses, parasites and both gram-positive and gram-negative bacteria and play important physiological roles^ref. It has been suggested that during an infectious process these IBPs may act as an evasion mechanism to divert specific antibody (Ab) responses^{ref1, ref2}]
Igs in mucosal secretions (tears, saliva, urine, nasal, bronchial, gastric and intestinal secretions, and even in colostrum, where they confer passive natural immunity). The mucosal surfaces can be reached by ...
• paracellular passive external transfer for theoretically all Ig classes
• IgGs are quite important in the respiratory tract (where they are less subjected to proteolytic degradation)
• IgDs are found only in traces
• IgEs can be carried by mast cells
• basolateral to apical transcytosis in epithelial cells thanks to a polymeric Ig receptor (pIgR) that binds to p14^{J chain} : on the luminal domain of plasma membrane pIgR is cleaved and only a p70^{secretory component (SC)}fragment remains bound between the 2 remaining Fcas, forming a 11S p390 SC-pIgA complex (S-IgA / SIgA). On the contrary of mouse hepatocytes, human hepatocytes don't express pIgR, so bile doesn't contain pIgs.
• dimeric IgAs (dIgA; expecially IgA₂)
• pentameric IgMs undergo
The retromer is a multimeric protein complex, originally described in yeast, which mediates intracellular sorting of Vps10p, a receptor that transports vacuolar enzymes. The yeast retromer contains 2 sub-complexes. One includes the Vps5p and Vps17p subunits, which provide mechanical force for vesicle budding. The other is the Vps35p–Vps29p–Vps26p subcomplex, which provides cargo specificity. The mammalian retromer binds to the mannose 6-phosphate receptor, which sorts lysosomal enzymes from the trans-Golgi network to the lysosomal pathway^ref
SIgA in saliva are undetectable at birth, approximate peak at age 7, consistently high during mid-life (25-900 mg/mL) and then decline during old age, no gender differences. SIgA in saliva is not directly related to serum levels of SIgA. A lower concentration of SIgA in saliva has been conceptualized as a risk factor for URT infection in children and the elderly, periodontal disease and caries. Variability in salivary flow rate should be taken into account when estimating saliva levels of SIgA and making comparisons between individuals.
The C region affects V region protein conformation, leading to differences in fine specificity and Id^ref. The C region can also have a major effect on the apparent affinity through its contribution to valency, and hence, avidity. Nevertheless, there is some evidence that C region interactions among Abs bound to Ags with repeating structural motifs can affect the types of complexes formed
Antibodies have the following biological activities :
• neutralize biological activities of the antigen (when this occurs on mucosal surfaces it is termed immune exclusion)
• activate the complement system by the classical pathway (only where complement factors are available)
• stimulate type II immune or opsonic phagocytosis by phagocytes (opsonization; only when phagocytes are available) and antibody-dependent cell-mediated cytotoxicity (ADCC) (only when killer cells are available) through Fc receptors (FcRs) : high affinity FcRs are referred to as FcRI, and low affinity FcRs are referred to as FcRII/III. High affnity FcRs can bind noncomplexed, monomeric Igs, while low affinity FcR bind aggregated Igs or Abs complexed to multivalent Ags. Please note IgE is the only isotype which can bind its receptors even when the antigen is not bound. The FcR is found also associated to the collagen receptor glycoprotein VI on platelets. Fc receptors can be divided into 2 groups based on their signaling capability and structure. The first and most common type of Fc receptor is a multichain heterocomplex composed of a ligand-binding a-chain and one or more signal-transducing g-chains. The second type of Fc receptor is a single-chain transmembrane receptor containing a signal-generating motif(s) in the cytoplasmic domain and, thus, not requiring another signal-transducing subunit
• C_m3 has the following receptor :
• FcamR (expressed in hematopoietic and non-hematopoietic tissues)
• C_g3 has the following receptors^ref :
• FcgRI / CD64 + FcRg_{FcgRI, FcgRIIIA, and FceRI} (70 kDa; high affinity : K_d = 10^-9 M for IgG monomers, IgG₁ >=IgG₃ > IgG₄ > IgG₂) : 3 immunoglobulin-like domains. The third domain of FcRI has been attributed to the high affinity binding of IgG by this receptor; however, since the extracellular region of FcRI has not been crystallized this has not been confirmed experimentally. Expressed constitutively on monocytes, macrophages, and some DCs, but also on IFN-g-, TNF-a-, or GM-CSF-stimulated neutrophils^{ref1, ref2}; => cell activation, opsonization) : a-chain gene expression is up-regulated by YY1, PU.1 and GATA1, down-regulated by Elf-1.
• FcgRIA
• FcgRIB
• FcgRIC
• FcgRII / CD32 (relatively low avidity (K_d > 10^-7 M) for monomeric IgG but of high avidity for complexed IgG) : 2 extracellular immunoglobulin-like domains; alternative splicing =>
• A isoform (40 kDa; IgG₃ > IgG₁ > IgG₂ > IgG₄; ITAM-containing) on neutrophils, monocytes and macrophages (=> cell activation by up-regulation of indoleamine 2,3-dioxygenase (IDO) and 3-monooxygenase; opsonization), natural killer cells, platelets, placental trophoblasts and endothelial cells ;
• B isoform (40 kDa; IgG₃ >= IgG₁ > IgG₄ > IgG₂; ITIM-containing) => alternative mRNA splicing =>
• b₁ isoform (FcgRIIb1) on B lymphocytes, placental trophoblasts and endothelial cells : a multivalent Ag can be bound by a soluble IgG but also from mIgs on a B cell with the same Ag specificity. If binding of immunocomplex to FcgRIIB and binding of a remaining epitope to mIg occur contemporarily, coaggregation induces rapid Tyr-phosphorylation on FcgRIIB => recruits SHIP-1 => recruitment of p62^dok and RasGAP1 => inhibition of ERK1 and ERK2 => inhibitory effects on cell activation (negative Ab feedback on humoral immune responses !).
• b₂ isoform (FcgRIIb2) on macrophages, mast cells, placental trophoblasts and endothelial cells : it lacks a domain encoded by the first intracytoplasmic exon and when coaggregated with FceRI, it is rapidly Tyr-phosphorylated and recruits SHIP-1 => recruitment of p62^dok and RasGAP1 => inhibition of ERK1 and ERK2 => inhibitory effects on cell activation (prevention of mast cells activation in presence of IgGs !). It internalizes IgG immune complexes for Ag presentation mainly in phagocytosis.
