Table of contents :

lymphocyte activation : stimulation of lymphocytes by specific antigen or nonspecific mitogens resulting in macromolecular synthesis (RNA, protein, and DNA) and production of lymphokines; it is followed by proliferation and differentiation of the progeny into various effector and memory cells. NFAT5 has an important role in enabling lymphocytes to adapt to osmotic stress in lymphoid tissues : the proliferation of Nfat5^+/D T cells was inhibited by > 60% under conditions  of hyperosmotic stress. Similar to T-cell responses, Nfat5^+/D B-cell proliferation after stimulation with LPS is markedly reduced in hypertonic but not isotonic media. Despite it is generally thought that unlike unicellular organisms, most mammalian cells are not normally subjected to extremes of hypertonic stress in vivo because of the maintenance of body-fluid homeostasis.
lymphocyte transformation : the morphologic changes accompanying lymphocyte activation, in which small, resting lymphocytes are transformed into large, active lymphocytes (lymphoblasts)
Lymphoid tissues have increased osmolarity compared with the serum, and brain and lung tissues : this might reflect high cell density or the presence of highly metabolically active cells in the lymphoid tissues^ref
A major challenge is understanding what endows the overall ensemble with both sensitivity and global reliability despite variations in the concentration, initial state, local behavior, or previse number of partecipating components. Emerging evidence suggests that these properties arise from a combination of :

• small alterations in the probabilistic behavior of a biochemical pathway, a cell, or a collection of cells
• the amplification of these induced changes by positive-feedback loops and/or exponential cell growth (nonlinear amplification or clonal expansion of antigen-specific lymphocytes)
• temporal control
• kinetic proofreading : relatives rates of ligand dissociation from the TcR and of coreceptor interaction with an engaged TcR complex as it moves within the membrane
• requirement for some minimal duration in one signaling state before the next input can be received
• spatial control : early in the response, individual lymphocytes, even those with equivalent antigen specificity, meet depots of antigen in discrete sites too far apart for locally secreted mediators to readily influence lymphocytes at other antigen foci. For this reason, the effects of feedback amplification and control are most often local, not global. This disperse nature of clonal immune responses prevents an inappropriate response from dominating the entire system
• stochastic gene expression : with the exception of antigen receptor genes that show allelic exclusion, most genes are thought to be expressed from both alleles when the differentiated state of the cell supports transcription from that locus. In mice, several cytokines have been reported to exhibit monoallelic transcription.
• IL-4 gene during any given activation cycle of effector T cells^ref : the activation of each individual allele is regulated by a stochastic process whose probability can be augmented by increasing the strength of signal delivered through the TcR^ref. The allelic pattern is transmitted as a stable epigenetic trait^ref. In the case of CD4⁺ T cells, this could limit full polarization of an expanding T cell clone, or leave cells within the clone susceptible to the influence of counterregulatory cytokines, providing the evolving response with the potential for greater functional diversity
• human IL-1a gene in CD4⁺ T cell clones. Differential methylation of CpGs in the proximal promoter region is associated with allele-specific expression of IL-1a in CD4⁺ T cells. The differential methylation pattern is already observed in naive T cells. In keratinocytes, which constitutively produce IL-1a, the proximal promoter is hypomethylated. CpGs located further upstream and in intron 4 were almost all methylated, irrespective of expression. Treatment of non-expressing cells and of T cell clones with 5-aza-2’ deoxycytidine induced IL-1a expression in the non-expressing cells and induced expression of the formerly silent allele in T cell clones. In addition, electrophoretic mobility shift assays showed that methylation of CpGs in the proximal promoter resulted in direct inhibition of binding of nuclear factor(s). Taken together, these results suggest that allele-specific expression of IL-1a in CD4⁺ cells is achieved at least in part, by differential methylation of the promoter^ref.
• this type of feedback poses the threat of full activation in response to low-level nonspecific inputs ("noise") arose from random fluctuations intrinsic to the system itself or from physiologically irrelevant environmental stimuli : counter-regulatory controls that modulate these potentially unidirectional, explosive processes include :
• tonic filters : their action carries the price of an overall blunting of the sensitivity of the system.
• mechanism that suppresses inadvertent stimuli while facilitating sensitivity include :
• negative feedback circuit provides a more flexible form of control : there is a time delay in which the input signal mediates some effect before the effect of negative feedback is manifest, allowing low-level, short-term responses followed by a return to baseline, or, in a more sophisticated manner, to funnel inputs along one, but not another, pathway because of differing requirements for the concentration of a second messenger.
• ultransensitivity circuits (eg MAPK activation) : going from 10 to 90% completion requires a change in input concentration smaller than 81-fold, a property that often arises from cooperativity in the behavior of the interacting components. The stepness of the dose-response curve reflects a parameter termed the Hill coefficient, which at high values (> 5) provides the system with nearly digital switch-like effects. A valuable property of certain forms of ultrasensitivity is that the response is negligible at low input levels. Thus, small spikes of input are suppressed and do not undergo positive amplification. However, a real signal that slightly exceeds in magnitude these incidental stimuli instead produces a maximal signal from the system.
• the summation of individual topographically dispersed cellular responses subject to these local control mechanisms

• innate immune recognition of mucosal infection with HHV-2 / HSV-2 occurs in 2 distinct stages, one at the level of the infected epithelial cells and the other at the level of the noninfected DCs. Importantly, both TLR-mediated recognition events are required for the induction of effector T cells. Virally infected tissues instruct DCs to initiate the appropriate class of effector T cell responses and reveal the critical importance of the stromal cells in detecting infectious agents through their own pattern recognition receptors^ref
• antibody-mediated immunity (AMI)
• activation of naive B cells

The B cell takes up the antigen to which it has bound, internalizes it, processes it and re-express it in the groove of class II MHC molecules, waiting for recognition by the T_h cell, which then sends helper (activating) signals (signal 2), which can be divided in ...
• competence signal (drive the cell from G₀ to G₁ phase)
• cross-linking of BcR by multivalent Ag
• cognate interaction with helper T cells through an immunological synapse
• CD40 --- CD40L
• progression signals (drive the cell from G₁ to S phase dividing within 48÷72 hrs.). The antibody response begins with induction of naïve mature B cells, which are naturally arrested in G₀/G₁ phase of the cell cycle, to enter the cell cycle in response to antigen and cytokine. BLyS (BAFF), a cytokine essential for mature B cell development and survival, is thought to act mainly by attenuation of apoptosis. BLyS alone induces cell-cycle entry and early G₁ cell-cycle progression, but not S-phase entry, in opposition to the CKIs p18^INK4c. Independent of its survival function, BLyS enhances the synthesis of cyclin D2, in part through activation of NF-kB, as well as CDK4 and RB protein phosphorylation. By convergent activation of the same cell-cycle regulators in opposition to p18^INK4c, BcR signaling induces cell-cycle entry and G₁ progression in synergy with BLyS, but also DNA replication. The failure of BLyS to induce S-phase cell-cycle entry lies in its inability to increase cyclin E and reduce p27^Kip1 expression. Antagonistic cell-cycle regulation by BLyS and p18^INK4c is functionally linked to apoptotic control and conserved from B cell activation in vitro to antibody response in vivo, further indicating a physiologic role in homeostasis^ref.
• IL-2
• IL-4
• IL-5
• IL-6
Caspase-6 and caspase-8 activity is required for stimulated B lymphocytes to upregulate expression of D-type cyclins and CDK4 and to enter the cell cycle, but not cytokine secretion.
The B cell can wait about 24 h after BcR engagement, after which it dies if no help appears^ref. The smaller the amount of antigen in absolute and local concentrations, the more directly dependent the B cell response is on T cell help. CD40 engagement alone induces proliferation followed by apoptosis. B-cells stimualted with BcR alone (mimicking the ansence of T-cell help) do not produce pignificatnt levels of cytokines. However, when BcR crosslinking is combined with stimulation through CD40, the B cells produce high levels of TNF-a, TNF-b / LT-a and IL-6 in a dose-dependent manner, which would promote an ongoing immune response. Staggered dual activation (24 hours of BcR crosslinking before addition of CD40L / CD154⁺ fibroblasts mimicking T-cell help)) amplified this cytokine response. This mimics the sequence of interactions in vivo, where a B cell typically encounters antigen in the B-cell zone of a lymph node before migrating to T-cell areas to receive help from CD40L⁺ T cells. By contrast, stimulation thorugh CD40 alone results in production of the immunosuppressive cytokine IL-10 by B cells, in the absence of TNF and lymphotoxin. CD40 is expreessed constitutively by B cells, so the production of IL-10 could help to ensure that bystander T cell help does not result in inappropriate B-cell responses in the absence of cognate antigen^ref
‡
+ type I IFNs from the plasmacytoid DCs / DC2
+ using an improved method to purify human naive B cells it was found that BCR stimulation and T cell help induced initial cell division but were not sufficient to promote survival and differentiation thus leading to abortive proliferation of naive B cells. Extensive B cell proliferation, isotypic switch and differentiation to Ig-secreting cells was induced by addition of microbial products that trigger any of the TLR that are up-regulated in naive B cells upon BCR triggering. TLR agonists acted directly on B cells and were required irrespective of the nature of the T_h cells present. Supernatants of DC stimulated by DC-specific TLR agonists were also capable of enhancing B cell responses although to a much lower and variable extent. These results indicate that human naive B cell activation is critically dependent on innate stimuli acting optimally on TLR expressed by B cells. The coupling of BCR stimulation to TLR expression endows the human system with a high degree of specificity since it allows focusing of innate signals only on antigen-stimulated B cells^ref.
: differentiate locally to ...
B-cell blast / plasmablast / centrocyte (CD10 / CALLA⁺15^-19⁺20^-21^-22^-23⁺24⁺38⁺39⁺57⁺FcgRIIb1^hi; high nucleocytoplasmic ratio, a large nucleus (>10 µm), thin chromatin, and the presence of a nucleolus (>2 µm)

It upregulates synthesis of CCL4(a chemokine for T_h3 lymphocytes). B-lymphocytes that are activated by antigen in the T-cell areas of lymphoid tissues (on the contrary of resting B-lymphocytes) increase processing and class II MHC presentation of BcR-associated Ags inducing T cell tolerance. They undergo 8 or 9 mitosis, each ~ 12 hrs. long, and produce IL-6 and TNF-a. High-density, resting, and low-density activated tonsillar B cells expressed TLR9 and TLR10 mRNA transcripts at the highest levels. Expression was higher in activated B cells than in resting cells. Analysis of a range of resting and activated human leukocyte populations revealed that mRNA expression of TLR10 was restricted to B cells. Stimulation of resting B cells with anti-m and anti-CD40 antibodies or with Staphylococcus aureus Cowan I bacteria (SAC) increased expression of TLR9 and TLR10. TLR1 and TLR4 expression were not significantly induced by B-cell activation. Interestingly, a CpG oligonucleotide, a TLR9 agonist, also stimulated TLR9 expression in B cells. Exposure to anti-m antibodies augmented TLR9 expression, concomitantly and dramatically increasing the responsiveness of B cells to CpG oligonucleotides in terms of proliferation and chemokine (CCL3 and CCL22) production. EBV-transformed cell lines and other cell lines representative of mature B-cell neoplasias (Burkitt lymphoma, follicular lymphoma, multiple myeloma) expressed TLR9 and/or TLR10, whereas pre-B cell lines were negative. Normal and neoplastic human B lymphocytes express a distinct TLR repertoire including TLR9 and TLR10 and expression is increased upon engagement of the antigen receptor complex or TLR9 itself. Regulated expression of selected TLRs in B cells is likely to play an important role in linking innate and adaptive immune responses in normal and pathologic conditions^ref. They have 2 possible fates :
‡
• proplasmacyte


+
• IL-6 from mature plasmacytoid DCs / DC2
• interferon-regulatory factor 4 (IRF4)
• BLIMP1^ref (which has also a role in T-cell differentiation^{ref1, ref2, ref3}) => XBP1 and other transcription factors
[inhibited by PAX5 and microphthalmia-associated transcription factor (MITF) A isoform (encoded by the common exons (exons 2–9) and 2 rarely studied exons (exons 1a and 1b)), which indirectly inhibits IRF4^ref]
• plasma cell / plasmacyte / antibody-secreting cell (ASC) / antibody-forming cell (AFC) (CD9⁺15^-19⁺20^-21 / CR2 / C3dR / EBVR^-22^-23⁺24^-38^bright56^weak/-72^-79a / Iga^-79b / Igb⁺syndecan-1^bright) : synthesize and secrete soluble antibody, undergo class switch recombination (CSR) and somatic hypermutation (SHR).


• some plasma cells present inclusions termed Russel bodies
• flaming plasma cell : an abnormal plasma cell that stains red to violet, probably because it contains immunoglobulins with a high carbohydrate content; it may be related to a thesaurocyte
After the activated B cells enter the bone marrow, they stop proliferating and differentiate into plasma cells, under the influence of adhesion molecules and factors such as IL-6. Plasma cells die by apoptosis after several weeks or months, but what initiates the process is unclear (MacLennan ICM, Hardie DL, Ball J, Drew M. Antibody-secreting cells and their origins. In: Malpas JS, Bergsagel DE, Kyle RA, eds. Myeloma: biology and management. Oxford, England: Oxford University Press, 1995:30-49). A small number of long-lived plasma cells in the bone marrow (< 1% of mononuclear cells) produce most of the IgG and IgA in the serum. The daily production of immunoglobulin by these cells can exceed 1 ng per cell.
or

+

• raft-dependent, GPI-linked IgD-mediated cAMP_i response
• BCL6 is a transcriptional repressor related to Drosophila Tramtrack that blocks AP1, a transcription factor required for expression of the other transcription factor BLIMP1, which would drive the development of plasma cells. BCL6 also directly inhibits transcriptionn of the CD80 gene by binding to its promoter enhancer and prevents the upregulation of expression of CD80 in response to CD40-CD40L interactions mediated by NFkB. CD80 has a crucial role ion T-cell-B-cell interactions required for GC development and T-cell-dependent antibody responses. Therefore, constitutive expression of BCL6 owing to a translocation might lead to lymphomagenesis by disrupting the normal GC differentiation pathway of B cells. MTA3, a cell-type-specific subunit of the Mi-2/NuRD

co-repressor complex, carboxyl terminus associates with the central region of BCL-6 to inhibit the terminal differentiation of B cells to plasma cells. Acetylation of the central domain of BCL-6 prevents interaction with MTA3 and abolishes repressive function,which indicates that such post-translational modification of BCL-6 might be one way in which signals for plasma-cell differentiation are transduced^ref. BCL6 also controls the transcription of genes lacking a BCL6 binding site and this mechanism is required for the prevention of tumor suppressor p53-independent cell cycle arrest in GC B cells. BCL6 interacted with the transcriptional activator ZBTB17 / Miz-1 and, via Miz-1, bound to the promoter and suppressed transcription of the cell cycle arrest gene CDKN1A. Through this mechanism, BCL6 may facilitate the proliferative expansion of GCs during the normal immune response and, when deregulated, the pathological expansion of B cell lymphomas^ref.
 : migrate to a B cell follicle to proliferate and form a germinal center (GC).

• follicular or germinal centre (GC) B cell (CD49d^-62L / L-selectin^-75⁺86 / B7-2⁺CLA^-CD162 / PSGL-1; CXCR4^-FcgRIIb1^loGL7 / Ly77⁺PNA-R⁺integrin b₇^-). Germinal centers (GC) are inducible lymphoid microenvironments that arise primarily in response to T-dependent Ags.
• centrocyte : a general term encompassing both large and small cleaved follicular center cells
• small cleaved follicular center cell :  it has a diameter of about 8 mm, a nucleus with a deep fold or cleft and clumped chromatin, and cytoplasm that is not pyroninophilic

‡
• large cleaved follicular center cell : it has a diameter of about 12 mm, a nucleus with deep folds or clefts and clumped chromatin, and cytoplasm that is not pyroninophilic but may have immunoglobulin inclusions.
‡
• centroblast : a general term encompassing both large and small noncleaved follicular center cells
• small noncleaved follicular center cell : it has a diameter of about 12 mm, a nucleus without folds or clefts that contains finely dispersed chromatin, and cytoplasm that is pyroninophilic and basophilic.

