Polarization of naive CD4+ T cells toward the Th1 subset by CTLA-4 costimulation

In this study, we examined in vitro the role of CTLA-4 costimulation in the polarization of naive CD4+ T cells toward the Th1 subset. When CTLA-4 costimulation was blocked by the inclusion of anti-CTLA-4 Fab in cultures during priming of naive CD4+ T cells with anti-CD3 in the presence of splenic adherent cells, they were polarized toward the Th2 subset. Conversely, the engagement of CTLA-4 with immobilized anti-CTLA-4 or with CD80-P815 cells polarized naive CD4+ T cells costimulated with anti-CD3 and anti-CD28 toward the Th1 subset. The CTLA-4 costimulation during priming augmented TGF-beta1 mRNA accumulation in naive CD4+ T cells, and the inclusion of anti-TGF-beta in cultures for priming suppressed the effect of CTLA-4 costimulation on the Th1 polarization. The addition of low doses of TGF-beta1 in cultures for priming of naive CD4+ T cells enhanced the production of Th1 cytokines upon secondary stimulation, although Th2 cytokine production was not affected by the doses of TGF-beta1. The CTLA-4 costimulation was also shown to suppress IL-4 production of naive CD4+ T cells upon priming. These results indicate that the costimulation against CTLA-4 drives polarization of naive CD4+ T cells toward the Th1 subset independent of IL-12 through, at least in part, the enhancement of TGF-beta1 production, and it also hampers Th2 subset differentiation by affecting IL-4 production of naive CD4+ T cells.     

Blockade of CTLA-4 signals inhibits Th2-mediated murine chronic graft-versus-host disease by an enhanced expansion of regulatory CD8+ T cells

CTLA-4 (CD152) is thought to be a negative regulator of T cell activation. Little is known about the function of CTLA-4 in Th2-type immune responses. We have investigated the effect of initial treatment with anti-CTLA-4 mAb on murine chronic graft-vs-host disease. Transfer of parental BALB/c splenocytes into C57BL/6 x BALB/c F1 mice induced serum IgE production, IL-4 expression by donor CD4+ T cells, and host allo-Ag-specific IgG1 production at 6-9 wk after transfer. Treatment with anti-CTLA-4 mAb for the initial 2 wk significantly reduced IgE and IgG1 production and IL-4 expression. Analysis of the splenic phenotype revealed the enhancement of donor T cell expansion, especially within the CD8 subset, and the elimination of host cells early after anti-CTLA-4 mAb treatment. This treatment did not affect early IFN-gamma expression by CD4+ and CD8+ T cells and anti-host cytolytic activity. Thus, blockade of CTLA-4 greatly enhanced CD8+ T cell expansion, and this may result in the regulation of consequent Th2-mediated humoral immune responses. These findings suggest a new approach for regulating IgE-mediated allergic immune responses by blockade of CTLA-4 during a critical period of Ag sensitization.     

Oxidative responses of human and murine macrophages during phagocytosis of Leishmania chagasi.

Leishmania chagasi, the cause of South American visceral leishmaniasis, must survive antimicrobial responses of host macrophages to establish infection. Macrophage oxidative responses have been shown to diminish in the presence of intracellular leishmania. However, using electron spin resonance we demonstrated that murine and human macrophages produce O2-during phagocytosis of opsonized promastigotes. Addition of the O2- scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl to cultures resulted in increased infection, suggesting that O2- enhances macrophage leishmanicidal activity. The importance of NO. produced by inducible NO synthase (iNOS) in controlling murine leishmaniasis is established, but its role in human macrophages has been debated. We detected NO. in supernatants from murine, but not human, macrophages infected with L. chagasi. Nonetheless, the iNOS inhibitor N(G)-monomethyl-L-arginine inhibited IFN-gamma-mediated intracellular killing by both murine and human macrophages. According to RNase protection assay and immunohistochemistry, iNOS mRNA and protein were expressed at higher levels in bone marrow of patients with visceral leishmaniasis than in controls. The iNOS protein also increased upon infection of human macrophages with L. chagasi promastigotes in vitro in the presence of IFN-gamma. These data suggest that O2- and NO. each contribute to intracellular killing of L. chagasi in human and murine macrophages.     

