HIV-Specific Ts and CTLA-4 Figure 3. CTLA-4 blockade decreases TGF-b and IL-10 expression by HIV-specific CD8+ T Cells. PBMC (n = 6) were stimulated with HIV peptides in the presence of anti-CTLA4 (or isotype control), then stained with anti-IFN-c FITC, anti-TGF-b PE, anti-IL-10 APC, anti-CD3 AmCyan, anti- CD4 PerCP CY5.5, anti-CD8 PE CY7, and analyzed by flow cytomerty. Samples were first gated on the CD3+/CD8+ lymphocyte population then the percent of TGF-b, IL-10, and IFN-c positive cells were determined. Results were expressed as percent of HIV-specific CD8+ T cells expressing TGF-b, IL- 10, or IFN-c after subtraction of the back ground. (A) Representative plots of HIV-specific CD8+ T cells expressing TGF-b, IL-10, or IFN-c in the presence or absence of anti-CTLA-4. (B-D) Dashed line represents the cutoff for significant TGF-b (B), IL-10 (C), and IFN-c (D) expression. Percentages in between brackets are median values. The two dots joined by a line represent the values obtained from the same individual and analysis was performed by paired t-test. doi:10.1371/journal.pone.0008194.g003 In this study we describe the presence of TGF-b positive CD8+ T cells with multiple antigen specificities that displayed an immunophenotype profile that is similar to our previously described IL-10 positive CD8+ Ts [2]. These TGF-b positive CD8+ T cells do not display the immunophenotypic patterns traditionally attributed to regulatory T cells. FOXP3 and CD25 surface expressions are the hallmark for identifying CD8+ Ts in diseases other than HIV infection [1,3,4,13,14,45–47]. The lack of CD127 expression on FOXP3 positive CD4+ T cells is used to distinguish between regulatory and effector T cells [48,49]. However, FOXP3 negative and CD25 negative CD8+ Ts have also been described [2]. The mechanisms leading to the induction of these TGF-b positive CD8+ T cells likely share similar pathways with other regulatory T cells. One key question is whether CD4+Treg are required for the generation and suppressive function of CD8+Ts. Inhibitory functions of regulatory T cells are mediated by direct binding of CTLA-4 and by cytokine production [28,50,51]. Surprisingly, engagement of CTLA-4 prevented the production of suppressive cytokines by the CTLA-4 negative HIV-specific CD8+ T cells. We propose a model by which CTLA-4 positive CD4+ T cells indirectly influence the inhibitory function of HIV-specific CD8+ Ts by CTLA-4 engagement. The abrogation of anti-CTLA-4 blockade in the absence of CD4+ T cells supports our hypothesis. CTLA-4 is upregulated on HIV-specific CD4+ T cells in advanced disease [29-31], and CTLA-4 is also constitutively expressed on regulatory CD4+ Treg [27]. Clearly, the presence of CD4+ Treg in HIV infection adds to the complexity of immune regulation and suppression. The influence of CTLA-4 expres- sion by populations other than CD4+ T cells on the effectiveness of CD8+ suppressor T cells remains to be determined. In summary, our study described an HIV-specific CD8+ T cell population that mediated in vitro suppression of HIV-specific effector function through the production of TGF-b. These TGF-b positive CD8+ T cells most likely confer regulatory properties. Nevertheless, the relative contribution of these TGF-b positive CD8+ T cells to the general immune dysfunction observed in HIV infection is currently unknown. The impact of HIV-specific CD8+ Ts remains an intriguing new research area that offers new direction into the consequence of HIV immunopathogenesis. PLoS ONE | www.plosone.org 5 December 2009 | Volume 4 | Issue 12 | e8194