‡
• large noncleaved follicular center cell : a follicular center cell considered to be the stage immediately preceding the development of the B lymphoblast and migration out of the follicle; it has a diameter of 15–20 mm, a nucleus without clefts that contains finely dispersed chromatin, and cytoplasm that is abundant and pyroninophilic.
During the GC response, a high frequency of mutations is introduced in the variable regions of the BcRs expressed by GC B cells. Ag present in the form of immune complexes (IC) is retained on follicular dendritic cells (FDC) within the GC is thought to drive the selection of high-affinity B cell mutants, ultimately resulting in the formation of the memory B cell compartment.
‡
+
• IL-2 (from activated T cells)
• IL-3
• IL-4 which controls the transcription of XBP1: but XBP-1 splicing depends on synthesis of immunoglobulins during B cell differentiation and the unfolded protein response (UPR).
• IL-5
• IL-6
• IL-14
• post-GC B cell migrate in the medulla of lymph nodes, as well as to the bone marrow
• lymphoblast : a morphologically immature lymphocyte, once thought to represent an early stage in lymphocyte development but now known to be an activated lymphocyte that has been transformed in response to antigenic stimulation


IgM are produced within 4÷7 days, but significant IgG concentrations are reached only at day 7÷10 from Ag challenge ("window period"). Please note anti-HDAg IgM are produced at day 30÷40 due to low immunogenicity. Differentiation of circulating B cells depends on where they are first activated. If they first see antigen in peripheral lymphoid tissue (lymph nodes and spleen) they appear to be biased toward expression of antibodies associated with "peripheral" immunity (IgG). If the first see antigen in mucosal sites (such as Peyer's patches) they are more likely to be committed to IgA expression. In the latter case, despite having made such a commitment, the switch to IgA is often delayed for several days. This cell leaves in the efferent lymph, passes through the mesenteric lymph node (where it may be subject to immunoregulation by T cells it encounters there) and returns to the blood. These cells selectively migrate to the spleen, which serves as an auxiliary site for clonal expansion before dissemination of the daughter cells via the blood into mucosal lamina propria. This process takes 5-7 days, at least 4 trips in the blood and 2-3 cell generations
Interactions for B-cell homing

molecules on naive B-cell	addressins on high endothelial venule (HEV) of B-cell zones of secondary lymphoid organs
step 1) : emperypolesis / reversible adhesion (few seconds) step 1a : tethering (bind loosely to endothelial cells) step 1b : slow rolling along the vessel (pushed forwards by the blood stream)
p90-110L-selectin / CD62L / MEL-14 / LAM-1 / LECAM-1 / Leu-8	peripheral node addressins (PNAd) (an O-linked carbohydrate moiety in SO42--GAGs, the main components of which are recognized in humans and mice by the mAb MECA-79ref1, ref2) :  GlyCAM-1  gp105-120CD34  ... In addition, some LN venules express structurally and antigenically distinct L-selectin ligands that are not detected by MECA-79ref1, ref2. The structure of L-selectin ligands in HEVs and the role of glycosyl- and sulphotransferases in the biosynthesis of these ligands is a topic of intense studyref1, ref2
CD49dCD? / a4b7 integrinref1, ref2	MAdCAM-1 (in mucosa-associated LNs, such as the mesenteric LNs) : as the a4b7 -MADCAM1 pathway can mediate selectin-independent rollingref, L-selectin deficiency compromises lymphocyte homing to peripheral LNs more markedly than to mesenteric LNsref, whereas b7 integrin deficiency has no effect in peripheral LNs, but causes reduced homing to mesenteric LNs and loss of homing to Peyer's patches where HEVs are PNAD-MADCAM+ref.
CD47 / w149	SHPS-1
step 2) : GiPCRs-mediated adhesion activation  (few seconds) : either pathway is sufficient to maintain B-cell homing (albeit at lower levels than in wild-type animals, in which both function simultaneously)
CCR7	CCL21 (constitutively secretedref1, ref2, ref3)  CCL19 is secreted by the lymphatic endothelium and interstitial cells in LNs, but not by HEVs. Perivascular CCL19 can be transported to the luminal surface of HEVs and induce integrin activation of rolling B cells
CXCR4	CXCL12 / SDF-1 induces tyrosine phosphorylation of ITK and RLK, which activate the actin-cytoskeleton regulators CDC42 and RACref
?	MAdCAM-1 (in marginal zone)
step 3) : adhesion / attachment & arrest (few seconds)
	vascular adhesion protein 1 (VAP-1)ref
CD11aCD18 / LFA-1 / aLb2 integrin (RhoH is required to maintain the integrin LFA-1 in a nonadhesive state on resting lymphocytesref; lymphocytes fail to home in one strain of LFA1-deficient miceref, whereas some residual homing was reported in a second strain, which was mediated by ...	CD54 / ICAM-1 (up-regulated by IL-1b, too)  CD102 / ICAM-2 (constitutive)ref1, ref2, ref3
CD49dCD? / a4b7 integrinref	CD106 / VCAM-1 / INCAM-110
step 4) : diapedesis / transmigration /  transendothelial migration (TEM) (10')

Following extravasation, B cells migrate to B-cell follicls - an event that depends on CXCR5 - CXCL13 / BLC (expressed on the luminal surface of HEVs) interactions
Even though B cells encounter 2 distinct integrin-activation signals in HEVs, B-cell homing to LNs is less efficient than that of T cells. This is because the level of L-selectin expression by B cells is 50% lower than the level of epxression by T cells, which express 7-10 x 10⁴ molecules per cell^ref. In experiments with stably transformed pre-B-cell clones that express human L-selectin, at least 50,0000 L-selectin molecules per cell are required for efficient rolling in LN HEVs^{ref1, ref2}. So its is important to keeop in mind that the presence of L-selectin on a leukocyte does not necessarily predict its potential to home to LNs.
In the absence of activating signals B cells undergo apoptosis within few days in the germinal center, leaving only Ag-reactive B cells.
‡
+
• SAP / SH2D1A in CD4⁺ T cells
• BLIMP1, a TF that downregulates genes associated with ...
• cell-cycle progression
• BCL6
• SPI-B
• ID3
• Ig class switching
• AID
• Ku70
• Ku80
• DNA-PK_CS / XRCC7
• STAT6
... and upregulates the transcriptional activator XBP1, which controls the production of IL-6, a cytokine essential for plasma cell survival.
• production of memory B cells (CD15^-19⁺23⁺24^-38^-44⁺62L / L-selectin⁺86 / B7-2⁺, CXCR4⁺, Ig(non-IgD, non-IgM)⁺). They arise from GC B lymphocytes and continues to express BCL6. The fact that protective immunity is long-lasting, coupled with the short half-life of Abs (< 3 wk), implies at least 1 of the following hypotheses :
• plasma cells are long-lived (and not dynamic dividing) cells : they are not intrisically long-lived, but rather plasma cells contact and modify bone marrow stromal cells to provide survival factors.
• plasma cell pool is continually replenished to maintain certain levels of secreted Ab
Despite the important role of plasma cells, they make up a small percentage of the total cells in secondary lymphoid tissue and bone marrow, making it difficult to isolate plasma cells for in vitro studies, so that most studies have used myeloma cell lines or patient material.
• pre-plasma memory B cells
• cell-mediated immunity (CMI)
• activation of NK cells

+
• IFN-g
• IL-2
• high doses of IL-12
• IL-15
• T-bet^ref
• algebric sum of interactions that signal the absence of self (e.g. MHC class I molecules, independently from the bound antigenic peptide, instead of from the presence of non-self (i.e. Ag)) exceeding a given threshold (on the contrary of CTL CD8ab⁺ T-cells). The mature activating NK cell immunological synapse / supramolecular activation cluster (SMAC)^ref consists of :
• pSMAC : CD2, CD11a, CD11b (polarization depends on actin polymerization and WASp, but not on microtubule function)
• cSMAC : perforin (polarization depends on actin polymerization, WASp and microtubule function, with a rate lower than that of pSMAC
• NK cells were efficiently activated by murine cytomegalovirus (MCMV)-infected CD11b⁺ DCs. NK cell cytotoxicity required IFN-a and interactions between the NKG2D activating receptor and NKG2D ligand, whereas the production of interferon- by NK cells relied mainly on DC-derived IL-18. Although TLR9 contributes to antiviral immunity, signaling pathways independent of TLR9 were important in generating immune responses to MCMV, including the production of IFN-a and the induction of NK cell cytotoxicity. Notably, adoptive transfer of MCMV-activated CD11b⁺ DCs resulted in improved control of MCMV infection, indicating that these cells participate in controlling viral replication in vivo^ref.
‡
lymphokine activated killer (LAK) cell (CD25 / p55^IL-2Ra+69⁺122 / IL-2Rb137 / 4-1BB⁺223 / LAG-3⁺CCR1⁺CCR2⁺CCR7⁺CCR8⁺CXCR1⁺CXCR2⁺CXCR3⁺CXCR4⁺CX₃CR1⁺XCR1⁺)
They allow an early killing of neoplastic cells and cells infected by viruses that down-regulate MHC class I presentation pathway. IL-2 and IL-12 alter NK cell responsiveness to CXCL10 by down-regulating CXCR3 expression. IFN-g up-regulates TRAIL expression. CD223 / LAG-3 is a class II MHC ligand that leads to complete maturation of DCs, which acquire the capacity to trigger naive T cells and drive polarized T_h1 responses.
Apart from those expressed also on resting NK cells, LAK express another activating receptor :
• NKG2D / NKG2F / KLRC4-S isoform (up-regulated by IL-15) --- MIC proteins, CMV-encoded UL16 binding proteins (ULBPs)
... which can use as adapter :
• DAP10 (pYXNM-containing) => PI(3)K STP (far less effective than ZAP70/Syk STP in inducing activation)
• 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
LAK cells act as a killer cell and produce :
• IFN-g (autocrine positive feedback; induced by T-bet)
• IL-13
• IL-22
• IL-26
• TNF-a
• TNF-b / LT-a
• GM-CSF
• CCL1
• CCL4
• XCL1
• NK2 cells (as for T_h2 cells) produce :
• IL-3
• IL-5
NK cells, which are normally excluded from lymph nodes, are rapidly recruited in a CCR7-independent, CXCR3-dependent manner to lymph nodes on stimulation by the injection of mature DCs. Recruitment of NK cells is also induced by some, but not all, adjuvants and correlates with the induction of T_h1 responses. NK cell depletion and reconstitution experiments show that NK cells provide an early source of IFN-g that is necessary for T_h1 polarization^ref
Activated NK cells induce the maturation of myeloid DCs into stable type-1 polarised DCs (DC1), characterised by up to a 100-fold enhanced ability to produce IL-12p70 in response to subsequent interaction with T_h cells. DC1, induced by NK cells or by NK cell-related soluble factors, are stable, resistant to tumour-related suppressive factors, and demonstrate a strongly enhanced ability to induce T_h1 and CTL responses in human in vitro and mouse in vivo models. Compared with the standard mature DCs that are used in clinical trials at present, human NK cell-induced DC1s act as superior inducers of anticancer CTL responses during in vitro sensitisation. This provides a strong rationale for the combined use of NK cells and DCs in the immunotherapy of patients with cancer and patients with chronic infections that are resistant to standard forms of treatment. Stage I/II clinical trials that are being implemented at present should allow evaluation of the immunological and clinical efficacy of combined NK-DC therapy of melanoma and other cancers^ref
• activation of bone marrow DC progenitors

It is inhibited by :
• 1a,25-(OH)₂-vitamin D₃ inhibits differentiation and maturation of DCs via vitamin D receptor (VDR)
• down-regulates expression of
• IL-12
• TNF-a
• class II MHC
• CD80 / B7-1
• CD115 / CSF-R
• ICAM-1
• c-Rel and Rel B
... without altering survival.
• up-regulates expression of
• CSF-1 / M-CSF (stimulate the proliferation and differentiation of circulating monocytes into macrophages, which form granulomas)
• CCL-2 / MCP-1
• CCL-3 / MIP-1a
Following terminal differentiation induced by a diverse set of maturation stimuli, there is marked transcriptional up-regulation of CYP27B1 / steroid 1a-hydroxylase, leading to increased intracellular concentrations of 1a,25-(OH)₂-vitamin D₃. On the contrary VDR expression is down-regulated as monocytes differentiate into immature DCs : in fact addition of 1a,25-(OH)₂-vitamin D₃ to moDC cultures at different time points indicates that its inhibitory effects are greater in monocyte precursors than in immature DCs^ref. A negative feedback normally exists as VDR activation then down-regulates steroid 1a-hydroxylase expression while up-regulating expression of the inactivating enzyme CYP24 / steroid 24 hydroxylase
• glucocorticoids via GR
• down-regulate expression of
• c-Rel
• Rel B
• potently attenuate pro-inflammatory cytokines and chemokines
• modest, reversible effects on T-cell stimulatory capacity
Additive inhibition is most striking for CCL5 / RANTES, CCR5, CCR7, and Rel B.
• thrombospondin 1 (TSP) is an autocrine negative regulator of human DC activation : human monocyte-derived immature dendritic cells (iDCs) spontaneously produce TSP, which is strongly enhanced by PGE₂ and to a lesser extent by TGF-b, 2 soluble mediators secreted by macrophages after engulfment of damaged tissues. Microbial stimuli increase TSP production, which is further enhanced by IL-10 or TGF-b. The endogenous TSP produced during early DC activation negatively regulates IL-12, TNF-a, and IL-10 release (transiently activated by danger signals) through its interactions with CD47 and CD36 / thrombospondin receptor / p95^{gpIV / gpIIIb}. After prolonged activation, DCs extinguish their cytokine synthesis and become refractory to subsequent stimulation, thereby favoring the return to steady state. Such "exhausted" DCs continue to release TSP but not IL-10. Disrupting TSP-CD47 interactions during their restimulation restores their cytokine production^ref
... while at sites of infection and inflammation DC-poietins ...
• GM-CSF (the most important) =>
• T_h1 effector pathways
• activation of macrophages, which results in the production of both O₂^2- and NO^. as tumoricidal effectors
• generation of CTL
• T_h2 effector pathways : activation of eosinophils
• IL-3
• IL-4
• FLT3L
• p43
... promote differentiation into precursor of dendritic cells 1 (pDC1) / immature or intermediate dendritic cell (iDCs / DC_imm) / immature bone marrow-derived dendritic cell (BMDC) (CD1a^int1b^int1c / BDCA-1^int4⁺5^-7^lo8a^-9⁺11b^lo11c^hi13⁺14⁺19^-23⁺33⁺35 / CR1⁺40^low43^hi45RA^-45RO⁺46⁺50 / ICAM-3⁺54 / ICAM-1⁺58/ LFA-3⁺68⁺80 / B7-1^lo83⁺86 / B7-2^lo89⁺91 / LRP / a₂-macroglobulin R⁺123 / IL-3Ra^lo153 / 30L⁺ 205 / DEC-205^lo206 / MMR^hi207 / DC-SIGN^hiGM-CSFR⁺⁺NKG2D^-, MHC I⁺MHC II^lo, BDCA-2^-, TLR1⁺⁺2⁺⁺3^-4⁺⁺5⁺⁺6⁺7^-8⁺⁺9^-10^-, CCR2⁺, CCR5⁺, MR⁺, MSR2⁺,
They produce :
• IL-6
• low levels of IFN-a and IFN-b
They have high phagocytic activity, efficient antigen uptake and processing, but peptide-MHC class II complexes are retained in endosomes and lysosomes. Lysosomal pH is 5.4 : a substantial fraction of cathepsin L and legumain (LGMN) / asparaginyl endopeptidase (AEP) is in the inactive proenzyme form. After 19 hours in the absence of LPS, immature DCs contain most (> 80%) of a model antigen (OVA, HEL or HRP) that they have accumulated initially : anyway BSA (half-time for digestion 5 min in macrophages) is readily degraded in immature DCs, suggesting that it is susceptible to lysosomal proteolysis even by the reduced proteolytic activity in immature DCs.
The name "dendritic" comes from the definition given in 1868 by German anatomist Paul Langerhans, who mistook long arms of Langerhans' cells for nerve endings in the skin. Normal blood concentration : 0.048÷0.064 ^. 10⁹ / weight kg ; 0.264÷0.904 ^. 10³ / mL blood ; 0.096÷0.128 % total blood cells ; 6÷8 % total WBCs.
Then ... :
• TNF family members
• CD40L / CD154
• TNF-a
• IFN-g
• TLRs ligand(s) (most drive T_h1 polarization) : TLR ligation transiently enhances the uptake of FITC–dextran by both mouse bone-marrow-derived DCs or spleen-derived DCs, peaking after 30 to 45 minutes of treatment with LPS. Co-administration of antigen with LPS markedly enhances the presentation of both MHC-class-I- and MHC-class-II-restricted antigen to T cells compared with administration of antigen before exposure to LPS. The accumulation of FITC-dextran after TLR ligation is associated with increased actin-dependent membrane-ruffling activity. In addition, there is TLR-, MEK1- and SAPK2-dependent destabilization in the F-actin-rich structures known as podosomes, which are involved in cell migration and tissue invasion, in most DCs after 30 minutes, although these structures were fully restored after 2 hours^ref.


TLR expression is variable and might identify subsets of innate immune cells, such as DCs, with specific functions^{ref1, ref2, ref3}. DC activation through TLRs results in increased production of pro-inflammatory cytokines and antimicrobial peptides, increased nitric oxide synthesis and enhanced bacterial killing, as well as increased antigen presentation^ref. The binding of TLR ligands is associated with the recruitment of intracellular adaptor proteins similar to those used by IL-1R and subsequent activation of the JUN N-terminal kinase (JNK) and NFkB signalling pathways^ref.
• ligation of human osteoclast-associated receptor (hOSCAR), a novel FcRg-associated receptor expressed by myeloid cells, by specific antibodies promotes dendritic cell (DC) survival by an ERK- and PI3K-dependent pathway, linked to expression of the Bcl-2 and Bcl-x_L antiapoptotic molecules. Crosslinking of hOSCAR leads to maturation of DCs, as demonstrated by up-regulation of maturation markers, decrease in dextran uptake capacity, and secretion of immune system effectors such as CXCL8 / IL-8, IL-12 p40, CCL2 / MCP-1 and CCL22 / MDC. Stimulation of hOSCAR acts in conjunction with the TLR ligands, LPS, R-848, and poly(I:C), to increase the expression of maturation markers, and to modulate cytokine release. A PI3K-dependent up-regulation of IL-10 release is observed with all the TLR ligands used, whereas regulation of IL-12 production is variable depending on the TLR stimulated. hOSCAR engagement on DCs did not significantly increase the proliferation of naive T cells; however, when co-incubated with TLR ligands, an enhanced proliferation was observed. The percentage of IFN-g-producing T cells is decreased when hOSCAR engagement is combined with LPS stimulation. Altogether, these data suggest that hOSCAR may modulate the responses of both innate resistance and adaptive immunity^ref. There is synergy hOSCAR and TLR ligands on both monocytes and neutrophils, with up to 8-fold increases in cytokine secretion when hOSCAR was cross-linked in the presence of LPS or R-848^ref.
• HMGB1^ref (a nuclear factor involved in chromatin organization recently identified as a cytokine; DCs matured by exposure to HMGB1 drive T_h1 polarization of activated resting allogeneic T cells; environments that are rich in HMGB1 for example, those containing necrotic cells — might facilitate the breaking of tolerance and lead to autoimmune responses)
• engagememt of vanilloid receptor 1 (VR1)^ref
... stimulate differentiation into :
activated mature dendritic cells  (mDC / M-DC / MDC / DC_mat) (CD1a^lo1b^hi1c^hi14^-32 / FcgRIIA⁺33⁺38⁺40^hi50 / ICAM-3⁺54 / ICAM-1⁺58 / LFA-3⁺64 / FcgRI⁺70⁺80 / B7-1^hi83⁺86 / B7-2^hi91 / LRP / a₂-macroglobulin R⁺205 / DEC-205^hi206 / MMR^lo207 / DC-SIGN^lo210 / IL-10Ra^hi, NKG2D-S^hi, NKG2D-L^-, ILT1⁺, MHC I⁺MHC II^hi, CCR7^hi, weak phagocytosis (=> can no longer acquire exogenous antigens) and are typically short lived^ref).

