CTLA-4 blockade decreases TGF-beta, IDO, and viral RNA expression in tissues of SIVmac251-infected macaques

Abstract

Regulatory T (T(reg)) cells are a subset of CD25(+)CD4(+) T cells that constitutively express high levels of cytotoxic T lymphocyte antigen-4 (CTLA-4) and suppress T-cell activation and effector functions. T(reg) cells are increased in tissues of individuals infected with HIV-1 and macaques infected with simian immunodeficiency virus (SIV(mac251)). In HIV-1 infection, T(reg) cells could exert contrasting effects: they may limit viral replication by decreasing immune activation, or they may increase viral replication by suppressing virusspecific immune response. Thus, the outcome of blocking T(reg) function in HIV/SIV should be empirically tested. Here, we demonstrate that CD25(+) T cells inhibit virus-specific T-cell responses in cultured T cells from blood and lymph nodes of SIV-infected macaques. We investigated the impact of CTLA-4 blockade using the anti-CTLA-4 human antibody MDX-010 in SIV-infected macaques treated with antiretroviral therapy (ART). CTLA-4 blockade decreased expression of the tryptophan-depleting enzyme IDO and the level of the suppressive cytokine transforming growth factor-beta (TGF-beta) in tissues. CTLA-4 blockade was associated with decreased viral RNA levels in lymph nodes and an increase in the effector function of both SIV-specific CD4(+) and CD8(+) T cells. Therefore, blunting T(reg) function in macaques infected with SIV did not have detrimental virologic effects and may provide a valuable approach to complement ART and therapeutic vaccination in the treatment of HIV-1 infection.

Figure 1.

CD25 depletion reduces virus-specific immune responses. (A) FACS analysis of CD4⁺ T cells following depletion of CD25⁺ T cells. (B) Production of cytokines by total or CD25-depleted CD4⁺ and CD8⁺ T cells following Gag-specific stimulation in vitro. Cells were obtained from lymph nodes or blood before the initiation of the study. (C) Schematic representation of the study design and numerical designation of the animals enrolled. *Mamu-A*01⁺ macaques.

Figure 2.

Ki-67, FoxP3, and CTLA-4 expression in treated macaques. Flow cytometry analysis of the Ki-67 marker on CD4⁺ or CD8⁺ T cells at the time of ART initiation (week 0) and 1 week after MDX-010 inoculation (week 6) in blood (A) or lymph nodes (B). FoxP3 expression measured by FACS in CD25⁺CD4⁺ T cells and CD25⁺CD8⁺ T cells in blood at weeks 4 and 13 (C). (D) Level of FoxP3 and CTLA-4 expression in lymph nodes of macaques before treatment (time 0) and 1 week after CTLA-4 treatment (week 6). For panels A-C, mean values plus standard error are shown. For panel D, horizontal bars within boxes correspond to the median; box limits correspond to the 25th and 75th percentiles; and vertical lines extend to the 10th and 90th percentiles.

Figure 3.

IDO mRNA expression and enzymatic activity. (A) IDO mRNA expression, analyzed by real-time PCR, on total RNA extracted from macaques' lymph nodes before initiation of ART (week 0), and after 6 weeks of treatment (ART) and MDX-010 treatment (ART and anti–CTLA-4) for each group. (B) Plasma kynureninetryptophan ratio measured by high-performance liquid chromatography in each group before initiation of ART (week 0), and after 6 weeks of treatment (ART) and MDX-010 (ART and anti–CTLA-4). Changes in IL-2 mRNA (C) and TGF-β (D) induced by ART and ART and anti–CTLA-4 after 6 weeks of treatment in each group. (E) Ratio between the level of IL-2 and TGF-β mRNA. Pre-ART indicates before initiation of ART. For panels A and E, dots represent individual values for each animal; horizontal bars correspond to the median. For panels B-D, horizontal bars within boxes correspond to the median; box limits correspond to the 25th and 75th percentiles; and vertical lines extend to the 10th and 90th percentiles.

Figure 4.

Viral RNA levels in plasma and lymph nodes. (A) Virus levels in plasma of the macaques treated with ART alone (top panel) or ART plus anti–CTLA-4 (bottom panel). (B) SIV_mac251 RNA quantified on 1 μg total RNA extracted from macaque lymph nodes before initiation of ART (pre-ART), and after 6 weeks of treatment (ART) and MDX-010 (ART and anti–CTLA-4). Horizontal bars within boxes correspond to the median; box limits correspond to the 25th and 75th percentiles; and vertical lines extend to the 10th and 90th percentiles. (C) Changes in SIV_mac251 RNA induced by ART and ART plus MDX-010 (ART and anti–CTLA-4) after 6 weeks of treatment. Mean values plus standard error are shown. (D-E) CD4⁺ (D) and CD8⁺ (E) absolute counts per cubic millimeter in blood of the macaques over time.

Figure 5.

Virus-specific T cell response. (A) Gag CM9 tetramer staining of blood lymphocytes over time in the Mamu-A*01⁺ animals of groups 1 and 2. (B) CD8⁺ and CD8^– T cells expressing CD107, TNF-α, and IFN-γ following stimulation with peptide pools encompassing the entire SIV Gag protein. The mean values and SD of the percentage of T cells positive for all markers are shown.