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The clinical manipulation of regulatory T cells (Tregs) represents a promising strategy for the regulation of unwanted immune responses. It is now becoming clear that Tregs exert multiple effects on different cell targets under particular conditions; however, the interplay between these different factors remains unclear. Using mouse Tregs of known Ag specificity, we report in this study two different levels of Treg-mediated suppression: one that targets T cell proliferation and one that targets dendritic cell-mediated proinflammatory chemokine (CCL3 and CCL4) production. These two effects can be dissociated, and whereas modulation of T cell proliferation depends on the strength of the antigenic stimulus, modulation of chemokine production by dendritic cells does not. We also provide evidence that the bystander effect of Tregs on immune responses observed in vivo may be in great part explained by a decrease in the recruitment of target T cells, and therefore in the magnitude of the response, rather than by a direct effect on their priming or proliferation. Overall, our results shed some light on the different aspects that need to be considered when attempting to modulate Tregs for clinical purposes.

The ability of NK cells to rapidly produce IFN-gamma is an important innate mechanism of resistance to many pathogens including Leishmania major. Molecular and cellular components involved in NK cell activation in vivo are still poorly defined, although a central role for dendritic cells has been described. In this study, we demonstrate that Ag-specific CD4(+) T cells are required to initiate NK cell activation early on in draining lymph nodes of L. major-infected mice. We show that early IFN-gamma secretion by NK cells is controlled by IL-2 and IL-12 and is dependent on CD40/CD40L interaction. These findings suggest that newly primed Ag-specific CD4(+) T cells could directly activate NK cells through the secretion of IL-2 but also indirectly through the regulation of IL-12 secretion by dendritic cells. Our results reveal an unappreciated role for Ag-specific CD4(+) T cells in the initiation of NK cell activation in vivo upon L. major infection and demonstrate bidirectional regulations between innate and adaptive immunity.

Although a great deal is known about T cell entry into lymph nodes, much less is understood about how T lymphocytes access the splenic white pulp (WP). We show in this study that, as recently described for lymph nodes, fibroblastic reticular cells (FRCs) form a network in the T cell zone (periarteriolar lymphoid sheath, PALS) of the WP on which T lymphocytes migrate. This network connects the PALS to the marginal zone (MZ), which is the initial site of lymphocyte entry from the blood. T cells do not enter the WP at random locations but instead traffic to that site using the FRC-rich MZ bridging channels (MZBCs). These data reveal that FRCs form a substrate for T cells in the spleen, guiding these lymphocytes from their site of entry in the MZ into the PALS, within which they continue to move on the same network.

Langerhans cells have been thought to play a major role as APCs for induction of specific immune responses to Leishmania major. Although their requirement for control of infection has been challenged recently, it remains unclear whether they can transport Ag to lymph nodes and promote initiation of T cell responses. Moreover, the role of dermal dendritic cells (DCs), another population of skin DCs, has so far not been addressed. We have investigated the origin and characterized the cell population responsible for initial activation of L. major-specific T cells in susceptible and resistant mice. We found that Ag presentation in draining lymph nodes peaks as early as 24 h after infection and is mainly mediated by a population of CD11c(high)CD11b(high)Gr-1-CD8-langerin- DCs residing in lymph nodes and acquiring soluble Ags possibly drained through the conduit network. In contrast, skin-derived DCs, including Langerhans cells and dermal DCs, migrated poorly to lymph nodes and played a minor role in early T cell activation. Furthermore, prevention of migration through early removal of the infection site did not affect Ag presentation by CD11c(high) CD11b(high) DCs and activation of Leishmania major-specific naive CD4+ T cells in vivo.

Technical difficulties in tracking endogenous CD4 T lymphocytes have limited the characterization of tumor-specific CD4 T cell responses. Using fluorescent MHC class II/peptide multimers, we defined the fate of endogenous Leishmania receptor for activated C kinase (LACK)-specific CD4 T cells in mice bearing LACK-expressing TS/A tumors. LACK-specific CD44(high)CD62L(low) CD4 T cells accumulated in the draining lymph nodes and had characteristics of effector cells, secreting IL-2 and IFN-gamma upon Ag restimulation. Increased frequencies of CD44(high)CD62L(low) LACK-experienced cells were also detected in the spleen, lung, liver, and tumor itself, but not in nondraining lymph nodes, where the cells maintained a naive phenotype. The absence of systemic redistribution of LACK-specific memory T cells correlated with the presence of tumor. Indeed, LACK-specific CD4 T cells with central memory features (IL-2(+)IFN-gamma(-)CD44(high)CD62L(high) cells) accumulated in all peripheral lymph nodes of mice immunized with LACK-pulsed dendritic cells and after tumor resection. Together, our data demonstrate that although tumor-specific CD4 effector T cells producing IFN-gamma are continuously generated in the presence of tumor, central memory CD4 T cells accumulate only after tumor resection. Thus, the continuous stimulation of tumor-specific CD4 T cells in tumor-bearing mice appears to hinder the systemic accumulation of central memory CD4 T lymphocytes.