• C isoform (ITAM-containing) on human natural killer cells, neutrophils, placental trophoblasts and endothelial cells?
• FcgRIII : 2 extracellular immunoglobulin-like domains; 50-80 kDa; K_d > 2 ^. 10^-7 M for IgG monomers and IgG₁ = IgG₃ > IgG₂ = IgG₄)
• FcgRIIIA / CD16a + FcRg_{FcgRI, FcgRIIIA, and FceRI} (medium affinity); transmembrane form : on NK cells, monocytes and macrophages (also on mouse neutrophils and myeloid precursor cells, including eosinophils^ref) => cell activation and ADCC
• FcgRIIIB / CD16b (low affinity; GPI-anchored; no homolog in Mus musculus; on neutrophils and natural killer cells => phagocytosis)
• neonatal FcR (FcRn) is an heterodimer formed by the association of an L (b₂-microglobulin) and H (a-chain) chain homologous to the MHC I molecules. It can form both non-covalent and covalent dimers in the absence of its ligand, IgG. The most notable feature of the interaction of FcRn with IgG is its pH dependancy : the Fc portion of IgGs binds FcRn with a high affinity at pH 6.0 and is released at pH 7.2. It is responsible for IgG transport across the intestinal epithelium in newborn rodents, is expressed in epithelial cells in adult humans and non-human primates. A role for FcRn in IgG homeostasis in humans has been hypothised, but to date, there is no direct evidence to invoke its role in such a process. This suggests, along with colocalization studies, that for cells bathed in near neutral pH, the IgG/FcRn complex forms within acidified endosomes. More precisely, FcRn acts as a salvage receptor, binding and transporting pinocytosed IgGs in intact form both within and across cells, and rescuing them from a default degradative pathway. Although the molecular mechanisms responsible for salvaging IgGs are still largely unknown, it is thought that unbound IgGs are directed toward proteolysis in lysosomes, whereas  bound IgGs are recycled to the surface of the cells and released. This control takes place within the endothelial cells located diffusely throughout adult tissues. In contrast to mouse FcRn, human FcRn is surprisingly stringent in its binding specificity for IgG derived from different species. Human FcRn binds to human, rabbit and guinea pig IgG, but not significantly to rat, bovine, sheep or mouse IgG (with the exception of weak binding to mouse IgG_2b). In contrast, mouse FcRn binds to all IgG analyzed. The lack of binding of human FcRn to mouse IgG₁ has been confirmed using transfectants that have been engineered to express human FcRn on the cell surface. These results provide a molecular explanation for the enigmatic observation that mouse IgG behave anomalously in humans^ref. FcRn is expressed within azurophilic and specific granules of neutrophils, and relocates to phagolysosomes upon phagocytosis of IgG-opsonized bacteria. FcRn enhances phagocytosis in a pH dependent manner which is independent of IgG recycling. IgG-opsonized bacteria were inefficiently phagocytosed by neutrophils from b₂M knock-out or FcRn a-chain knock-out mice, which both lack expression of FcRn. Similarly, low phagocytic activity was also observed with mutated IgG (H435A), which is incapable of binding to FcRn, while retaining normal binding to classical leukocyte FcgRs. Finally, a TAT peptide representing intracellular endocytosis- and transport motifs within FcRn, strongly inhibited IgG-mediated phagocytosis. These findings support a novel concept in which FcRn fulfills a major role in IgG-mediated phagocytosis^ref.
The crystal structures of the extracellular region of FcgRIIA, FcgRIIB, FcgRIIIA, and FcgRIIIB have been determined. The 2 Ig-like domains of these receptors, D1 (membrane distal) and D2 (membrane proximal), are bent at an approximately 50–70° angle relative to each other depending on the algorithm used^ref. All FcgRs are structurally very homologous, with their domains oriented in a steep angle to each other. While most studies have shown FcgRs existing in the membrane as a monomer, some conflicting reports have suggested that FcgRIIa forms a homodimer, with a 2:1 stoichiometry of receptor to IgG^ref. However, the crystallization of FcgRIIIa in complex with the Fc region of IgG₁ suggests a 1:1 stoichiometry of receptor to IgG^ref. It is believed that the other FcgRs display similar binding stoichiometry due to their high degree of homology^ref. Also, as a dimer is able to crosslink FcgRs to induce signaling, it may not be efficient for cells to be able to induce signaling by binding a single IgG molecule. However, the conformation of both bound and unbound FcgRIIa remains a matter of debate. The binding sites of the FcgRs for IgG are fairly well established. From the crystallization studies as well as some mutational studies, the D2, or membrane proximal, domain of the FcgR is important for the binding of IgG. The binding of an IgG by the receptor induces approximately a 10° shift in the angle between the D1 and D2 domains^ref. The FcgR binds asymmetrically to an IgG molecule in the lower hinge region of the C2 domains of an IgG heavy chain. The asymmetrical binding may result from a conformational change in one of the C2 domains of IgG in relation to the other. A factor that could affect the IgG binding ability of the FcgR is the glycosylation state of the receptor and IgG. One example of this is found in the crystal structure of FcgRIIIa/IgG-Fc, whereby the glycosylation of the IgG₁ at 297N was not involved in the binding site. However, 2 groups have shown that glycosylation of an IgG may be involved with the formation of a stable C2-C2 interaction of the 2 heavy chains of IgG₁. When the carbohydrates are removed, there is decreased binding of IgG₁ to both FcgRIII and FcgRIIb^{ref1, ref2}. The glycosylation of FcgRs can also affect binding of IgG. Expression of FcgRIIIa on different cell types leads to different glycosylation patterns. The differences in the ability to bind monomeric IgG by FcgRIIIa have been attributed to the cell type specific glycosylation of the receptor^ref. Also, one particular glycosylation site in the D2 domain of FcgRIIIa and FcgRIIIb, 163N, is located in the IgG-binding site. Receptors lacking glycosylation at this amino acid have an increased affinity for IgG^ref. Thus, the glycosylation state of the receptor, which may vary in cases of inflammation, could affect the binding of IgG. Another example of affinity modulation is through polymorphism. As such, a polymorphism in FcgRIIa (131R or 131H) affects the binding affinity of FcgRIIa for human IgG₂; FcgRIIa (131H) binds IgG₂ but FcgRIIa (131R) does not^ref. Results of a recent meta-analysis show that the FcgRIIa polymorphism is important in genetic susceptibility for systemic lupus erythematosus (SLE)^ref. There is also a polymorphism in FcgRIIIa (158V or 158F) that affects binding to human IgG₁ and human IgG₃. NK cells from patients with FcgRIIIa (158F) are unable to bind IgG₃ and bind IgG₁ with lower affinity, whereas NK cells from patients with FcgRIIIa (158V) are able to do so^ref. In addition, a triallelic polymorphism in