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CD103 (alpha(E)) integrin expression distinguishes a population of dendritic cells (DCs) that can be found in many if not all lymphoid and non-lymphoid organs. CD103(+) DCs display distinct functional activities. Migratory CD103(+) DCs derived from skin, lung, and intestine efficiently present exogenous antigens in their corresponding draining lymph nodes to specific CD8(+) T cells through a mechanism known as cross-presentation. On the T cells they prime, intestinal CD103(+) DCs can drive the induction of the chemokine receptor CCR9 and alpha(4)beta(7) integrin, both known as gut-homing receptors. CD103(+) DCs also contribute to control inflammatory responses and intestinal homeostasis by fostering the conversion of naive T cells into induced Foxp3(+) regulatory T cells, a mechanism that relies on transforming growth factor-beta and retinoic acid signaling. This review discusses recent findings that identify murine CD103(+) DCs as important regulators of the immune response.

During their final maturation in the medulla, semimature single-positive (SP) thymocytes downregulate activation markers and subsequently exit into the periphery. Although semimature CD4+ SP cells are sensitive to negative selection, the timing of when negative selection occurs in the CD8 lineage remains elusive. We show that the abundance of terminally matured CD8+ SP cells in adult thymus is modulated by the genetic background. Moreover, in BALB/c mice, the frequency of terminally matured CD8+ SP cells, but not that of CD4+ SP cells present in thymus, varies depending on age. In mice lacking expression of the adhesion receptor CD155, a selective deficiency of mature CD8+ SP thymocytes was observed, emerging first in adolescent animals at the age when these cells start to accumulate in wild-type thymus. Evidence is provided that the mature cells emigrate prematurely when CD155 is absent, cutting short their retention time in the medulla. Moreover, in nonmanipulated wild-type mice, semimature CD8+ SP thymocytes are subjected to negative selection, as reflected by the diverging TCR repertoires present on semimature and mature CD8+ T cells. In CD155-deficient animals, a shift was found in the TCR repertoire displayed by the pool of CD8+ SP cells, demonstrating that CD155 is involved in negative selection.

International audience; Intestinal intraepithelial lymphocytes carrying the γδ TCR (γδ iIEL) are involved in the maintenance of epithelial integrity. γδ iIEL have an activated phenotype, characterized by CD69 expression and increased cell size compared with systemic T lymphocytes. As an additional activation marker, the majority of γδ iIEL express the CD8αα homodimer. However, our knowledge about cognate ligands for most γδ TCR remains fragmentary and recent advances show that γδ T cells including iIEL may be directly activated by cytokines or through NK-receptors, TLR and other pattern recognition receptors. We therefore asked whether the TCR of γδ iIEL was functional beyond its role during thymic selection. Using TcrdH2BeGFP (Tcrd, T-cell receptor δ locus; H2B, histone 2B) reporter mice to identify γδ T cells, we measured their intracellular free calcium concentration in response to TCR-crosslinking. In contrast to systemic γδ T cells, CD8αα(+) γδ iIEL showed high basal calcium levels and were refractory to TCR-dependent calcium-flux induction; however, they readily produced CC chemokine ligand 4 (CCL4) and IFN-γ upon TCR triggering in vitro. Notably, in vivo blocking of the γδ TCR with specific mAb led to a decrease of basal calcium levels in CD8αα(+) γδ iIEL. This suggests that the γδ TCR of CD8αα(+) γδ iIEL is constantly being triggered and therefore functional in vivo.

Dendritic cells (DCs) residing in the lung are known to acquire inhaled Ag and, after migration to the draining bronchial lymph node (brLN), to present it to naive T cells in an either tolerogenic or immunogenic context. To visualize endogenous lung-derived DCs, we applied fluorescent latex beads (LXs) intratracheally, thereby in vivo labeling the majority of phagocytic cells within the lung. Of note, LX-bearing cells subsequently arriving in the draining brLN were found to represent lung-derived migratory DCs. Imaging explanted brLN by two-photon laser-scanning microscopy, we quantitatively analyzed the migration and interaction behavior of naive CD4+ T cells and endogenous, lung-derived DC presenting airway-delivered Ag under inflammatory or noninflammatory conditions. Ag-specific naive CD4+ T cells engaged in stable as well as transient contacts with LX-bearing DCs in both situations and displayed similar overall motility kinetics, including a pronounced decrease in motility at 16–20 h after antigenic challenge. In contrast, the comparative analysis of T cell–DC cluster sizes as well as contact durations strongly suggests that lung-derived migratory DCs and naive CD4+ T cells form more stable, long-lasting contacts under inflammatory conditions favoring the induction of respiratory immunity.

International audience; Induced antigen-specific Foxp3(+) T cells (iTreg) are being discussed as a promising alternative to polyclonal natural Foxp3(+) T cells (nTreg) for cell-based therapies, particularly to achieve transplantation tolerance. Using Foxp3eGFP-reporter mice, we here establish an efficient protocol to induce and expand alloantigen-specific iTreg from Foxp3(-)CD4(+) T cells with cluster-disrupted DC. These iTreg were mainly CD62L(+) and showed efficient suppressive activity in vitro. However, in contrast to nTreg, adoptively transferred iTreg entirely failed to prevent lethal graft versus host disease (GVHD). Within irradiated recipients, the majority of adoptively transferred Foxp3(+) iTreg, but not Foxp3(+) nTreg quickly reverted to Foxp3(-)CD4(+) T cells. We therefore suggest that therapeutic approaches to treat GVHD should rely on nTreg, whereas the use of de novo alloantigen-induced iTreg should be handled with caution since the stability of the regulatory phenotype of the iTreg could be of major concern.

Gammadelta T cells are a potent source of innate IL-17A and IFN-gamma, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24(low) CD44(high) effector gammadelta T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6+ gammadelta T cells produced IL-17A, while NK1.1+ gammadelta T cells were efficient producers of IFN-gamma but not of IL-17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1+ gammadelta T cells. Accordingly, both gammadelta T-cell subsets were rare in gut-associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL-17A and IFN-gamma in response to TCR-specific and TCR-independent stimuli. IL-12 and IL-18 induced IFN-gamma and IL-23 induced IL-17A production by NK1.1+ or CCR6+ gammadelta T cells, respectively. Importantly, we show that CCR6+ gammadelta T cells are more responsive to TCR stimulation than their NK1.1+ counterparts. In conclusion, our findings support the hypothesis that CCR6+ IL-17A-producing gammadelta T cells derive from less TCR-dependent selection events than IFN-gamma-producing NK1.1+ gammadelta T cells.