Preferential costimulation by CD80 results in IL-10-dependent TGF-beta1(+) -adaptive regulatory T cell generation

Costimulatory ligands CD80 and CD86 have different binding preferences and affinities to their receptors, CD28 and CTLA-4. Earlier, we demonstrated that CD80 binds to CTLA-4 with higher affinity and has a role in suppressing T cell response. The current study demonstrates that not only did blockade of CD86 upon Ag presentation by bone marrow-derived dendritic cells (DC) to OVA-specific T cells result in induction of hyporesponsive T cells but also that these T cells could suppress the proliferative response of effector T cells. These T cells showed TGF-beta1 on their surface and secreted TGF-beta1 and IL-10 upon restimulation. Although blockade of CTLA-4 and neutralization of IL-10 profoundly inhibited the induction of these TGF-beta1(+) T cells, their ability to suppress the effector T cell proliferation was abrogated by neutralization of TGF-beta1 alone. Induction of TGF-beta1(+) and IL-10(+) T cells was found to be independent of natural CD4(+)CD25(+) regulatory T cells, demonstrating that preferential ligation of CTLA-4 by CD80 induced IL-10 production by effector T cells, which in turn promoted the secretion of TGF-beta1. Treatment of prediabetic NOD mice with islet beta cell Ag-pulsed CD86(-/-) DCs, but not CD80(-/-) DCs, resulted in the induction of TGF-beta1- and IL-10-producing cells, significant suppression of insulitis, and delay of the onset of hyperglycemia. These observations demonstrate not only that CD80 preferentially binds to CTLA-4 but also that interaction during Ag presentation can result in IL-10-dependent TGF-beta1(+) regulatory T cell induction, reinstating the potential of approaches to preferentially engage CTLA-4 through CD80 during self-Ag presentation in suppressing autoimmunity.     

TGF-beta requires CTLA-4 early after T cell activation to induce FoxP3 and generate adaptive CD4+CD25+ regulatory cells

Although positive CD28 costimulation is needed for the generation of natural CD4+CD25+ regulatory T cells, we report that negative CTLA-4 costimulation is necessary for generating phenotypically and functionally similar adaptive CD4+CD25+ suppressor cells. TGF-beta could not induce CD4+CD25- cells from CTLA-4(-/-) mice to express normal levels of FoxP3 or to develop suppressor activity. Moreover, blockade of CTLA-4 following activation of wild-type CD4+ cells abolished the ability of TGF-beta to induce FoxP3-expressing mouse suppressor cells. TGF-beta accelerated expression of CTLA-4, and time course studies suggested that CTLA-4 ligation of CD80 shortly after T cell activation enables TGF-beta to induce CD4+CD25- cells to express FoxP3 and develop suppressor activity. TGF-beta also enhanced CD4+ cell expression of CD80. Thus, CTLA-4 has an essential role in the generation of acquired CD4+CD25+ suppressor cells in addition to its other inhibitory effects. Although natural CD4+CD25+ cells develop normally in CTLA-4(-/-) mice, the lack of TGF-beta-induced, peripheral CD4+CD25+ suppressor cells in these mice may contribute to their rapid demise.     