FcgRIIIa (48 H, R, or L) was originally reported to affect the binding affinity for human IgG. Subsequently, this difference in binding affinity was determined to be due to the polymorphism at amino acid 158^ref. This is consistent with structural studies as 48 H/R/L is located in the D1 immunoglobulin-like domain whereas 158 V/F is located in the D2, IgG-binding domain. Finally, in FcgRIIIb there are 2 alleles (NA1 and NA2). These 2 alleles have minimal differences in binding affinity for IgG, but patients homozygous for the NA1 allele have a higher level of phagocytosis of IgG-coated particles^ref. Given that most FcgRs are transmembrane proteins, the first step of receptor activation and subsequent phagocytosis is binding of IgG containing complexes to the receptor extracellular domain. Binding and phagocytosis by human FcgRs has been observed both in normal human leukocytes and in model systems such as transfected COS-1 cells^ref. Initial FcgR activation takes place upon ligand binding to the extracellular domain. Since each FcgR has a structurally distinct extracellular domain, the receptor binds to IgG with varying affinity. FcgRs traditionally signal through an ITAM or through ITIM^{ref1, ref2}. Both AIHA and ITP are mediated by FcgRs found on phagocytes (macrophages) as part of the reticuloendothelial system (RES) in the spleen and liver and may lead to thrombocytopenia or anemia, respectively^ref. Various observations by multiple laboratories have determined the role of FcgRs in these diseases most conclusively in murine models lacking the common g-chain (KO). Experimentally induced AIHA and ITP are markedly decreased in mice lacking the g-chain^ref. Additionally, administration of mAb 2.4G2, which binds to and blocks mouse FcgRII and FcgRIII, allows a rapid recovery after induction of AIHA or ITP. Similarily, administration of GM-CSF has been shown by various groups to accelerate AIHA potentially due to upregulation of FcgRI^ref. These reports show that altering the balance of stimulatory to inhibitory FcgRs has a marked effect on disease progression and susceptibility. As such, viral infection has been shown to stimulate or increase susceptibility to both AIHA and ITP. However, the mechanism(s) by which viral infection does so has yet to be fully elucidated. Potential scenarios of viral infection that could lead to this outcome include stimulated production of IFN during viral infection that causes upregulation of FcgRI. Alternatively, viral infection may cause a change in the expression pattern of FcgRs due to transcriptional activation or other mechanisms.
Treatment for ITP and AIHA has recently been reviewed elsewhere^{ref1, ref2}. Many of the therapies used involve altering FcgR expression or phagocytosis. One of the first treatments is the administration of glucocorticoids. Prednisone and dexamethasone have been shown to downregulate FcR expression, thus potentially decreasing the phagocytosis of IgG-coated particles. Other treatments involve administration of anti-D IgG to Rh-D positive patients. The rationale behind this treatment is that the IgG will coat erythrocytes with subsequent binding by FcgRs. Binding of the IgG-coated red blood cells will prevent the binding and phagocytosis of the IgG-coated platelets. However, self-limited hemolytic anemia is to be expected after anti-D treatment. High-dose intravenous immunoglobulin (IVIG) is also used as a treatment for both ITP and AIHA. However, the mechanism by which this inhibits the clearance of the IgG-coated platelets or IgG-coated red blood cells is still unknown. It is believed to work through a number of different mechanisms, which may include anti-idiotypic antibodies and decreased autoantibody production^ref, and, of interest here, its effects on FcgR function. One principal theory for a mechanism of IVIG’s function is through a blockade of FcgRs. At high local concentrations of IgG, the IVIG may either bind to the low-affinity FcgRs, preventing the binding of IgG-coated cells, or outcompete binding of the high avidity immune complexes. Also, the possibility of IgG dimers existing in IVIG preparations would even further enhance this blockade^ref. Another possibility involves the inhibitory FcgRIIb, which is able to inhibit phagocytosis mediated by activating Fc receptors^ref. Injection of IVIG into a mouse model of ITP increased circulating platelet counts^ref. Also in this model was an increased percentage of non B cell splenocytes expressing FcgRIIb. Increased expression of this receptor relative to the expression of activating receptors could decrease the phagocytosis of IgG-coated platelets. The mechanism for the IVIG-mediated increase in FcgR–expressing cells is still unknown.
10-20% of the IgG isolated from non-immune rat sera is asymmetrically glycosylated (asymmetric IgG antibodies (AAb)) with a mannose-rich oligosaccharide residue : in such a way that it fails to trigger immune effector mechanisms. The proportion of asymmetric antibodies is increased after prolonged immunization with particulate antigens like cellular spleen cells. Upon being purified from normal rat sera, 78% of the asymmetric IgG sub-population showed self-reactivity. About 14% of rat asymmetric IgG-F(ab')₂ fragments was able to react with bacteria isolated from the intestine of uninfected rats. Asymmetric antibodies may exert a beneficial action by protecting self-antigens as well as normal intestinal flora from a deleterious immune response. The asymmetric:symmetric IgG ratio increases during pregnancy and with ageing.
The mechanism underlying the long-standing observation of substantial differences between IgG subclasses in the ability to mediate effector responses, contributing to variable activity of antibodies against microbes and tumors (subclass dominance), is provided by the differential affinities of IgG subclasses for specific activating FcgRs compared with their affinities for the inhibitory FcgRs. The significant differences in the ratios of activating-to-inhibitory receptor binding predicted the in vivo activity^ref.
• C_a13 and C_a23 have the following receptors :
• gp50÷75^{CD89 / FcaRI} (medium affinity : K_d = 10^-6 M : expressed on neutrophils, monocytes/macrophages, interstitial DC, immature and mature monocyte-derived DC^ref, and eosinophils => cell activation, ADCC). The FcaRI-Fca complex consists of 2 FcaRI molecules and a single Fca dimer, with one receptor binding to each of the Ca2-Ca3 junctions. As the pIgR ectodomain of SIgA seems to interact with residues of IgA that are required for binding of the FcaRI-binding site, SIgA requires the presence of an integrin co-receptor for signalling through FcaRI, and even then, cannot trigger phagocytosis. Its expression and function can be up-regulated by fMLP, GM-CSF, IL-8, IL-1b, TNF-a, CD11bCD18 / a_Mb₂ integrin / Mac1 / CR3 and LPS, and down-regulated by TGF-b₁. The FcaRI ligand-binding chain contains a short cytoplasmic tail and its expression and function are dependent on FcR g-chains, which contain ITAMs.