Naive T lymphocytes continuously recirculate through secondary lymphoid organs such as lymph nodes until they are eventually activated by recognizing cognate peptide/MHC-complexes on the surface of antigen-protecting cells. The intranodal T cell migration behavior leading to these crucial--and potentially rare--encounters during the induction of an adaptive immune response could not be directly addressed until, in 2002, the use of two-photon microscopy also allowed the visualization of cellular dynamics deep within intact lymph nodes. Since then, numerous studies have confirmed that, by default, naive T cells are extremely motile, scanning the paracortical T cell zone for cognate antigen by means of an apparent random walk. This review attempts to summarize the current knowledge of factors influencing the basal migration behavior of naive T lymphocytes within lymph nodes during steady state. Extracellular cues, such as the motility-promoting influence of CCR7 ligands and the role of integrins during interstitial migration, as well as intracellular signaling pathways involved in T cell motility, will be discussed. Particular emphasis is placed on structural features of the lymph node environment orchestrating T cell migration, namely the framework of fibroblastic reticular cells serving as migration "highways." Finally, new approaches to simulate the cellular dynamics within lymph nodes in silico by means of mathematical modeling will be reviewed.

Solitary intestinal lymphoid tissue (SILT) comprises a spectrum of phenotypically diverse lymphoid aggregates interspersed throughout the small intestinal mucosa. Manifestations of SILT range from tiny lymphoid aggregates almost void of mature lymphocytes to large structures dominated by B cells. Large SILT phenotypically resemble a single Peyer’s patch follicle, suggesting that SILT might contribute to intestinal humoral immune responses. In this study, we track the fate of individual SILT in vivo over time and analyze SILT formation and function in chemokine receptor CXCR5-deficient mice. We show that, in analogy to Peyer’s patches, formation of SILT is invariantly determined during ontogeny and depends on CXCR5. Young CXCR5-deficient mice completely lack SILT, suggesting that CXCR5 is essential for SILT formation during regular postnatal development. However, microbiota and other external stimuli can induce the formation of aberrant SILT distinguished by impaired development of B cell follicles in CXCR5-deficient mice. Small intestinal transplantation and bone marrow transplantation reveal that defect follicle formation is due to impaired B cell homing. Moreover, oral immunization with cholera toxin or infection with noninvasive Salmonella fail to induce efficient humoral immune responses in CXCR5-deficient mice. Bone marrow transplantation of CXCR5-deficient recipients with wild-type bone marrow rescued B cell follicle formation in SILT but failed to restore full humoral immune responses. These results reveal an essential role of CXCR5 in Peyer’s patch and SILT development and function and indicate that SILT do not fully compensate for the lack of Peyer’s patches in T cell-dependent humoral immune responses.

T cells are imprinted to express tissue-specific homing receptors upon activation in tissue-draining lymph nodes, resulting in their migration to the site of Ag entry. Expression of gut-homing molecules α4β7 and CCR9 is induced by retinoic acid, a vitamin A metabolite produced by retinal dehydrogenases, which are specifically expressed in dendritic cells as well as stromal cells in mucosa-draining lymph nodes. In this study, we demonstrate that mesenteric lymph node stromal cell-derived retinoic acid can directly induce the expression of gut-homing molecules on proliferating T cells, a process strongly enhanced by bone marrow-derived dendritic cells in vitro. Therefore, cooperation of sessile lymph node stromal cells with mobile dendritic cells warrants the imprinting of tissue specific homing receptors on activated T cells.

Naive T lymphocytes continuously recirculate through secondary lymphoid organs such as lymph nodes until they are eventually activated by recognizing cognate peptide/MHC-complexes on the surface of antigen-presenting cells. The intranodal T cell migration behavior leading to these crucial – and potentially rare – encounters during the induction of an adaptive immune response could not be directly addressed until, in 2002, the use of two-photon microscopy also allowed the visualization of cellular dynamics deep within intact lymph nodes. Since then, numerous studies have confirmed that, by default, naive T cells are extremely motile, scanning the paracortical T cell zone for cognate antigen by means of an apparent random walk. This review attempts to summarize the current knowledge of factors influencing the basal migration behavior of naive T lymphocytes within lymph nodes during steady state. Extracellular cues, such as the motility-promoting influence of CCR7 ligands and the role of integrins during interstitial migration, as well as intracellular signaling pathways involved in T cell motility, will be discussed. Particular emphasis is placed on structural features of the lymph node environment orchestrating T cell migration, namely the network of fibroblastic reticular cells serving as migration “highways.” Finally, new approaches to simulate the cellular dynamics within lymph nodes in silico by means of mathematical modeling will be reviewed.

Background. The chemokine receptor CCR7 plays a pivotal role in the homing of naive T cells and regulatory T cells to secondary lymphoid organs and the migration of dendritic cells into afferent lymphatic vessels. Antigen presentation, T cell recruitment, and expansion of regulatory cells are crucial events in establishing and controlling chronic allograft dysfunction. In this study, we report an important role for chemokine receptor CCR7 in the development of transplant arteriosclerosis. Methods. Fully major histocompatibility complex-mismatched CBA (H2 k ) donor aortas were transplanted into BALB/ c-CCR7 -/- (H2 d ), BALB/c-CCR7 +/- (H2 d ), or BALB/c-CCR7 +/+ (H2 d ) recipients. Grafts were analyzed by histology, morphometry, and immunofluorescence on day 30 after transplantation. Intragraft cytokine mRNA production was analyzed by realtime polymerase chain reaction on day 14 after transplantation. Results. After implanting fully major histocompatibility complex-mismatched donor aortas into CCR7-deficient recipients, transplant arteriosclerosis was significantly elevated. CD4 depletion resulted in a reduction of intima proliferation in CCR7 -/- recipients whereas CD8 depletion had no effect. Analysis of aortic grafts from CCR7 -/- recipients revealed high numbers of infiltrating CD4 + , F4/80 + , and CD205 + cells. Furthermore, intragraft cytokine production showed higher levels of interleukin-4, interleukin-12, and eotaxin mRNA expression, whereas significantly lower Foxp3 mRNA expression was observed in CCR7 -/- recipients. Conclusion. These data suggest that although alloantigen presentation in secondary lymphoid organs is hampered in CCR7-deficient recipients, this process may take place within the allograft itself, leading to increased formation of transplant arteriosclerosis. The decrease in Foxp3 expression despite increased in CD4 + T cell infiltration indicates a reduction in T regulatory cells possibly influencing the intensity of the graft rejection.