Activation of TGF-beta by Leishmania chagasi: importance for parasite survival in macrophages

TGF-beta is a potent regulatory cytokine that suppresses expression of inducible NO synthase and IFN-gamma, and suppresses Th1 and Th2 cell development. We examined whether functionally active TGF-beta is present in the local environment surrounding the invading protozoan Leishmania chagasi. Our prior data showed that TGF-beta levels are significantly increased in L. chagasi-infected mice. In the current study, we found TGF-beta was also abundant in bone marrows of humans with acute visceral leishmaniasis but not in those of uninfected controls. Furthermore, L. chagasi infection caused an increase in biologically active TGF-beta in human macrophage cultures without changing the total TGF-beta. Therefore, we investigated the means through which leishmania could augment activated but not total TGF-beta. Incubation of latent TGF-beta with Leishmania sp. promastigotes caused active TGF-beta to be released from the latent complex. In contrast, the nonpathogenic protozoan Crithidia fasciculata could not activate TGF-beta. TGF-beta activation by leishmania was prevented by inhibitors of cysteine proteases and by the specific cathepsin B inhibitor CA074. Physiologic concentrations of TGF-beta inhibited killing of intracellular L. chagasi in macrophages, although the phagocytosis-induced respiratory burst remained intact. In contrast, supraphysiologic concentrations of TGF-beta had no effect on parasite survival. We hypothesize that the combined effect of abundant TGF-beta stores at extracellular sites during infection, and the ability of the parasite to activate TGF-beta in its local environment, leads to high levels of active TGF-beta in the vicinity of the infected macrophage. Locally activated TGF-beta could, in turn, enhance parasite survival through its effects on innate and adaptive immune responses.     

The dual role of CTLA-4 in Leishmania infection

The role of CTLA-4 in inducing the production of transforming growth factor beta (TGF-beta) from T cells during a Leishmania infection has only recently been recognized. However, CTLA-4 and TGF-beta affect T helper cells differently, depending on the maturation. This review discusses the data obtained from different experimental models and demonstrates that CTLA-4 is a target molecule for vaccination and therapy against leishmaniasis.     

DNA damage and nitric oxide production in mice following infection with L. chagasi

Leishmania chagasi, which causes visceral leishmaniasis in South America, is an obligate intracellular protozoan. Production of nitric oxide by macrophages during the inflammatory response is one of the main microbicidal mechanisms against this parasite. The goal of this study was to evaluate whether L. chagasi infection causes DNA damage in peripheral blood and spleen cells of Balb/c mice and whether such damage may be related to NO production. Balb/c mice were either infected with L. chagasi or maintained as controls. The single-cell gel electrophoresis (comet) assay was used to measure DNA damage in peripheral blood and spleen cells, and the Griess reaction was used to measure NO production in the spleen. L. chagasi infection induced DNA damage in peripheral blood and spleen cells of infected mice. Macrophages from the control group, challenged with L. chagasi, showed significantly (p<0.05) greater NO production, compared to non-challenged cells. Treatment of spleen cells with N(G)-monomethyl-l-arginine (LNMMA) caused a significant reduction of NO production and DNA damage (p<0.05). Our results indicate that L. chagasi induces DNA damage in the peripheral blood and spleen cells and that NO not only causes killing of the parasite but also induces DNA damage in adjacent cells.     

TGF-beta 1 plays an important role in the mechanism of CD4+CD25+ regulatory T cell activity in both humans and mice

In previous studies, we have shown that murine CD4+CD25+ regulatory T cells produce high levels of TGF-beta1 in a cell surface and/or secreted form, and blockade of such TGF-beta1 by anti-TGF-beta curtails the ability of these cells to suppress CD25- T cell proliferation and B cell Ig production in in vitro suppressor assays. In further support for the role of TGF-beta1 in suppression by CD4+CD25+ T cells, we show in this study that another TGF-beta1-blocking molecule, recombinant latency-associated peptide of TGF-beta1 (rLAP), also reverses suppression by mouse CD4+CD25+ T cells as well as their human counterparts, CD4+CD25(high) T cells. In addition, we show that CD25- T cells exposed to CD4+CD25+ T cells in vitro manifest activation of Smad-2 and induction of CD103, the latter a TGF-beta-inducible surface integrin. In further studies, we show that while CD4+CD25+ T cells from TGF-beta1-deficient mice can suppress CD25- T cell proliferation in vitro, these cells do not protect recipient mice from colitis in the SCID transfer model in vivo, and, in addition, CD4+LAP+, but not CD4+LAP- T cells from normal mice protect recipient mice from colitis in this model. Together, these studies demonstrate that TGF-beta1 produced by CD4+CD25+ T cells is involved in the suppressor activity of these cells, particularly in their ability to regulate intestinal inflammation.     