The Rho family GTPases Rac1 and Rac2 but not Rho itself control the formation of dendrites in mature DCs, their polarized short-range migration toward T cells, and T cell priming^ref.
Expression of receptor for inflammatory chemokines is lost (immature DC expressed CCR5 and migrated in response to CCL3 / MIP-1a), whereas expression of those for lymphoid chemokines, particularly CCR7 (respond exclusively to CCL19 and CCL21), CXCR4, and CCR4 is upregulated^{ref1, ref2}. CCR7 mediates traffic out of the tissue space and into draining lymph nodes through afferent lymphatics. CCR7-deficient DCs fail to migrate from the skin into lymphatics^ref, whereas DCs in plt/plt mice still gain access to lymphatics (which express the CCL21Leu isoform), but they fail to penetrate the cortex after they have arrived in the draining LNs^ref. Lymph vessels also express CCL19, but DCs require autocrine production of cysteinylated leukotrienes to respond to this chemokine^ref. Access to lymphatics probably requires (presumably chemokine induced) the activation of b₂ integrin on DCs, because DC entry is compromised in ICAM1-deficient lymphatics^ref. Changes include, for example, expression of "empty" class II MHC proteins at the surface in iDC, whereas a much larger amount of peptide-loaded MHC-II is expressed at the surface in mDC. In cultured iDC derived from murine bone-marrow precursors, a number of molecules involved in intracellular trafficking are present in a cleaved form, degraded by caspase-like proteases. Cleavage is either inhibited or reduced significantly during maturation of DC induced by either LPS and TNF-a or by peptides that inhibit caspase activities. iNOS up-regulated by LPS is essential for inhibiting the caspase-like activity during the maturation of DC. Moreover, treatment with LPS or caspase inhibitor results in expression of MHC-II/peptide complexes at the cell surface. Thus, the alteration of the endosomal trafficking pathways during the development of DC that parallels the changes in surface expression of MHCII is regulated at least in part by the activities of caspases, iNOS, and its product NO^ref.
Maturation allows DCs to enter lymph vessels and to access the T-cell area in draining LNs^ref. While in transit, DCs upregulate the expression of apparatus for antigen presentation and T-cell stimulation, and begin to generate :
• G-CSF
• GM-CSF
• MIF : tThe intraperitoneal injection of antigen into delayed hypersensitive animals or of preformed lymphokines into unimmunized animals can lead to changes in the distribution and content of inflammatory cells within the peritoneal cavity. If a preexisting macrophage-rich exudate is present (usually induced with paraffin oil 5 days before antigen challenge^ref), then these challenges will lead to a diminution of the number of macrophages recoverable (macrophage disappearance reaction (MDR) / reaction of macrophage disappearance (RMD) (a.k.a. tumor disappearance reaction^ref)). If the peritoneal cavity has been unprepared, then the same stimuli will lead to an increase in the number of macrophages (macrophage appearance reaction (MAR)). MDR is an in vivo analogue of in vitro tests for delayed hypersensitivity : it is mediated by MIF^ref and consists of both acute nonspecific inflammatory and specific DTH^ref in response to antigens (e.g. Coxsackie B3 virus^ref, bovine gamma globulin or human scrum albumin^ref, MDP^ref) injected in the peritoneal compartment^ref, to an extent able to suppress cutaneous DTH^ref. MDR is an in vivo manifestation of macrophage activation^ref. Hyaluronic acid accumulates in the peritoneal cavity of mice during the macrophage disapperance reaction. Macrophages reappear in the exudates during the depletion of hyaluronate. The disappearance reaction is simulated by the intraperitional (i.p.) injection of hyaluronic acid into normal mice. The disapperance reaction largely is inhibited by the simultaneous injection (i.p.) of specific hyaluronidase with the challenge dose of BCG^{ref1, ref2}. L-fucose can inhibit the ability of lymphokine-containing supernatants to induce skin reactions or cause reductions in the macrophage content of peritoneal exudates. Moreover, L-fucose can inhibit the cutaneous delayed hyoersensitivity reaction and the peritoneal macrophage disappearance reaction (MDR) induced by antigen in actively immunized guinea pigs. The effect of L-rhamnose, another sugar with in vitro inhibitory activity, was investigated in the MDR, and was also shown to be inhibitory. L-arabinose, which has no in vitro effects on lymphokines, had no suppressive effect on any of the in vivo systems studied. No monosaccharide inhibition of Arthus reactions or nonspecific inflammation could be found in these studies. The results demonstrate that monosaccharides capable of inhibiting lymphokine activity in vitro are effective in suppressing in vivo manifestations of cellular immunity^ref. Injection of a variety of irritants (saline, ovalbumin, compound 48/80 and powdered glass) into the rat pleural cavity induced the disappearance of pleural leucocytes during the first two hours of the reaction. This phenomenon, termed the leucocyte disappearance reaction (LDR), was suppressed by treatment with the anticoagulants heparin and warfarin. The in-vitro incubation of normal, or inflammatory pleural leucocytes resulted in the deposition of dense interconnecting meshwork of fibrin only upon addition of fibrinogen to the culture medium. It is suggested from these results that the LDR is related to the clotting system, involving leucocyte-derived enzyme(s) analogous to those of the clotting system (e.g., tissue thromboplastin), which convert fibrogen to fibrin, resulting in cell-trapping and subsequent "disappearance" of pleural leuococytes. Similarities were observed betweeen the LDR in non-immune inflammation and the macrophage disappearance reaction of cell-mediated immunity^ref.
• TNF-a
• IL-1a
• IL-1b
• IL-1RA
• IL-2
• IL-6 renders effector T cells unresponsive to suppression by T_Reg lymphocytes.
• IL-10, mildly upregulated by TLR signaling, strongly upregulated by Fcgs binding to FcgRI and/or FcgRIIA : it activates T_Reg lymphocytes and inhibits (autocrine signaling) production of pro-inflammatory cytokines such as TNF-a, IL-6 and IL-12.
• IL-12 p70, necessary for T_h1 polarization. Monocytes are licensed to synthesize IL-12p70 through type I IFN provided via the Toll/IL-1R domain-containing adaptor inducing IFN-b pathway and the inhibition of IL-10, both provided by combined stimulation with TLR4 and TLR8 ligands, triggering a potent T_h1 response before T cell help is established^ref.
• up-regulated by :
• priming signals
• GM-CSF
• IFN-g (T_h1 cytokine: positive feedback for T_h1 polarization !)
preceding a second signal (e.g. LPS)
• IL-4 (a T_h2 cytokine, via the IL-4R type I : negative feedback for T_h2 polarization ; IL-4R type II instead up-regulates MHC class II and costimulatory surface markers)
• low amount of receptor bound antigen
• down-regulated (shift from T_h1 toward T_h2 response) by :
• PGE₂
• IL-3
• IL-10
• signals coming before the priming signal
• IFN-b
• CD154 / 40L / gp39 interaction
• TRANCE / OPG-L / RANK-L interactions
• high amount of receptor bound antigen
• IL-18 (essential for antiviral immunity)
• IL-23
• up-regulated by :
• IFN-g
• down-regulated by :
• IL-4
• IFN-a (essential for antiviral immunity)
• IFN-b (essential for antiviral immunity)
• chemokines that allow them to attract T cells : the high production of CCR4 ligands by activated DCs suggests their capacity to selectively recruit at sites of inflammation T_Reg lymphocytes or T_h2 lymphocytes
• CCL2 / MCP-1
• CCL3 / MIP-1a
• CCL4
• CCL17 / TARC attracts naive T lymphocytes.
• CCL18 / PARC preferentially attracts naive T cells^{ref1, ref2}
• CCL19^ref
• CCL22^ref
• CXCL1 / Gro-a
• CXCL8 / IL-8
• TXA₂, which might increease chance encounters with T cells by enhancing their random motility^ref
Evidence is provided that macrophages can make M-1 or M-2 responses. The concept of M-1/M-2 fomented from observations that macrophages from prototypical T_h1 strains (C57BL/6, B10D2) are more easily activated to produce NO with either IFN-g or LPS than macrophages from T_h2 strains (BALB/c, DBA/2). In marked contrast, LPS stimulates T_h2, but not T_h1, macrophages to increase arginine metabolism to ornithine. Thus, M-1/M-2 does not simply describe activated or unactivated macrophages, but cells expressing distinct metabolic programs. Because NO inhibits cell division, while ornithine can stimulate cell division (via polyamines), these results also indicate that M-1 and M-2 responses can influence inflammatory reactions in opposite ways. Macrophage TGF-b₁, which inhibits inducible NO synthase and stimulates arginase, appears to play an important role in regulating the balance between M-1 and M-2. M-1/M-2 phenotypes are independent of T or B lymphocytes because C57BL/6 and BALB/c NUDE or SCID macrophages also exhibit M-1/M-2. Indeed, M-1/M-2 proclivities are magnified in NUDE and SCID mice. Finally, C57BL/6 SCID macrophages cause CB6F1 lymphocytes to increase IFN-g production, while BALB/c SCID macrophages increase TGF-ß production. Together, the results indicate that M-1- or M-2-dominant macrophage responses can influence whether T_h1/T_h2 or other types of inflammatory responses occur^ref
Functional classes of macrophages (Mp) :
• reductive macrophages (RMp) with a high intracellular content of glutathione
• oxidative macrophages (OMp) with a reduced content
The T_h1/T_h2 balance is regulated by the balance between RMp and OMp due to the disparate production of IL-12 versus IL-6 and IL-10. RMp were induced by in vivo application of N-acetyl-L-cysteine or glutathione monoethylester and OMp by L-cystine derivatives, diethyl maleate or L-buthionine-[S,R]-sulfoximine. The Mp arbitrarily called OMp showed elevated IL-6 and IL-10 production, and reduced NO and IL-12 production. The RMp elicited a reciprocal response, i.e. elevated IL-12 and NO production, and reduced IL-6 and IL-10 production. The cytokine propensities of OMp or RMp were inter-converted to each other. The results were also confirmed by using auto-MACS purifiedF4/80⁺ Mp without adherence. Interestingly, IFN- induced RMp and augmented NO generation with decreased production of IL-6, whilst IL-4 induced OMp and augmented IL-6 production. CD4⁺CD44^- naive T_h0 cells were differentiated preferentially either to T_h1 or T_h2 cells, depending on the presence of RMp or OMp during the initial 24 h of culture, from ovalbumin-specific TcR-transgenic mouse spleen cells in the presence of IL-2. Taken together, RMp induction may generate the amplification loop of a RMp/T_h1 circuit and OMp that of OMp/T_h2. The findings implicate that the alteration in Mp functions because altered intracellular glutathione may play a relevant role in the pathological progression of inflammation^ref.

As they arrive in the paracortical regions of the draining lymph nodes, DCs upregulate their expression of co-stimulatory molecules, and peptide-loaded MHC class II molecules transport from the intravesicular processing compartment to the cell surface. Maturation induces a more efficient recruitment of preexisting V1 sectors of vacuolar proteon pump (V-ATPase) onto lysosomal membranes : the 37 and 70 kDa subunits of the V1 sector are enriched (80%) on the membrane fraction of mature DCs versus immature DCs (45%). Enhanced lysosomal acidification (pH = 4.5-4.6) provides a pH environment closer to the optimal pH of H-2M activity and most lysosomal enzymes (including cathepsin L, legumain (LGMN) / asparaginyl endopeptidase (AEP), and gamma-interferon-inducible lysosomal thiol reductase (GILT)), whose activation increases efficient formation of peptide-MHC class II complexes (pMHC). Mature DCs cultured in the presence of LPS contain 10% of a model antigen (OVA, HEL or HRP) initially internalized. Low proteolytic activity in immature DCs would allow a substantial fraction of the internalized antigen to escape defgradation inlysosomes, facilitating efficient conversion into pMHC after DC maturation is induced : in fact they can present peptides from antigens internalized hours or even days before maturation. Trafficking of peptide-MHC complexes is increased by development of tubules that extend from late endosomes or lysosomes towards the plasma membrane.
DCs position themselves close to the high endothelial venules (HEVs), at the site of T-cell entry into the LN to maximize the chance of an immunogenic CD4⁺ T lymphocytes-DC interaction occurring : only antigen-specific T lymphocytes are retained in the proximity of HEVs. Throughout the twentieth century, the migratory behaviour of lymphocytes and DCs in the LN parenchyma has been inferred from increasingly sophisticated examination of histological sections. These studies indicated that T cells were initially activated in the T-cell area, especially the superficial paracortex, whereas the antigen-specific B cells were scattered throughout the B cell follicles. Later on, CD4⁺ T cells localized to the follicle edges where they provided help to antigen-specific B cells and promoted the formation of germinal centres^{ref1, ref2}. A limitation of histology-based techniques is that they provide snapshots of cellular localization, but cannot resolve the true dynamics of interstitial cell migration. Ongoing technological innovations, in particular multi-photon microscopy, are revolutionizing our ability to track individual migrating cells in living tissue^{ref1, ref2}. Strategies to visualize lymphocyte and DC migration in mouse LNs were recently described^{ref1, ref2}. In experiments by Stoll et al^ref, fluorescent-labelled TcR-transgenic lymphocytes were injected intravenously and antigen-pulsed DCs labelled with a different fluorophore were injected in into the skin. The draining LNs were then removed and maintained in a culture dish in normal air while they were visualized by confocal microscopy at an imaging depth of up to 80 mm below the surface^ref. Within a day, the tagged DCs and lymphocytes had homed to LNs through afferent lymphatics and HEVs, respectively. In this study, naive T cells were virtually stationary and migrated relatively slowly (5-7 mm/min) after prolonged exposure to antigen. In the presence of antigen, T cells and DCs formed stable clusters at 1:1 ratio for over 15 hours^ref. By contrast, in a parallel study by Miller et al.^ref, removed LNs were maintained in a 95% oxygen environment and lymphocytes were visualized up to 350 mm below the surface using multi-photon microscopy. These authors found that T cells migrate faster, on average, than follicular B cells (10.8 mm/min versus 6.4 mm/min). Both subsets migrated in random directions, a behaviour that is inconsistent with the idea that lymphocyte migration is governed over long distances by chemotactic gradients. Although random migration was also observed by Stoll et al., the T cells observed observed by Miller et al. were more dynamic. Moreover, although DCs were not directly visualized by Miller et al. the kinetics and geometry of T-cell migration in response to antigen indicated that ech DC might interact with many T cells and that only a minority of T-cell-DC interactions are long-lived. This idea was recently substantiated in another multi-photon study of excised LNs by Bousso and Robey who observed that a single DC might contact as many as 500 different T cells per hour in the absence of antigen and engage more than 10 T cells simultaneously. The duration of these interactions is of the order of hours, not minutes. The overall avidity of the interaction influences the probability that T cells will be stably captured by DCs, providing a possible basis for T cell competition^ref. The discrepancies between the studies by Stoll et al^ref and Miller et al^ref could have several reasons, including the difference in imaging depth or tissue oxygenation^ref. Cardiovascular arrest is followed within minutes by a marked loss of lymphocyte motility in the LNs of anaesthetized mice. Given that oxygen diffusion through metabolically active tissues is only sufficient to a distance of beyond 100 mm^ref, LNs that are removed and cultured in ambient air will probably have hypoxic regions in which cellular motility might be compromised. Hyperoxic culture conditions might alleviate this problem^{ref1, ref2}. However, supraphysiological oxygen concentrations enhance oxidative stress in living tissues and by shifting redox balances might alter lymphocyte behaviour^{ref1, ref2}. So, it remains to be seen whether studies of excised LNs can faithfully reproduce the multi-faceted events that occur in vivo. Moreover the LN environment is modulated by afferent lymph flow, and LN homeostasis is also influenced by the autonomous nervous system^ref. Clearly, the definitive experiments will be intravital observations of LNs of live animals. The first report of multi-photon intravital microscopy in surgically exposed mouse inguinal LNs has been published recently^ref. This study found that naive CD4⁺ T cells migrate in vivo with high speed and random directionality^ref. So, it was proposed that naive T cells are preprogrammed to scan the T-cell areas for the presence of antigen-presenting DCs randomly^ref. The study also visualized T cells immediately after they entered the LNs across HEVs. The homed cells migrated rapidly away from the entry site in random directions and without apparent delay. This is somewhat surprising given that HEVs are surrounded by an elaborate system of channels and corridors^ref. It had been proposed that emigrating lymphocytes might be delayed during their passage through perivenular channels and that homed cells would initially move in a spiralling motion around HEVs, following preformed corridors^ref. Such an orchestrated migratory behaviour was not detected in vivo^ref. Observations in explanted LNs indicate that B cells, too, have a similar random walk, at least in follicles^ref. However, it is probable that directed migration occurs in LNs in other situations, for example when newly homed B cells traverse the T-cell area to access follicles. This process is mediated by CXCL13 - a chemokine that is generated in B-cell follicles and stimulates CXCR5^{ref1, ref2}. Most T cells do not express CXCR5, but when CD4⁺ T cells encounter antigen they transiently upregulate the expression of this receptor and down-regulate the expression of CCR7, which allows them to disengage from the T-cell area and migrate to the B-cell follicles^ref. As CXCR5-expressing CD4⁺ T cells express prerequisite cell-surface molecules for B-cell help, the term follicular B helper T (T_FH) cell has been proposed for them^ref : they are characterized by high expression of the costimulatory molecule ICOS, but not CD57 expression. CXCR5^hiICOS^hi CD4 T cells are the most potent inducers of IgG production that also secrete large amounts of the B cell-attracting chemokine CXCL13. CXCR5^hiICOS^hi CD4 T cells differ from other tonsillar CD4 T cell subsets in their stimulatory activity, proliferative capacity and susceptibility to apoptosis. Large-scale gene expression analysis revealed that T_FH cells are only distantly related to CXCR5^- and CXCR5⁺ central memory T (T_CM) as well as effector memory T (T_EM) cells present in the periphery. CXCR5^hiICOS^hi CD4 T cells appear to be terminally differentiated T_h cells that express a unique set of transcription factors related to the Notch signaling pathway and thus differentiate independent of other T_h cell populations^ref. Unlike other effector T cells, a significant number of CD4⁺45RO⁺57⁺ T cells, which are the major T_h cells residing in the GCs, undergo apoptosis in vivo. CD4⁺45RO⁺57⁺ GC T cells exhibit similar sensitivities to apoptotic signals and to caspase inhibitors as immature thymocytes. Moreover, CD4⁺45RO⁺57⁺ GC T cells express a unique profile of genes that control apoptosis and cell cycle, providing possible molecular mechanisms for the high rates of apoptotic death of these T_h cells in the GCs^ref.
2-photon microscopy has been used to study T-cell-DC interactions in vivo in the intact popliteal lymph nodes (PLN) of anesthetized mice, in which blood and lymph flow are maintained^ref : mice are injected with fluorescently labelled DCs into the footpad, and by the following day, DCs have accumulated in the PLNs. LPS is co-administered with DCs to induce the expression of CCR7 by the DCs, which allows them to home to lymph nodes. 18 hours after DC injection, TcR-transgenic CD4⁺ or CD8⁺ T cells are injected i.v.. 2 hours after injection, the transgenic T cells have migrated to the PLNs and constituted 1-2% of T cells. Many of the DCs localize around HEVs. Analysis of the T-cell dynamics reveals that T cells interact with DCs in 3 distinct phases :
• phase 1 (covering the first 8 hours after T-cell entry to the PLNs) : T cell formed brief contacts with DCs that last < 30', resulting in increased expression of early activation markers by the T cells
• phase 2 (covers the 8-24 hours period after T-cell transfer) : T cells form stable contacts with DCs that last > 1 hr
• phase 3 (starts after 24 hours) : T cells dissociate from DCs, and undergo rapid migration and proliferation
Different DC populations can have preferential stimulatory or suppressive functions. The rate of DC migration into LNs might have a role in deciding whether and in what way a T cell responds to a DC that express its cognate antigen. Local pathogen-induced inflammation usually attracts large numbers of monocytes and/or immature DCs to tissues. These newly recruited cells differentiate into mature DCs and, together with activated resident DCs, migrate to the draining LNs, allowing for strong and sustained T-cell stimulation. By contrast, without inflammation DCs with weak stimulatory capacity migrate to LNs in numbers too low to drive T-cell activation. The resulting sub-threshold stimulation of autoreactive T-cells could promote and sustain T-cell tolerance to self-antigen^{ref1, ref2}. At 15–20 h after the induction of immunity, T cells stopped moving and established prolonged interactions with DCs. In tolerogenic conditions, despite effective initial T cell activation and proliferation, naive T cells remained motile and established serial brief contacts with multiple DCs. Thus, stable DC–T cell interactions occur during the induction of priming, whereas brief contacts may contribute to the induction of T cell tolerance^ref. Most of the steady-state DCs were enmeshed in an extensive network and remained in place while actively probing adjacent T cells with their processes. Mature DCs were more motile than steady-state DCs and were rapidly dispersed and integrated into the sessile network, facilitating their interaction with migrating T cells^ref.
Knockdown of the Gb2 subunit in the mouse macrophage-cell line J744A.1 abolished migration towards C3a and C5a, whereas cells lacking other Gb or Ga subunits migrated normally. Gb2 knockdown diminishes the C5a-induced Ca²⁺ signal and eliminates sustained Akt phosphorylation, providing evidence that PL-Cb and Akt are downstream effectors of this signalling pathway. However, in the absence of Gb2, levels of both Gg2 and Gg5 are down-regulted, indicating that fujrether studies are required to define the precise molecular mechanisms by which Gb2 regulates C5a-induced chemotaxis^ref.
Killing may occur through induction of apoptosis or phagocytosis followed by oxidative burst.
‡
+ inflammatory stimuli
• epitheloid cell
• or fuse each other to create giant cell (e.g. Langhans cellduring infection with Mycobacterium tuberculosis, ...)