Inactivation of members of the nuclear factor-kappaB (NF-kappaB) family results in the decrease or defect of marginal zone B (MZB) cells. It is not known which inhibitors of the NF-kappaB family (IkappaB) are required for MZB cell development. Here, we show that mice with B cell-specific inactivation of the main NF-kappaB inhibitor IkappaBalpha have a marked decrease of MZB cells and their presumed precursors. They exhibited increased mortality rates after blood-borne bacterial infection, indicating the importance of MZB cells for bacterial clearance. In contrast, response to T cell-dependent and -independent antigens resulted only in minor changes in immunoglobulin production. Our data demonstrate the importance of the intact NF-kappaB/IkappaBalpha pathway for proper MZB cell development.

Chemokine receptors and their ligands are crucial for lymphocyte trafficking under both homeostatic and inflammatory conditions. The chemokine receptor CXCR5 controls B cell migration and the organization of B cell follicles. The aim of this study was to investigate the impact of CXCR5 on the development of transplant arteriosclerosis. Fully MHC mismatched BALB/c (H2(d)) donor aortas were transplanted into C57BL/6-CXCR5(-/-) (H2(b)), C57BL/6-CXCR5(+/-) (H2(b)) or C57BL/6-CXCR5(+/+) (H2(b)) recipients. Grafts were analysed by morphometry and immunofluorescence and intra-graft cytokine mRNA production was analysed by RT-PCR. Transplant arteriosclerosis was evident in CXCR5+/+ and CXCR5+/- mice and only mildly reduced in CXCR5-/- recipients indicating that absence of CXCR5 had no substantial effect on the development of transplant arteriosclerosis. Analysis of the cellular infiltrate of aortic grafts implanted in CXCR5-/- recipients revealed no differences in the number of T-cells, macrophages and B cells as compared to controls. Intra-graft cytokine production showed no significant changes in Th1 (IL-12) and Th2 (IL-4) cytokines as well as in TGF-beta and iNOS production. These data suggest that lack of CXCR5 expression by recipient T- and B-cells has little effect on the development of transplant arteriosclerosis.

All metazoan cells carry transmembrane receptors of the integrin family, which couple the contractile force of the actomyosin cytoskeleton to the extracellular environment. In agreement with this principle, rapidly migrating leukocytes use integrin-mediated adhesion when moving over two-dimensional surfaces. As migration on two-dimensional substrates naturally overemphasizes the role of adhesion, the contribution of integrins during three-dimensional movement of leukocytes within tissues has remained controversial. We studied the interplay between adhesive, contractile and protrusive forces during interstitial leukocyte chemotaxis in vivo and in vitro. We ablated all integrin heterodimers from murine leukocytes, and show here that functional integrins do not contribute to migration in three-dimensional environments. Instead, these cells migrate by the sole force of actin-network expansion, which promotes protrusive flowing of the leading edge. Myosin II-dependent contraction is only required on passage through narrow gaps, where a squeezing contraction of the trailing edge propels the rigid nucleus.

With the advent of two-photon microscopic imaging, the last few years have witnessed remarkable progress regarding our understanding of the movement behavior and interaction dynamics of different immune cells within lymphoid organs. The basal intranodal motility of naive T lymphocytes in the absence of antigenic or inflammatory stimuli, although at first glance representing a phenomenon of apparent simplicity, is far from being completely understood. The most important open question in this context relates to the origin of intranodal T-cell motility itself: how are these highly dynamic cells 'motivated' to carry out their relentless scanning of dendritic cells present in the lymph node (LN)? This review summarizes recent advances in the search for factors governing the intranodal migration behavior of naive T lymphocytes, focusing in particular on the role of extracellular pro-migratory cues, intracellular signaling components, and the influence of the structural LN environment on intranodal T-cell motility.

Journal of experimental medicine : JEM 205(11), 2483-2490 (2008). doi:10.1084/jem.20080039, Published by Rockefeller Univ. Press, New York, NY

The CD155 ligand CD96 is an immunoglobulin-like protein tentatively allocated to the repertoire of human NK receptors. We report here that the CD96/CD155-interaction is preserved between man and mouse although both receptors are only moderately conserved in amino acid sequence. Moreover, murine CD96 (mCD96) binds to nectin-1, a receptor related to CD155. Applying newly generated monoclonal antibodies specifically recognizing mCD96, an expression profile is revealed resembling closely that of human CD96 (hCD96) on cells of hematopoietic origin. A panel of anti-mCD96 but also recently established anti-mCD155 antibodies effectively prevents formation of CD96/CD155-complexes. This was exploited to demonstrate that the only available receptor for mCD96 present on thymocytes is mCD155. Moreover, T cell adhesion to insect cells expressing mCD155 is blocked by these antibodies depending on the T cell subtype. These results suggest a function of the CD96/CD155-adhesion system in T cell biology.

The chemokine receptor CCR7 has been implicated in maintenance of thymus morphology and establishment of tolerance to self-antigens. In this study, we provide direct evidence that negative selection of maturing thymocytes is defective in CCR7-deficent mice. Impaired negative selection was observed after TCR/CD3 complex stimulation in vivo as well as in vitro and was prominent in both double-positive and semimature single positive cells (CD4+CD8−CD24high). It is noteworthy that thymocytes of CCR7−/− mice display defective negative selection in response to endogenous superantigens, demonstrating that the defect also occurs under physiological conditions. Disturbed negative selection was correlated with delayed activation kinetics and decreased calcium flux response of CCR7−/− thymocytes after in vitro TCR/CD3 stimulation, suggesting that an impaired response of CCR7−/− thymocytes via TCR-mediated signaling is responsible for defective negative selection in these mice.

Background Signaling through the T cell receptor (TCR) leads to profound changes in the function and properties of T cells, including integrin activation. Adhesion and degranulation promoting adapter protein (ADAP) is an adapter protein linking T cell receptor stimulation to integrin activation. We aim to clarify how disruption of TCR-mediated integrin activation affects alloreactive immune responses. Methods In vitro T cell proliferation and the cytokine production was determined. In vivo cytotoxic T lymphocyte (CTL) activity was measured as well. Allogenic skin and heart transplantation was used to test the in vivo role of ADAP in alloimmune responses. Histology and flow cytometry was applied to analyze the graft infiltrating lymphocytes. Results Upon stimulation with allogenic dendritic cells ADAP-deficient T cells displayed impaired proliferative responses compared to wild type (WT) T cells. This was accompanied by significantly decreased production of the cytokine interleukin-2. In contrast, the in vivo CTL activity in ADAP-deficient mice was comparable to that of WT mice. Consistently, we observed a prolongation of fully major histocompatibility complex (MHC)-mismatched heart transplants in ADAP deficient mice. Protection of allogenic heart grafts in ADAP-deficient mice was accompanied by a decrease in the infiltration, proliferation and activation of T cells in the allograft. However, no effect was observed after fully MHC-mismatched skin transplantation. Conclusions We have shown that although ADAP is dispensable for the rejection of allografts, ADAP function plays an important role for the efficacy of graft rejection. ADAP's main function appears to affect the induction phase of the immune response.