• FcamR (expressed on hematopoietic and non-hematopoietic tissues)
• CD71 / TfR (expressed on immature monocyte-derived DC^ref)
• C_a1 and C_a2 have the following receptor :
• unknown receptor (on M cell apical surfaces)
• C_e3 has the following receptors :
• FceRI (high affinity : K_d > 10^-10 M) is a heterotetramer made up of a-b-(FcRg_{FcgRI, FcgRIIIA, and FceRI})₂ subunits, expressed consitutively on mast cells, basophils, and Langerhans cells, and inducible on eosinophils (a(FcRg_{FcgRI, FcgRIIIA, and FceRI})₂ trimer on monocytes and eosinophils from atopic patients) : the a-chain is the only Fce-binding subunit, while aand g ones are signal transducing subunits. The a-chain can be bound and activated by :
• free IgE in an Ag-independent manner: upon Ag-binding, cell activation, degranulation and ADCC occur.
• membrane IgEs in an Ag-independent manner. Noteworthily, human peripheral blood basophils and monocytes also were activated upon contact with cells bearing membrane IgE. In humans, the presence of FceRI in several cellular entities suggests a possible membrane IgE-FceRI-driven cell-cell dialogue, with likely implications for IgE homeostasis in physiology and pathology^ref.
Relatively subtle structural differences in ligand binding and receptor cross-linking can affect the efficacy of STP. Usually upon FceRI cross-linking the a subunit-associated PTK Lyn is activated by transphosphorylation. Activated Lyn phosphorylates the Tyr residues in the ITAMs of the cytoplasmic region of FceRI b an g subunits, enabling the recruitment of further Lyn and Syk, respectively, through SH2 domain-pY interactions : newly recruited PTKs are activated by transphosphorylation of Tyr residues in their activation loops and by conformational changes in the case of Syk. Active Lyn and Syk phosphorylate themselves and other protein substrates such as PL-Cg₁, PL-Cg₂, PI(3)K and Btk. Lysophospholipids are fusogenic and allow degranulation within 1'. Genes induced after FceRI stimulation include : CCL-2 / MCP-1, CCL-4 / MIP-1b, IL-4, IL-5, IL-6, TNF-a, GM-CSF,b_A subunit of inhibin/activin, IL-1ra, and (after 24 hours) kynurenine 3-monooxygenase.
• CD23 / FceRII (low affinity) :
• A isoform (IL-4-inducible) on B lymphocytes
• B isoform on monocytes and macrophages, platelets, neutrophils and eosinophils, some B and T lymphocytes, and intestinal epithelial cells^ref
Affinity maturation(i.e. CDRs diversification : even FRs mutate, but their mutations are often deleterious to Ab stability) is obtained ...
• in humans, frogs and sharks through activation-induced cytidine deaminase (AID)-dependent somatic hypermutation (SHM). In Escherichia coli, as in eukaryotic cells, the mutation frequency is directly proportional to the transcription of target genes. Transcription enhances mutation of the nontemplate DNA strand, which is exposed as ssDNA during the elongation reaction, but not mutation of the template DNA strand, which is protected by E. coli RNA polymerase. The role of transcription in facilitating mutation is to provide AID with access to ssDNA. However, although transcription through Ig V region exons is required for SHM, it does not generate stable ssDNA, which leaves the mechanism of AID targeting unresolved. Although AID purified from B cells can by itself deaminate ssDNA templates, it cannot by itself deaminate an actively transcribed dsDNA SHM substrate (that is, a substrate containing multiple RGYW motifs that cannot form R loops — structures generated during transcription that displace the nontemplate strand as ssDNA). Replication protein A (RPA) 2 / 32 kDa subunit of RPA (RPA32) is a heterotrimeric ssDNA-binding protein — consisting of a 70 kDa (RPA70) subunit / RPA1 and a 14 kDa (RPA14) subunit / RPA3, in addition to RPA32 — and has previously been implicated in DNA replication, recombination and repair. AID-mediated deamination leads to the release of AID from the AID–RPA–DNA complex and the DNA-bound RPA might enable the recruitment of DNA-repair proteins. The AID–RPA interaction is activated B-cell specific (AID that was purified from the 293 non-lymphoid cell line ectopically expressing AID was unable to mediate deamination of the actively transcribed SHM or CSR substrate in the presence of RPA, and RPA and AID did not associate in this cell line) and this specificity probably depends, at least in part, on the post-translational modification of AID by PKA^ref (more highly phosphorylated in B cells than in the 293 cell lines). B-cell-specific AID–RPA complexes preferentially bind to ssDNA of small transcription bubbles at SHM 'hotspots', leading to AID-mediated deamination and RPA-mediated recruitment of DNA repair proteins^ref. N-terminus mutations in AID impair SHM but not CSR, while the C-terminus is not required for SHM^ref. Rheumatoid factor⁺ B cells undergo SHM at the T-zone-red-pulp border due to abundance of self-antigen that prolongs the proliferation of B cells at extrafollicular sites, allowing them to reach a threshold number of cell divisions for the initation of mutation (it is not known whether there might be normal immune responses that follow similar rules). Mutational activity could result from a specific mechanism that actively introduces nucleotide changes or an ineffective repair mechanism that does not correct errors efficiently or precisely or both. The net result is hypermutational activity that is highly targeted (> 50%) to RGYW (purine, G, pyrimidine, A/T) motifs on each DNA strand and provides the observed G:C bias of mutations. In contrast, the acquisition of mutations also occurs frequently in nontargeted sequences. There might be 2 separate mechanisms for SHM : the first is MMR-independent, targets RGYW motifs, and occurs in germinal center (GC), whereas the second is MMR dependent, does not target specific motifs, and does not require germinal center formation. Variable but not constant regions of Ig genes contain a high frequency of DSBs near RGYW hotspots, suggesting that several error-prone DNA polymerases (h, i, z and m) might be involved in creating mutations during the repair of these DSBs. Transcription through mammalian switch regions, because of their GC-rich composition, generates stable R-loops, which provide ssDNA substrates for AID. However, the Xenopus laevis switch region S, which is rich in AT and not prone to form R-loops, can functionally replace a mouse switch region to mediate CSR in vivo. X. laevis S–mediated CSR occurred mostly in a region of AGCT repeats targeted by the AID–replication protein A complex when transcribed in vitro. AGCT is a primordial CSR motif that targets AID through a non-R-loop mechanism involving an AID–replication protein A complex^ref. SHM has been difficult to study in vivo because the DNA in and around rearranged V genes undergoes random point mutation so that many different mutations occur. Furthermore, cells expressing mutated Igs are then subjected to antigen selection, which selects a subset of the mutated Igs that have increased affinity for the antigen. Mice transgenic for a reporter transgene (HuG-X) that encodes a chimeric Ig heavy chain (composed of a

murine VDJ segment and a human IgG₁ constant region reporter gene) were created with the VDJ containing a TAG stop codon in the D segment, so that the transgene is thus transcribed but not translated. Point mutation of the stop codon results in expression of the chimeric H chain, which is readily, detected as human IgG₁ expression. The transgene undergoes somatic mutation at a low
frequency in vivo. Treatment of the animals with anti-IgD greatly increases the frequency of somatic mutation.  The observed mutation frequency in anti-IgD treated mice increases with age until adulthood, then plateaus, and finally declines in aged mice.  Mutations in the HuG-X transgene occur in germinal centers. Sequence analysis demonstrates that the majority of mutations are point mutations; more rarely single base pair deletions are noted. Consistent with currently proposed mechanisms for somatic mutation, the mutations in TAG codon located in D segment are associated with increased double stranded breaks (DSB) within and around TAG site. It is noteworthy that the decreased frequency of somatic mutation in 24-month-old mice is associated
with a decrease of AID activity, These mice may prove to be a valuable model for studying somatic mutation in vitro and in vivo.
