HIV-Specific Ts and CTLA-4 Figure 1. TGF-b positive, IL-10 positive, and IFN-c positive HIV-specific CD8+ T cell populations are distinct. PBMC were stimulated with HIV peptides then stained with anti-IFN-c FITC, anti-TGF-b PE, anti-CD3 AmCyan, anti-CD4 PerCP Cy5.5, and anti-CD8 PE Cy7, anti-IL-10 APC, and analyzed by flow cytometry. Samples were first gated on the CD3+/CD8+ lymphocyte population then the percent of TGF-b, IFN-c, and IL-10 positive CD8+ T cells were determined. (A) Data from individuals with significant cytokine expression and analysis were performed by Mann-Whitney U test. (B) Representative plots of the number of HIV-specific CD8+ T cells expressing TGF-b, IFN-c, and IL-10 after subtraction of the back ground values. doi:10.1371/journal.pone.0008194.g001 stimulated with HIV peptides and the extent of FOXP3, CD25, CD127, and CD27 expression by the HIV-specific TGF-b positive CD8+ T cells was assessed. Representative plots are shown in Fig. 2. HIV-specific TGF-b positive CD8+ T cells were FOXP3 negative and CD25 negative. Similar to the IL-10 positive CD8+ Ts, Gag-specific TGF-b positive CD8+ T cells displayed heterogeneous expression of CD27 and CD127. In contrast, HIV-specific IFN-c positive CD8+ T cells were CD27 and CD127 negative (data not shown). These results indicate that the HIV- specific TGF-b positive CD8+ T cells are different from the population of suppressor CD8+ T cells in HIV negative individuals [1,4,11,12,39]. CTLA-4 Blockade Decreases TGF-b and IL-10 Expression by HIV-Specific CD8+ T Cells In vitro blockade of CTLA-4 engagement augments HIV- specific CD4+ T cell proliferation, IL-2, and IFN-c production [29]. We explored whether this inhibitory mechanism is involved in HIV-specific TGF-b and IL-10 CD8+ T cells. PBMC from 6 HIV-positive volunteers with demonstrated Gag- and Nef-specific TGF-b and IL-10 positive CD8+ T cell responses were incubated with anti-CTLA-4 Ab (or isotype control). Representative plots are shown in Fig. 3A. CTLA-4 blockade resulted in a significant decrease in the frequency of HIV-specific TGF-b (Fig. 3C) and IL- 10 (Fig. 3D) positive CD8+ T cell responses. In contrast, blockade of CTLA-4 led to a significant increase in the frequency of HIV- specific IFN-c positive CD8+ T cell responses (Fig. 3B). These results suggest that engagement of CTLA-4 regulates TGF-b and IL-10 production by HIV-specific CD8+ T cells. HIV-Specific TGF-b and IL-10 Positive CD8+ T Cells Are CTLA-4 Negative CTLA-4 is upregulated in HIV-specific CD4+ T cells [29], particularly in patients with chronic HIV-1 infection [33]. We next determined whether CTLA-4 is also expressed on TGF-b and IL- 10 positive CD8+ T cells. Representative plots are shown in Fig. 4. We found no evidence of CTLA-4 expression on CD8+ T cells, including TGF-b and IL-10 positive CD8+ T cells (Fig. 4B) as well as on CD8+ IFN+ T cells (data not shown). This is in contrast to the high CTLA-4 expression detected on CD4+ T cells (median PLoS ONE | www.plosone.org 3 December 2009 | Volume 4 | Issue 12 | e8194