Background The chemokine receptor CCR7 is a key organizer of the immune system. Gene targeting in mice revealed that Ccr7-deficient animals are severely impaired in the induction of central and peripheral tolerance. Due to these defects, Ccr7-deficient mice spontaneously develop multi-organ autoimmunity showing symptoms similar to those observed in humans suffering from connective tissue autoimmune diseases. However, it is unknown whether mutations of CCR7 are linked to autoimmunity in humans. Results DNA samples were collected from 160 patients suffering from connective tissue autoimmune disease (Sjogren's syndrome, n = 40; systemic lupus erythematosus, SLE, n = 20 and systemic sclerosis, n = 100) and 40 health subjects (n = 40). All participants in this study were of German descent. Samples were screened for single nucleotide polymorphisms (SNP) by sequencing the coding region of the CCR7 gene as well asthe exon flaking intron sites and parts of the regions encoding for the 5'- and 3'-UTR. CCR7 variants were rare. We identified six different sequence variants, which occurred in heterozygosis. The identified SNP were observed at position -60 C/T (observed 1x), +6,476 A/G (7x), +6,555 C/T (15x), +6,560 C/T (6x), +10,440 A/G (3x) and +11,475 C/A (1x). Four of these variants (+6,476 A/G, +6,555 C/T, +6,560 C/T and +10,440 A/G) display allelic frequencies between 1% and 5 % and were present in both patients and control groups. The variants +6,476 A/G, +6,555 C/T, +6,560 C/T are located in the intron 2, while the +10,440 A/G variant corresponds to a silent mutation in exon 3. The variants -60 C/T and +11,475 C/A which are located at the 5'-UTR and 3-UTR respectively, display allelic frequencies below 1%. No correlation between these variants and the autoimmune diseases investigated could be observed. However, reporter gene expression assay demonstrated that the mutation at the -60 C/T position in homozygosis leads to reduced luciferase activity. Conclusion These results suggest that variants of CCR7 gene occur at an extremely low frequency in the German population and that neither Sjogren's syndrome, systemic lupus erythematosus, nor systemic sclerosis are associated with these variants. Nevertheless, the decreased luciferase activity observed in cells transfected with the promoter region bearing the -60 C/T mutation suggests that this CCR7 variant could potentially lead to increased susceptibility to autoimmunity.

Mucosal surfaces are exposed continuously to a flood of foreign antigens demanding a tightly controlled balance between immunity and tolerance induction. Tolerance toward food and inhaled antigens, known as oral and respiratory tolerance, respectively, evokes a body-wide nonresponsiveness against the plethora of environmental antigens. Key issues in understanding the induction of mucosal tolerance relate to the site of antigen entrance, the mechanisms of antigen transport, and the exact anatomical location where lymphocytes meet their cognate antigens. In this regard, opposing ideas have been put forward: In one scenario, antigens taken up at mucosal surfaces are considered to spread throughout the body, thus potentially evoking tolerogenic immune responses in all secondary lymphoid organs. Alternatively, tolerance induction might be confined to the draining regional lymph nodes (LN). Recent observations strongly supported the latter scenario, emphasizing the importance of regional LN and their network of afferent lymphatics in this process. In this model, air-borne and intestinal antigens are captured at mucosal sites by dendritic cells, which then migrate exclusively in a CCR7-dependent way to draining regional LN. Tolerance is then induced actively by the activation of antigen-specific T cells, which are subsequently deleted, become anergic, or alternatively, differentiate into regulatory T cells. Thus, the concept of local induction of immune responses seems to hold true for the majority of immune reactions, regardless of whether they are tolerogenic or defensive in their outcome.

Small intestine plasmacytoid dendritic cells (pDC) are poorly characterized. Here, we demonstrate that intestinal pDC show the characteristic plasma cell-like morphology, and are recognized by antibodies against B220, Ly6c, 120G8, and PDCA-1, markers that are typically expressed by pDC. Furthermore, intestinal pDC carry high levels of CCR9 and are largely absent in the intestine, but not in lung, liver, or secondary lymphoid organs of CCR9-deficient animals. Competitive adoptive transfers reveal that CCR9-deficient pDC are impaired in homing to the small intestine after i.v. transfer. In a model of cholera toxin-induced gut inflammation, pDC are recruited to the intestine in WT but not CCR9-deficient animals. Furthermore, after oral application of a Toll-like receptor (TLR) 7/8 ligand, myeloid DC of the lamina propria are rapidly mobilized in WT but not in CCR9-deficient animals. Mobilization of myeloid DC can be completely rescued by adoptively transferred WT pDC to CCR9-deficient mice before oral challenge. Together, our data reveal an essential role for CCR9 in the homing of pDC to the intestine under homeostatic and inflammatory conditions and demonstrate an important role for intestinal pDC for the rapid mobilization of lamina propria DC.

Development of autoimmunity is a multi-factorial process involving genetic predisposition as well as environmental and stochastic factors. Although the mechanisms responsible for the initiation of autoimmunity remain only partially understood, several studies have demonstrated that genetic predisposition plays a major role in this process. In the present study, we analyzed the influence of CCR7 signaling in the development of autoimmunity, because this chemokine receptor is essentially involved in the functional organization of thymus architecture. We demonstrate that CCR7-deficient mice are prone to develop generalized multi-organ autoimmunity. The autoimmune phenotype of CCR7-/- mice encompasses the presence of lymphocyte infiltrates in several peripheral organs, circulating autoantibodies against a multitude of tissue-specific antigens and IgG deposition on renal glomeruli. Additionally, CCR7-deficient mice show increased susceptibility to streptozotocin-induced diabetes and spontaneously display signs of chronic autoimmune renal disease. Thus, this study identifies CCR7 as a genetic factor involved in the regulation of autoimmunity.