• BcR revision : peripheral mature B cells (including malignant B cell lymphomas) occasionally undergo secondary V(D)J recombination of immunoglobulin genes and modify their antigen specificity by V_H segment replacements during the germinal centre reaction to diversify the immune response, or to overcome self reactive antigen receptors. Excessive secondary rearrangements, especially in the periphery where tolerance mechanisms are less effective, can result in the production of autoantibodies by edited B cells
• chickens (Gallus gallus), pigs (Sus scrofa) and rabbits (Oryctolagus cuniculus) use gene conversion as first choice and somatic hypermutation as second choice. In chickens, prebursal stem cells (e.g. the B lymphoma DT40 cell line) are committed to a particular Ig gene rearrangement in the intraembryonic mesenchyme and colonize the follicles of the bursa of Fabricius. All chicken Ig L chains are rearranged by the same V and J gene segments and only one-third of the L chains derived from bursal B cells up to day 13 of embryonic development are in-frame. No further V-J rearrangements are ongoing in the embryonic bursa. A major role of the bursa is to provide the necessary microenvironment for the somatic diversification of rearranged V-J genes through a program of segmental gene conversion with a pool of noncoding pseudogenes being used as donors and for the selective amplification of lymphocytes with productive gene rearrangements. In posthatching bursal cells, nearly all V-J joints are in-frame. The bursa is GALT and a major trapping site for environmental Ags from the gut. Exogenous and gut-derived Ags are actively transported across the bursal epithelium into the lymphoid follicles of the bursa. Anyway the preimmune repertoire in the bursa is generated by gene conversion during Ag-independent B cell proliferation, and antigenic stimulation from the bursal epithelium to bursal B cells plays roles in the selection of clones with a productive V-J joint : ligation of the bursal duct before hatching blocks the transport of gut-derived antigens into the bursa and results in reduced proliferation of bursal cells after hatching.
All together these differentiating mechanisms allow the immune system to create > 10¹¹ different BcRs. The B-cell-specific transcription repressor BACH2, expressed by IgM⁺ cells within the lymphoid follicles of the spleen (the site of CSR and SHM), is crucial for CSR and SHM. By contrast, no BACH2 was detected in IgM⁺ marginal-zone B cells. BACH2 deficiency doesn't impair transcription step of CSR and prevents cleavage and/or ligation of the switch regions^ref
The multi-chain immune recognition receptor (MIRR) family includes :
• TcR
• BcR
• FcRs
Web resources : ImMunoGeneTics (IMGT) : LIGM Database
• Classical activation pathway (CP) for complement (C' ; a.k.a. alexin)
Activators :
• adjacent C_g2 domains in at least 2 IgG molecules (IgG₃ >> IgG₁ > IgG₂ : please note IgG₄ instead activates the alternative pathway) : so at least bivalent Ags are necessary (e.g. there is no complement activation with anti-Rh IgG)
• C_m4 domains in 1 IgM pentamer which has adopted a "staple" conformation due to binding to an antigen which is at least bivalent.
• myelin
• bacterial surfaces of Escherichia coli and Klebsiella pneumoniae
• DNA
• CRP-phosphorylcholine complexes
• trypsin-like enzymes (plasmin, kallikreins)
They bind p410^C1q subcomponent (a 11S thermolabile complex made up of 3 different polypeptides arranged as 6 p29^{A / a}-p27^{B / b} heterodimers and 3 p24^{C / g} homodimers = 18 polypeptides) to a S1 site on plasma membrane.
[C1q globular heads may be inhibited by C1q-binding protein (C1qBP)]
C1q changes its conformation and induces an activating autocleavage in p190^C1r subcomponent (a 7S b₁-globulin), which in turn cleaves and activates p87^C1s subcomponent (a 4.1S a-globulin). Such subunits are connected by Ca²⁺.
[activated C1r and C1s may be inhibited by C1 inhibitor (C1-INH), a serpin produced by hepatocytes, monocyte/macrophages, fibroblasts, plateletes, and endothelial cells. C1-INH also inhibits kallikrein and the coagulation factors XIa, XIIa, and plasmin. Its deficiency leads to high C2a levels, causing hereditary angioneurotic oedema (HANE)]
C1s in the resulting C1qrs complex is an active Ser protease that cleaves soluble C4A (acidic form) or C4B (basic form) (a 10S p95^a-p78^b-p33^gcomplex derived from cleavage of a single chain proprotein before secretion) into p20^C4a (arising from a subunit) and p190^C4b, exposing an internal thioester moiety in the C4b fragment. Thioester groups are highly reactive, and are rapidly attacked by a nucleophilic amine or hydroxyl group on a neighbouring molecule, to form a covalent bond. Thus, C4b can bind to :
• the Fab of the antibody binding C1
• the membrane of the target cell
• the membrane of nearby cell
Surface-bound C4b acts as a binding site for the b-globulin p115^C2 in the presence of Mg²⁺ ions.