(reproduced with permission from Nature Reviews Immunology (Vol 4, No. 6, pp 432-444 (2004)) Macmillan Magazines Ltd)


(reproduced with permission from Nature Reviews Immunology (Vol 4, No. 6, pp 432-444 (2004)) Macmillan Magazines Ltd)
Types of activated dendritic cells :
• dendritic cell 1 (DC1) / myeloid dendritic cell (MDC / mDC) (TLR1⁺⁺2⁺⁺3^-4⁺⁺5⁺⁺6⁺7^-8⁺⁺9^-10^-)
• myeloid dendritic cell 1 (MDC1 / mDC1) (CD1a⁺8a^-11b^lo11c^hi13⁺14⁺25^hi32A / FcgRII⁺33⁺35 / CR1⁺36 / thrombospondin receptor / p95^{gpIV / gpIIIb+}40^hi43^hi46⁺50 / ICAM-3⁺54 / ICAM-1⁺58 / LFA-3⁺68⁺80 / B7-1^hi83^hi86 / B7-2^hi88 / C5aR⁺89⁺91 / LRP / a₂-macroglobulin R⁺205 / DEC-205^hi206 / MMR^lo207 / DC-SIGN^lo210 / ILR10a^lo, NKG2D-S^-, NKG2D-L^-, MHC I⁺MHC II^hi, CCR7⁺, NKG2D^-, TRANCER^hi, Bcl-2^lo, weak phagocytosis, myeloid-related proteins (MRPs) S100A8, S100A9, and S100A12 (Ca²⁺-binding proteins that belong to the S100 protein family : S100A8 and S100A9 associate noncovalently to form homodimers and the heterodimer S100A8/A9)). Most of them express HLA-DP and HLA-DR, but not HLA-DQ class II molecules. They are found in lymphoid tissues where induce T_h1 polarization and produce self-tolerance by killing or anergizing T cells or by stimulating T_Reg lymphocytes. At later stages of the immune response they are killed by T_s lymphocytes. Since macrophages do not generally divide, they are radioresistant and can carry out some essential functions following irradiation.
• mature interstitial DC (iDC) / dermal DC (CD1a^-14^-) : they migrate from the skin to draining lymph nodes in a MMP-2 / gelatinase A- and MMP-9 / gelatinase B-dependent manner. Their development is impaired by corticosteroids.

 
• mature activated Langerhans dendritic cell (DC)
• "veiled cells" in lymph (CD83⁺CCR7^hi) : they enter lymph nodes reaching paracortex, where they lose Birbeck granules and ...

‡
+ TGF-b :
• interdigitated dendritic cell / Langerhans cell-derived dendritic cell (LCDC) (CD83⁺86 / B7-2⁺) in the paracortex of lymph nodes
• myeloid dendritic cell 2 (MDC2 / mDC2) (CD1a^-8a^-11c^hi40^hi80 / B7-1^hi86 / B7-2^hiMHC II^hi) induce T_h2 polarization.
Mature myeloid DCs express CCR7 and CD11aCD18 / LFA-1 / a_Lb₂ integrin, but not p90-110^{L-selectin / CD62L / MEL-14 / LAM-1 / LECAM-1 / Leu-8}, so they fail to roll in HEVs and cannot home to LNs. BDCA-3 is expressed on small population of CD11c⁺ CD123^- dendritic cells. All three Ags are not detectable on a third blood dendritic cell population, which is CD1c⁺ CD11c^bright CD123^dim, or on any other cells in blood. BDCA-4 is expressed on monocyte-derived and CD34⁺ cell-derived dendritic cells. Upon in vitro maturation, expression of BDCA-3 is up-regulated on both plasmacytoid CD11c^- CD123^bright DCs and CD1c⁺ CD11c^bright CD123^dim dendritic cells, and expression of BDCA-4 is up-regulated on CD1c⁺ CD11c^bright CD123^dim DCs^ref.
• dendritic cell 2 (DC2) / plasmacytoid DC (PDC / P-DC) / IFN-producing cell (IPC) (CD4^lo8a^-11b^{- / dull}11c^-/lo13^-33^-40^high45RA^lo62L / L-selectin^hi80^high86^high117^-123 / IL-3Ra^high/bright205 / DEC-205⁺, MHC II^hi, BDCA-2^-, BDCA-3⁺, BDCA-4^+ref, MPO^-, CXCR3⁺, CXCR4⁺, CCR5⁺, NKG2D^-, TLR1⁺2^-3^-4^-5^-6⁺7⁺⁺8^-9⁺⁺10^+/-) :

Upon stimulation with pathogenic stimuli (IL-3 and CD154 / 40L / gp39-containing medium^{ref1, ref2, ref3, ref4}), IPC up-regulate CCR7 and migrate from the blood to inflamed lymph nodes through HEV. They produce :
• very high levels of type I IFNs in response to certain viruses : they drive differentiation of B cells into plasmablasts. Secretion depends on osteopontin^ref
• CCL3
• CCL4
• CXCL8 / IL-8
• no IL-6
• no IL-12
The preferential secretion of CCR5 ligands suggest their capacity to selectively recruit at sites of inflammation effector T cells, and, in particular, T_h1 lymphocytes (e.g. when activated by influenza virus or CpG motifs^ref). Anyway they can also induce T_h2-type responses (e.g. after CD40L/IL-3 stimulation^ref) or generate T_Reg lymphocytes. As T lymphocytes respond by secreting IL-2 and expressing CD40L / CD154, pDCs are then stimulated to secrete IL-6, which promotes the transition of plasmablasts into plasma cells. Influenza virus or TLR agonists induce TRAIL-mediated cytotoxic activity of PDCs^ref.
• TNF-a/iNOS-producing DCs (Tip-DCs) (CD11b^mid11c^midCCR2^hi) are esssential for host defence against bacterial infections and are probably recruited to the spleen by CCR2 signalling.
Virus infection induces bone marrow pDCs to differentiate into mDCs, thereby undergoing profound phenotypic and functional changes including the acquisition of enhanced antigen-presenting capacity and the ability to recognize different microbial structures through TLR4. The conversion of pDCs into mDCs is also induced by the injection of dsRNA and requires type I interferons^ref.
... or : previous studies had shown that once mature DCs complete their journey from the peripheral tissues to the secondary lymphoid organs, they succumb to activation-induced apoptosis through agonists of opiate receptors, but new evidences suggest that mature DCs can differentiate (in vitro and in vivo) further under the influence of the secondary lymphoid microenvironment ... :
• mimicked by ...
• direct fibronectin-dependent contact with fixed endothelial-like splenic stromal cells (ESSCs), required for both proliferation and differentiation
• ESSC–derived TGF-b , required for differentiation
• IL-10
• TGF-b₁
• HGF^ref
into ... :
• regulatory DC (diffDCs) (CD40^hi80^hiMHCII^lo): proliferate in a fibronectin-dependent way and secrete ...
• more T_h2-biased cytokine profile : IL-10 and NO (which mediates the suppression of T cell proliferation in response to antigen presentation by mature DCs)
• less TGF-b₁ and IL-12 than mature DCs.
Ag-pulsed regulatory DCs do not promote proliferation of Ag-specific CD4⁺ T lymphocytes and can inhibit T-lymphocyte proliferation induced by Ag-pulsed mature DCs. The increased production of NO by ESSC-differentiated DCs directly inhibits T-cell proliferation, without the generation of regulatory T cells that has been shown for other DC subtypes^ref. Upon activation with HGF-DCs, allogeneic CD4⁺CD25^- T cells expressed the T_reg-associated transcription factor FoxP3, proliferated poorly and released high levels of IL-10^ref. Unlike immature DCs, diffDCs secrete more IL-10 but little IL-12p70 in response to LPS or other TLR agonists. Upregulation of ERK1/2 activation was shown to be responsible for IL-10 preferential production, and suppression of p38 activation was for impaired IL-12p70 production in diffDCs. Interestingly, LPS treatment could not reverse the inhibitory effect of diffDCs on the proliferation of antigen-specific CD4⁺ T cells. However, diffDCs could activate NK cells through diffDCs-derived IL-10, and even more markedly after TLR agonists stimulation. These diffDCs-activated NK cells could in turn kill surrounding diffDCs^ref.



DCs carry antigen from peripheral tissues via lymphatics to lymph nodes : differentiated DCs can also travel from the periphery into the blood. Circulating DCs migrated to the spleen, liver and lung but not lymph nodes. They also homed to the bone marrow, where they were retained better than in most other tissues. Homing of DCs to the bone marrow depended on constitutively expressed VCAM1 and endothelial selectins in bone marrow microvessels. 2-photon intravital microscopy in bone marrow cavities showed that DCs formed stable antigen-dependent contacts with bone marrow-resident central memory T cells. Moreover, using this previously unknown migratory pathway, antigen-pulsed DCs were able to trigger central memory T cell-mediated recall responses in the bone marrow^ref.
Web resources :

• European Macrophage Society (EMS)
• The Macrophage Homepage by Siamon Gordon
• activation of NKT lymphocytes: NKT cells use a unique activation mechanism not requiring their recognition of microbial antigens. Weak responses to CD1d-presented self antigens are amplified by IL-12 made by activated DCs in response to microbial products, resulting in potent IFN-g secretion. NKT cells are among the first lymphocytes to respond during Salmonella typhimurium infection, and their activation in vivo also depends on IL-12 and CD1d recognition.

Necessary co-stimulatory signals for such activation are :
• CD28 --- CD80 / B7.1 & CD86 / B.7.2
• CD40 --- CD40L
• low doses of IL-12
• T-bet^ref
• [in experimental studies : anti-CD3 mAbs]
Only 5-15 cells per clone are created upon stimulation due to rapid activation-induced cell-death (AICD). Most of the adult population is non-responsive and non-divinding. Within 90' of TcR stimulation in vivo they produce large quantities of :
• IL-4 that induces DCs to produce IL-12 , which in turn activates NK cells
• IFN-g, that activates NK cells
• directly
• indirectly, by inducing DCs to produce IL-12
• CCL5
Upon maturation the cytokine secretion profile changes from a T_h2 to a T_h1 pattern.
• activation of granulocytes : they express p90-110^{L-selectin / CD62L / MEL-14 / LAM-1 / LECAM-1 / Leu-8} and CD11aCD18 / LFA-1 / a_Lb₂ integrin, but not CCR7 or CXCR4, so they roll but fail to arrest and cannot home to LNs. Csk negatively regulates granulocyte activation, modulating cellular adhesion and preventing inappropriate granulocyte recruitment^ref
• activation of neutrophils: the eicosanoid hepoxilin A₃ (hepA₃) (enriched on the apical side of epithelial-cell monolayers after infection) promotes the migration of neutrophils across epithelial-cell barriers at sites of mucosal inflammation, but do not cause degranulation of^ref.
• bacteria are phagocytosed and fusion of phagocytic vacuoles with neutrophil granules exposes the bacteria to a collection of antimicrobial reagents, including lytic enzymes, cationic peptides that disrupt bacterial membranes and reactive oxygen species (ROS)
• neutrophils can also kill bacteria extracellularly by concentrating antimicrobial reagents in a fibrous extracellular network termed neutrophil extracellular traps (NETs), released by the neutrophil as early as 10 minutes after activation with IL-8, PMA or LPS, detected with high-resolution scanning electron microscopy (SEM), composed of DNA, histones and granule enzymes, such as neutrophil elastase, able to trap both Gram-positive and Gram-negative bacteria. Blocking the function of neutrophil proteases showed that they were required for the deactivation of bacterial virulence factors. NETs are also observed in vivo in samples taken from shigellosis infection of rabbits and appendicitis in humans. NETs amnplify the activity of antimicrobial components by concentrating them in the fibrous network, which should also reduce exposure of host tissue to these components. It remains to be determined whether NETs are formed by an active process or are an early stage in neutrophil apoptosis^ref.
• activation of eosinophils

+ :
• IL-3
• IL-5
• GM-CSF
• EAF
• ECF-A (up-regulates CR1)
• histamine
• TNF-a
Upon activation they express FceRI.
They migrate into tissues thanks to chemokines :
• CCL11 / eotaxin 1
• CCL24 / eotaxin 2
They produce :
• positive feedback cytokines :
• IL-3
• IL-5
• GM-CSF : Pin1, a cis-trans isomerase, is an essential component of the ribonucleoprotein complex responsible for GM-CSF mRNA stabilization, cytokine secretion and the survival of activated eosinophils. Pin1 regulates the association of the AU-rich element–binding proteins AUF1 and hnRNP C with GM-CSF mRNA, accelerating or slowing decay, respectively^ref
• IL-6
• IL-8
• IL-13
• activation of basophils

+
• IgE crosslinking
They up-regulate CD40L expression and produce IL-4 and basophil kallikrein of anaphylaxis (BK-A). Basophils degeneration produces Charcot-Leyden crystals, consisting of eosinophil lysophospholipase related to galectin family of b-galactoside binding proteins.
• activation of mast cells