The distinct combination of homing receptors such as selectins, chemokine receptors, and integrins directs the migration of lymphocytes throughout the body. Upon activation lymphocytes irreversibly switch their set of homing receptors, now guiding them to entirely different destinations. Here we report that exposure of naive B cells to the microenvironment of the peritoneal cavity modulates their migration propensities in the absence of antigenic stimulation. B1 and B2 cells isolated from the peritoneal cavity reenter this compartment more efficiently compared with splenic follicular B cells. Moreover, when kept in the peritoneal cavity splenic follicular B cells gain such increased capability to reenter this compartment. These altered migratory capacities are reflected by an up-regulation of the chemokine receptors CXCR4 and CXCR5 and β7 integrin by the peritoneum-experienced splenic B cells, among which CXCR5 is instrumental in directing B cells into the peritoneal cavity. Moreover, intraperitoneal transfer of plasma blasts favors their migration into the small intestine presumably before class switch recombination occurs, demonstrating that a reconfigured transient migration pattern is not restricted to naive cells. In conclusion, these data demonstrate a hitherto unrecognized role for tissue-specific cues, altering the migratory capacity of B1, naive B2, as well as antigen-experienced B2 cells.

Early studies identified the CC-chemokine receptor (CCR)7 as an important homing molecule controlling the lymph node entry of naive T cells through high endothelial venules and of activated mature dendritic cells through afferent lymphatics. Consequently, these properties initially branded CCR7 as a central organizer of the primary immune response. However, recent studies have demonstrated that a variety of immune cells crucially rely on CCR7-directed migration not only for the induction of protective immunity but also for the establishment of immunological tolerance. In this review, we therefore highlight some of the recent advances in understanding the multiple roles of CCR7 in the induction and maintenance of central and peripheral tolerance.

Unserer Ergebnisse zeigen, dass die T Zell-Rezeptor-vermittelte Integrinaktivierung durch das »Adhesion and Degranulation promoting Adapter Protein« (ADAP) eine wichtige Rolle bei alloreaktiven Abstosungsreaktionen spielt. ADAP konnte daher eine neuartige therapeutische Zielstruktur fur das Targeting der Induktionsphase von Abstosungsreaktionen darstellen.

Accumulating evidence indicates that regulatory T (Treg) cells control development of various diseases both systemically and locally. However, molecular mechanisms involved in Treg cell homing remain elusive. We have shown previously that αβTCR+CD3+CD4−CD8− double-negative (DN) Treg cells selectively accumulate in tolerant allografts to maintain localized immune regulation. However, the molecular mechanism leading to the accumulation of DN Treg cells in tolerant grafts was not known. Our cDNA microarray analysis revealed significant up-regulation of chemokine receptor CXCR5 mRNA in DN Treg clones compared with nonregulatory clones. In this study, we examined the importance of CXCR5 in mediating DN Treg migration. Compared with CD4 and CD8 T cells, both primary DN Treg cells and clones constitutively express high levels of CXCR5 protein, enabling them to migrate toward increasing CXCL13 gradients in vitro. After infusion into recipient mice, CXCR5+ DN Treg clones, but not their CXCR5− mutants, preferentially accumulated in cardiac allografts and could prevent graft rejection. Furthermore, we found that allogeneic cardiac allografts express high levels of CXCL13 mRNA compared with either recipient native hearts or nontransplanted donor hearts. Ab neutralization of CXCL13 abrogated DN Treg cell migration in vitro and prevented in vivo homing of DN Treg clones into allografts. These data demonstrate that DN Treg cells preferentially express CXCR5, and interaction of this chemokine receptor with its ligand CXCL13 plays an important role in DN Treg cell migration both in vitro and in vivo.

Oral tolerance induction is a key feature of intestinal immunity, generating systemic nonresponsiveness to ingested antigens. In this study, we report that orally applied soluble antigens are exclusively recognized in the intestinal immune system, particularly in the mesenteric lymph nodes. Consequently, the initiation of oral tolerance is impeded by mesenteric lymphadenectomy. Small bowel transplantation reveals that mesenteric lymph nodes require afferent lymph to accomplish the recognition of orally applied antigens. Finally, oral tolerance cannot be induced in CCR7-deficient mice that display impaired migration of dendritic cells from the intestine to the mesenteric lymph nodes, suggesting that immunologically relevant antigen is transported in a cell-bound fashion. These results demonstrate that antigen transport via afferent lymphatics into the draining mesenteric lymph nodes is obligatory for oral tolerance induction, inspiring new therapeutic strategies to exploit oral tolerance induction for the prevention and treatment of autoimmune diseases.

Summary: Maintenance of the peripheral T-cell pool throughout the life requires uninterrupted generation of T cells. The majority of peripheral T cells are generated in the thymus. However, the thymus does not contain hematopoietic progenitors with unlimited self-renewing potential, and continuous production of T cells requires importation of such progenitors from the bone marrow into the thymus. Thymus-homing progenitors enter the thymus and subsequently migrate throughout distinct intrathymic microenvironments while differentiating into mature T cells. At each step of this scheduled journey, developing thymocytes interact intimately with the local stroma, which allow them to proceed to the next stage of their differentiation and maturation program. Undoubtedly, thymocyte/stroma interactions are instrumental for both thymocytes and stroma, because only their ongoing interplay generates and maintains a fully operational thymus, able to guarantee unimpaired T-cell supply. Therefore, proper T-cell generation intrinsically involves polarized cell migration during both adult life and embryogenesis when the thymus primordium develops into a functional thymus. The molecular mechanisms controlling cell migration during thymus development and postnatal T-cell differentiation are beginning to be defined. This review focuses on recent data regarding the role of cell migration in both colonization of the fetal thymus and T-cell development during postnatal life in mice.

The immunomodulatory drug FTY720 interferes with sphingosine-1-phosphate (S1P) receptor signaling leading to lymphocyte retention in secondary lymphoid organs and consequently to profound lymphopenia in the peripheral blood. The molecular mechanisms transduced by S1P receptors upon being triggered by its native ligand, S1P, or by FTY720, are largely unknown. In this study we analyze the role of beta2 and beta7 integrin and their ligands ICAM-1, VCAM-1, and MadCAM-1 on lymphocyte homing in the presence of FTY720. We demonstrate that this drug facilitates homing of lymphocytes single-deficient of either beta2 or beta7 integrin but not of beta2-deficient lymphocytes, which in addition were blocked by anti-beta7 integrin Abs. Enhanced lymphocyte homing is preceded by increased adherence of integrin-deficient as well as wild-type lymphocytes to high endothelial venules (HEV) in FTY720-treated animals. Elevated adherence to HEV requires intact lymphocyte Galphai signaling that cannot be stably imprinted on lymphocytes even after prolonged exposure to FTY720. Thus, FTY720 influences lymphocyte homeostasis not only by suppressing lymphocyte egress from lymph nodes but also by facilitating lymphocyte homing across HEV in an integrin-dependent fashion.