Dendritic cells (DC) are short-lived, professional APCs that play a central role in the generation of adaptive immune responses. Induction of efficient immune responses is dependent on how long DCs survive in the host. Therefore, the regulation of DC apoptosis in vivo during infection remains an important question that requires further investigation. The impact of Escherichia coli bacteremia on DCs has never been analyzed. We show here that i.v. or i.p. administration of live or heat-killed E. coli in mice induces splenic DC migration, maturation, and apoptosis. We further characterize which TLR and Toll-IL-1R (TIR)-containing adaptor molecules regulate these processes in vivo. In this model, DC maturation is impaired in TLR2(-/-), TLR4(-/-) and TIR domain-containing adapter-inducing IFN-beta (TRIF)(-/-) mice. In contrast, DC apoptosis is reduced only in TLR4(-/-) and TRIF(-/-) mice. As expected, DC apoptosis induced by the TLR4 ligand LPS is also abolished in these mice. Injection of the TLR9 ligand CpG-oligodeoxynucleotide (synthetic bacterial DNA) induces DC migration and maturation, but only modest DC apoptosis when compared with LPS and E. coli. Together, these results suggest that E. coli bacteremia directly impacts on DC maturation and survival in vivo through a TLR4-TRIF-dependent signaling pathway.

Type 1 diabetes is an autoimmune disorder caused by autoreactive T cells that mediate destruction of insulin-producing beta cells of the pancreas. Studies have shown that T cell tolerance can be restored by inducing a partial or altered signal through the TCR. To investigate the potential of bivalent peptide-MHC class II/Ig fusion proteins as therapeutics to restore Ag-specific tolerance, we have developed soluble peptide I-A(g7) dimers for use in the nonobese diabetic mouse model of diabetes. I-A(g7) dimers with a linked peptide specific for islet-reactive BDC2.5 TCR transgenic CD4(+) T cells were shown to specifically bind BDC2.5 T cells as well as a small population of Ag-specific T cells in nonobese diabetic mice. In vivo treatment with BDC2.5 peptide I-A(g7) dimers protected mice from diabetes mediated by the adoptive transfer of diabetogenic BDC2.5 CD4(+) T cells. The dimer therapy resulted in the activation and increased cell death of transferred BDC2.5 CD4(+) T cells. Surviving cells were hypoproliferative to challenge by Ag and produced increased levels of IL-10 and decreased levels of IFN-gamma compared with cells from control I-A(g7) dimer-treated mice. Anti-IL-10R therapy reversed the tolerogenic effects of the dimer. Thus, peptide I-A(g7) dimers induce tolerance of BDC2.5 TCR T cells through a combination of the induction of clonal anergy and anti-inflammatory cytokines.

A diverse population of MHC class II-restricted CD4 lineage T cells develops in mice that lack expression of the CD4 molecule. In this study, we show that the TCR repertoire selected in the absence of CD4 is distinct, but still overlapping in its properties with that selected in the presence of CD4. Immunization of mice lacking CD4 caused the clonal expansion of T cells that showed less breadth in the range of Ag-binding properties exhibited by their TCRs. Specifically, the CD4-deficient Ag-specific TCR repertoire was depleted of TCRs that demonstrated low-affinity binding to their ligands. The data thus suggest a key role for CD4 in broadening the TCR repertoire by potentiating productive TCR signaling and clonal expansion in response to the engagement of low-affinity antigenic ligands.

