Supplementary Figure 1. Enhanced detection of CTLA-4 on the surface of HIV-specific

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2 SUPPLEMENTARY FIGURE LEGEND Supplementary Figure 1. Enhanced detection of CTLA-4 on the surface of HIV-specific CD4 + T cells correlates with intracellular CTLA-4 levels. (a) Comparative CTLA-4 levels in HIV Gag-specific CD4 + T cells observed in the same individual by standard direct fluorescent antibody staining, enhanced surface CTLA-4 staining and intracellular staining. Filled trace: CTLA-4 expression; open trace: fluorescence minus one (FMO). (b) enhanced surface and intracellular CTLA-4 expression in Gag-specific CD4 + T cells of three representative HIV-infected individuals. Subject 1: elite controller, CD4 count 900 cell/mm 3, viral load<50 copies/ml; Subject 2: chronically infected subject with CD4 count 900 cell/mm 3, viral load<50 copies/ml on antiviral therapy; Subject 3: untreated chronically infected subject with CD4 count 370 cell/mm 3, viral load 28,000 copies/ml. (c) There is a strong correlation of CTLA-4 expression levels measured by intracellular staining and enhanced surface staining (11 HIV-infected subjects, correlation was assessed with the Spearman test), although the intensity of the latter is much smaller.

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4 Supplementary Figure 2. CTLA-4 is not upregulated by HIV-specific CD8 + T cells. (a) Comparative intracellular CTLA-4 expression in the HIV-specific CD4 + and CD8 + T cells stimulated with an Gag peptide pool of the same 4 representative untreated subjects infected with HIV illustrated in Fig 1a. MFI: median fluorescence intensity. MFI in the No antigens plots correspond to intracellular CTLA-4 level in the total CD4 + or CD8 + T cell population, respectively. MFI in the HIV Gag plots correspond to intracellular CTLA-4 expression in the IFN-γ + CD4 + or CD8 + T cell population. (b) Expression of CTLA-4 in IFN-γ-secreting HIV Gag-specific CD8 + T cells. Groups of subjects: 12 elite controllers : viral load<50 copies/ml without therapy (ELITE); 8 viremic controllers : viral load > 400 and <2,500 in the absence of antiviral treatment (VC); 14 untreated chronically HIV-infected subjects with viral load >5,000 (CXUN); 5 subjects with acute HIV infection (ACUTE); and 7 chronically HIV-infected individuals treated with antiviral therapy and viral load <50 (CXRX). Horizontal bars indicate the mean performed on log 10 MFI of CTLA- 4 expression within each group. Comparison between all groups of subjects was made with ANOVA on log 10 CTLA-4 MFI. (c) Expression of CTLA-4 in HIV Gag-specific CD8 + T cells does not correlate with expression of CTLA-4 in HIV-specific CD4 + T cells in the same subjects (P= 0.08, Spearman). (d) The enhanced detection of CTLA-4 based on the recycling of CTLA-4 molecules described in Supplementary Figure 1 does not show expression of CTLA-4 on the surface of HIV-specific CD8 + T cells, whereas this technique easily detect upregulation of CTLA-4 on the HIV-specific CD4 + T cells of the same samples, thus showing that the differences observed in intracellular levels of CTLA-4 correspond to differences in cell surface expression as well. Subject 1: chronically infected individual treated with antiviral therapy: CD4 510/mm 3, viral load < 50 copies/ml. Subject 2: chronically infected, untreated individual: CD4 357/mm 3, viral load 28,500/copies/mL.

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6 Supplementary Figure 3. CTLA-4 is pre-expressed in HIV-specific CD4 + T cells before peptide stimulation and further rapidly induced upon encounter with the cognate antigen. (a) Comparative intracellular CTLA-4 levels in HIV-specific Class II tetramer + CD4 + T cells (filled trace) and in the general CD4 + T cell population (open trace) of two HIV-infected subjects, in the absence of pre-stimulation with synthetic peptide. Subject A: elite controller, CD4 count 1,024 cell/mm 3, viral load<50 copies/ml; Subject B: untreated chronically infected subject with CD4 count 648 cell/mm 3, viral load 19,564 copies/ml. (b) Resting HIV-specific CD4 + T cells identified by Class II tetramers in PBMC express significantly higher levels of CTLA-4 than the general CD4 + T cell population (n=7; Wilcoxon signed rank test). (c) Comparative CTLA-4 levels in HIVspecific Class II-tetramer + CD4 + T cells before and after a 6-hour stimulation with the cognate epitope (untreated chronically infected subject; CD4 count 511 cell/mm 3, viral load 31,500 copies/ml). The data demonstrate that the CTLA-4 MFI of the Tet + cells before stimulation is already four-fold higher than in the general CD4 T cell population, and further rapidly increases eight-fold after encounter with the antigen.