The chemokine receptor CXCR5 and its ligand CXCL13 define the structure of B cell follicles within secondary lymphoid organs. Here, we examined the impact of CXCR5 on antiviral B cell responses in vivo. CXCR5−/− mice showed a normal production of IgM and IgG acutely after infection with vesicular stomatitis virus (VSV) and developed VSV-specific germinal centers. However, impaired Ig class switch and Ab production were observed under conditions of limited availability of Ag (i.e., after immunization with nonreplicating viral particles or soluble Ag). Adoptive transfer of CXCR5-deficient, VSV-specific B and Th cells demonstrated that CXCR5 expression on both B and Th cells is required for an efficient Ig class switch. These experiments revealed that CXCR5 is critical for the coordinated interaction of antiviral T and B cells through its impact on initial B cell expansion and the recruitment of Ag-specific B and Th cells to germinal centers.

Gut-associated lymphoid tissue (GALT) dendritic cells (DCs) display a unique ability to generate CCR9 + α 4 β 7 + gut-tropic CD8 + effector T cells. We demonstrate efficient induction of CCR9 and α 4 β 7 on CD8 + T cells in mesenteric lymph nodes (MLNs) after oral but not intraperitoneal (i.p.) antigen administration indicating differential targeting of DCs via the oral route. In vitro, lamina propria (LP)–derived DCs were more potent than MLN or Peyer's patch DCs in their ability to generate CCR9 + α 4 β 7 + CD8 + T cells. The integrin α chain CD103 ( α E) was expressed on almost all LP DCs, a subset of MLN DCs, but on few splenic DCs. CD103 + MLN DCs were reduced in number in CCR7 − / − mice and, although CD8 + T cells proliferated in the MLNs of CCR7 − / − mice after i.p. but not oral antigen administration, they failed to express CCR9 and had reduced levels of α 4 β 7. Strikingly, although CD103 + and CD103 − MLN DCs were equally potent at inducing CD8 + T cell proliferation and IFN- γ production, only CD103 + DCs were capable of generating gut-tropic CD8 + effector T cells in vitro. Collectively, these results demonstrate a unique function for LP-derived CD103 + MLN DCs in the generation of gut-tropic effector T cells.

Background. The clinical application of small bowel transplantation (SBTx) is hampered by its pronounced immunogenicity. We aimed to test the hypothesis that prolonged sequestration of lymphocytes in secondary lymphoid organs may enhance the alloprotective effect of costimulation blockade. Methods. For this purpose, recipients of intestinal allografts were treated with MR1, FTY720, combined FTY720 plus MR1, or were left untreated. Grafts were examined 6 and 14 days after transplantation by applying a histologic rejection score, multiparameter-immunofluorescent staining, and flow cytometry. Results. FTY720 or MR1 monotherapy did not prevent the rejection of mouse intestinal allografts, whereas combined therapy with FTY720 plus MR1 profoundly inhibited rejection at day 6 and day 14 after transplantation. In FTY720-treated mice infiltration of host lymphocytes in graft mesenteric lymph nodes, Peyer's patches, intraepithelial lymphocytes, and lamina propria lymphocytes (LPLs) was reduced on day 6. Anti-CD40L antibody improved the rejection score at day 14 but had no effect at day 6. Importantly, host CD8 + T-cell infiltration in graft LPLs was significantly reduced compared with all other groups. Conclusion. FTY720 plus MR1 effectively inhibited intestinal allograft rejection in mice, possibly by enhancing the alloprotecitve effects of costimulation blockade by prolonged sequestration of lymphocytes in secondary lymphoid organs.

The aetiology of primary B-cell lymphomas of the thymus is enigmatic. Although thymic follicular lymphoid hyperplasia (TFH) is commonly associated with myasthenia gravis (MG), lymphoma is not a complication of this condition. The present paper reports a high frequency of monoclonal B-cell populations (6 of 18 cases; 33%) in micronodular thymoma (MNT), a peculiar thymic epithelial neoplasm with a B-cell-rich stroma, while B cells were consistently polyclonal in TFH (25 cases) and other types of thymomas (15 cases) (p < 0.001). An intratumoural lymphoma could be identified in three of the six monoclonal MNTs. Sequencing of the monoclonal IgH chain revealed partially overlapping VDJ gene usage in MNT and thymic mucosa-associated lymphoid tissue (MALT) lymphomas. The neoplastic epithelium of MNTs, but not of TFH and other types of thymoma, expressed high levels of dendritic cell, T-cell, and B-cell chemoattractants, such as CCL18, CCR6, and CCL20. It is concluded that abnormal chemokine expression in an epithelial tumour, MNT, can promote the recruitment of MALT, the emergence of monoclonal B cells, and, eventually, the subsequent development of mediastinal lymphomas. More generally, the concept that expression of a 'high-risk' spectrum of chemokines due to local or genetic factors may interfere with B-cell homeostasis and may contribute to MALT lymphoma development in chronic inflammatory states is proposed.

Cytoadherence of Plasmodium falciparum -infected erythrocytes (PRBC) to endothelial cells causes severe clinical disease, presumably as a of result perfusion failure and tissue hypoxia. Cytoadherence to endothelial cells is increased by endothelial cell activation, which is believed to occur in a paracrine fashion by mediators such as tumor necrosis factor alpha (TNF-α) released from macrophages that initially recognize PRBC. Here we provide evidence that PRBC directly stimulate human endothelial cells in the absence of macrophages, leading to increased expression of adhesion-promoting molecules, such as intercellular adhesion molecule 1. Endothelial cell stimulation by PRBC required direct physical contact for a short time (30 to 60 min) and was correlated with parasitemia. Gene expression profiling of endothelial cells stimulated by PRBC revealed increased expression levels of chemokine and adhesion molecule genes. PRBC-stimulated endothelial cells especially showed increased expression of molecules involved in parasite adhesion but failed to express molecules promoting leukocyte adhesion, such as E-selectin and vascular cell adhesion molecule 1, even after challenge with TNF-α. Collectively, our data suggest that stimulation of endothelial cells by PRBC may have two effects: prevention of parasite clearance through increased cytoadherence and attenuation of leukocyte binding to endothelial cells, thereby preventing deleterious immune reactivity.

