Synthetic peptides for Gag (total = 123), and Nef (total = 49), used for all T cell assays were pooled into one single pool of peptides with final concentration of 1 mg/ml per peptide [35]. Flow-Based Intracellular Cytokine Staining Detection of HIV-specific TGF-b, IL-10, and IFN-c production was performed as previously described [36]. PBMC (16106) were incubated with Gag or Nef peptide pools for 2 hours at 37uC in 5% CO2 in the presence of one of these following antibodies: anti- TGF-b R II (2 mg/ml, R&D systems, Minneapolis MN), anti- CTLA-4 (5 mg/ml, BD Phamingen), or the respective isotype control for 12–14 hours at 37uC in 5% CO2 in the presence of co- stimulatory anti-CD49d (1 mg/ml, Becton-Dickinson) and Golgi stop (BD Pharmigen). Phorbol myristate acetate (PMA, 50 ng/ml) and ionomycin (1 mg/ml; Sigma-Aldrich, St. Louis, MO), Lypopolysacccharide (LPS, 1 ng/ml; Sigma-Aldrich), and phyto- haemagglutinin (PHA, 10 mg/ml; Sigma-Aldrich) were used as positive controls for TGF-b, IL-10 and IFN-c production respectively. Media alone without antigen stimulation was used as negative control. All samples were stained with an amine reactive viability dye as a dead cell exclusion marker (Molecular probes, Eugene, OR) [37]. In some experiments depletion of CD4+ T cells was performed using the MACS CD4 depletion kit following the manufacturer’s protocol (Miltenyi Biotec, Germany). Cells were then stained with specific combinations of the following antibodies: CD27 FITC, CD4 PerCP CY5.5, CD127 Alexa Fluor 647, CD8 PE CY7, CD3 AmCyan (BD Pharmigen). PBMC were permeabilized and stained with different combinations of the following antibodies: CTLA-4 APC, IL-10 PE, IL-10 APC, IFN-c FITC (BD Pharmigen), TGFb PE (Biotest Diagnostics, Denville, NJ), and analyzed by flow cytometry. A minimum of 30,000 CD3+ T cells per sample were acquired using a 6-color flow cytometer (LSRII, BD Biosciences) and analysis was performed by FLOWJO software (TreeStar, San Carlos, CA). Results were expressed as: Percent TGF-b, IL-10, or IFN-c positive CD8+, T cells (Percent positive = % antigen-specific - % negative control). Responses greater than or equal to 0.1% and 2 times the background were considered positive. The extent of CTLA-4, CD27, and CD127 expression was also assessed. All volunteers demonstrated significant TGF-b, IL-10, and IFN-c production following PMA/Ionomycin, LPS, and PHA stimulation respectively. Background expression was ,0.1 percent. Flow-Based FOXP3 Staining Assay PBMC were stained for FOXP3 expression following the manufacturer’s protocol (eBioscience, San Diego, CA) with some modifications [2]. Briefly, PBMC (16106) were incubated with HIV peptide pools for 12–14 hrs as described above for intracellular cytokine staining. Cells were then stained with an amine reactive viability dye as a dead cell exclusion marker (Molecular probes) [37], and the following antibodies: FOXP3 FITC (eBioscience), TGF-b PE, CD4 PerCP Cy5.5, CD8 PE CY7, CD25 allophycocyanin, and CD3 AmCyan (BD Pharmi- gen). Analysis was performed by flow cytometry as described above. The percentage of TGF-b positive CD8+ T cells was determined and the extent of FOXP3 and CD25 expression was also assessed. Results were expressed as the fraction of TGF-b positive cells that expressed FOXP3 or CD25 over the total number of TGF-b positive cells (equivalent to 100%). Statistical Analysis Groups were compared using the Mann-Whitney U test or the paired t test. Analysis was performed with PRISM software version 4.02 (Graph-Pad). Statistical significance was