Leishmania donovani: evolution and architecture of the splenic cellular immune response related to control of infection.

Infection with the protozoan Leishmania donovani in humans is usually subclinical. Parasites probably persist for the life of the host and the low-level infection is controlled by the cellular immune response. To better understand the mechanisms related to the control of infection, we studied the evolution and architecture of the splenic cellular immune response in a murine model that is most representative of human subclinical infection. Following systemic inoculation with L. donovani, the parasites were primarily localized to the macrophage-rich splenic red pulp. There was an initial increase in the numbers of T cells and dendritic cells in the periarteriolar lymphoid sheath and marginal zone, but the red pulp (where parasitized macrophages were prominent) remained free of these cells until later in the course of infection. Thus, T cells did not colocalize with parasitized red pulp macrophages until later in the course of infection. Early in the course of infection, IL-10 production within the marginal zone and TGF-beta production by cells in the red pulp were prominent. These macrophage-inhibitory cytokines may contribute to the establishment of the infection and early parasite replication. By day 28 of infection, when the visceral parasite burden began to decline, the number of IL-10-producing spleen cells was back to the baseline level, but IFN-gamma production was higher and the number of IL-12-producing cells was increased dramatically. At this time T cells and dendritic cells had moved out of the lymphoid follicle and marginal zone into the red pulp where the parasites were located. These findings therefore suggest that control of infection is associated with IFN-gamma and IL-12 production and migration of T cells and dendritic cells to the site of chronic parasitism.     

Systemic and local characterization of regulatory T cells in a chronic fungal infection in humans

The long-term persistence of pathogens in a host is a hallmark of certain infectious diseases, including schistosomiasis, leishmaniasis, and paracoccidioidomycosis (PCM). Natural regulatory T (Treg) cells are involved in control of the immune responses, including response to pathogens. Because CTLA-4 is constitutively expressed in Treg cells and it acts as a negative regulator of T cell activation in patients with PCM, here we investigated the involvement of Treg cells in the control of systemic and local immune response in patients with PCM. We found that the leukocyte subsets were similar in patients and controls, except for CD11c+CD1a+ cells. However, a higher frequency of CD4+CD25+ T cells expressing CTLA-4, glucorticoid-inducible TNFR, membrane-bound TGF-beta, and forkhead-box 3 were observed in PBMC of patients. In accordance, these cells exhibited stronger suppressive activity when compared with those from controls (94.0 vs 67.5% of inhibition of allogeneic T cell proliferation). In addition, the data showed that CD4+CD25+ T cells expressing CTLA-4+, glucocorticoid-inducible TNFR positive, CD103+, CD45RO+, membrane-bound TGF-beta, forkhead-box 3 positive, and the chemokines receptors CCR4 and CCR5 accumulate in the Paracoccidioides brasiliensis-induced lesions. Indeed, the secreted CCL17 and CCL22, both associated with the migration of Treg cells to peripheral tissues, were also detected in the biopsies. Moreover, the CD4+CD25+ T cell derived from lesions, most of them TGF-beta+, also exhibited functional activity in vitro. Altogether, these data provide the first evidence that Treg cells play a role in controlling local and systemic immune response in patients with a fungal-induced granulomatous disease advancing our understanding about the immune regulation in human chronic diseases.     