+
• IgE crosslinking (not by LPS) : high levels of IgE bound to multivalent antigen [IgE(+Ag)] crosslinks FceRI and results through the common g-subunit (FcRg) in less prolonged Erk activation, followed by degranulation in the absence of cell survival ; the threshold for survival induced by IgE in the absence of antigen [IgE(-Ag)]) without degranulation is lower than that for degranulation and involves more prolonged ERk activation^ref
They undergo degranulation, up-regulate CD154 / 40L / gp39 expression and produce :
• LTB₄
• LTC₄
• PGD₂
• PAF
• T_h2-associated cytokines^{ref1, ref2}
• IL-1b
• IL-2
• IL-3
• IL-4
• IL-5
• IL-6
• IL-8 / CXCL8 / NCF
• IL-9
• IL-10
• IL-13
• IL-16
• TNF-a, whose level in the draining lymph nodes increases markedly 3 hours after infection (or mast-cell activation with 48/80) is the main mediator of the 3-fold increase in the number of circulating T cells recruited to the lymph nodes, leading to remote nodal hypertrophy, whereas tryptase and histamine have no effect^ref : draining lymph nodes of mast cell–deficient mice do not demonstrate swelling after intradermal bacterial challenge
• TGF-b
• GM-CSF
RabGEF1 is a negative regulator of cytokine and lipid-mediator production on FceRI aggregation, and a lack of RabGEF1 results in the development of skin inflammation in vivo^ref
• activation of naive T cells : naive T cells (CD45 / B220 / LCARA⁺w197 / CCR7⁺TLR3⁺9⁺) recirculate continuously from blood to lymph through specialized T cell zones (TCZ) in the secondary lymphoid compartment, attracted by CCL4 / MIP-1b. Enough lymphocytes recirculate from lymph to blood to replace the total blood lymphocyte pool from 10 to 48 folds every 24 hours. On average naive lymphocytes spend less than a half hour in the circulation before homing to another lymphoid organ^ref. The limited recirculation of T lymphocytes pattern means that many pathogens enter the body at sites where they will not directly encounter naive T cells : naive T cells can scan the entire body for the presence of pathogens simply by scanning antigens presented on DCs that migrate to the secondary lymphoid compartment. Primary T cell responses are initiated in secondary lymphoid organs by mature DCs. Recognition of 300 immunogenic peptide-MHC complexes (pMHC) on the surface of a DC in the T cell zone causes activation and sequestration ("trapping") of antigen-specific recirculating T cells entering lymphoid tissue from the blood^ref; the trapped cells are then induced to proliferate leading to > 1000-fold expansion of the responding cells within a few days^ref. As well as transporting antigen, DCs express co-stimulatory molecules that allow them to activate naive T-cells. Most of them fall broadly into 3 families : the B7 family, the TNF family, and cytokines (which are important in determining the direction of T lymphocyte differentiation). The TNF receptor family members are upregulated on T-cell activation, indicating that the role of this family might be to amplify responses once initial T-cell activation has occurred. Because lymphocytes continually enter and leave normal LNs, the resident lymphocyte pool is composed of non-synchronized cells with different dwell times that display heterogeneous behaviour in mouse LNs in vitro. In vivo 2-photon fluorescence light microscopy can be used to study antigen-presenting DCs and naive T cells whose dwell time in LNs has been synchronized. T-cell priming by DCs occurs in 3 successive stages^ref :
• transient serial encounters during the first activation phase (first 8 h after entering from the blood)
• long-lasting stable contacts culminating in IL-2 and IFN-g production and upregulation of activation markers (h 8-20)
• high motility and migration, onset of rapid proliferation, and short DC contacts (day 2)
Immature immunological synapse forms within 5 to 15 minutes after contact with an APC in naive CD4⁺ T lymphocytes and evolves to mature IS within 30 minutes, but by 60 to 90 minutes, the whole synapse has dissipated^ref
T lymphocyte --- APC signals can be subdivided into :
• obliged activation signals (if absent => clonal anergy; if present without TcR-MHC binding => no T-cell activation occurs) :
• CD28 (homodimer) --- B7
• CD80 / B7-1 and CD86 / B7-2 / B7.2 : expression on APCs is induced by IL-18 and inhibited by PGE₂ through EP₂ and EP₄ receptors).
• B7 homolog 4 (B7-H4) / B7X / B7S1
CD28 costimulatory signals promote cell division independent of its effects on the initial TcR ligation-induced G₀ => G₁ transition. The interaction stimulates IL-2 production thanks to IkBb inactivation, that allows increase of CD137 / 4-1BB expression. IL-2 concentration has been shown affect T cell doubling time and the difference in output typically seen upon adding CD28 signaling to TcR stimulation can be readily explained by a modest change in cycle time, iterated over many cell divisions. No individual T cell requires 2 signals to divide or to produce IL-2 at levels necessary for cell division : rather, the probability that many cells will divide more often is increased by costimulation as a result of a minor increase in IL-2 production. T cell-APC interaction can cause APC-derived surface molecules to adhere to the surface of T cells and to be internalized through TcR endocytosis : so CD80 molecules are physically acquired by naive as well as memory T cells from APCs shortly after T cell activation and this acquisition plays a role in enhancing the proliferation of bystander T cells and can thus, in essence, themselves act as APCs ! This interaction results in up-regulation of CD154 / 40L / gp39 on T cells : the T cells may then engage CD40 on DCs and trigger a burst of cytokine expression. When antigen-presenting cells (APCs) encounter inflammatory stimuli, they up-regulate their expression of B7. Constitutively expressed B7 on APCs function to suppress T cell activation and maintain self-tolerance, principally by sustaining a population of regulatory T cells : responses are much greater in the absence of basal B7 expression.
Bidirectional signaling along the B7–CD28 coreceptor pathway enables reciprocal conditioning of T cells and DC : mouse dendritic cells are induced by soluble CD28 to express IL-6 and IFN-g. Production of IL-6 requires B7-1 (CD80), B7-2 (CD86) and p38^MAPK and prevents IFN-g-driven expression of immunosuppressive tryptophan catabolism. In vivo, an adjuvant activity of soluble CD28 is demonstrated as enhanced T cell-mediated immunity to tumor and self peptides and protection against microbial and tumor challenge^ref.
• neuropilin-1 --- neuropilin-1 (homophilic interaction)
• CD153 / 30L (activated T cells, monocytes, B cells, neutrophils, eosinophils) --- CD30 / Ki-1 Ag (APCs) => induces IL-13 production in a TRAF/p38^MAPK1 -dependent mechanism
• CD50 / ICAM-3 --- CD209 / DC-SIGN (APCs)
• CD81 / TAPA-1 --- ?  (APCs)
• facultative activation signals :
• p50^{CD2 / LFA-2 / T11 / E-rosette receptor} --- CD58 / LFA-3 (endothelial cells)
• CD11a-18 / LFA-1 / a_Lb₂ integrin --- CD54 / ICAM-1 (induced by IL-18 ; inhibited by PGE₂ through EP₂ and EP₄ receptors)
• inducible T-cell co-stimulator (ICOS) --- B7 homolog 2 (B7-H2) / B7-related protein 1 (B7RP-1) / GL50 / ICOS ligand (ICOSL / ICOS-L / ICOSLG / LICOS)
• ? ---
• PD-L1 / B7-H1
• PD-L2 / B7-DC
• ? --- B7 homolog 3 (B7-H3)
• CD27 --- CD27L
• CD134 / OX40 --- CD134L / OX40L / gp34
• TNFSF9 / CD137L / 4-1BBL --- CD137 / 4-1BB
• CD150 / signalling lymphocyte activation molecule (SLAM) functions as a co-receptor for signalling through the TcR via SLAM-associated protein (SAP)^ref
• stimulation with TLR3 or TLR9 agonists increased the survival (via upregulation of anti-apoptotic molecule BCL-X_L) without any change in proliferation via both MyD88-dependent and -independent pathways of NFkB activation^ref. Therefore, TLR signalling in T cells might improve memory responses by promoting the survival of T cells after primary stimulation. The ability of T cells to respond directly to PAMPs, without using DCs as an intermediary, might be one way in which we have evolved to deal with pathogens that try to evade the immune system by inhibiting DC function.
• signals mediated by chemokine receptors may compete with T cell receptor stop signals and determine the duration of T cell-APC interactions. During T cell stimulation by APCs, T cell chemokine receptors coupled to G_q and/or G₁₁ protein are recruited to the immunological synapse by a G_i-independent mechanism. When chemokine receptors are sequestered at the immunological synapse, T cells become insensitive to chemotactic gradients, form more stable conjugates and finally respond with enhanced proliferation and cytokine production. Chemokine receptor trapping at the immunological synapse enhances T cell activation by improving T cell-APC attraction and impeding the 'distraction' of successfully engaged T cells by other chemokine sources^ref.
• inhibitory signals
• HVEM --- LIGHT^ref

[HHV-1 / HSV-1 envelope glycoprotein D binds both LIGHT and BTLA, potentially nullifying the HVEM pathway. On the contrary HHV-5 / CMV UL144 binds BTLA, but not LIGHT, and inhibits T cell proliferation, selectively mimicking the inhibitory cosignaling function of HVEM^ref]
• programmed death 1 (PD-1) (ITIM-containing) (selectively upregulated by the exhausted T cells^ref) ---
• --- PD-like 1 (PD-L1) / B7 homolog 1 (B7-H1) (on a subset of PHA-activated T cells, splenic T cells, B cells, macrophages, and myeloid dendritic cells (expecially from tumor environments), on IFN-g- and LPS-activated monocytes, on IFN-g-treated keratinocytes, and on IFN-g- and TNF-a-induced endothelial cells and most human cancer cells). Bt-H1 might have different roles depending on the nature and stage of disease pathogenesis : despite most studies have found that ligation results in production of the regulatory cytokine IL-10 and many studies have supported its role as an inhibitor of T-cell responses in in vivo tumour, transplant and autoimmune models, local expression of B7-H1 promotes organ-specific autoimmunity and transplant rejection^ref and blockade of B7-H1 suppresses the development of chronic intestinal inflammation^ref.
• --- PD-like 2 (PD-L2) / B7-DC (on macrophages and DCs upon stimulation with IFN-g, GM-CSF, or IL-4). Although not eliminated, IFN-g production by CD4 T cells and IFN-g–dependent humoral responses were reduced in B7-DC KO mice relative to wild type mice. Antigen-specific CD8 T cell responses and cytotoxic T lymphocyte (CTL) activity were also diminished in B7-DC KO mice. Hepatic tumors grew more quickly in B7-DC KO mice, associated with a decrease in intrahepatic tumor-specific CD8 T cells. These results highlight the contrasting in vivo roles of B7-DC and B7-H1 and indicate that B7-DC functions as a tuning molecule, selectively augmenting T_h1 and CTL responses^ref.
Interaction inhibits cytokine secretion, causes cell-cycle arrest and/or apoptosis (mediated by both IL-10 and CD178 / FasL).
• CD152 / CTLA-4 --- B7 (i.e. CD80 / B7-1 and CD86 / B7-2 / B7.2 : expression on APCs is induced by IL-18 and inhibited by PGE₂ through EP₂ and EP₄ receptors). Bidirectional signaling along the B7–CTLA-4 coreceptor pathway enables reciprocal conditioning of T cells and DC : T cells can instruct DCs to manifest tolerogenic properties after CTLA-4 engagement of B7
• TP --- TxA₂ (produced by DCs) enhances random cell movement (chemokinesis) of naive but not memory T cells, impairs DC–T cell adhesion, and inhibits DC-dependent proliferation of T cells
• early growth response gene 2 (Egr-2) and Egr-3 are associated with an increase in the E3 ubiquitin ligase Cbl-b^ref
As during T-cell homing interactions (see above), steric hindrance is avoided by displacing large molecules such as CD43. Naive T lymphocytes activation is detectable after 2÷3 weeks but full activity occurs only after 4 weeks. BCL3 is the T lymphocyte gene, of those tested, with the greatest differential transcriptional response to adjuvant administration in vivo^ref. A single CD4⁺ T-cell clone has been shown to respond to as many as 10⁶ 11-mer peptides at low nM concentration. For both CD4⁺ and CD8⁺ T cells during this stage the protective effect of IL-7 on naive T lymphocytes is replaced by other cytokines, especially autocrine IL-2, which act as growth factors for potentiating clonal expansion following stimulation with Ag and costimulation. This need is particularly stringent for T lymphocytes because many of their effector functions are executed on a cell-to-cell basis.
TcR stimulation induces 3 separable events generating reactive oxygen species (ROS)^ref :
• rapid hydrogen peroxide production independent of Fas or NADPH oxidase
• sustained hydrogen peroxide production dependent on both Fas and phagocyte-type NADPH oxidase that is activated after TcR crosslinking through the recruitment of preformed Fas ligand and Fas. NADPH oxidase–deficient T cells show enhanced activation of the kinase Erk and a relative increase in T_h1 cytokine secretion
• delayed superoxide production that was dependent on Fas ligand and Fas yet independent of NADPH oxidase
Activation of the TcR by ligand initiates a rapid influx of Ca²⁺ into T cells. This leads to the activation of a phosphatase called calcineurin and the subsequent nuclear import and assembly of NFAT transcription complexes, which switch on and off the expression of genes essential for T cell development and function^ref. Calcineurin is the target of the immunosuppressive drugs cyclosporine A and FK506, the discovery of which ushered in the modern era of organ transplantation. The importance of Ca²⁺/calcineurin in immune responses and transplantation therapy has generated great interest in how Ca²⁺ signals are regulated. Badou and colleagues examined the lethargic mouse^ref, which carries a mutation in the b₄ subunit of the L-type voltage-gated Ca²⁺ channel (Ca_V1). Previous work showed that L-type Ca²⁺ channels are essential for calcineurin activation and NFAT function in neurons^ref. Hence it was possible that these channels might also be important for NFAT-dependent transcription in lymphocytes. Indeed, CD4⁺ T cells from b₄-mutant mice exhibit defects in Ca²⁺ entry in response to TCR signaling, are defective in NFATc1 and NFATc2 dephosphorylation, and exhibit severe defects in cytokine production. But what role do these voltage-gated channels play in the activation of nonexcitable cells such as T lymphocytes? T cells express many different ion channels in their plasma and endoplasmic reticulum (ER) membranes. These channels coordinately regulate the initial Ca²⁺ spike and sustained Ca²⁺ elevation after antigenic stimulation of T cells. In a resting T cell, K_V channels regulate the flow of potassium ions (K⁺) out of the cell, and sarco-endoplasmic reticulum Ca²⁺-ATPases (SERCA) transport Ca²⁺ into the intracellular stores. The resulting resting potential is about -65 mV. When T cells are stimulated by binding of antigenic peptide associated with major histocompatibility molecules to their TCRs, the lipid second messenger inositol 1,4,5-trisphosphate (InsP₃) is generated and triggers the release of Ca²⁺ from intracellular stores through the InsP₃ receptor. This increase in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) activates the Ca²⁺ release-activated Ca²⁺ (CRAC) channels in the plasma membrane. Once the CRAC channel is activated, the Ca²⁺ influx is driven by the cell's negative resting potential, and the positively charged Ca²⁺ ions flow into the cell. This constitutes the initial rise in [Ca²⁺]_i observed during the early stages of T cell activation. L-type channels, and specifically b₄-containing L-type calcium channels, may be involved in the initiation of this calcium response in T cells^ref. These channels are composed of a₁, g, and d transmembrane subunits as well as an extracellular a₂ and an intracellular b subunit^ref. L-type Ca²⁺ (Ca_V1) channels have distinct biophysical properties that enable them to participate in signal transduction. The a₁ subunit can associate with any of 4 distinct cytoplasmic b subunits (b₁ to b₄), all of which may be expressed in T cells^ref. The b₁ subunit influences the trafficking of the pore-forming a₁ subunit and modulates the characteristics of the channel^ref.

The ebb and flow of calcium ions. Ion channels involved in the initiation and modulation of the calcium response in T lymphocytes after binding of the TCR to ligand and activation of T cell signaling. In a resting lymphocyte, K_V1.3 channels transport K⁺ out of the cell, and the SERCA channels pump Ca²⁺ into stores in the ER lumen. The resulting resting potential is about -65 mV. TCR stimulation leads to the activation of phospholipase C- (PLC-) producing the second messenger inositol 1,4,5-trisphosphate (InsP₃). InsP₃ binds to the InsP₃ receptor (A) in the ER membrane, which results in the release of intracellular Ca²⁺. Ca_V1 channels (B) are activated by an undefined mechanism after TCR stimulation and may contribute to the initial increase in [Ca²⁺]_i. Following stimulation of the TCR, Ca²⁺ release-activated Ca²⁺ (CRAC) channels (C) open and extracellular Ca²⁺ flows into the cell. When [Ca²⁺]_i reaches a threshold level, TRPM4 (D) is activated and the CRAC channels are inhibited. TRPM4 activation allows Na⁺ to enter the cell, which reduces Ca²⁺ flux by depolarizing the membrane. Ca²⁺-activated K_Ca channels (E) are also activated, which repolarizes the cells and leads to a subsequent spike in [Ca²⁺]_i. (Inset) Diagram of a typical [Ca²⁺]_i profile following TCR stimulation (arrow). The channels that are active during each stage are indicated under the profile.
These investigators suggest that signaling via L-type Ca²⁺ channels influences the initial Ca²⁺ influx based partly on the currents induced after TCR stimulation. The interpretation that these currents are from Ca_V1 channels would be better supported if b₄-mutant T cells failed to produce the TCR-induced currents, which the authors did not test. The presumed coupling of TCR to b₄ raises the possibility that the b₄ or b₁ subunits are regulated by phosphorylation, as has been shown in other cell types, and that this modification serves to facilitate the activation of these channels. Determining the phosphorylation status of the endogenous channel components after antigen receptor signaling may contribute to further understanding of this mechanism. Given that between 10 and a few hundred channels of each type are expressed per T cell, this will not be an easy task. The phenotype of b₄-mutant CD4⁺ T cells also indicates that the other 3 b subunits cannot substitute for b₄. Therefore, specific Ca_V1 channels may have specific functions in T cells. b₄-containing Ca_V1 channels are clearly not CRAC channels because Ca²⁺ store-operated entry of Ca²⁺ is normal in 4-mutant CD4⁺ T cells. This places the Ca_V1 channel in a pathway that is upstream of, or parallel to, that containing the InsP₃ receptor. Treatment of T cells with L-type calcium-channel inhibitors indicates that Ca_V1 channels may operate during both the initial and sustained Ca²⁺ influx, hinting at additional roles for the other Ca_V1 channels^{ref1, ref2}. Patients with mutations in the human ortholog of the Ca_V1 b₄ subunit exhibit symptoms of epilepsy and ataxia consistent with the neurological phenotype of the 4-deficient mice^ref. Analysis of immune cells from these patients also may be informative. After the initial release of intracellular Ca²⁺ and the activation of the CRAC channel, [Ca²⁺]_i remains elevated. This sustained elevation of [Ca²⁺]_i is necessary to regulate gene expression^ref. As Ca²⁺ levels increase, intracellular Ca²⁺ inhibits the CRAC channel and activates K_Ca channels, which transport K⁺ out of the cell (see the figure). This combination of negative and positive feedback is important for initiating [Ca²⁺]_i oscillations with specific characteristics. Launay et al.^ref provide multiple lines of evidence implicating TRPM4, a Ca²⁺-activated monovalent cation channel, in Ca²⁺ oscillations. They show that a version of TRPM4 with a truncated amino terminus (N-TRPM4) inhibits sodium ion (Na⁺) transport into stimulated lymphocytes. Disruption of TRPM4 activity resulted in an increase in [Ca²⁺]_i after TCR stimulation without the characteristic Ca²⁺ oscillation, and also increased cytokine production. This ion channel may decrease the electrochemical driving force for Ca²⁺ entry, thereby allowing oscillations in [Ca²⁺]_i. Complementary data from cells in which TRPM4 expression has been decreased by RNA interference (RNAi) strengthen the conclusion that the dominant-negative form of TRPM4 (N-TRPM4) is specific for TRPM4 channels among the large TRP channel family^ref. However, studies of mice lacking TRPM4 will be required to determine the importance of TRPM4 in vivo. It is possible that TRPM4-deficient lymphocytes might either produce unsurpassed immune responses or deadly autoimmunity. The results of Badou et al.^ref and Launay et al.^ref raise the exciting possibility of developing therapeutics that target these newly described channels. For example, inhibitors of K_v channels attenuate the initial Ca²⁺ influx and early gene induction after T cell stimulation^ref. Specific and high-affinity inhibitors of the Ca_V1 channels, or of the b₄ subunit only, may inhibit lymphocyte activation in vivo. Pharmacological activation of TRPM4 channels would decrease the driving force for Ca²⁺ entry and lead to immunosuppression, whereas TRPM4 inhibitors might lead to more rapid pathogen clearance or more robust responses to immunization. Small changes in channel expression can have profound effects on [Ca²⁺]_i. For example, RNAi of TRPM4 mRNA, which reduces TRPM4 protein levels by half, has a dramatic effect on Ca²⁺ flux after TCR stimulation. Interestingly, the expression of many of these ion channels dramatically changes after lymphocyte activation, indicating that effector and memory T cells are likely to express distinct combinations and different amounts of these ion channels^{ref1, ref2}. The natural differences in ion-channel expression among different lymphocyte populations may also permit specific cell types to be targeted owing to their unique channel profiles. Whereas this research area will likely become more complex before it becomes clear, the work by Badou et al.^ref and Launay et al^ref provides insight into the most poorly understood step in T lymphocyte activation, and sets the stage for developing practical ways to modulate lymphocyte activity.
Details :
• in vivo priming of naive CD4⁺ T helper lymphocytes (T_h0): the Lck and ZAP-70 tyrosine kinases are rapidly activated within 2 min, while both CTLA-4 induction and the first mitosis occur around 36 hours after first contact for a naive T_H cell. On engagement of the TcR on Thp cells, rapid co-polarization of IFNGR with the TcR occurs within the developing immunological synapse. For CD8⁺ T cells, a relatively short antigen pulse seems sufficient for antigen-presenting cells to drive clonal expansion and differentiation. It is unknown whether the requirement for antigen is similarly ephemeral for CD4⁺ T cells. To study the dependence of a CD4⁺ T cell response on antigen persistence in a quantitatively and temporally controlled manner in vivo, a mouse line expressing a MHC class II–restricted epitope in dendritic cells under the control of a tetracycline-inducible promoter was engineered : experiments tracking the proliferation of CD4⁺ T cells exposed to their cognate antigen in various amounts for different time periods revealed that the division of such cells was contingent on the presence of antigen throughout their expansion phase, even in the presence of an inflammatory stimulus. This previously unrecognized feature of a CD4⁺ T cell response contrasts with the proliferative behavior of CD8⁺ T cells that has been documented, and it implies that the 2 T cell subsets might require different strategies for efficient vaccination^ref. Thp cells from the intrinsically T_h1-like C57BL/6 mouse strain have significantly more receptor co-polarization than T_h2-prone BALB/c Thp cells. Remarkably, in the presence of IL-4, a cytokine required for T_h2 differentiation, IFNGR co-polarization with TcR is prevented. This inhibition depends on STAT6, the transcription factor downstream of IL-4R that is required for T_h2 differentiation. This cytokine receptor crossregulation provides an explanation for the effect of IL-4 in inhibiting T_h1 differentiation^ref. Functional nonresponsiveness or clonal anergy is observed in CD4⁺ T cell clones that are stimulated through the TcR in the absence of costimulation. These cells are characterized by an inability to produce IL-2 and proliferate following restimulation through the TcR, even if costimulation is present. Furthermore, studies using in vivo models have shown that a transient clonal expression can precede the induction of Ag-specific nonresponsiveness in CD4⁺ T cells. Although these studies suggest that a nonresponsive state similar to AINR might occur in CD4⁺ T cells, they do not clearly distinguish whether the refractoriness to restimulation is a consequence of inappropriate initial stimulation or a regulatory mechanism that is inherent to the cells, as appears to be the case for CD8⁺ T cells. Various mechanisms of tolerance induction have been described for CD4⁺ T cells, including AICD, functional nonresponsiveness, or active suppression by T_Reg lymphocytes or cytokines. These mechanisms are not necessarily mutually exclusive. CD4⁺ T cells, unlike their CD8⁺ counterparts, do not become AINR following optimal stimulation through the TcR and costimulatory receptors. They do, however, undergo a rewiring similar to that seen in reversed AINR CD8 T cells. Thus, they retain the ability to rapidly up-regulate IL-2 production (with no longer requirement for a costimulatory signal) upon re-encountering Ag to provide help to AINR CD8 T cells, but may then be eliminated due to higher sensitivity to Fas-mediated AICD upon restimulation, again differing from activated CD8 T lymphocytes in this respect. If suitably and extensively stimulated, 2 polarized, antagonistic patterns of cytokine production can emerge (anyway in balanced immune responses, individual T_h cells show a range of cytokine production potentials, not just these extreme forms !). In these conditions human naive T cells acquired stable histone hyperacetylation at either the IFN-g or IL-4 promoter. Fox (forkhead)-family transcription factor Foxj1 maintains naive CD4⁺ T_H cells in a quiescent state by inhibiting NFkB activity : its downregulation (e.g. in mice that are prone to systemic lupus erythematosus (SLE)) can make naive T_h cells more responsive to the activating signals that wake them from their slumber by downregulating IkBb removing inhibition on NFkB^ref. This adds to the known role of other Fox-family members in the immune system, such as Foxp3 in T_Reg lymphocytes, Foxo proteins in lymphocyte proliferation and apoptosis, and Foxn1 in thymic epithelial-cell development.
• germinal-centre T helper (GC-T_H) cells are specifically localized in the germinal centres of lymphoid follicles to help B cells produce antibodies. They are characterized by expression of CXCR5 and CD57, and are non-polarized in terms of cytokine production. GC-TH cells are unique in their expression of the chemokine CXCL13, which is crucial for B-cell entry to lymphoid follicles but was previously thought to be produced by non-T cells, such as follicular dendritic cells (FDC). There are also marked differences in the expression of transcription factors between GC-T_H cells and other T-cell subsets, which supports the theory that GC-TH cells are a novel functional lineage^ref.
They need :
• high-affinity peptide ligands for TcR
• intermediate dose of antigen
• IL-12 (from macrophages without Fcgs bound ; => STAT4 => SOCS1)
• IL-18
• IL-23
• IL-27 (IL-27Ra / WSX1 signalling is required for STAT1 activation, and subsequent T_h1-cell differentiation, only when IL-12 is limiting, and in situations in which IL-12 is plentiful - such as during T.gondii infection - WSX1 is not required for the generation of a T_h1-cell response and is, in fact, required to prevent overproduction of cytokines^{ref1, ref2})
• IFN-g (=> up-regulation of IL-12Rb₂)
• MIF
• CD28-- CD80 / B7-1 interaction
• CCR2--- CCL2 interaction
• ? --- CXCL10 interaction
• ? --- M150 / LAMP-1 / CD107a (activated macrophages)
• CD26 (T cells) --- adenosine deaminase (ADA)-A₁ and A_2B adenosine receptors complexes (DCs), reducing by 3-fold the EC₅₀ for the antigen and increasing (3- to 34-fold) the production of the T_h1 and proinflammatory cytokines IFN-g, TNF-a, and IL-6^ref. ADA deficit leads to ADA-SCID
• JNK2
• transcription factors :
• STAT1 => upregulation of T-bet :
• positive regulator of
• IL-12R expression
• IFN-g expression  : a T-bet-responsive unit is located within the -565 to -410 region of the IFN-g promoter. The -225 promoter is silent due to Yin Yang 1 (YY1 : Mus musculus) binding. The p38^MAPK1-RAC-1 signaling pathway regulates the -70 to -44-bp promoter
• negative regulator of T_h2 cytokines expression and of IL-2 during differentiation
• T-helper-inducing POZ/ Krüppel factor (Th-POK) : in the thymus, thymocytes receive positive selection signals when their TcRs recognize 'self' peptides (red circles) bound to molecules of the major histocompatibility complex (MHC) displayed on thymic epithelial cells. This interaction is helped by the CD4 or CD8 proteins.
• a, The thymocyte recognizes class II MHC, and receives a moderate, prolonged signal. This leads to production of Th-POK, a gene-regulatory protein that in turn controls other genes (including repression of the CD8 gene), causing the cell to adopt a CD4 fate. TCR signalling may also work independently of Th-POK (dashed line) to promote the differentiation or survival of CD4 cells.
• b, The thymocyte recognizes class I MHC, and receives a weak, transient TCR signal that fails to increase production of Th-POK. TCR-induced events in the absence of Th-POK lead to changes in gene expression (including repression of the CD4 gene), causing the cell to adopt the CD8 fate.

