TGF-b and IL-10 Production by HIV-Specific CD8+ T Cells Is Regulated by CTLA-4 Signaling on CD4+ T Cells Mohamed Elrefaei1, Candace M. Burke1, Chris A. R. Baker1, Norman G. Jones1, Stephanie Bousheri1, David R. Bangsberg2, Huyen Cao1* 1 California Department of Public Health, Richmond, California, United States of America, 2 Harvard Initiative for Global Health, Massachusetts General Hospital, Cambridge, Massachusetts, United States of America Abstract Immune dysregulation in HIV-1 infection is associated with increased expression of inhibitory molecules such as CTLA-4, TGF-b, and IL-10. In this study we examined one potential mechanism for regulating TGF-b and IL-10 expression by HIV- specific suppressor CD8+ T cells. No overlap between TGF-b, IL-10, and IFN-c cytokine production by HIV-specific CD8+ T cells was observed. TGF-b positive and IL-10 positive cells were FOXP3 negative, CD25 negative, and displayed a heterogeneous surface expression of CD127. TGF-b and IL-10 positive CD8+ T cells did not express CTLA-4. Nevertheless, CTLA-4 blockade resulted in a significant decrease in HIV-specific TGF-b positive and IL-10 positive CD8+ T cell responses, and a concomitant increase in HIV-specific IFN-c positive CD8+ T cell responses. Depletion of CD4+ T cells abrogated the impact of CTLA-4 on HIV-specific TGF-b positive and IL-10 positive CD8+ T cells. Our study suggests that CTLA-4 Signaling on CD4+ T cells regulates the inhibitory functions of the HIV-specific suppressor CD8+ T cells. Citation: Elrefaei M, Burke CM, Baker CAR, Jones NG, Bousheri S, et al. (2009) TGF-b and IL-10 Production by HIV-Specific CD8+ T Cells Is Regulated by CTLA-4 Signaling on CD4+ T Cells. PLoS ONE 4(12): e8194. doi:10.1371/journal.pone.0008194 Editor: Fu-Sheng Wang, Beijing Institute of Infectious Diseases, China Received July 7, 2009; Accepted November 5, 2009; Published December 14, 2009 Copyright: 2009 Elrefaei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Work was supported by NIH grants AI43885, MH54907, AI71772, and California Research Center for the Biology of HIV in Minorities grant, UC Davis. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hcao@dhs.ca.gov Introduction Suppressor CD8+ T cells (CD8+ Ts) exert their inhibitory effects on T cell proliferation and cytokine production through a cell-cell contact-dependent mechanism [1–4], and by secreting the immunosuppressive cytokines interleukin-10 and TGF-b [5,6]. CD8+ Ts mediate non-antigen specific suppression of T cell responses via IL-10 [7–10]. These suppressor CD8+ T cells are typically CD28 negative [11,12] and express CD25, FOXP3, and CTLA-4 [1,4,13,14]. Transforming growth factor b (TGF-b) is another immunosuppressive cytokine that inhibits T cell functions [15–18]. TGF-b up-regulates CTLA-4 expression [19–21] and suppresses T cell proliferation and IL-2 production [22]. Increased TGF-b and IL-10 is associated with disease progression in HIV-1- infected patients [23,24], and likely influences the antiviral immune responses [23–25]. Cytotoxic T lymphocyte antigen 4 (CTLA-4) is overexpressed on activated T cells and inhibits IL-2 production and cell cycle progression by binding to its ligands B7-1 (CD80) and B7-2 (CD86) [26,27]. CTLA-4 is also constitutively expressed on regulatory CD4+ T cells (Treg) and mediates T cell suppression [27]. Treg inhibits T cells via direct cell-cell interaction or indirectly by an antigen-presenting cell–mediated mechanism [28]. High CTLA-4 expression correlates with markers of HIV disease progression [29–31]. CTLA-4 is upregulated on HIV- specific CD4+, but not CD8+, T cells and in vitro blockade of CTLA-4 augments HIV-specific CD4+ T cell functions [29]. Upregulation of CTLA-4 also increases CCR5 expression and enhances susceptibility of CD4+ T cells to HIV infection [32]. The frequency of CTLA-4 positive Treg is increased in patients with chronic HIV-1 infection and is suspected to play a critical immunomodulatory role leading HIV-associated immune dys- function [33]. In this study, we explore the relationship between CTLA-4 positive CD4+ T cells and the production of TGF-b and IL-10 by HIV-specific CD8+ T cells. Materials and Methods Study Subjects and Samples HIV positive volunteers (n = 37) were recruited from the ‘‘The Research in Access to Care in the Homeless (REACH)’’ cohort in San Francisco as previously described [2,34]. Demographic information and CD4+ T cell count was obtained at the time of enrollment and blood draw. Institutional Review Board approvals were obtained from the California Department of Public Health and UCSF Committee on Human Research, and all study participants gave written informed consent. None of the study participants have received antiretroviral therapy (ART) for at least 6 months. HIV RNA level was determined from plasma using the Roche Amplicor 1.5 (Roche, Branchburg, New Jersey), as per manufacturer’s recommendations. Peripheral blood mononuclear cells (PBMC) were separated and cryopreserved in liquid nitrogen until assay time. Antigens Peptides corresponding to the clade B consensus sequences of HIV-1 for Gag and Nef were synthesized as 15 amino acids (a.a.) overlapping by 11 a.a. (Mitochor Mimotopes,Victoria, Australia). PLoS ONE | www.plosone.org 1 December 2009 | Volume 4 | Issue 12 | e8194