IL-10 and TGF-beta redundantly protect against severe liver injury and mortality during acute schistosomiasis

The cytokines IL-10 and TGF-beta regulate immunity and inflammation. IL-10 is known to suppress the extent of hepatic damage caused by parasite ova during natural infection with Schistosoma mansoni, but the role of TGF-beta is less clear. Cytokine blockade studies in mice revealed that anti-IL-10R mAb treatment during acute infection modestly increased cytokine production and liver damage, whereas selective anti-TGF-beta mAb treatment had marginal effects. In contrast, mice administered both mAbs developed severe hepatic inflammation, with enlarged, necrotic liver granulomas, cachexia, and >80% mortality by 8 wk postinfection, despite increased numbers of CD4(+)CD25(+)Foxp3(+) T regulatory cells. Blocking both IL-10 and TGF-beta at the onset of egg production also significantly increased IL-4, IL-6, TNF, IFN-gamma, and IL-17 production and markedly increased hepatic, peritoneal, and splenic neutrophilia. In contrast, coadministration of anti-IL-10R and TGF-beta mAbs had little effect upon parasite ova-induced intestinal pathology or development of alternatively activated macrophages, which are required to suppress intestinal pathology. This suggests that inflammation is controlled during acute S. mansoni infection by two distinct, organ-specific mechanisms: TGF-beta and IL-10 redundantly suppress hepatic inflammation while intestinal inflammation is regulated by alternatively activated macrophages.     

Co-stimulation of T cell proliferation by transforming growth factor-beta 1.

Transforming growth factor-beta (TGF-beta) exhibits diverse regulatory roles in the immune system and has been described as a potent inhibitor of lymphocyte and hemopoietic progenitor cell growth. The present studies investigated the effects of TGF-beta 1 on murine T cell proliferation triggered through the T cell receptor/CD3 complex. In contrast to previously reported T cell growth inhibition, TGF-beta 1 costimulated splenic T cell proliferation in the presence of immobilized anti-CD3 antibody 2C11, with maximal effect at anti-CD3 concentration of 50 micrograms/ml. Although TGF-beta 1 induced a modest increase in IL-2R display, TGF-beta 1 co-stimulated proliferation was largely independent of IL-2 and/or IL-4. Anti-IL-2 and/or anti-IL-4 antibody did not significantly block the TGF-beta 1 co-stimulated T cell growth, and no IL-2 or IL-4 bioactivity was detected in TGF-beta 1 co-stimulated cultures. TGF-beta 1 did not enhance IL-2 mRNA expression beyond control levels. However, TGF-beta 1 co-stimulation caused an accelerated evolution of a memory or mature T cell population phenotype. Both CD4+ and CD8+ T cell subsets were significantly enriched for cells of the mature CD45RBloPgp-1hi phenotype, in comparison with T cells stimulated by anti-CD3 alone or with PMA, and CD8+ T cells predominated. These results thus provide initial evidence that TGF-beta 1 is capable of bifunctional T cell growth regulation, which occurs largely via an IL-2- and IL-4-independent pathway.     

Role of costimulatory molecules in immune response of patients with cutaneous leishmaniasis

T cell-mediated immunity is critical in resistance against Leishmania parasites, and T cell activation requires signals provided by costimulatory molecules. Herein we evaluated the role of costimulatory molecules on cytokine production and T cell surface molecule expression by peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis (CL) patients. PBMC from CL patients were stimulated with soluble Leishmania antigen (SLA, 10 microg/ml), in the presence or absence of soluble CTLA4-Ig to block CD28-B7 interaction or in the presence or absence of anti-human CD40L to block CD40-CD40L interaction. Supernatants were harvested to evaluate tumor necrosis factor alpha (TNF-alpha), interleukin 10 (IL-10), transforming growth factor beta (TGF-beta) and interferon gamma (IFN-gamma) production by ELISA. Cells were harvested after 48 h of culture, stained for specific activation markers and analyzed by flow cytometry. Results show that the blockade of CD28-B7 interaction by CTLA4-Ig downmodulated IFN-gamma, IL-10, and TNF-alpha secretion by PBMC from CL patients. No alteration was detected on either TGF-beta production or the expression of CTLA44 or CD25 on CD4+ and CD8+ T cells. When the CD40-CD40L interaction was blockade using anti-CD40L, we did not observe changes in cytokine production or in surface molecule expression. The blockade of the CD28-B7 interactions by CTLA4-Ig also did not alter cytokine production in volunteers immunized against tetanus toxoid (TT). Taken together, these data suggest that the interaction of CTLA4 and CD28-B7 is a TGF-beta-independent mechanism that specifically downmodulates the immune response in cutaneous leishmaniasis patients.     

