Aim 1 We have designed a study in macaques to assess more directly the role of CD8 SIV-specific responses in control of viral replication. Depletion of CD4+ cells was performed in the immunized macaques at the peak of SIV-specific CD4+ T-cell responses following DNA prime. An additional group of macaques immunized in normal condition was depleted of CD8+ T-cells prior challenge exposure to SIVmac251. Analysis of the quality and quantity of vaccine induced CD8+ T cells demonstrated that SIV-specific CD8+ T-cells generated in conditions of CD4+ T-cell deficiency expressed low levels of Bcl2 and interleukin-2 (IL-2) and plasma virus level increased over time in these animals. Depletion of CD8+ T-cells prior challenge exposure abrogated vaccine-induced protection as expected. These data support the notion that adaptive CD4+ T-cells are critical for the generation of effective CD8+ T-cell responses to SIV that in turn contribute to protection from AIDS. Importantly, they also suggest that long-term protection from disease will only be afforded by T-cell vaccines for HIV that provide a balanced induction of CD4+ and CD8+ T-cell responses and protect against early depletion of CD4+ T-cells post-infection.
Aim 2 Negative regulatory molecules (CTLA-4 ,IDO ,FoxP3 and PD-1) and regulatory cells (Th17) at systemic and mucosal compartment of SIV infected macaques. High levels of viral replication occur in gut-associated lymphoid tissue (GALT) and other lymphoid tissues (LT) since the early phase of human/simian immunodeficiency virus (HIV/SIV) infection. Regulatory T cells (Treg), a subset of immunosuppressive T cells expressing CTLA-4 and the FoxP3 transcription factor, accumulate in LT during HIV/SIV infection. We have shown that FoxP3 and CTLA-4 mRNA are increased in leukocytes from the spleens, lymph nodes (LN), and mucosal sites of chronically SIV-infected macaques with high viremia (SIVHI) compared to animals with low viremia (SIVLO). FoxP3 and CTLA-4 correlated with SIV RNA levels in tissues; SIV virus levels in the spleen, inguinal LN, mesenteric LN, colon, and jejunum directly correlated with the plasma virus level. Importantly, CTLA-4 and FoxP3 mRNA were predominantly increased in the CD25 subpopulation of leukocytes from SIVHI, further challenging the classical definition of Treg as CD4+ CD25+ T cells. Similar to CTLA-4 and FoxP3, expression of indoleamine 2,3-dioxygenase (IDO), an immunosuppressive enzyme induced by Treg in antigen-presenting cells, was increased in the spleens, mesenteric LN, colons, and jejuna from SIVHI compared to SIVLO and directly correlated to SIV RNA in the same tissues. Accordingly, plasma kynurenine/tryptophan, a marker for IDO enzymatic activity, was significantly higher in SIVHI compared to SIVLO and correlated with plasma viral levels. Collectively these data suggested two alternative hypothesis: a) the increased Treg and IDO in LT of SIV-infected macaques may be an attempt of the host to diminish tissue inflammation and virus replication in the chronic phase of SIV infection (Tregs help the host); b) the increased in Tregs may decrease the immune response and allow viral replication (tregs help the virus). Programmed death-1 (PD-1) is a critical mediator of virus-specific CD8+ T-cell exhaustion. Here, we examined the expression of PD-1 on simian immunodeficiency virus (SIV)-specific CD8+ T cells and its possible involvement in regulation of cytokine production, proliferation, and survival of these cells. The majority of SIV-specific CD8+ T cells expressed a PD-1(high) phenotype, independent of their differentiation status, in all tissues tested. PD-1 expression gradually declined on CD8+ T cells specific for SIV-derived epitopes that had undergone mutational escape, indicating that antigen-specific TCR stimulation is the primary determinant of PD-1 expression. Loss of CD4+ T-cells in the gut is necessary but not sufficient to cause AIDS in animal models, raising the possibility that a differential loss of CD4+ T-cell subtypes may be important. We found that CD4+T-cells that produce IL-17 , a recently identified lineage of effector CD4+ T-helper cells, are infected by SIVmac251 in vitro and in vivo, and are found at lower frequency at mucosal and systemic sites within a few weeks from infection . In animals that progress to disease TH1 cells predominates on Th17 T-cells whereas in SIVmac251-infected elite controllers macaques a normal balance between these CD4+T subtypes is maintained. Indeed regression analysis of the frequency of TH17 cells at mucosal sites and plasma virus level, demonstrates a negative correlation suggesting their importance in HIV/SIV pathogenesis. Because Th17 cells play a central role in innate and adaptive immune response to extracellular bacteria, our finding may explain the chronic enteropathy in HIV infection.
Aim 3 : Pharmacological Manipulation of T-cells, with cytokines, IL7 , IL-15 and IL-21 ,or with drugs that target the immune regulatory molecules CTLA-4 ,IDO, PD-1 of SIV infected macaques. IL7&IL-15: The loss of CD4+ T cells and the impairment of CD8+ T cell function in HIV infection suggest that pharmacological treatment with IL-7 and IL-15, cytokines that increase the homeostatic proliferation of T cells and improve effector function, may be beneficial. However, these cytokines could also have a detrimental effect in HIV-1-infected individuals, because both cytokines increase HIV replication in vitro. We assessed the impact of IL-7 and IL-15 treatment on viral replication and the immunogenicity of live poxvirus vaccines in SIVmac251-infected macaques (Macaca mulatta). Neither cytokine augmented the frequency of vaccine-expanded CD4+ or CD8+ memory T cells, clonal recruitment to the SIV-specific CD8+ T cell pool, or CD8+ T cell function. Vaccination alone transiently decreased the viral set point following antiretroviral therapy suspension. IL-15 induced massive proliferation of CD4+ effector T cells and abrogated the ability of vaccination to decrease set point viremia. In contrast, IL-7 neither augmented nor decreased the vaccine effect and was associated with a decrease in TGF- expression. These results underscore the importance of testing immunomodulatory approaches in vivo to assess potential risks and benefits for HIV-1-infected individuals. CTLA-4: We directly addressed the impact of immune activation by inhibiting cytotoxic T-lymphocyte antigen-4 (CTLA-4), an immunoregulatory molecule expressed on activated T-cells and a subset of regulatory T-cells (Treg). We found that CTLA-4 blockade significantly increased T-cell activation and viral replication in primary SIVmac251 infection, particularly at mucosal sites, and increased indoleamine 2,3-dioxygenase (IDO) expression and activity. Accordingly, protracted treatment with anti-CTLA-4 antibody of macaques chronically infected with SIVmac251 decreased esponsiveness to antiretroviral therapy (ART) and abrogated the ability of therapeutic T-cell vaccines to decrease viral set point. These data provide the first direct evidence that immune activation drives viral replication, and suggest caution in the use of therapeutic approaches for HIV infection in vivo that increase CD4+ T-cell proliferation. IDO:An additional study using 1-Methyl Tryptophan (1MT) an inhibitor [summary truncated at 7800 characters]
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