TGF-beta and IL-10 production by HIV-specific CD8+ T cells is regulated by CTLA-4 signaling on CD4+ T cells

Abstract

Immune dysregulation in HIV-1 infection is associated with increased expression of inhibitory molecules such as CTLA-4, TGF-beta, and IL-10. In this study we examined one potential mechanism for regulating TGF-beta and IL-10 expression by HIV-specific suppressor CD8+ T cells. No overlap between TGF-beta, IL-10, and IFN-gamma cytokine production by HIV-specific CD8+ T cells was observed. TGF-beta positive and IL-10 positive cells were FOXP3 negative, CD25 negative, and displayed a heterogeneous surface expression of CD127. TGF-beta 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-beta positive and IL-10 positive CD8+ T cell responses, and a concomitant increase in HIV-specific IFN-gamma positive CD8+ T cell responses. Depletion of CD4+ T cells abrogated the impact of CTLA-4 on HIV-specific TGF-beta 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.

Figure 1. TGF-β positive, IL-10 positive, and IFN-γ positive HIV-specific CD8+ T cell populations are distinct.

PBMC were stimulated with HIV peptides then stained with anti-IFN-γ FITC, anti-TGF-β 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-β, IFN-γ, 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-β, IFN-γ, and IL-10 after subtraction of the back ground values.

Figure 2. Analysis of regulatory surface markers expression by HIV-specific TGF-β positive CD8+ T cells.

PBMC were stimulated with HIV peptides, then stained for various memory and regulatory markers and the percentage of TGF-β positive CD8+ T cells was determined by flow cytometry. Samples were first gated on the CD3+/CD8+ lymphocyte population and then the percentages of TGF-β positive cells were determined and the extent of FOXP3, CD127, CD25, and CD27 expression was also examined. Gating was performed using the fluorescence-minus-one (FMO) control for each marker. Representative plots of the phenotype of the HIV-specific CD8+ T cells expressing TGF-β. The values marked with an asterisk represent the fraction of TGF-β positive cells that express FOXP3, CD127, CD25, or CD27 over the total number of TGF-β positive cells (equivalent to 100%).

Figure 3. CTLA-4 blockade decreases TGF-β and IL-10 expression by HIV-specific CD8+ T Cells.

PBMC (n = 6) were stimulated with HIV peptides in the presence of anti-CTLA4 (or isotype control), then stained with anti-IFN-γ FITC, anti-TGF-β PE, anti-IL-10 APC, anti-CD3 AmCyan, anti-CD4 PerCP CY5.5, anti-CD8 PE CY7, and analyzed by flow cytomerty. Samples were first gated on the CD3+/CD8+ lymphocyte population then the percent of TGF-β, IL-10, and IFN-γ positive cells were determined. Results were expressed as percent of HIV-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ after subtraction of the back ground. (A) Representative plots of HIV-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ in the presence or absence of anti-CTLA-4. (B-D) Dashed line represents the cutoff for significant TGF-β (B), IL-10 (C), and IFN-γ (D) expression. Percentages in between brackets are median values. The two dots joined by a line represent the values obtained from the same individual and analysis was performed by paired t-test.

Figure 4. HIV-specific TGF-β and IL-10 positive CD8+ T cells are CTLA-4 negative.

PBMC were stimulated with HIV peptides then stained with anti-TGF-β PE (or IL-10 PE), anti-CD3 AmCyan, anti-CD4 PerCP Cy5.5, anti-CD8 PE Cy7, anti-CTLA-4 APC, and analyzed by flow cytometry. Gating on the CTLA-4 positive cells was performed using the fluorescence-minus-one (FMO) control for CTLA-4. (A) Representative plots of samples that were first gated on the CD3+/CD4+ and CD3+/CD8+ lymphocyte population and then the percentages of CTLA-4 positive cells were determined. (B) Representative plots of samples that were first gated on the CD3+/CD8+ lymphocyte population and then the percent of TGF-β and IL-10 positive cells that express CTLA-4 was determined after subtraction of the back ground values. The values marked with an asterisk represent the fraction of TGF-β (or IL-10) positive cells that express CTLA-4 over the total number of TGF-β (or IL-10) positive cells (equivalent to 100%). Plots are from three independent experiments yielding similar results.

Figure 5. Removal of CD4+ T cells abrogates the effects of CTLA-4 blockade on HIV-specific CD8+ T Cells.

PBMC (or CD4 negative PBMC) were stimulated with HIV peptides in the presence of anti-CTLA4 (or isotype control), then stained with anti-IFN-γ FITC, anti-TGF-β PE, IL-10 APC, anti-CD3 Am Cyan, anti-CD4 PerCP CY5.5, anti-CD8 PE CY7, and analyzed by flow cytomerty. Samples were first gated on the CD3+/CD8+ lymphocyte population then the percent of TGF-β, IL-10, and IFN-γ positive cells were determined. Results were expressed as percent of HIV-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ after subtraction of the back ground. Representative plots of (A) Gag and (B) Nef-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ in the presence or absence of anti-CTLA-4. Data plots shown are representative of three volunteers examined in three independent experiments yielding similar results.