[Schistosoma mansoni produces CRIT, a C2 decoy receptor]
When bound to C4b, C2 becomes a substrate for the Ser esterase activity of C1s, which generates p73^C2a and p34^C2b. A C2a4b complex (a.k.a. classical pathway C3 convertase) arises.
[Joining between C2a and C4b may be prevented by ...
• C4b-binding protein (C4BP / C4bp) is present in human plasma at concentrations of 200-250 mg/mL and is found in several forms, the major one being (p70^a)₇(p45^b). Most C4BP in blood exists in complexes with protein S, which interacts with the b-chain of C4BP.

[C4BP is bound by some bacterial surfaces :
• N-terminal loops of OmpA, which is a 35-kDa highly conserved protein among Gram-negative Bacteria, bind to CCP3 of the a-chain of C4BP, which leads to a decrease in serum killing, allowing a certain threshold of bacteremia
• Por1A, Por1B , and type IV pili of Neisseria gonorrhoeae
• NH₂-terminal, highly variable region of several members of the M family proteins in many isolates of Streptococcus pyogenes
• FHA in Bordetella pertussis]
• CD55 / decay accelerating factor (DAF) [produced also by Trypanosoma cruzi as T-DAF] : Daf dissociates C3/C5 convertases that assemble on host cells and thereby prevents complement activation on their surfaces. During primary T cell activation, the absence of Daf on antigen-presenting cells (APCs) and on T cells enhances T cell proliferation and augments the induced frequency of effector cells. The effect is factor D- and, at least in part, C5-dependent, indicating that local alternative pathway activation is essential. Cognate T cell–APC interactions are accompanied by rapid production of alternative pathway components and down-regulation of Daf expression. The findings argue that local alternative pathway activation and surface Daf protein function respectively as a costimulator and a negative modulator of T cell immunity and explain previously reported observations linking complement to T cell function. The results could have broad therapeutic implications for disorders in which T cell immunity is important^ref.
• [staphylococcal complement inhibitor, an excreted 9.8-kilodalton protein, blocks human complement by specific interaction with C3 convertases (C4b2a and C3bBb). Staphylococcal complement inhibitor bound and stabilized C3 convertases, interfering with additional C3b deposition through the classical, lectin and alternative complement pathways. This led to a substantial decrease in phagocytosis and killing of Staphylococcus aureus by human neutrophils. As a highly active and small soluble protein that acts exclusively on surfaces, staphylococcal complement inhibitor may represent a promising anti-inflammatory molecule^ref.]
Each C2a4b complex cleaves up to 200 (amplification !!) p180^C3 molecules (a 9.5S b-globulin made up of p110^a and p70^b subunits), creating p9^C3a and a larger, thioester-containing p171^C3b: activation of component C3 (1,641 residues) is central to the 3 complement pathways and results in inflammation and elimination of self and non-self targets. C3 is secreted by proximal-tubule epithelial cells, endothelial cells, and mesenchymal glomerular cells. Crystal structures of native C3 and its final major proteolytic fragment C3c reveals 13 domains, 9 of which were unpredicted, and suggest that the proteins of the a₂-macroglobulin family evolved from a core of 8 homologous domains. A double mechanism prevents hydrolysis of the thioester group, essential for covalent attachment of activated C3 to target surfaces. Marked conformational changes in the b-chain, including movement of a critical interaction site through a ring formed by the domains of the a-chain, indicate an unprecedented, conformation-dependent mechanism of activation, regulation and biological function of C3^ref. Human C3 exists as 2 allotypic variants, C3F (fast) (present in about 20% of white patients) and C3S (slow), according to electrophoretic mobility. The arginine substitution to glycine at position 80, that discriminates between the C3F and the C3S allele, lies in the first a₂-macroglobulin domain (MG1) at the C3 terminal part of the C3b light chain^ref :

In our current understanding of the molecular portrait of C3, MG1, which is conserved in C3, C4, C5, and other members of the a₂-macroglobulin superfamily, does not appear to be a critical C3 functional domain, as is the case for the thioester, the anaphylatoxin (which recruits inflammatory cells), or the proteolytic cascade sites in the molecule. Differences that are not yet understood could affect binding to complement receptor on antigen-presenting cells, B cells, or T cells. The effect of an unknown gene proximal to the C3 gene cannot be excluded, but research has not yielded pertinent candidates. No information is yet available on possible differences in the production of alloantibody (or C3F antibody) in the at-risk population of transplant recipients. A partial histologic analysis of late-deteriorating transplants did not reveal C4d deposition in the C3S/S grafts — a finding that does not suggest chronic stimulation of T-cell–dependent alloantibody^ref. The newly exposed thioester group concentrates C3b onto the surface of the antigen as described for C4b, causing opsonisation of the surface of the targeted cell and of bystander cells and further complement amplification by the alternative pathway.
Further C3b binds non-covalently to p190^C5 (a b₁-globulin made up of p115^a and p75^b subunits) : the C2a activity of C2a(3b)4b complex (a.k.a. classical pathway C5 convertase) cleaves C5 to p17^C5a and p163^C5b.
In the genetic absence of C3, thrombin substitutes for the C3-dependent C5 convertase. This linkage between the complement and coagulation pathways may represent a new pathway of complement activation. Plasma from C3^-/- mice contained threefold higher levels of thrombin activity compared to plasma from C3^+/+ mice^ref.
• Some "byproducts" of all the 3 complement activation pathways have biological activities through binding to respective GPCRs :
• C1q (it is considered a lectin although devoid of a lectin-like domain) --- C1qR / collectins receptor : opsonin
• Ba --- ? : chemotaxin
• C2a --- ? : vasoactive activity
• C3a --- C3aR : strong anaphylotoxin and chemotaxin
• C3b ---
• CD35 / CR1 (on B lymphocytes, 10÷15% of T lymphocytes, neutrophils, macrophages, FDCs, RBCs, eosinophils, some astrocytes, glomerular podocytes : in non primates also on platelets, where binding causes degranulation)
• CD21 / CR2 / EBVR (on B lymphocytes, neutrophils, eosinophils, macrophages, FDCs, platelets, uterus cervix and nasopharyngeal epithelium)
opsonin (induces erythrocyte-antibody-complement (EAC) rosettes) and involved in immune adherence.