The chemokine receptor CCR7 is a key factor in the coordinate migration of T cells and dendritic cells (DC) into and their localization within secondary lymphoid organs. In this study we investigated the impact of CCR7 on CD8+ T cell responses by infecting CCR7−/− mice with lymphocytic choriomeningitis virus (LCMV). We found that the absence of CCR7 affects the magnitude of an antiviral CTL response during the acute phase, with reduced numbers of virus-specific CTL in all lymphoid and nonlymphoid organs tested. On the single cell level, CCR7-deficient CTL gained full effector function, such that antiviral protection in CCR7-deficient mice was complete, but delayed. Similarly, adoptive transfer experiments using DC from CCR7-deficient or competent mice for the priming of CCR7-positive or CCR7-negative CD8+ T cells, respectively, revealed that ectopic positioning of DC and CTL outside organized T cell zones results in reduced priming efficacy. In the memory phase, CCR7-deficient mice maintained a stable LCMV-specific CTL population, predominantly in nonlymphoid organs, and rapidly mounted protective CTL responses against a challenge infection with a vaccinia virus recombinant for the gp33 epitope of LCMV. Taken together, the CCR7-dependent organization of the T cell zone does not appear to be a prerequisite for antiviral effector CTL differentiation and the sustenance of antiviral memory responses in lymphoid or peripheral tissues.

CCR7-deficient (CCR7 / ) mice. Methods. Heterotopic heart and skin allotransplantation was performed in CCR7 / and wild-type (WT) recipients. Graft survival was monitored daily. Grafts and draining lymph nodes were analyzed by immunohistology and flow cytometry at different time points. Groups of mice were splenectomized at the day of allotransplantation. Results. A significant though modest prolongation of allograft survival in CCR7 / recipients was observed for heart grafts (WT, 7.30.5 days; CCR7 / , 10.72.8 days) and skin grafts (WT, 8.90.9 days; CCR7 / , 12.30.9 days). This was accompanied by a delay in the cellular infiltration of allografts. T-cell accumulation and expansion in the draining lymph nodes in CCR7 / recipients was severely impaired. Splenectomy had only a moderate prolongation effect on allograft survival in CCR7 / mice. Conclusions. These results suggest that CCR7-dependent processes support allograft rejection yet are dispensable for the rejection response. Initiation of organ allograft rejection is thought to occur in secondary lymphoid organs (lymph nodes, spleen), where alloreactive T lymphocytes and antigen-presenting cells convene (1). Migration of T cells and antigen-presenting cells (APC) to secondary lymphoid organs is controlled by the homeostatic chemokine receptor CCR7 and its ligands, CCL19 (ELC) and CCL21 (SLC) (2). To prevent lymphocyte homing and activation during allograft rejection, CCR7 thus represents an ideal target. CCR7-deficient mice have been generated by one of us, and show a severe defect in T-cell and dendritic cell migration to secondary lymphoid organs, as well as diminished delayed-type hypersensitivity responses in vivo (3). They differ from mouse models, in which a different family of chemokine receptors, the inflammatory chemokine receptors, has been inactivated. Inflammatory chemokine receptors are involved in the effector phase of the immune response. CXCR3-, CCR1-, CCR5-, and CXC3CR1deficient mice show a limited prolongation of allograft survival that is significantly augmented by the addition of the immunosuppressive drug cyclosporine A (CsA) ( 4– 7). The present study was performed to investigate the role of T-cell and APC migration to secondary lymphoid organs during the initiation of the immune response. We demonstrate that CCR7 deficiency extends skin and heart allograft survival but cannot ultimately prevent allograft rejection. Supporting the proposed mechanism of a defect in T-cell and APC recirculation, activation of T cells in graft draining lymph nodes was severely impaired in CCR7-deficient recipients. In contrast to the effects observed in inflammatory chemokine receptor-deficient mice, the immunosuppressive drug CsA did not augment but rather abrogated the prolongation effect of CCR7 deficiency on allograft survival.

SUMMARY Initiation of an antitumour immune response is characterized bya complex process of chemokine-mediated cell migration and cell–cell interactions. Overexpression of chemokine CCL19 in tumour cells has been shown to result in accelerated tumour rejection under certain experimental conditions, suggesting a novel approach in the therapyof neoplastic malignancies. To investigate CCL19-mediated modulations of cellular immune responses in vivo, we generated a chimeric CCL19-immunoglobulin G2b (IgG2b) Fc fusion protein, which binds to the chemokine receptor CCR7 comparable to native CCL19. CCL19-IgG2b possesses a long-lasting potent chemotactic activityas a result of the extended half-life of Fc fusion proteins. Stable overexpression of CCL19-IgG2b in BALB ⁄c-derived J558L tumour cells fails to support tumour cell rejection following transplantation in syngeneic mice. Moreover, overexpression of CCL19IgG2b hinders tumour rejection in allogeneic C57BL ⁄6 mice. This phenomenon was accompanied bya six-fold increase of dendritic cells (DCs) isolated from CCL19-IgG2b-secreting tumours when compared to the number of DCs isolated from control parental J558L tumours. While mice bearing the allogeneic parental tumour showed an intense hypercellularity in the draining lymph nodes, no such response could be observed in the draining lymph nodes of mice carrying the CCL19-IgG2b-secreting tumour. We could demonstrate that overexpression of CCL19-IgG2b in tumour cells retains antigen-presenting cells in the tumour mass and prevents DCs from migrating into draining lymph nodes to present antigens and to activate T cells, resulting in an impaired immune response against the tumour.

T cell differentiation in the adult thymus depends on sequential interactions between lymphoid progenitors and stromal cells found in distinct regions of the cortex and medulla. Therefore, migration of T cell progenitors through distinct stromal environments seems to be a crucial process regulating differentiation and homeostasis inside the thymus.Here we show that CCR7-deficient mice are distinguished by a disturbed thymic architecture, impaired T cell development, and decreased numbers of the thymocytes. Analysis of developing double negative (CD4−CD8−) pool of wild-type thymus reveals that CCR7 expression is restricted to a CD25intCD44+ subpopulation. Correspondingly, CCR7 deficiency results in an accumulation of this population in mutant thymus. Furthermore, immunohistology shows that in CCR7-deficient mice CD25+CD44+ cells accumulate at the cortico-medullary junction, suggesting that CCR7 signaling regulates the migration of early progenitors toward the outer thymic cortex, thereby continuing differentiation. Results obtained from mixed bone marrow chimeras support this view, since the development of CCR7-deficient thymocytes is also disturbed in a morphologically intact thymus. Thus, our findings establish an essential role for CCR7 in intrathymic migration and proper T cell development.