The work by Kappes and colleagues^ref defines the molecular trail from the thymocyte–MHC interaction to the change in gene expression that guides the cell to its ultimate fate. While screening mice bred from two lines carrying different genetically engineered mutations, the authors noted^ref that some of the progeny had an unusual characteristic that was not the result of either mutation — a complete absence of mature CD4 T cells. A spontaneous mutation, which they termed 'helper deficient', or HD, was responsible for the defect. Further analysis revealed that HD did not simply cause loss of CD4 cells, but rather produced a 'cell-fate switch' by which thymocytes that would normally have matured into CD4 T cells instead gave rise to CD8 T cells^ref. Although there are a number of engineered mutations that can also redirect the CD4 versus CD8 fate decision^{ref1, ref2}, most of these mutations lead to proteins that have abnormal functions (rather than causing disruption of a normal function), and thus they may not reflect the true functions of the unmutated genes. Furthermore, most of these engineered mutations cause only a fraction of thymocytes to make the wrong cell-fate choice. HD is the only mutation known that not only is a loss-of-function mutation, but also causes a wholesale conversion of thymocytes from the CD4 to the CD8 lineage, and so it seems to hold the key to understanding this cell-fate decision. 7 years after the original description of the HD mutation, Kappes and colleagues identified the gene affected by it. It encodes a transcription factor called Th-POK. Transcription factors regulate gene activity, often in a tissue-specific manner. The discovery, therefore, that the HD mutation changes an amino acid of a transcription factor made perfect sense. The authors also show that, in the thymus, Th-POK is found specifically in mature CD4 T cells, and not in CD8 T cells. Moreover, enforced expression of the Th-POK gene in thymocytes transforms cells that would have been CD8 into CD4 cells, the opposite effect to the HD mutation. Thus, Th-POK is necessary and sufficient to induce the gene-expression programme for a CD4 cell fate and to suppress the CD8 programme. Other transcription factors have been implicated in T-cell development, including GATA3, which is required for CD4 T-cell maturation^ref1,ref2, and Runx3, which silences the gene encoding CD4 in CD8 T cells^ref. However, mutation of their corresponding genes does not cause a cell-fate switch, but merely impairs the development of one cell type. So Th-POK seems to be at the top of a gene-regulation hierarchy that controls T-cell fate. Many tantalizing questions remain. What controls expression of the Th-POK gene in developing thymocytes? The observation that Th-POK synthesis is increased in thymocyte precursors that recognize class II MHC, but not class I MHC, implies that the gene's expression is regulated by engagement of class II MHC. This may be controlled by differences in T-cell receptor signalling on recognition of class I compared with class II MHC, as predicted by a popular model for CD4 versus CD8 lineage commitment^{ref1, ref2}. Further analysis of the regulation of Th-POK by T-cell-receptor signalling will be needed to test this idea. What genes are regulated directly by Th-POK? The gene-expression programmes of CD4 and CD8 cells differ in terms of the production of GATA3, perforin (a T-cell effector protein), and CD4 and CD8 themselves. However, it is not yet clear whether these differences in gene expression are direct or indirect consequences of Th-POK activity. Finally, self-reinforcing feedback loops are often used to 'lock in' cell-fate decisions, and there are hints that Th-POK is part of such a regulatory circuit. HD mice expressing class II MHC, but not class I MHC, show increased synthesis of messenger RNA for Th-POK in thymocyte precursor cells. However, the redirected CD8 T cells that ultimately develop in these mice lack Th-POK. Whether the requirement for Th-POK function to maintain Th-POK gene expression involves auto-regulation of the gene or involves other components of the gene regulatory hierarchy remains to be seen. The quest to understand the CD4 versus CD8 lineage decision has focused mostly on the question of whether MHC recognition instructs the cell-fate decision (the instructive model), or whether cells choose their fate randomly and are then tested for the presence of the appropriate CD4 or CD8 molecule (the stochastic or selection model)^{ref1, ref2}. These models, although useful, assumed that T-cell fate determination occurs as a single discrete step, but it has become clear that it is a multi-step process involving feedback and reinforcement. The identification of Th-POK as a key in the T-cell fate decision should open the way to deeper mechanistic insights into how cells are guided to their destiny.
• ERM
• TXK
• delta-like 4 (DLL4)expression on APCs^ref
• T_h cell differentiation involves silencing as well as activation of gene expression. A conserved silencer of the gene encoding IL-4 marked by DNase I hypersensitivity (HS IV) and permissive chromatin structure in all T_h cells. Deletion of HS IV increased IL-4 and IL-13 transcription by naive T cells and led to T_h2 skewing in vitro. HS IV controlled Il4 silencing during T_h1 differentiation, as HS IV–deficient T_h1 cells that expressed IFN-g also produced abundant IL-4 in vitro and in vivo. Despite mounting a vigorous IFN-g response, HS IV–deficient mice were more susceptible to Leishmania major infection than were wild-type littermate control mice, showing a critical function for Il4 silencing in T_h1–mediated immunity^ref.
... to become a :
• T_h1 or T_H1 or T_DTH or T_D cell (CD10^-13^-14^-15^-19^-52⁺183 / CXCR3⁺195 / CCR5⁺223 / LAG-3⁺, CD94/NKG2A, -NKG2C, or -NKG2E⁺, NKG2D^-, T cell immunoglobulin domain, mucin domain 3 (Tim-3) / HAV cellular receptor 2⁺CCR1⁺CXCR6⁺CX₃CR1⁺, LIGHT⁺, BLT₁ / P2Y₇^hi) undergoes IL-12-driven up-regulation of IL-12Rb2 and IL-18Ra : IFN-g up-regulates IL-12Rb2 expression only and limits the negative effects of IL-4 on IL-12 STP. They produce :
• IL-2 (autocrine signalling)
• IL-3
• low IL-6
• low IL-10
• IL-12 (autocrine positive feedback and inhibition of T_h2)
• IL-18 (autocrine positive feedback)
• TNF-a
• TNF-b
• IFN-g (that prevents class switch to IgE in plasma cells)
• high levels of GM-CSF
On the contrary of T_h2 lymphocytes, T_h1 ones express B and T lymphocyte attenuator (BTLA), an activatory ligand which binds to.. :
• B7X / B7-H4 : crosslinking with antigen receptors induces its tyrosine phosphorylation and association with SHP-1 / PTPN6 and SHP2
• HVEM