‡ factor I / C3b inactivator (+ factor H as cofactor) -mediated proteolysis
• inactivated C3b (C3bi / iC3b) / C3b[H₂O] (normal plasma concentration = 8.0 mg/mL) ---
• CD21 / CR2 / EBVR  (on B lymphocytes as part of the B cell coreceptor complex, neutrophils, eosinophils, macrophages, FDCs, platelets, uterus cervix and nasopharyngeal epithelium)
• CD11bCD18 / a_Mb₂ integrin / Mac1 / CR3 or CD11cCD18 / a_Xb₂ integrin / CR4
opsonin.
‡
• C3c

and
• C3d,g / C3dg ---
• CD21 / CR2 / EBVR  (on B lymphocytes as part of the B cell coreceptor complex, neutrophils, eosinophils, macrophages, FDCs, platelets, uterus cervix and nasopharyngeal epithelium)
• CD11cCD18 / a_Xb₂ integrin / CR4
opsonin
‡ plasmin or kallikrein proteolysis
• C3d --- CD21 / CR2 / EBVR  (on B lymphocytes as part of the B cell coreceptor complex, neutrophils, eosinophils, macrophages, FDCs, platelets, uterus cervix and nasopharyngeal epithelium) : opsonin

Web resources : C3d's role in the immune response (animation)
• C3d1 / C3d-k

‡  plasmin proteolysis
• C3d2
and
• C3g
• C4a --- ? : weak anaphylotoxin
• C4b --- CD35 / CR1 (on B lymphocytes, 10÷15% of T lymphocytes, neutrophils, macrophages, FDCs, RBCs, platelets, eosinophils, some astrocytes, glomerular podocytes) : opsonin and involved in immune adherence

‡ factor I / C3b inactivator+ factor H as cofactor proteolysis
• C4c (free)

and
• C4d (bound on cell surface)
• C5a --- CD88 / C5aR : strong anaphylotoxin and chemotaxin


(reproduced with permission from Nature Reviews Immunology (Vol 4, No. 2, pp 133-142 (2004)) copyright Macmillan Magazines Ltd)
All the above listed anaphylotoxins may be inactivated by removal of the C-terminus Arg by p300^{N-carboxypeptidase} (an a-globulin), leading to production of desArg derivatives. Please note CR3 also mediates nonopsonic phagocytosis of zymosan (Z) and Mycobacterium kansasii through engagement of distinct sites : pseudopodia formation, Rac- and Cdc42-, and Hck-dependance with no Rho activity are the differential features with opsonic phagocytosis.
Opsonization and complement-mediated phagocytosis of immunocomplexes allows plasma clearing preventing type III hypersensitivities from occurring.
The genes coding for C2, C4 and B proteins are in the MHC class III region on chromosome 6. The complement proteins are produced mainly by hepatocytes (excepts for C1), enterocytes (C1), RES cells (macrophages, ...), and fibroblasts.
Web resurces :
• International Complement Society
• The Complement Genetics Home Page
• The common terminal or lytic complement pathway (although nowadays it is also used by the alternative and lectin activation pathways, it arose together with the classical activation pathway as an effector system of acquired immunity)

C5b recruits p120^C6 (a 5.7S heat-stable b₂-globulin):  if no C6 binding occurs within ~ 2 minutes, the C6-binding site on C5b decays and the C5b becomes inactive. The binding of C6 stabilises the complex and provides a binding site for p110^C7 (a 5.6S heat-stable b₂-globulin). The binding of C7 allows the C5b67 complex to dissociate from the C3/5 convertase and to bind cell membranes through hydrophobic interaction sites predominately in C7.
[complement S-protein / PA-I-BP1 / vitronectin / somatomedin B binds soluble C5b67]
The C5b67 complex has a high affinity forp163^C8, a g-globulin made up of a p63^{b chain} non covalently linked to a disulfide bound p13.7^a-p77^gdimer. C8a and C8b are homologous to C6, C7 and C9 and together these proteins comprise what is referred to as the 'MAC protein family'. By comparison, C8g is distinct in that it belongs to the lipocalin family of small, secreted proteins which have the common ability to bind small hydrophobic ligands. While specific roles have been identified for C8a and C8b in the formation and function of the MAC, a function for C8g and the identity of its ligand are unknown. The binding of C8 increases the penetration of the complex into the membrane.
Binding of p79^C9 (a heat-labile a-globulin: 58 mg/mL) to the C5b678 complex causes a major conformational change in the C9 molecule, causing it to unfold (hydrophilic-amphiphilic transition) and insert into and through the phospholipid bilayer.
[CD59 / MIRL / protectin / homologous restriction factor of 20 kDa (HRF-20 / HRF20) / MACIF is membrane glycoprotein anchored via galactosyl phosphatidyl inositol which binds both C8 and C9 of autologous cells and by preventing C9 insertion into the membrane and subsequent polymerization so inhibits the final stages of complement activation]
Multiple (up to 18) C9 molecules sequentially bind in the complex and polymerise into a ring, forming pores. The C5b678(9)_1-18 complex is a.k.a. the membrane attachment complex (MAC) :
• the ‘doughnut’ hypothesis : the channel (external Ø = 20 nm; internal Ø = 10 nm ; height = 16 nm) allows leakage of cellular contents, and water & small ions to flow into the cell along an osmotic gradient, causing the cell to undergo osmotic burst. Further Ca²⁺ influx allows calpains activation => cell autolysis.
• the ‘leaky patch’ model : the MAC causes a wide area of membrane perturbation through localised phospholipid disorder. This theory therefore suggests that the number of C9 molecules in the complex is irrelevant and the formation of a physical pore is unneccessary. On RBCs, on average, only 3-4 C9 molecules bind per C5b678 complex, and it has been shown that a single C9 molecule bound to the C5b678 complex is sufficient to cause efficient lysis. However, RBCs are metabolically inactive and cannot repair themselves. Metabolically active cells display multi-hit kinetics before cell death occurs, suggesting that more than one MAC is necessary for lysis. Nucleated cells can prevent lysis by 2 major mechanisms:
• ion pumps can counteract the influx of ions and water; this allows the cell to survive long enough for the second mechanism to occur :
• endocytosis or exocytosis of the MAC, thus removing the pore from the cell surface. This is a temperature dependent process which occurs rapidly, leading to a 90% decrease in surface MAC on neutrophils after 10 minutes at 37°C.
