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 duratio