Patients with short bowel syndrome require life-long parenteral nutrition with potentially life-threatening complications. Intestinal transplantation is the only curative treatment, but is hampered by the pronounced immunogenicity of intestinal allografts that necessitates potent immunosuppressive therapy. This paper examines a novel therapeutic strategy to prevent intestinal allograft rejection in the mouse. Costimulation blockade and the novel immunosuppressive drug FTY720 pronouncedly inhibited intestinal allograft rejection compared to untreated mice, or mice treated with either compound alone. Costimulation blockade and FTY720 inhibited allograft infiltration by both CD4 and CD8 T cells. This therapeutic regimen might prove a promising new strategy to prevent intestinal allograft rejection in the clinics.

CD155 is a member of the immunoglobulin superfamily also known as the human receptor for poliovirus (PVR). Transmembrane glycoproteins related to CD155, the nectins, are well-characterized cell adhesion receptors displaying a high degree of sequence conservation across species. In contrast, CD155 belongs to the category of rapidly evolving genes wherefore a mouse CD155 gene distinguished by an affirmative extent of amino acid conservation as observed for nectins is absent. Consequently, the existing genetic evidence by itself is an inferior indicator to consider whether Tage4, a mouse orphan receptor, represents the murine orthologue of CD155. In the present study Tage4 cDNA was cloned from mouse lung and further characterized genetically. CD155 and Tage4 possess an identical genomic organization and reside in syntenic chromosomal regions. The Tage4 expression pattern was explored applying a newly generated antibody. Both receptors, CD155 in human and Tage4 in mouse, are expressed by intestinal epithelia as well as by follicle associated epithelium and follicular dendritic cells inside Peyer's patches of the gut associated lymphoid tissue. Furthermore, Tage4 lacks self-adhesion capacity but binds to vitronectin, two known features of CD155. These data indicate that Tage4 represents the functional orthologue of CD155 in mouse. Therefore, we suggest to rename Tage4 into rodent CD155.

Dendritic cells (DCs) are potent antigen-presenting cells that are able to initiate and modulate immune responses and are hence exploited as cellular vaccines for immunotherapy. Their capacity to migrate from peripheral tissues to the T-cell areas of draining lymph nodes is crucial for the priming of T lymphocytes. In this study, we investigated how the maturation of human monocyte-derived DCs (MoDCs) by several different stimuli under serum-free conditions affected their T-cell stimulatory function, cytokine secretion, and migratory behavior. Surprisingly, we found that for all maturation stimuli tested, the addition of prostaglandin E2 (PGE2) was required for effective migration of MoDCs toward the lymph node-derived chemokines CCL19 (EBI1 ligand chemokine/macrophage inflammatory protein--3beta) and CCL21 (secondary lymphoid tissue chemokine [SLC]/6Ckine). Costimulation with PGE2 enhanced the expression of the CCL19/CCL21 receptor CCR7 on the cell surface of MoDCs when they were matured with soluble CD40 ligand or proinflammatory cytokines, but did not affect CCR7 expression of polyI:C-stimulated MoDCs. The effects of PGE2 on MoDCs were mediated through increased cyclic adenosine monophosphate by 2 of the known PGE2 receptors, EP2 and EP4, which are expressed and down-regulated after PGE2 binding in these cells. In conclusion, our results suggest that signals provided by the proinflammatory mediator PGE2 are crucial for MoDCs to acquire potent T-helper cell stimulatory capacity and substantial chemotactic responsiveness to lymph node-derived chemokines. This is a new and important parameter for the preparation of MoDCs as cellular vaccines in tumor immunotherapy. (Blood. 2002;100:1354-1361)

B cell entry to lymph nodes and Peyer's patches depends on chemokine receptor signaling, but the principal chemokine involved has not been defined. Here we show that the homing of CXCR4−/− B cells is suppressed in CCL19 (ELC)- and CCL21 (SLC)-deficient paucity of lymph node T cells mice, but not in wild-type mice. We also find that CXCR4 can contribute to T cell homing. Using intravital microscopy, we find that B cell adhesion to high endothelial venules (HEVs) is disrupted when CCR7 and CXCR4 are predesensitized. In Peyer's patches, B cell entry is dependent on CXCR5 in addition to CCR7/CXCR4. CXCL12 (SDF1) is displayed broadly on HEVs, whereas CXCL13 (BLC) is found selectively on Peyer's patch follicular HEVs. These findings establish the principal chemokine and chemokine receptor requirements for B cell entry to lymph nodes and Peyer's patches.

Monitoring of CD8+ T-cell responses in cancer patients during peptide vaccination is essential to provide useful surrogate markers and to demonstrate vaccine efficacy. We have longitudinally followed CD8+ T-cell responses in 3 melanoma patients who were immunized with peptides derived from Melan-A/MART-1. Recombinant HLA-A2 tetramers loaded with the naturally presented Melan-A/MART-1 nonamer peptide (AAGIGILTV) and the Melan-A/MART-1 analog (ELAGIGILTV) were used in combination with phenotypical analysis for different T-cell subsets including naive T cells, effector T cells, "true memory" T cells and "memory effector" T cells, based on CD45RA/RO and CCR7-expression. At least in a single patient, T cells binding to the AAGIGILTV epitope were detected in naive, precursor (CD45RA+/CCR7+) CD8+ T cells, and CD8+ T cells binding to the analog ELAGIGILTV peptide were identified in the terminally differentiated (CD45RA+/CCR7-) T-cell subset. Molecular and functional analysis of tetramer-binding T cells revealed that the T-cell receptor (TCR) repertoire was oligo/polyclonal in AAGIGILTV-reactive T cells, but different and restricted to a few TCR clonotypes in ELAGIGILTV-reactive T cells prior to vaccination. The TCR repertoire reactive with Melan-A/MART-1 peptide epitopes was broadened during vaccination and exhibited a different profile of cytokine release after specific stimulation: tetramer-binding T cells from 2/3 patients secreted granulocyte/macrophage colony-stimulating factor (GM-CSF) and interferon-gamma but not interleukin-2 (IL-2) in response to Melan-A/MART-1 peptides. In the third patient, tetramer-binding T cells secreted IL-2 exclusively. Our results show that T-cell responses to peptide vaccination consist of different T-cell subsets associated with different effector functions. Complementary analysis for TCR CDR3 and cytokine profiles may be useful to define the most effective CD8+ T-cell population induced by peptide vaccination.