[HHV-1 / HSV-1 envelope glycoprotein D binds both LIGHT and BTLA, potentially nullifying the HVEM pathway. On the contrary HHV-5 / CMV UL144 binds BTLA, but not LIGHT, and inhibits T cell proliferation, selectively mimicking the inhibitory cosignaling function of HVEM^ref]
Tim-3 also exists in a secreted isoform that contains only the immunoglobulin (Ig) variable (V) domain of the full-length molecule. Interaction of both Tim-3 isoforms with an undefined Tim-3 ligand on CD4⁺ T cells may serve to inhibit effector T_h1 cells during a normal immune response and may be crucial for the induction of peripheral tolerance, in part due to effects on CD4⁺CD25⁺ regulatory T cells, preventing autoimmunity^{ref1, ref2}. Galectin 9 binds to Tim-3 on T_h1 lymphocytes and induces intracellular calcium flux, aggregation and death^ref
... or :
• IL-1b
• IL-4 (=> STAT6 => transcription and negative feedback via SOCS3) : anyway T_h2 lymphocytes still develop in STAT6-deficient mice. If T_h2 cells produce the cytokine required for their own differentiation, how is a T_h2 response initiated? One possibility is that T_h2 cells arise by default when a strong T_h1 stimulus is lacking, but DCs induce T_h2 responses directly, even in the absence of IL-4 signals^ref
• IL-17E / IL-25
• TGF-b₁
• 9-cis retinoic acid (RA) (=> RXR => increases IL-4 and IL-5)
• low-affinity peptide ligands for TcR (altered peptide ligands (APLs))
• low or very high dose (> 10 mM) of antigen
• low signal strength at the immunological synapse :
• CD134 / OX40 --- CD134L / OX40L / gp34 interaction
• CD154 / 40L / gp39 --- CD40 (on DCs) interaction
• ? --- B1 (LPS-activated B cells)
• CD91 / LRP / a₂-macroglobulin R --- B2 / gp96 (LPS-activated B cells, thioglycollate-elicited macrophages and BMDCs)
• ? --- B3 (LPS-activated B cells)
• ICOS --- B7H / B7RP-1 / GL50 (expressed constitutively on naive B cells; up-regulated by CD40 signals and, in nonlymphoid tissues, by TNF-a; down-regulated by IL-4 and BcR activation) interaction
• mDCs can upregulate expression of cell-surface CLIP–MHC class II complexes as a result of downregulation of HLA-DM activity (which would normally exchange CLIP for an exogenous antigen). The CLIP–MHC class II complexes are localized to immunological synapses with CD4⁺ T cells — together with MHC class II molecules carrying exogenous peptide — where they influence the type of T_h-cell response to the exogenous peptide by favouring T_h2-cell polarization^ref.
... resulting in reduced secretion of IL-12
• IL-1R-like 1 / T1 / ST2-- IL-1R-like 1 ligand / ST2Linteraction. It also mediates endotoxin tolerance (the reduced production of cytokines after re-exposure to the bacterial cell-wall component LPS) by inhibiting signalling through IL-1RI when either Myd88 or Mal is co-expressed, but not when TRIF or the downstream signalling molecule IRAK is co-expressed^ref
• ITK
• SLAM-associated protein (SAP) / SH2D1A (whose mutations cause X-linked lymphoproliferative syndrome (XLP) / Duncan disease), an adaptor that recruits FynT to CD150 / signalling lymphocyte activation molecule (SLAM)^{ref1, ref2}
• ligation of Notch on T_h0 lymphocyte by jagged-1 on APC^ref directly upregulates IL-4 transcription through upregulation of RBPJ-k sites in a 3' enhancer region^ref (but is unclear how this distinction is made: in some settings at least, both classes of ligand are able to activate RBPJk-dependent signals^ref. The Notch pathwayis an evolutionarily conserved signalling mechanism that regulates lineage choices in a variety of cell types, including T cells^ref. Divergent signals could result from the specificity of Notch ligands for different Notch family members. Or Notch ligands might transduce distinct signals through a single receptor, for example through RBPJk-dependent of independent pathways. The importance of retaining 2 classes of Notch ligands is underscored by the fact that simpler organisms, which possess a single Notch receptor, also have 2 classes of ligands that do not signal equivalently^ref) => upregulation of GATA3 (whose expression is inhibited by RUNX1 / AML1)
• negative regulator of IFN-g expression
• positive regulator of
• C-terminal zinc finger : IL-4 and IL-13 expression
• N-terminal zinc finger : IL-5 expression
GATA-3 binds to a conserved noncoding sequence (CNS)-1 in the IL-4/IL-13 intergenic region containing the HSS1 and HSS2 DNase I hypersensitive (DH) sites. The conserved noncoding element in the intergenic region regulates replication initiation from a distant replication origin (downstream from exon 4 of IL-13) in a manner independent from its effect on lineage-specific transcription but not the replication timing of the segment surrounding this origin^ref. As deletion of Gata3 results in an early block in T-lineage commitment, conditional deletion of Gata3 in stage-3 DN thymocytes leads to both CD4⁺ and CD8⁺ T cells that are unable to produce T_h2 cytokines. Furthermore the persistent expression of Gata3 is required for T_h2 cells to maintain their appropriate cytokine production^ref. In vitro deletion of Gata3 diminishes both IL-4–dependent and IL-4-independent T_h2 cell differentiation; without GATA-3, T_h1 differentiation occurs in the absence of IL-12 and IFN-g. Gata3 deletion limits the growth of T_h2 cells but not T_h1 cells. Deletion of Gata3 from established T_h2 cells abolished IL-5 and IL-13 but not IL-4 production. In vivo deletion of Gata3 using OX40-Cre eliminated T_h2 responses and allowed the development of IFN-g-producing cells in mice infected with Nippostrongylus brasiliensis. Thus, GATA-3 serves as a principal switch in determining T_h1-T_h2 responses^ref.
• c-Maf (positive regulator of IL-4 expression)
• SOCS3
• SWAP70-like adaptor of T cells (SLAT) : after antigen stimulation of the TcR, it translocates to the immunological synapse, where it inhibits ZAP70
• down-regulation of ...
• T-bet
• IL-12R
• IFN-g (which otherwise prevents IL-4 transcription via IRF1 and IRF2)
• SHP-1 / PTPN6
... to become a :
• T_h2 or T_H2 cell (CD13^-14^-15^-19^-30 / Ki-1 Ag⁺134 / OX40⁺CCR1⁺CCR3⁺ CCR4⁺CCR8⁺CRTH2 / GPR44⁺IL-1R-like 1 / T1 / ST2⁺, LIGHT⁺, NKG2D^-, BLT₁ / P2Y₇^hiTIM1 / HAV cellular receptor 1⁺). They produce :
• IL-3
• IL-4 (autocrine positive feedback; the most important T_h2 cytokine; it also inhibits T_h1 differentiation and stimulates class-switch recombination to IgE in plasma cells). DNMT1 recruitment is blocked, histone H3 Lys4 methylation is enhanced (indicative of accessible chromatin) and DNA demethylation of the locus is initiated : although 6 NFAT-binding sites (P0 to P5) and 3 STAT6-binding sites (overlapped to P1, P2, and P4) exist in the IL-4 promoter, only STAT6 modulates IL-4 production under TcR triggering.
• IL-5
• high levels of IL-6
• IL-9 (stimulator of TGF-b₁ synthesis in monocytes (positive feedback !) => autoinhibition of oxidative burst, TNF-a and IL-10 release)
• high levels of IL-10 (that inhibits T_h1 lymphocytes (both directly and by generating T_Reg cells) and synthesis of pro-inflammatory cytokines such as IL-1b, IL-12 and TNF-a )
• IL-13
• IL-31
• TIM1 / HAV cellular receptor 1 --- TIM-4 (on APCs)^{ref1, ref2}
• soluble CD30 (sCD30) : CD30 is preferentially expressed on CD4⁺ and CD8⁺ T-cell clones that produce T_h2-type cytokines, and is also released in a soluble form by these cells. Elevated serum levels of sCD30 have been found in some conditions in which a pathogenic role for T_h2 cells has been suggested, such as atopy, Omenn's syndrome, systemic lupus erythematosus, as well as following infection with measles virus or HIV^ref.
• low levels of GM-CSF (see also T_h1 and T_h2 cytokines and cross-regulation).
• VEGF-A, which increases the number of activated DC2 dendritic cells. It is a mediator of vascular and extravascular remodeling and inflammation that enhances antigen sensitization^ref
They suppress CMI.
The T_h2 locus control region (LCR) is important in the regulation of the genes encoding the cytokines IL-4, IL-5 and IL-13. In T cells, NK cells, B cells and fibroblasts, the promoters for the genes encoding T_h2 cytokines are located in close spatial proximity, forming an initial chromatin core configuration. In CD4⁺ T cells and NK cells, but not B cells and fibroblasts, the T_h2 locus control region participates in this configuration. The transcription factors GATA3 and STAT6 are essential for the establishment and/or maintenance of these interactions. Intrachromosomal interactions in the T_h2 cytokine locus may form the basis for the coordinated transcriptional regulation of cytokine-encoding genes by the T_h2 locus control region^ref.
Human T cells, when repeatedly stimulated in vitro, will often develop a T_h2 phenotype regardless of their origin^ref1,ref2.
... or :
• CD223 / LAG-3 upregulation^ref
• minute antigen doses with suboptimal dendritic cell activation, addition of TGF-b or the absence of IL-2 production : populations induced in subimmunogenic conditions could subsequently be expanded by delivery of antigen in immunogenic conditions^ref
... to become a :
• T_reg lymphocyte
• T_h1-like T_R cells are induced by CD8a⁺ DCs and develop in vivo from naive CD4⁺CD25^- T cells during a T_h1–polarized response, distinct from CD25⁺ T_R cells. They produce both IL-10 and IFN-g, and potently inhibit the development of airway hyper-reactivity (AHR). These T_R cells express the transcription factors Foxp3 and T-bet, indicating that these T_R cells are related to T_h1 cells^ref. Scurfy mice, which are deficient in a functional Foxp3, exhibit a severe lymphoproliferative disorder and display generalized overproduction of cytokines. Among the Foxp transcriptional factor family, which includes Foxp1, Foxp2, and Foxp3, only Foxp3 has the ability to inhibit IL-2, IL-4, and IFN-g production by primary T_h cells. Foxp3 physically associates with the Rel family transcription factors, NFAT and NF-kB, and blocks their ability to induce the endogenous expression of their target genes, including key cytokine genes. More importantly, T cells derived from scurfy mice have a dramatic increase in NFAT and NF-kB transcriptional activity compared with the T cells derived from WT mice. Furthermore, complementation of Foxp3 in scurfy-derived T cells lowers the NFAT and NF-kB transcriptional activity to the physiological level. Finally, myelin proteolipid protein-specific autoreactive T cells transduced with Foxp3 cannot mediate EAE, providing further support that Foxp3 suppresses the effector function of autoreactive T cells. Foxp3 has already been associated with the generation of CD4⁺CD25⁺ regulatory T cells; our data additionally demonstrate that Foxp3 suppresses the effector functions of T helper cells by directly inhibiting the activity of two key transcription factors, NFAT and NF-kB, which are essential for cytokine gene expression and T cell functions^ref.
• T_h2-like T_R cells, which express Foxp3 and are induced during the development of respiratory tolerance by CD8a^- DCs.
By using CD4⁺ T cell-driven experimental autoimmune encephalomyelitis as a model, it was found that depletion of CD4⁺CD25⁺Foxp3⁺ T_reg allowed pathology to develop in response to suboptimal T cell stimulation^ref.
Islet antigen–specific CD4⁺25^- T_h and T_reg cells swarm and arrest in the presence of autoantigens. These T_h cell activities are progressively inhibited in the presence of increasing numbers of T_reg cells. There were no detectable stable associations between T_reg and T_h cells during active suppression. In contrast, T_reg cells directly interacted with DCs bearing islet antigen. Such persistent T_reg cell–DC contacts preceded the inhibition of T_h cell activation by DCs, supporting the idea that DCs are central to T_reg cell function in vivo^ref.
... or :
• lack of IFN-g and IL-4^ref
• costimulatory molecules CD28 and ICOS
• TGF-b₁^ref and IL-6^ref
• IL-23 independently of the transcription factors  required for T_h1 or T_h2 differentiation (T-bet, STAT1, STAT4 and STAT6); but IL-23 is not the differentiation factor^ref; upregulate IL-23R expression, thereby conferring responsiveness to IL-23. Although dispensable for the development of IL-17-producing T cells in vitro and in vivo, IL-23 is required for host protection against a bacterial pathogen, Citrobacter rodentium^ref
... to become a :
• T_h17 lymphocyte : are associated with autoimmunity. In vivo, antibody to IL-17 inhibited chemokine expression in the brain during experimental autoimmune encephalomyelitis, whereas overexpression of IL-17 in lung epithelium caused chemokine production and leukocyte infiltration^ref. Committed T_h-17 cells were resistant to suppression by T_h1 or T_h2 cytokines^ref. They produce :
• IL-17E / IL-25
• in vivo priming of naive CD8⁺ CTLs : after immunization but before antigen recognition, naive CD8⁺ T cells in immunogen-draining lymph nodes upregulate the chemokine receptor CCR5, permitting these cells to be attracted to sites of antigen-specific dendritic cell–CD4⁺ T cell interaction where the cognate chemokines CCL3 and CCL4 (also known as MIP-1a and MIP-1b) are produced. Interference with this actively guided recruitment markedly reduces the ability of CD4⁺ T cells to promote memory CD8⁺ T-cell generation, indicating that an orchestrated series of differentiation events drives nonrandom cell–cell interactions within lymph nodes, optimizing CD8⁺ T-cell immune responses involving the few antigen-specific precursors present in the naive repertoire^ref.
• responses against some Ags require help from CD4⁺ T_h lymphocytes via ...
• paracrine secretion of IL-2 (required by CTL during primary activation) while both T_h and CTL are in sufficient proximity to each other at the surface of the same APC
• CD4⁺ T lymphocytes-dependent activation of APC to a state in which they are able to directly prime CTL ("licence to kill") in the absence of CD4⁺ T lymphocytes. This model allows the obligate interaction of 3 cell types to be accomplished through serial 2-cell interaction, thus avoiding the temporal and statistical probability drawbacks implicit in the first model. In support of the second model, cross-priming studies have shown that the CD4⁺ T lymphocytes requirement in CTL priming can be overcome using an agonistic mAb to CD40 on APC and that blockade of CD40L / CD154 on CD4⁺ T cells prevents them from providing help to CTL. Activation induces a number of functional and phenotypic changes within APC, including their migration to lymphoid organs, secretion of cytokines/chemokines, and expression of a range of costimulatory molecules. Both B7 and CD28 are absolutely required for CTL cross-priming by CD40-activated APC and indicate that CD28 can function as a kind of molecular rheostat through which up-regulation of B7 molecules on APC is perceived as help by CTL, leading to their primary activation and functional development.
Initial cell division occurs after 48 hours and 3-4 divisions per day lead to > 1000-fold expansion of the responding cells within a few days. Anyway the initial helper-dependent, autocrine IL-2-induced CTL response to full stimulation with Ag and costimulation is transient, because within 3-4 days the cells become activation-induced nonresponsive (AINR), unable to up-regulate IL-2 mRNA and protein upon further stimulation. That's why an active process of demethylation of sites 2-6 in the IL-2 promoter within 20' after stimulation is necessary and sufficient to enhance IL-2 gene transcription, in the absence of cell division. As the demethylation is stable, this process could increase the amount and/or rate of IL-2 transcription in memory T cells. Unless exogenous IL-2 or CD4 T cells help is available to these AINR cells, their clonal expansion ceases and survival declines. Biochemical analyses have revealed that the defect in IL-2 production in AINR CD8 T cells is at least partly attributable to a block in activation of the MAPKs, ERK, JNK, and p38 (not the result of CTLA-4 engagement or down-regulation of TcR, CD28, or LFA-1). TcR-mediated signaling can still occur, however, because AINR CD8 T cells can produce IFN-g and carry out cytotoxic effector function upon recognition of Ag-bearing target cells. The AINR state is reversed when the cells are allowed to undergo a brief period of expansion (1-2 days) in response to exogenous IL-2. Once reversed, the cells again can make a sustained and effective response to Ag. Upon reversal, the cells regain the ability to up-regulate MAPKs and produce IL-2 to drive a sustained response to Ag in the absence of any further help. Furthermore, some rewiring occurs upon reversal so that unkile naive cells, these reversed cells do not require costimulation to activate the MAPKs and IL-2 production : TcR engagement is sufficient.
• other responses, notably those against certain viruses (LCMV and vaccinia virus) which may achieve the same functional activation of APC through direct infection or replication within lymphoid tissues, can apparently be generated in absence of help from CD4⁺ T_h lymphocytes thanks to cross-priming. If an APC is infected it produces MHC I co-presented Ags and can directly activate T_c-cells to kill itself. However, once cross-primed in the absence of T_h, these viral CTL responses wane over time and become effective in controlling viral persistence and replication.
• immune complex–mediated complement activation is crucial for the priming of vaccine-specific CTLs^ref
They need :
• IL-2
• IL-12 (not for activation of memory CD8⁺ T cells)
• IL-15
• IL-21
• eomesodermin homolog (Xenopus laevis) (Eomes) , a T-box factor that is important for initiating the fate of mesodermal cells in vertebrates, and whose expression is restricted to activated CD8+ T cells^ref
• positive regulator of
• IFN-g (complemented by T-bet)
• perforin
• granzyme B
... to become activated single-positive (SP8) cytotoxic T-lymphocyte (CTL) (CD10^-13^-14^-15^-19^-25 / IL-2Ra⁺26^hi27^-30 / Ki-1 Ag⁺38⁺45 / B220 / LCARA^-RO⁺48⁺49d⁺62L / MEL-14^-98⁺101⁺107a / LAMP1⁺152 / CTLA-4⁺153 / 30L⁺ 154 / 40L / gp39⁺223 / LAG-3⁺, NKG2D-S^hi, NKG2D-L^hi, LIGHT⁺, BLT₁ / P2Y₇^hi). Both NKG2D isoforms signal via DAP10 adapter (pYXNM-containing => PI(3)K STP, far less effective than ZAP70/Syk STP in inducing activation, so relying on the TcR for primary specificity !) only as DAP12 is not expressed here. They produce IFN-g and act as killer cells. High avidity CTLs express a greater proportion of CD8ab heterodimers relative to low avidity CTLs. According to their cytokine secretion profile, they are classified as :
• T_c1 lymphocytes, having a cytokine secretion profile similar to T_h1 lymphocytes.
• T_c2 lymphocytes, having a cytokine secretion profile similar to T_h2 lymphocytes.
Surface mobilization of CD107a / LAMP1—an integral membrane protein in cytolytic granules—is a marker for degranulation after antigen stimulation : FACS monitoring is useful not only for immune monitoring of vaccine trials, but also for adoptive cellular immunotherapy after ex vivo expansion.
Antigen-specific CD8⁺ T cells acquire peptide-MHC (pMHC) clusters through TcR–mediated endocytosis after specific antigen stimulation.
Activated effector CTLs lose expression of both p90-110^{L-selectin / CD62L / MEL-14 / LAM-1 / LECAM-1 / Leu-8} and CCR7, so they fail to roll in HEVs and cannot home to LNs.
Activated antigen-specific CD8⁺ T cells activated by localized intestinal viral infection are found not only in the intestine but also in other non-lymphoid tissues. This widespread distribution is observed for both the primary effector cells and virus-specific memory cells. Furthermore, memory CD8⁺ T cells from non-lymphoid tissues, other than the intestinal mucosa, retained the ability to disseminate into multiple extranodal sites, albeit with some preference for their site of isolation^ref.
Antigen-specific lysis of target cells, mediated by recognition of abTCR of HLA and cognate antigen, by CD8⁺ T cells preloaded with cytotoxic granules can occur within 5 minutes, although the killing process can stretch out over hours^ref.
[lymphotropic herpesvirus saimiri (HVS) —which induces T-cell lymphomas in infected primates —tyrosine-kinase-interacting protein (Tip) disrupt signalling through the TcR inhibiting LCK ability to phosphorylate tyrosine residues in the CD3 subunits important for the recruitment and activation of ZAP70 through its phosphorylation. In addition, Tip expression inhibited immunological synapse formation^ref]
Interactions for T-cell homing

molecules on newly activated effector T-lymphocytes whose expression is induced by the microenvironment in tissue-draining lymph nodes	addressins on high endothelial venule (HEV) of thymus and T-cell zones of secondary lymphoid organs
step 1) : emperypolesis / reversible adhesion step 1a : tethering (bind loosely to endothelial cells) step 1b : slow rolling along the vessel (pushed forwards by the blood stream)
p90-110L-selectin / CD62L / MEL-14 / LAM-1 / LECAM-1 / Leu-8 (mainly on naive T-cells). Its ectodomain is proteolitically shed from the cell surface after cross-linking : L-selectin shedding prevents the re-entry of activated T-cells into the PLNs by downregulating L-selectin expression, but is not required for naive T-cell homing to PLNsref.	peripheral node addressins (PNAd) (an O-linked carbohydrate moiety in SO42--GAGs, the main components of which are recognized in humans and mice by the mAb MECA-79ref1, ref2) :  GlyCAM-1  gp105-120CD34  MAdCAM-1  ... In addition, some LN venules express structurally and antigenically distinct L-selectin ligands that are not detected by MECA-79ref1, ref2. The structure of L-selectin ligands in HEVs and the role of glycosyl- and sulphotransferases in the biosynthesis of these ligands is a topic of intense studyref1, ref2. Carbohydrate (keratan sulfate Gal-6) sulfotransferase 1 (CHST1) / GlcNAc6ST-1 and carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 2 (CHST2) / GlcNAc6ST-2 are essential for biosynthesis of syalyl Lewis X : mutant mice had approximately 75% less homing of lymphocytes to the peripheral lymph nodes than did wild-type miceref1, ref2 CD31 / PECAM1a
step 2) : GiPCRs-mediated adhesion activation  (few seconds) : integrin must be activated to mediate arrest
CCR7 (on naive and central memory T-cells)ref1, ref2	CCL21 (constitutively secretedref1, ref2, ref3)  CCL19 is secreted by the lymphatic endothelium and interstitial cells in LNs, but not by HEVs. Perivascular CCL19 can be transported to the luminal surface of HEVs and induce integrin activation of rolling T cellsref. The realtive contribution by CCL19 versus CCL21 to T-cell homing remains to be determined.
( CXCR4 )	( CXCL12 / SDF-1 : although it potently activates integrins on rolling naive T cells in vitroref1, ref2, it does so poorly in vivoref. So, despite low-level expression of CXCL12 by mouse HEVs, naive T cells depend on CCR7 signals, at least in BALB/c and DDD/1 genetic backgroundsref1, ref2. By contrast, CXCL12-CXCR4 interactions have a modest, but detectable role in T-cell homing in C57BL/6 mice, indicating some chemokine pathways are regulated by strain-specific genetic modifiersref)
CD49dCD29 / VLA-4 / a4b1 integrin	CD106 / VCAM-1 / INCAM-110  complement S-protein / PA-I-BP1 / vitronectin / somatomedin B fibronectin 1
cutaneous lymphocyte antigen (CLA) / HECA-452+ (a carbohydrate determinant created by a(1,3)fucosyltransferase VII (FT7) adding fucose residues to a2,3-sialylated lactosamine acceptors to form the SLex epitope, defined by reactivity with the unique mAb HECA-452) CD162 / PSGL-1 (on a subset of Th1 memory cells) ;  other sialyl-Lewis A and B molecules (e.g. CD15u). The microenvironment in skin-draining lymph nodes promotes the expression of CLAref only by newly activated effector T cells and CCR10+ memory T cells associated with cutaneous inflammatory responses (e.g. circulating memory T cells specific for nickel or house-dust mite in allergic or atopic individuals, respectively, express high levels of CLAref, presumably because these antigens were encountered through the skin. Similarly, circulating CD8+ effector T cells specific for skin-associated viruses express CLA, whereas those specific for non-cutaneous viruses do notref). CLA is expressed by 30% of circulating memory T cells and is virtually absent on naive T cellsref. T cells found in inflammatory skin lesions are mainly CD45RO+CLA+, whereas few T cells that accumulate in inflammatory sites other than the skin express CLA. CLA is reproducibly found on most T cells present in cutanoeus lymphocyhtic infiltrates of almost all skin diseasesref, including psoriasisref1, ref2, atopic dermatitisref1, ref2, allergic contact dermatitis, erythema multiformeref, cutaneous GvHDref, skin-associated viral infectionsref, and cutaneous T-cell lymphoma (CTCL)ref1, ref2 (both malignant T cells and those that respond to the presence of tumour cells : CLA also seems to be a good marker of malignant CTCL cells in the peripheral blood of some patients with Sezary syndromeref). Studies of CLA induction in vitro have indicated that expression is enhanced by CD3 activation in the presence of IL-12 and is not restricted to functional and phenotypic T-cell subsetsref1, ref2. However, the factors that regulate the induction of expression in vivo and the maintenance of expression by resting circulating cells have not been determined.	p110CD62E / E-selectin / ELAM-1 / LECAM-2 (blocked by CGP69669Aref)  p110CD62P / P-selectin / GMP-140 / PADGEM (which is expressed by high endothelial cellsref and circulating activated plateletsref ; deficitary in LAD-2) (blocked by mAb RB40.3ref)
step 3) : adhesion / attachment & arrest(few seconds)
CD11aCD18 / LFA-1 / aLb2 integrin (CD18 is selectively required for Th2, but not Th1, homing at inflammatory sites and has a minimal influence on T-effector development) : LFA-1 activation induces phosphorylation of the b2 integrin chain and release of Jun-activating binding protein 1 (JAB-1), and mediates signaling of kinase Erk1/2 through cytohesin-1. LFA-1 stimulation lowers the threshold of Th1 cell activation.  Despite it is widely believed that rolling lymphocytes require successive chemokine-induced signaling for LFA-1 to achieve a threshold avidity that will mediate lymphocyte arrest, endothelium-presented but not soluble chemokines trigger instantaneous extension of bent LFA-1 in the absence of LFA-1 ligand engagement. To support lymphocyte adhesion, this extended LFA-1 conformation required immediate activation by ICAM1ref. Lymphocytes failed to home in one strain of LFA1-deficient miceref, whereas some residual homing was reported in a second strain, which was mediated by ...	CD54 / ICAM-1 (up-regulated by IL-1b, too)  CD102 / ICAM-2 (constitutive)ref1, ref2, ref3
CD49dCD? / a4b7 integrin (only on gut-homing T cells, e.g. those specific for rotavirusref)ref : mouse studies indicate that DCs derived from Peyer's patches can preferentially induce the expression of a4b7 by newly generated effector cells in vitroref1, ref2	peripheral node addressins (PNAd) (SO42--GAGs recognized by the mAb MECA-79) :  MAdCAM-1  CD106 / VCAM-1 / INCAM-110 complement S-protein / PA-I-BP1 / vitronectin / somatomedin B
DC-SIGN	CD102 / ICAM-2 (constitutive)
CD49eCD29 / VLA-5 / a5b1 integrin	fibronectin 1
CD49fCD29 / VLA-6 / a6b1 integrin	laminin
CD43ref1, ref2 : conflicting reports
gp85CD44 / H-CAM / Hermes / ECMR III / HUTCH-1ref1, ref2 : conflicting reports
	vascular adhesion protein 1 (VAP-1)ref
step 4) : diapedesis / transmigration / transendothelial migration (TEM) (10')
gp85CD44 / H-CAM / Hermes / ECMR III / HUTCH-1	hyaluronic acid (HA)