Immunotherapy for visceral leishmaniasis: ability of factors produced during anti-leishmania responses of skin test positive adults to inhibit peripheral blood mononuclear cell activities associated with visceral leishmaniasis

The course of human Leishmania chagasi infections appears to be determined by the balance between type 1 (Tl) CD4+ and CD8+ T suppressor (Ts) cell activities. Skin test positive adults living in hyperendemic areas who have no history of visceral leishmaniasis (VL) have Tl CD4+ T cell immunodominant responses against L. chagasi. The cytokines they secrete during anti-leishmania responses are a probable source of cytokines which inhibit the CD8+ Ts cells associated with VL. The ability of supernatants generated from peripheral blood mononuclear cells derived from skin test positive adults to reverse immune responses which appear to be mediated by CD8+ Ts cells was assessed in three sets of screening assays. The supernatants displayed three candidate factors. One, which could be explained by Leishmania antigens in the supernatant, decreased high endogenous IL-10 secretion characteristic of one class of VL patients. A second activity decreased high endogenous proliferation characteristic of the same class of patients without decreasing antigen specific proliferation. The third activity inhibited or killed CD8+ T cells but not CD4+ T cells. These activities might be useful in treating VL.     

Expression of a soluble TGF-beta receptor by tumor cells enhances dendritic cell/tumor fusion vaccine efficacy

Dendritic cell (DC)-based antitumor immunotherapy is a promising cancer therapy. We have previously shown that tumor-derived TGF-beta limits the efficacy of the DC/tumor fusion vaccine in mice. In the current study we investigated the effect of neutralizing tumor-derived TGF-beta on the efficacy of the DC/tumor fusion vaccine. An adenovirus encoding human TGF-beta receptor type II fused to the Fc region of human IgM (Adv-TGF-beta-R) or a control adenovirus encoding LacZ (Adv-LacZ) was used to express a soluble form of the neutralizing TGF-beta receptor (TGF-beta-R). Murine breast carcinoma cells, 4T1, but not bone marrow-derived DCs, were successfully transfected with Adv-TGF-beta-R (4T1+Adv-TGF-beta-R) using a multiplicity of infection of 300. Immunization with irradiated 4T1+Adv-TGF-beta-R tumor cells conferred enhanced antitumor immunity compared with immunization with irradiated 4T1+Adv-LacZ tumor cells. The DC/4T1+Adv-TGF-beta-R fusion vaccine offered enhanced protective and therapeutic efficacy compared with the DC/4T1-Adv-LacZ fusion vaccine. Because TGF-beta is known to induce regulatory T cells (Tregs), we further showed that the DC/4T1+Adv-TGF-beta-R fusion vaccine induced fewer CD4(+)CD25(+)Foxp3(+) Tregs than the DC/4T1+Adv-LacZ fusion vaccine in vitro and in vivo. The suppressive role of splenic CD4(+)CD25(+) Tregs isolated from mice immunized with DC/4T1+Adv-LacZ was demonstrated using a CTL killing assay. Similar enhanced therapeutic efficacy was observed in murine renal cell carcinoma, RenCa, which expresses a high level of TGF-beta. We conclude that the blockade of tumor-derived TGF-beta reduces Treg induction by the DC/tumor fusion vaccine and enhances antitumor immunity. This may be an effective strategy to enhance human DC-based antitumor vaccines.     

