HIV-Specific Ts and CTLA-4 Figure 2. Analysis of regulatory surface markers expression by HIV-specific TGF-b positive CD8+ T cells. PBMC were stimulated with HIV peptides, then stained for various memory and regulatory markers and the percentage of TGF-b positive CD8+ T cells was determined by flow cytometry. Samples were first gated on the CD3+/CD8+ lymphocyte population and then the percentages of TGF-b positive cells were determined and the extent of FOXP3, CD127, CD25, and CD27 expression was also examined. Gating was performed using the fluorescence-minus-one (FMO) control for each marker. Representative plots of the phenotype of the HIV-specific CD8+ T cells expressing TGF-b. The values marked with an asterisk represent the fraction of TGF-b positive cells that express FOXP3, CD127, CD25, or CD27 over the total number of TGF-b positive cells (equivalent to 100%). doi:10.1371/journal.pone.0008194.g002 = 2.8%; range = 1.3%27.0%; Fig. 4A). The lack of CTLA-4 expression on the CD8+ T cells suggests that the effect of CTLA-4 blockade on the TGF-b and IL-10 positive CD8+ T cells is mediated by an indirect mechanism. We next attempted to further elucidate the role of CTLA-4 on TGF-b and IL-10 production by HIV-specific CD8+ T cells. We depleted CD4+ T cells in order to examine the effect of CTLA-4 expression by these CD4+ T cells on TGF-b and IL-10 production. Removal of CD4+ T cells fully abrogated the effect of CTLA-4 blockade on TGF-b, IL-10, and IFN-c production by HIV-specific CD8+ T cells that we described above (Fig. 5). Adding back the autologous CD4+ T cells fully restored the effect of CTLA-4 blockade on TGF-b-, IL-10-, and IFN-c-positive HIV- specific CD8+ T cells. While a relatively small number of volunteers were examined in the study, these results strongly support the role of CTLA-4 positive CD4+ T cells in the regulation of TGF-b, IL-10, and IFN-c production by HIV- specific CD8+ T cells. Discussion The mechanisms leading to the progressive loss of immune function and eventual failure of T cell responses in chronic HIV-1 infection remain unresolved. One hypothesis is that effector T cells succumb to an immunosuppressive environment. Multiple regu- latory pathways are functionally altered and different CD4+ Treg subpopulations are induced in different stages of HIV infection [reviewed in [40]]. Similarly, distinct subpopulations of CD8+ Ts may also contribute to the immune dysregulation observed in HIV infection [1,2,4,38]. Suppressor CD8+ T cells produce IL-10 and TGF-b, and mediate non-antigen specific suppression of T cell responses [41–43]. Our current study focuses on the mechanisms of suppression utilized by CD8+ Ts in inhibiting HIV-specific CD8+ T cell responses. The inhibition of HIV-specific T-cell responses by CD8+ Ts has been attributed to TGF-b and IL-10 production [18,38]. We have previously described the presence of IL-10 positive CD8+ Ts that inhibited HIV-specific cytolysis and IL-2 production by direct cell-cell interaction [2], and indirectly by releasing IL-10 [38]. The presence of these suppressor IL-10 positive CD8+ T cells was associated with increased expression of the inhibitory receptor programmed cell death-1(PD-1) on exhausted HIV-specific effector CD8+ T cells [36]. However, blockade of the PD-1/PDL-1 pathway did not prevent suppression, suggesting that PD-1 engagement is not a mechanism utilized by the IL-10 positive CD8+ T cells. We now describe an HIV-specific TGF-b positive CD8+ T cell population that is distinct from IL-10 positive CD8+ Ts. Blocking the binding of TGF-b to its receptor resulted in increased IFN-c expression by HIV-specific CD8+ T cells. We postulate that these TGF-b positive CD8+ T cells exert an inhibitory effect on HIV-specific effector function in vitro. Nevertheless, the suppressive impact of these TGF-b CD8+ T cells in vivo remains unknown, and direct cell-cell contact may be required for the these TGF-b positive CD8+ T cells to exert their maximum inhibitory effect. The intriguing role by which distinct antigen-specificity of these TGF-b positive CD8+ T cells contrib- utes to the level of suppression is remains to be explored [18]. We have previously shown that IL-10 positive CD8+ Ts are observed in late HIV disease [38]. The induction and detection of TGF-b positive CD8+ T cells in the course of HIV disease is still unknown but may develop earlier in HIV disease [44]. Because TGF-b is a key regulator in the suppressive signaling pathways implicated in immune regulation, signaling through TGF-b receptor may promote differential and temporal effect on T cells in different stages of HIV disease. We have shown previously that the concomitant presence of Gag- and Nef-specific IL-10 positive CD8+ Ts augmented the in vitro suppressive effect on effector CD8+ T cells [38]. We postulate that the concomitant presence of HIV-specific IL-10 positive and TGF-b positive CD8+ T cells will promote additive suppression of effector CD8+ T cell responses. PLoS ONE | www.plosone.org 4 December 2009 | Volume 4 | Issue 12 | e8194