Infection of BALB/c mice with Leishmania major results in the rapid accumulation of IL-4 transcripts within CD4(+) T cells that react to the parasite Leishmania homologue of mammalian RACK1 (LACK) Ag. Because memory/effector cells secrete IL-4 more rapidly than naive cells, we sought to analyze the phenotype of these lymphocytes before infection. Indeed, a fraction of LACK-specific CD4(+) T cells expressed a typical CD62 ligand(low)CD44(high)CD45RB(low) phenotype in uninfected mice. LACK-specific T cells were primed in gut-associated lymphoid tissues by cross-reactive microbial Ags as demonstrated by their reactivity with bacterial extracts and by the ability of APCs from the mesenteric LN of BALB/c mice to induce their proliferation. Also, mice in which the digestive tract has been decontaminated exhibited a reduced proportion of LACK-specific T cells expressing a memory/effector phenotype and did not exhibit the early accumulation of IL-4 transcripts induced by L. major. Thus, LACK-specific T cells represent a subset of CD4(+) T cells which have acquired the ability to rapidly secrete IL-4 as the result of their priming by cross-reactive microbial Ags. Tracking the fate of these cells may provide information about the regulation of cell-mediated immune responses to gut Ags in physiological and pathological situations.

Protective immunity against Leishmania major generated by DNA encoding the LACK (Leishmania homologue of receptor for activated C kinase) Ag has been shown to be more durable than vaccination with LACK protein plus IL-12. One mechanism to account for this may be the selective ability of DNA vaccination to induce CD8+ IFN-gamma-producing T cells. In this regard, we previously reported that depletion of CD8+ T cells in LACK DNA-vaccinated mice abrogated protection when infectious challenge was done 2 wk postvaccination. In this study, we extend these findings to study the mechanism by which CD8+ T cells induced by LACK DNA vaccination mediate both short- and long-term protective immunity against L. major. Mice vaccinated with LACK DNA and depleted of CD8+ T cells at the time of vaccination or infection were unable to control infection when challenge was done 2 or 12 wk postvaccination. Remarkably, it was noted that depletion of CD8+ T cells in LACK DNA-vaccinated mice was associated with a striking decrease in the frequency of LACK-specific CD4+ IFN-gamma-producing T cells both before and after infection. Moreover, data are presented to suggest a mechanism by which CD8+ T cells exert this regulatory role. Taken together, these data provide additional insight into how Th1 cells are generated and sustained in vivo and suggest a potentially novel immunoregulatory role for CD8+ T cells following DNA vaccination.

Macrophages are apparently the only cells that in vivo allow the growth of the intracellular pathogen Leishmania. They are thus generally considered as likely candidates for the presentation of parasite Ag to CD4+ T lymphocytes known to be involved in protective and counterprotective immune responses. In the present study, we examined whether mouse macrophages infected with Leishmania were capable of stimulating T cell hybrids and a T cell clone reacting with the previously identified protective Ag LACK (Leishmania homologue of receptors for Activated C Kinase). This parasite protein is expressed in both promastigote and amastigote stages of Leishmania. We found that IFN-gamma-treated macrophages recently infected with live Leishmania promastigotes were fully competent to activate LACK-reactive T cells. However, at later times of infection, permissive macrophages infected with promastigotes were no longer able to present LACK, in spite of the presence of numerous intracellular parasites. This punctual presentation of LACK was apparently linked with the destruction, at least partial, of the intracellular parasites. In contrast, macrophages infected with live Leishmania amastigotes were always unable to stimulate the LACK-specific T cells. Amastigote-infected macrophages could, however, reactivate the T cells if LACK-delta(1), a recombinant form of LACK, was added as an exogenous protein in the culture medium. Similar results were obtained with all combinations tested involving macrophages from various origins, different activating cytokines (IFN-gamma, granulocyte-macrophage CSF, IL-4), several Leishmania species (L. amazonensis, L. major, L. donovani), and 15 different LACK-reactive T cell hybrids and clones. From these data, it is tempting to propose that the differentiation of promastigotes into amastigotes, which leads to a better survival of the parasites within macrophages, also allows them to go unnoticed by the immune system.

Mutant T cell lines that do not express the endogenous alpha- and/or beta-chain genes of the TCR were generated from the alpha beta TCR/CD3+ tumor cell line C6VL with a combination of classical mutagenesis methods and selection of somatic hybrid variants. This novel strategy obviated the need for repeated mutagenesis and screening of a large number of individual clones. The loss of either the alpha- or the beta-chain expression in the mutant cells was associated with the loss of surface TCR/CD3 complex, which could be rescued by the transfection of appropriate exogenous alpha- and/or beta-chain gene constructs. Because these cells express a single TCR molecule on the cell surface, they are useful for the study of the assembly and function of the alpha beta TCR. This strategy is also generally applicable for the generation of homozygous mutant cell lines lacking other gene products.