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8 Supplementary Figure 4. Regulatory T cells do not play a major role in modulation of HIV-specific CD4 + T cell proliferation by CTLA-4 blockade in vitro. (a) Impact of CD25 + cell depletion on proliferative responses to recombinant HIV p24 in 4 representative subjects (out of 6 individuals) who responded to CTLA-4 blockade in the standard assay. Depletion of CD25 + cells usually increased the proliferative response but did not result in abrogation of the response to CTLA-4 blockade in any of the 6 subjects examined. (b) When CTLA-4 had no impact in the standard assay, this lack of effect could also be observed when CD25-depleted PBMC are used (2 representative subjects out of 4 examined). (c) Representative example showing comparative expression levels of intracellular CTLA-4 and FoxP3 in Gagspecific CD4 + T cells (defined by IFN-γ production) and regulatory T cells (defined by CD25 expression). Empty traces: general CD4 + T cell population. Pale grey filled traces: Gag-specific CD4 + T cells. Dark grey filled traces: T reg cells. Both HIV-specific CD4 + T cells and T reg cells express CTLA-4, but T reg cells express much higher levels of FoxP3 than HIV-specific CD4 + T cells. (d) Summary data on nine subjects. T reg cells express significantly higher levels of FoxP3 than Gag-specific CD4 + T cells, whereas there is no significant difference in FoxP3 expression between the HIV Gag-specific CD4 + T cells and the general CD4 + T cell population. (e) Tetramers do not identify a significant population of HIV-specific CD4 + T cells expressing markers characteristic of regulatory T cells. T reg cells are identified as the CD127 low CD25 high CD4 + T cell population in an elite controller and in a chronic viremic untreated subject. (f) There is a significantly greater frequency of CD127 low CD25 high cells in the general CD4 + T cell population than in the Tet + CD4 + T cells (n=7, P=0.016, Wilcoxon signed rank test). In Tet + CD4 + T cells the proportion of cells with this staining pattern was insignificantly small (<1 % of CD127 low CD25 high cells in the Tet + population in all 7 subjects).

9 SUPPLEMENTARY METHODS Enhanced detection of CTLA-4 expression on the cell surface of antigen-specific T cells. In order to detect the expression of CTLA-4 on the cell surface with high sensitivity, we have adapted the technique recently described to enhance detection of CD40L on the surface of antigen-specific CD4 cells 1. This technique takes advantage of the trafficking of some molecules between intracellular stocks and the cell surface after antigen stimulation and is based on addition of a fluorochrome-labeled antibody to the culture medium in the presence of monensin. For this purpose, the ICS protocol is modified as follows: carefully titrated fluorochrome-conjugated CTLA-4 antibody is added to the culture medium at the time of antigen stimulation and monensin (0.6 g/ml, BD) is used instead of brefeldin, as previously described (Chattopadhyay et al, Nat Med 2005). Class II tetramer staining. Commercially available HLA Class II tetramers (Beckman Coulter) conjugated to PE were combined with dominant HIV epitopes (DRB1*0101- DRFYKTLRAEQASQEV, DRB1*0301- PEKEVLVWKFDSRLAFHH, DRB1*0401- DRFYKTLRAEQASQEV, DRB1*1501- PEKEVLVWKFDSRLAFHH) and used to stain PBMC (20 min at 37 ºC, 5% CO2). Tetramer + cells were subsequently enriched on magnetic columns (Miltenyi Biotec), as previously described (Day et al, J Clin Invest 2003). After recovery of the tetramer-enriched fraction, surface staining and intracellular staining for CTLA-4 were completed. Exclusion channels for dead cells and CD14 and CD19 were used, as described for ICS. Identification of regulatory T cells by FoxP3 and CD25 co-staining.. A FoxP3 staining set (Ebiosciences; FoxP3 antibody: clone PCH101 conjugated to APC) was used according the manufacturer s instructions simultaneously with CD25 staining to identify

10 regulatory T cells. This assay was performed at the time of ICS staining for IFNγ after stimulation with no antigen, a Gag peptide pool or CMV lysate. CFSE proliferation assays with or without regulatory T cell depletion. Fresh CD8- depleted PBMC were stained with CFSE, split and either left untouched or depleted of CD25 cells with anti-cd25 beads (Dynal), before being submitted to stimulation with recombinant HIV p24in the presence or absence of CTLA-4 blockade, as described above.