Sublytic MACs activate PK-C, ERKs, JNK, cPL-A₂, and COX-2.
• immune cytolysis : cell lysis produced by antibody with the participation of complement.
• Summary of killer (K) cells : they all express CX₃CR1.
• CD8ab⁺ab-TcR⁺ T_c lymphocytes (CTL)
• NK cells
• NKT cells
• gd-TcR⁺ T-cells
Summary of killing mechanisms : induction of apoptosis is preferred to necrotic death. For killing to be selective the killer cells needs to be near the targeted cells : proximity can be achieved through
• binding of Fc (on opsonized cells) to FcRs (on the killer cell) : this is referred as antibody-dependent cell-mediated cytotoxicity (ADCC)
• binding of complement opsonins (on opsonized cells) to CRs (on the killer cell)
Apoptosis may be induced by ...
• early granule exocytosis (see video (1.5 Mb ; local cache) at the immunological synapse (the contact zone between the killer cell and target cell due to the specific reorganization of cell-surface membrane proteins). Granules contain ...
• p70^{pore-forming protein (PFP) / perforin / cytolysin / NK cytotoxic factor (NKCF)} : its expression is under the control of the Ets-family member MEF. Perforin undergoes polymerization (up to 20 monomers) in presence of [Ca²⁺]_ex and creates a membrane pore for ions and small proteins. Perforin, unlike complement, lyses target cells across a variety of species, including the homologous one, while the same target cell populations resist the attack by homologous complement.

[UL16 protein frm HHV-4 / CMV mediates an increased protection against the action of cytolytic proteins released by activated NK cells, possibly by a membrane-stabilizing mechanisms, rather than by delivering negative signals to NK cells^ref]
• lysosomal enzymes
• cathepsin B : this associates with killer cell surface and protects it from autolysis by cleaving perforin in its intermediate membrane-associated stage that is highly susceptible to proteolysis
[chondroitinsulphate A protects NK cell from autolysis]
• granzymes (Ser-esterases)

They can enter the cell through ...
• perforin pores on plasma membrane
• CD222 / mannose 6-phosphate receptor-mediated endocytosis (RME) of  ...
• serglycin chondroitin sulfate proteoglycan (CSPG)-granzyme B complexes
• serglycin chondroitin sulfate proteoglycan (CSPG)-granzyme B-perforin complexes
... followed by perforin-mediated endosomolysis.
Once inside the cell ...
• granzyme A / granzyme 1 / Hanukah factor serine protease (HFSP) / cytotoxic T lymphocyte-associated serine esterase-3 (CTLA3) is a homodimeric, trypsin-like serine protease (Arg/Lys specific) of 60 kDa that cleaves fibronectin 1, collagen type IV, thrombin receptor, nucleolin, IL-1b, pro-urokinase-type plasminogen activator (pro-u-PA), the nucleosome assembly protein PHAPII, histone H1 family members and lamin A/C.
• granzyme B / granzyme 2 (Asp/Glu specific) cleaves procaspase 3 (after the initial cleavage event, autoactivation is controlled by the self-catalytic activity of caspase), procaspase 8, [procaspase 10 ?], Bid (not in studies when precautions are taken to minimize proteolysis during processing of cells for immunoblots) and ICAD. The predominant granzyme B death pathway starts with the activation of pro-caspase 3, and the death signal is maximized by mitochondria through caspase-mediated engagement of the BH3-only/BAX/BAK pathway. The BH3-only protein that's cleaved by caspase 3 might be BID, but there might be other participants, too. Maybe it also directly triggers the opening of the mitochondrial permeability transition pore (PTP).

[normal killer cells, activated DCs and some tumour cells express the protease inhibitor PI9]
[granzyme B is inhibited by the adenovirus L4-100K protein]
• granzyme H (Phe specific, orthologous to murine granzyme C) cleaves ?. It causes apoptosis (single-stranded DNA nicking, swelling and depolarization of mitochondria) but did not involve caspase activation, cleavage of BID or activation of the CAD nuclease.
• granzyme K / granzyme 3 (Arg/Lys specific) cleaves ?
• [granzyme M (Met/Leu specific) cleaves ?]
• granulysin : it is a member of the saposin-like protein family of molecules, including amoebapores from Protozoa, pulmonary surfactant protein B, saposins, and NK-lysins (from porcine lymphocytes). The 9-kDa active isoform is the product of processing of the 15-kDa isoform at the carboxyl and amino termini. Granulysin interacts with lipids in the cell membrane and also activates lipid-degrading enzymes, which leads to lesions in the cell membrane that result in death of the targets. It also has potent anti-microbial activity against many extracellular pathogens, including Bacteria, Fungi and helminths, inducing increased permeability and osmotic lysis. CTLs express granulysin after stimulation by IL-15 and this expression is abrogated after treatment with SrCl₂.
• TNF-a, which binds to TNFRs on the surface of the targeted cell.
• binding of CD178 / FasL on their surface with CD95 / Fas / Apo-1 on surface of the target cell. Only when the granule pathway is compromised are significant levels of Fas-mediated killing observed. Fas is up-regulated by IFN-g secreted by the killer cell.
• Summary of innate lymphocytes : they predominantly express recurrent families of germ-line-encoded (i.e. not requiring addition of N nucleotides often due to absence of TdT in fetal and neonatal period), semi-invariant BcR or TcR with borderline autoreactivity (allowing escape from negative selection) to a small set of conserved self-structures induced or exposed in conditions of cell stress or death :  most innate lymphocytes express distinct NK-lineage inhibitory and activating receptors that are not generally found on conventional lymphocytes and that fine-tune the threshold of Ag-receptor signalling. They have a "natural activated/memory or effector" phenotype(CD45 / B220 / LCARO⁺), rather than a naive phenotype, which indicates prior exposure to these self-Ags, even in germ-free conditions. They reside in tissues rather than in lymph nodes.
• B-1 B cells
• marginal zone (MZ) B cells
• hVg9/Vd2 gd-TcR⁺ T-cells
• NKT cells
• fourth circulation : the continuous movement of lymphocytes from their sources in all the hematopoietic and connective tissues to the blood passing through all the tissues and organs, then to the lymph nodes, then into the lymph of the thoracic duct, and into the blood again.
• ecotaxis : the “homing” of recirculating lymphocytes to specific compartments of peripheral lymphoid tissues, with B cells going to B-dependent areas and T cells to T-dependent areas.