T lymphocytes use 2 different chemokine-triggered actin remodeling programs: the first, DOCK2 dependent, to locomote laterally along apical and basal endothelial surfaces; the second, DOCK2 independent, to cross through a chemokine-bearing endothelial barrier^ref.
GnRH-I and GnRH-II induce adhesion to laminin and chemotaxis toward CXCL12 / SDF-1, and augment entry in vivo of metastatic T-lymphoma into the spleen and bone marrow.
CCL19 and CCL21 are localized to HEVs and are involved in controlling T-cell trafficking to lymph nodes or Peyer's patches.
The GTPase RAP1 (which is known to activate integrins, such as LFA1 (CD11aCD18) and VLA4 (a₄b₁ integrin), and invoke cell polarization and motility) effector molecule RAPL has crucial functions in lymphocyte and dendritic cell trafficking^ref.
LT-B₄ from activated mast cells induces rapid integrin-mediated arrest of rolling lymphocytes in postcapillary venules via BLT₁ / P2Y₇ signaling : naive and central memory CD8⁺ CTLs home to secondary lymphoid tissues, whereas effector T_h1 and T_h2 lymphocytes migrate efficiently to inflamed tissues. After differentiating into effector cells, the progeny of the responding cells reenter the circulation through efferent lymph and disseminate throughout the body. By means of expression of new cell surface-homing molecules, the effector cells acquire the capability to migrate preferentially to tissues that are connected to the lymphoid organs in which they first encountered antigen, penetrate capillary blood vessels and thus come into direct contact with parenchymal cells. T-cell activation by DCs from mesenteric LNs and Peyer's patches, but not from the spleen or cutaneous LNs, induces the expression of gut-homing receptors by effector cells (integrin a₄b₇^ref and CCR9^ref), indicating that DCs assume a lymphoid tissue-specific phenotype, which instructs T cells about where to find their antigen in the periphery. Whether this imprinting is an instructive or selective process remains to be determined.
Parenchymal cells are either induced to undergo apoptosis by killer cells or instructed to destroy the intracellular pathogen via cytokines such as IFN-g. Allowing all these T lymphocytes to survive en masse would lead to congestion in the lymphoid tissues and thereby  compromise subsequent immune responses. To cope with this problem, over the weeks that follow pathogen clearance, > 90% of effector T cells die. Contraction is controlled by early inflammation but is not essential for the generation of protective memory CD8⁺ T cells after infection : antibiotic treatment before Listeria monocytogenes infection induced numbers of protective memory CD8+ T cells similar to those in control infected mice, by a pathway without contraction, thanks to decreased early inflammation and IFN-g production and an increased fraction of CD8⁺ T cells expressing the IL-7R at the peak of the response^ref. Rather than reflecting a default pathway, the elimination of effector cells appears to reflect a tightly regulated instructional process involving multiple cell death-inducing mechanisms acting in consort. The sequence of molecular events required to initiate the death of effector cells is still unclear, but is thought to involve negative signaling by CTLA-4 and PD-1 receptors for costimulatory signals, activation of the Fas death pathway by dissociation of cFLIP from Fas, and onset of sensitivity to inhibition by several cytokines, notably IL-2 : prominent and progressive hypertrophy of the secondary lymphoid tissue occurs with deletion or mutation of Fas/FasL, PD-1, CTLA-4, NFAT, CD25, TGF-b₁ and several other molecules. Shutdown of an acute virus-specific CD8⁺ T cell immune response to viral infection is mediated by the proapoptotic BH3-only protein BIM^ref. Previous stidues have shown that BIM is essential for the deletion of autoreactive B and T cells.
• production of memory T lymphocytes : they represent the descendants of proliferating cells, which, at least transiently, express effector functions such as perforin^ref, granzyme B^ref, and cytokine production^ref.

There is a strong correlation between memory cell production and the intensity of the primary response : irrespective of the dose of antigen eliciting the response and the TcR-MHC-peptide affinities involved, the proportion of CD8⁺ cells that survive the response to become memory cells is remarkably constant, i.e., about 5 to 10% of the peak numbers of cells generated at the height of the response. One explanation for this apparent paradox is that all "successful" primary immune responses that lead to rapid rejection of pathogens may have essentially similar kinetics. Thus, whatever the initial dose or route of infection, the pathogen replicates untilthe concentration of antigen in the lymphoid tissue is sufficiently high to induce a powerful immune response, which then leads to rejection of the pathogen, usually after 7 to 10 days. As T cells are mobile cells, it is therefore quite likely that virtuall all T precursor cells will come into contact with antigen, probably in high concentrations, before the pathogen is cleared. However, the duration of exposure to antigen is likely to vary considerably, depending upon the anatomical distribution of the precursor cells. For typical infections occurring in mucosal sites, e.g., the lung and gut, the immune response is initiated in the draining lymph nodes and involved blood-borne recruitment of recirculating lymphocytes from throughout the body. Here, it should be borne in mind that lymph nodes are small structures with a limited blood supply and that the tempo of blood-to-lymph recirculation of lymphocytes is quite slow, 12 to 24 hours. Hence, the time taken for individual recirculating lymphocytes to enter an infected lymph node randomly varies enormously, from seconds for some cells to many days for others. Consequently, cells recruited late in the immune response will see antigen for a much briefer period than the cells initially recruited. The duration of exposure to antigen could be an important factor in memory-cell generation. Thus, the cells destined to die at the end of the immune response could be the progeny of cells undergoing a prolonged response, protracted to switch on the death pathways. Conversely, for latecomer cells arriving near the end of the response, brief exposure to antigen could be sufficient to cause these cells to proliferate and differentiate into effector cells, but could be inadequate to activate the various cell death pathways that are required for effector-cell elimination. According to the above model, generation of memory cells reflects incomplete differentiation of a subset of cells that avoids death (overstimulation) by arriving late in the immune response. It would follow that subjecting all responding lymphocytes to prolonged antigenic stimulation would induce full differentiation and death, thereby precluding memory-cell generation. The finding that overwhelming infection from DOCILE (LCMV-D) or Cl-13 Armstrong (Cl-13)) strains of LCMV can lead to total elimination ("exhaustion" or high zone tolerance) of the responding CTLs after a prolonged proliferative response is consistent with this idea^{ref1, ref2}.
They persist  in secondary lymphoid organs (lymphoid memory cells) but also in a variety of peripheral tissues (peripheral memory cells), including the lungs. Effector memory CTLs lose expression of both p90-110^{L-selectin / CD62L / MEL-14 / LAM-1 / LECAM-1 / Leu-8}, so they fail to roll in HEVs and cannot home to LNs. While lymphoid memory cells number remains relatively stable for the life, the number of peripheral ones declines within months after antigen clearance. Homeostatic proliferation to maintain T cell memory requires the presence of antigen^ref but memory CD8 T cells survive well after transfer to MHC I^-/- hosts^ref. Hence, the longevity of memory cells, in contrast to that of naive T cells, does not seem to depend on TcR ligation of trace amounts of antigen left over from the primary response and/or contact with cross-reactive environmental antigens. A notable feature of memory T lymphocytes is that the normal rate of division (turnover) of these cells in vivo is substantially higher than for naive T lymphocytes. Significantly, the relatively rapid turnover of memory T cells occurs after transfer into MHC^-/- hosts, indicating that the stimulus for proliferation is MHC-independent. The antigen-dependent phase of CD4⁺ and CD8⁺ T lymphocyte differentiation may have a stem-cell-like stage of development analogous to the BCL6⁺ memory B cell thanks to the transcriptional repressor homolog BCL6B / BAZF, and this phase may enable the life-long generation of antigen-specific effector T cells. Expression of serine protease inhibitor 2A (SPI2A), which inhibits programmed cell death (PCD) triggered by cathepsins, is upregulated by memory T cells but not by naive T cells^ref.
• production of CD4⁺5⁺25 / IL-2Ra^-27⁺44⁺45 / B220 / LCARA^-RO⁺122 / IL-2Rb⁺NKG2D^-197 / CCR7⁺memory T lymphocytes : in mice they appear only 3 weeks after the antigen has gone. So they don't appear in chronic infections (e.g. HIV-1, Mycobacterium tuberculosis, Mycobacterium leprae, ...). Vaccine boosters should be timed for the period when the first antigen has been cleared, and a stable population of memory CTL has been developed.
• CD43^hi : fully matured memory phenotype
• CD43^int : memory functionality is marginal
• CD43^lo : anergic cells that undergo spontaneous apoptosis via a caspase-independent pathway or Fas-mediated apoptosis via a caspase-dependent pathway, even in the resting stage.
They persist in the lungs of mice over the course of ~ 3 months after experimental infection. Effector memory T lymphocytes show polarized cytokine gene acetylation patterns in vivo, whereas central memory T cells have hypoacetylated cytokine genes but acquire polarized acetylation and expression after appropriate stimulation. However, hypoacetylation of the nonexpressed cytokine gene doesn't lead to irreversible silencing because most T_h1 and T_h2 cells acetylate and expresse the alternative gene when stimulated under opposite T_h conditions. Such cytokine flexibility is absent in a subset of T_h2 lymphocytes that fail to up-regulate T-bet and to express IFN-g when stimulated under T_h1 conditions. Thus, most human CD4⁺ T lymphocytes retain both memory and flexibility of cytokine gene expression. The stimulus for proliferation is likely to be a CD132 / g_c-independent cytokine : IL-7 is an important survival factor for CD4⁺ memory T lymphocytes that together with TcR signals promotes survival of both central memory (CD62L⁺, lymph-node homing) and effector memory (CD62L^-) CD4⁺ lymphocytes in lymphopenic conditions and in the intact immune system, concomitant with upregulation of the anti-apoptotic protein Bcl-2 and memory cell markers^{ref1, ref2}.
• production of CD8⁺ memory T lymphocytes from effector CTL (5-10% of all activated CTLs, those CD8aa⁺IL-7R^hi) : the presence of cognate, antigen-specific CD4⁺ T cells is important only after, not during, the early CD8⁺ T cell programming phase (in the context of an acute infection, in the absence of CD4⁺ T cells, memory CD8⁺ T cells become functionally impaired and decrease in quantity over time)^ref. DC licensing by CD4⁺ T helper cells is rapid and essential for the formation of effector and memory CTLs. In situations in which DCs are poorly licensed by pathogen-derived signals, CTL immunity may be heavily dependent on cognate DC licensing^ref. They are more resistant to apoptosis than naive T lymphocytes. CD8aa is transiently induced on a selected subset of CD8ab⁺ T lymphocytes from the spleen upon stimulation through CD3, after stimulation of OT-1 TcR-transgenic splenocytes with their cognate antigen (ovalbumin) or after infection of mice with LCMV. This effect is greatest when OT-1 splenocytes were stimulated with OVA presented by TL-expressing APCs, indicating that interaction between CD8aa and TL is necessary to maintain CD8aa expression. These CD8aa molecules promote the survival for up to 60 days after infection (resistance to apoptosis via induction of the anti-apoptotic proteins Bcl-2 and Bcl-X_L) and differentiation of activated lymphocytes into memory CD8 T lymphocytes. An enhancer deletion in the mouse Cd8 locus that prevents the high-level expression of CD8a required to form CD8aa homodimers, but still allows CD8aa heterodimer formation, did not affect the primary response to LCMV but led to low numbers and a poor in vitro response of LCMV-specific T cells 50 days after infection. Thus, memory precursors can be identified among primary effector cells and are selected for survival and differentiation by CD8aa. E8₁ knockout mice challenged with LCMV have considerably reduced numbers of antigen-specific memory T lymphocytes^ref. The amount of CD8aa⁺ T lymphocytes responding to the vaccine will reflect the size and effectiveness of the memory the patient will form against that particular pathogen
• "resting" or nonpolarized "central" memory T lymphocytes (T_CM) (CD5⁺25 / IL-2Ra^-27⁺44⁺45 / B220 / LCARA^-RO⁺62L / MEL-14^hi122 / IL-2Rb⁺195 / CCR5^-197 / CCR7⁺NKG2D⁺, BLT₁ / P2Y₇^int). They continue to recirculate through peripheral lymph nodes where have as stem-cell like function (consistent with the relatively small fraction of the total antigen-specific T lymphocytes pool that these cells represent) in mounting strong recall responses whenever the antigen returns. They are smaller than peripheral effector memory T lymphocytes, and produce :
• much IL-2
• low IFN-g
• low IL-4
• low IL-5
They don't contain perforin; after IL-15 stimulation they home in inflammed tissues, need to be stimulated with Ag before producing cytokines (T_h1) or acquiring cytolytic activity (CD8ab⁺) at the second challenge. T_CM have a greater capacity than T_EM to persist in vivo and are more efficient in mediating protective immunity because of their increased proliferative potential. CD8 memory is CD4 independent and there is no requirement for long term retention of immune complexes on FDCs, nor for B cells as APCs^ref.  In normal unstimulated mice IL-15 is directly stimulatory for gp85^{CD44 / H-CAM / Hermes / ECMR III / HUTCH-1hi}CD8⁺ lymphocytes^ref but not for CD44^hiCD4⁺ lymphocytes (because CD122 / IL-2Rb is expressed at a much higher level on CD44^hiCD8⁺ than on other subsets, including CD44^hiCD4⁺ lymphocytes).
‡
+ IL-7, IL-15 and IL-15Ra; T-bet and eomesodermin are responsible for inducing enhanced expression of CD122 / IL-2Rb^ref
• early or polarized"effector" memory T lymphocytes (T_EM / T_EFF) (CD5⁺25 / IL-2Ra^-27^-28⁺44⁺45 / B220 / LCARA^-RO⁺62L / MEL-14^lo122 / IL-2Rb⁺195 / CCR5⁺CCR6⁺197 / CCR7^-NKG2D⁺) : larger, home to peripheral tissues (liver and lungs), more differentiated (produce effector cytokines :
• many IFN-g
• many IL-4
• many IL-5
• low IL-2
..., contain perforin; after IL-2 stimulation they home to lymphoid organs, readily secrete cytokines (T_h1 lymphocytes) or have direct cytolytic activity (CD8ab⁺) at the second challenge)
‡  the duration of this differentiation is programmed within the first week after immunization
• CD27⁺28⁺45 / B220 / LCARA^-RO⁺195 / CCR5⁺197 / CCR7⁺

‡
• intermediate CD8⁺ memory T-lymphocytes
• CD5⁺25 / IL-2Ra^-27⁺28^-44⁺45 / B220 / LCARA^-RO⁺122 / IL-2Rb⁺195 / CCR5⁺CCR6⁺w197 / CCR7^-NKG2D⁺
‡
• CD27^-28^-45 / B220 / LCARA^-122 / IL-2Rb⁺195 / CCR5⁺CCR6⁺w197 / CCR7^-NKG2D⁺
• late CD8⁺ memory T-lymphocytes (CD5⁺25 / IL-2Ra^-27^-28^-44⁺45 / B220 / LCARA^-RO⁺122 / IL-2Rb⁺195 / CCR5^-CCR6⁺w197 / CCR7^-NKG2D⁺)
‡
CD5⁺25 / IL-2Ra^-27^-28^-45 / B220 / LCARA⁺RO^-57⁺122 / IL-2Rb⁺195 / CCR5^-CCR6⁺w197 / CCR7^-NKG2D⁺
They persist in the lungs of mice over the course of ~ 6 months after experimental infection.
Homeostatic proliferation–induced memory (HP-memory) CD8⁺ T cells controlled bacterial infection as effectively as 'true' memory CD8⁺ T cells, but their protective capacity required the presence of CD4⁺ T cells during homeostatic proliferation. The necessity for CD4 help was overcome, however, if the HP-memory CD8⁺ T cells lacked expression of TRAIL. Thus, like conventional CD8⁺ memory T cells, the protective function of HP-memory CD8⁺ T cells shows dependence on CD4⁺ T cell help^ref

(reproduced with permission from Nature Reviews Immunology (Vol 3, No. 11, pp 867-878 (2003)) copyright Macmillan Magazines Ltd)

S1P₁, which is highly expressed by T and B cells, is essential for lymphocyte recirculation^ref (i.e. T-cell exit from the thymus and B cell leave from peripheral lymphoid organs into the circulation to mediate their effector functions : agonism inhibits transendothelial migration of medullary T cells to lymphatic sinuses^ref). RAPL is indispensable in the integrin-mediated adhesion and migration of lymphocytes and dendritic cells, essential for immunosurveillance^ref. Sphingosine kinase and sphingosine-1-phosphate regulate migration, endocytosis and apoptosis of dendritic cells via S1P₁ > S1P₂ = S1P₃^ref

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