Synergistic Reversal of Intrahepatic HCV-Specific CD8 T Cell Exhaustion by Combined PD-1/CTLA-4 Blockade

Viral persistence is associated with hierarchical antiviral CD8 T cell exhaustion with increased programmed death-1 (PD-1) expression. In HCV persistence, HCV-specific CD8 T cells from the liver (the site of viral replication) display increased PD-1 expression and a profound functional impairment that is not reversed by PD-1 blockade alone. Here, we report that the inhibitory receptor cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is preferentially upregulated in PD-1⁺ T cells from the liver but not blood of chronically HCV-infected patients. PD-1/CTLA-4 co-expression in intrahepatic T cells was associated with a profound HCV-specific effector dysfunction that was synergistically reversed by combined PD-1/CTLA-4 blockade in vitro, but not by blocking PD-1 or CTLA-4 alone. A similar effect was observed in circulating HCV-specific CD8 T cells with increased PD-1/CTLA-4 co-expression during acute hepatitis C. The functional response to combined blockade was directly associated with CTLA-4 expression, lost with CD28-depletion and CD4-independent (including CD4⁺FoxP3⁺ Tregs). We conclude that PD-1 and CTLA-4 pathways both contribute to virus-specific T cell exhaustion at the site of viral replication by a redundant mechanism that requires combined PD-1/CTLA-4 blockade to reverse. These findings provide new insights into the mechanisms of virus-specific T cell dysfunction, and suggest that the synergistic effect by combined inhibitory receptor blockade might have a therapeutic application against chronic viral infection in vivo, provided that it does not induce autoimmunity.     

Targeted CTLA-4 engagement induces CD4+CD25+CTLA-4high T regulatory cells with target (allo)antigen specificity

CTLA-4 (CD152) is actively involved in down-regulating T cell activation and maintaining lymphocyte homeostasis. Our earlier studies showed that targeted engagement of CTLA-4 can down-modulate T cell response and suppress allo- and autoimmune responses. In this study, we report that targeted CTLA-4 engagement can induce immune tolerance to a specific target through selective induction of an Ag-specific CD4(+)CD25(+)CTLA-4(high) regulatory T cell (Treg cell) population. Allogeneic cells coated with anti-CTLA-4 Ab induced immune hyporesponsiveness through suppression of proinflammatory cytokines IFN-gamma and IL-2, and up-regulation of the regulatory cytokines IL-10, TGF-beta1, and IL-4, presumably through the engagement of CTLA-4 on activated T cells. Although rechallenge with alloantigen failed to break the unresponsiveness, a transient recovery from tolerance was observed in the presence of high concentrations of exogenous IL-2, saturating concentrations of neutralizing anti-TGF-beta1 and anti-IL-10 Abs, and blocking anti-CTLA-4 Ab, and upon depletion of CD4(+)CD25(+) Treg cells. The CD4(+)CD25(+)CTLA-4(high) Treg cells from tolerant mice suppressed the effector function of CD25(-) T cells from Ag-primed mice. Adoptive transfer of these Treg cells into Ag-primed mice resulted in a significantly reduced alloantigen-specific response. Further characterization demonstrated that the Treg cells with memory phenotype (CD62L(-)) were more potent in suppressing the alloantigen-specific T cell response. These results strongly support that the targeted engagement of CTLA-4 has therapeutic potential for the prevention of transplant rejection.     

Making an anti-amastigote vaccine for visceral leishmaniasis: rational, update and perspectives

Visceral leishmaniasis is a major health problem in Latina America, as well as the Mediterranean region of Europe and Asia. We aimed to develop a vaccine against visceral leishmaniasis targeting the intracellular amastigotes, which is the parasite stage that persists throughout infections with Leishmania parasites. With this in mind, we identified an amastigote specific antigen (A2) that contains an immunogenic epitope for CD4+ T helper (Th) cells and multiple repetitive units encoding CD8+ cytotoxic T lymphocyte (CTL) epitopes. Vaccine formulations containing the recombinant A2 associated with saponin, alum and IL-12 or expressed by attenuated adenovirus were shown to be protective in mice, dogs and nonhuman-primates. We are currently identifying novel amastigote specific immunogenic proteins that could be aggregated to A2 to further improve the level of vaccine-induced cell-mediated immunity and protection against visceral leishmaniasis.