Identification of Tregs. Freshly isolated PBMCs from HIV-positive patients were first negatively selected and then stained with anti-CD62L fluorescein isothiocyanate, anti-CD25 phycoerythrin, and anti-CD4 tricolor. After gating on CD4⁺ T cells, Tregs were defined as CD25^hi and CD62L^hi.

Peripheral blood Tregs are infected in vivo. Cell-associated viral loads for sorted CD4⁺ FoxP3⁺ CD25⁺ and CD4⁺ FoxP3⁻ CD25⁻ T cells. The average viral load for the two subsets is shown in the bar chart. (A) At least three different HAART-treated and ES patients are analyzed. (B) Representative graph of viral load in Tregs versus that in CD4⁺ CD25⁻ resting cells is shown for viremic patients.

Depletion of FOXP3⁺ Tregs from progressive patients. (A) Real-time qRT-PCR of FOXP3 expression in peripheral blood CD4⁺ T cells from treatment-naive (black), HAART-treated (blue), ES (red), or uninfected (green) patients. FOXP3 expression was significantly decreased in CD4⁺ T cells from HAART-treated and treatment-naive patients compared to that for ES and uninfected patients. The copy number of FOXP3 in each sample was normalized to GAPDH. Horizontal lines represent the mean for each patient group. (B) Correlation between FOXP3 copy number in CD4⁺ T cells from HIV-infected donors (treatment-naive [black], HAART-treated [blue], or ES [red]) and CD4⁺ T-cell count. The P value indicates the level of significance.

Depletion of FoxP3-expressing CD4⁺ T cells in HAART-treated patients inversely correlates with immune activation. (A) Flow cytometric analysis of CD25- and FoxP3-expressing CD4⁺ T cells from HAART-treated, ES, and uninfected patients. Representative FACS profiles of two patients from each group are shown. (B) CD4⁺ T cells expressing the FoxP3 protein were depleted for patients on HAART compared to levels for ES and uninfected patients (P < 0.01). HLA-DR expression on CD4⁺ T cells was significantly increased for patients on HAART over levels for ES and uninfected patients (top graph). No significant differences in coexpression of FoxP3 and CD69, HLA-DR, or Ki67 were observed (bottom graph). At least four different patients from each group were analyzed. (C) Correlation between FOXP3 copy number and HLA-DR expression on CD4⁺ T cells from HIV-infected donors (P < 0.05).

Treg functional activities are comparable for HAART-treated and ES patients. (A) CD4⁺ CD25⁻ CD62L^hi naïve T cells from either HAART-treated or ES patients were labeled with CFSE and cultured with anti-CD3 stimulation and irradiated autologous PBMCs. To measure suppressive activity, CD4⁺ CD25^hi CD62L^hi Tregs were added at day zero. Dilution of CFSE was measured by flow cytometry at day 6 poststimulation. Representative FACS profiles from each patient group are shown. (B) Summary of the percent suppression of proliferation of CD4⁺ CD25⁻CD62L^hi naive T cells by CD4⁺ CD25^hi CD62L^hi Tregs for three different HAART-treated or ES patients and two viremic patients is provided in graph form. All suppression assays were set up in triplicate, and results are expressed as means of triplicates. (C) Proliferative responses of CD4⁺ CD25^hi CD62L^hi Tregs to cytokine signals are comparable for HAART-treated and ES patients. Sort-purified CD4⁺ CD25^hi CD62L^hi Tregs were maintained in transwell cocultures for 6 days with anti-CD3- and anti-CD28-stimulated autologous PBMCs in the bottom wells. Proliferative responses induced by either SEB or p24 were compared. All proliferation assays were set up in triplicate, and at least three different HAART-treated and ES patients were analyzed.