defined as p,0.05. HIV-Specific Ts and CTLA-4 Results TGF-b, IL-10, and IFN-c Producing HIV-Specific CD8+ T Cells Are Distinct Populations Specific populations of HIV-specific CD8+ T cells have been shown to express high TGF-b and IL-10 levels [18,25,38]. We first measured the frequency of Gag and Nef-specific TGF-b positive and IL-10 positive CD8+ T cells in 37 HIV-1 infected volunteers (Fig. 1A). IFN-c production by Gag- and Nef-specific CD8+ T cells was determined concurrently. Volunteers had a median age of 45 years (range, 31–61), CD4+ T cell count of 168 cells/mm3 (range, 19–233), and median HIV plasma RNA of 132200 copies/ ml (range, 19000–780000). Representative plots of the frequency of the HIV-specific TGF-b positive, IL-10 positive, and IFN-c positive CD8+ T cells are shown in Fig. 1B. No overlap between HIV-specific CD8+ T cells producing TGF-b, IL-10 or IFN-c was observed. All volunteers demonstrated significant Gag- (median = 0.48%; range = 0.1%23.5%) and Nef- (median = 0.4%; range = 0.1%22.2%) specific IFN-c positive CD8+ T cell responses. Fourteen and fifteen volunteers demonstrated significant Gag-, and Nef-, specific TGF-b positive CD8+ T cell responses respectively (median = 0.6%; range = 0.15%22.0%, and median = 0.4%; range = 0.1%22.0% for Gag and Nef, respectively). Sixteen volunteers demonstrated significant Gag- (median = 0.13%; range = 0.1%20.45%) and Nef- (median = 0.19%; range = 0.1%20.6%) specific IL-10 positive CD8+ T cell responses. Differences in the frequencies of Gag- and Nef-specific TGF-b (or IL-10) positive CD8+ T cell responses was not significant (p = 0.22 and p = 0.45 for TGF-b and IL-10 respectively). We have previously demonstrated that HIV-specific IL-10 positive CD8+ T cells are increased in advanced HIV disease and are positively correlated with plasma HIV RNA [38]. No correlation was found between the frequency of the HIV-specific TGF-b positive CD8+ T cells and the CD4 count or plasma HIV RNA (for Gag: p = 0.07 and p = 0.1, for Nef: p = 0.68 and p = 0.13, for CD4 count or plasma HIV RNA respectively; data not shown). However, the relatively small number of volunteers in the study limited the full examination of this relationship. TGF-b Receptor II Blockade Increases IFN-c Expression by HIV-Specific CD8+ T Cells We have previously described the presence of suppressor HIV- specific IL-10 positive CD8+ T cells [2,38]. We hypothesized that HIV-specific TGF-b positive CD8+ T cells also exert immuno- suppressive functions [15–18,23,24]. To determine the effect of TGF-b on the HIV-specific effector functions (as measured by IFN-c production), PBMC from three HIV-positive volunteers with evidence of Gag- and Nef-specific TGF-b positive CD8+ T cells were co-cultured in the presence of anti-TGF-b receptor II Ab (or isotype control). Blocking of the TGF-b receptor II led to increased Gag- and Nef-specific IFN-c positive CD8+ T cell responses (data not shown). These results suggest that TGF-b production by CD8+ T cells inhibits HIV-specific effector functions. Analysis of the Immunophenotypic Profile of HIV-Specific TGF-b Positive CD8+ T Cells We have previously shown that the suppressor HIV-specific IL- 10 positive CD8+ T cells were FOXP3 and CD25 negative [2], and displayed a heterogenous memory phenotype that was distinct from the HIV-specific effector CD8+ T cell population [38]. In this study, we examined the immunophenotypic profile of the HIV-specific TGF-b positive CD8+ T cells. PBMC were PLoS ONE | www.plosone.org 2 December 2009 | Volume 4 | Issue 12 | e8194