Simian immunodeficiency virus (SIV) infection influences the level and function of regulatory T cells in SIV-infected rhesus macaques but not SIV-infected sooty mangabeys

Abstract

Differences in clinical outcome of simian immunodeficiency virus (SIV) infection in disease-resistant African sooty mangabeys (SM) and disease-susceptible Asian rhesus macaques (RM) prompted us to examine the role of regulatory T cells (Tregs) in these two animal models. Results from a cross-sectional study revealed maintenance of the frequency and absolute number of peripheral Tregs in chronically SIV-infected SM while a significant loss occurred in chronically SIV-infected RM compared to uninfected animals. A longitudinal study of experimentally SIV-infected animals revealed a transient increase in the frequency of Tregs from baseline values following acute infection in RM, but no change in the frequency of Tregs occurred in SM during this period. Further examination revealed a strong correlation between plasma viral load (VL) and the level of Tregs in SIV-infected RM but not SM. A correlation was also noted in SIV-infected RM that control VL spontaneously or in response to antiretroviral chemotherapy. In addition, immunofluorescent cell count assays showed that while Treg-depleted peripheral blood mononuclear cells from RM led to a significant enhancement of CD4+ and CD8+ T-cell responses to select pools of SIV peptides, there was no detectable T-cell response to the same pool of SIV peptides in Treg-depleted cells from SIV-infected SM. Our data collectively suggest that while Tregs do appear to play a role in the control of viremia and the magnitude of the SIV-specific immune response in RM, their role in disease resistance in SM remains unclear.

FIG. 1.

Expression of FoxP3 on CD4⁺ T cells from humans and the nonhuman primates RM and SM. PBMCs from uninfected human volunteers, RM, and SM were stained for cell surface expression of CD4 and CD25 or CD127. The stained cells were fixed and stained intracellularly with anti-FoxP3. Shown are representative profiles for each species. The number in each quadrant indicates the frequency of gated CD4⁺ T cells that express the relevant marker. APC, allophycocyanin.

FIG. 2.

Cross-sectional study of Treg levels in chronically SIV-infected SM and RM. (A) Representative flow cytometric profile illustrating the FoxP3⁺ populations within memory (CD95⁺) and naïve (CD95⁻) CD4⁺ subsets in RM (n = 12) and SM (n = 12). (B) Data obtained for uninfected monkeys were used to determine the change in ABS for each Treg subset in SIV-infected SM and RM. (C) The frequencies of Tregs (means ± SD) in the axillary lymph nodes (AxLN), mesenteric lymph nodes (MesLN), and colon were determined for uninfected (n = 5) and SIV-infected (n = 4) RM.

FIG. 3.

Longitudinal study of Tregs during the acute phase of SIV infection in RM and SM. A total of 26 RM and three SM were experimentally infected intravenously with SIVmac239 and SM SIV isolate FUo, respectively, and the mean frequencies (±SD) of Tregs within the total CD4⁺ population and within the memory and naïve CD4⁺ T-cell subsets were determined by flow cytometry at the indicated time points from preinfection (pre) to 12 weeks (wk) p.i.

FIG. 4.

Longitudinal study of the levels of peripheral Tregs during acute and chronic SIV infection in RM. A total of 26 RM were experimentally infected intravenously with SIVmac239. (A) Plasma VL and (B) frequencies and ABS of Tregs were determined over the course of infection. Data shown are for eight such animals, four with high VL and four with low VL. This group of animals was administered the nucleoside reverse transcriptase inhibitor PMPA at ∼16 weeks p.i. (20 mg/kg daily for 30 days). (C) The correlation between VL and ABS of Tregs during the chronic phase was determined.

FIG. 5.

Analysis of Treg levels and frequencies of activated cells in PBMCs from chronically SIV-infected RM (group III, n = 3) that spontaneously control VL in the absence of ART. Shown for comparison are data for PBMCs from monkey RHk10, an untreated SIV-infected RM with high VL. (A) The change in ABS of each Treg subset was determined ∼8 months after experimental infection with SIVmac239. Means ± SD are shown. (B) The frequencies of CD4⁺ and CD8⁺ T cells expressing the activation markers CD25, CD69, and HLA-DR were determined. The RM with high VL is RHk10.

FIG. 6.

In vitro MLR assay to demonstrate the effect of Tregs on cell proliferation in uninfected RM and SM. (A) Treg-depleted CD4⁺ responder T cells were isolated from PBMCs from uninfected RM (n = 9) or SM (n = 9) and cocultured with a fixed number of allogeneic irradiated stimulators in the absence or presence of the indicated ratios of autologous Tregs. The allo-proliferative response was determined 5 days later by measuring ³[H]thymidine uptake; shown for each species are data representative of at least three independent assays. (B) In vitro proliferation of undepleted and CD25-depleted PBMC fractions from SIV⁻ and SIV⁺ SM (n = 10) and SIV⁻ and SIV⁺ RM (n = 10) was determined by MLR assay. Responders (undepleted or CD25-depleted PBMC fractions) were incubated with irradiated stimulators in a 96-well plate for 5 days at 37°C and 5% CO₂. Sixteen hours prior to harvest, plates were pulsed with 1 μCi/well ³[H]thymidine. Thymidine uptake was determined, and the precursor frequency was calculated. Data shown are means ± SD, representative of at least three independent assays.

FIG. 7.

Effect of Treg depletion on the SIV Env or Gag peptide-specific CD4⁺ T-cell response in PBMCs from SIV⁻ or SIV⁺ RM and SM. Shown are unfractionated PBMCs from SIV⁻ and SIV⁺ animals (black and gray bars, respectively) that are paired with their corresponding CD4⁺CD5⁺ T-cell-depleted fraction (white bars). Cells were cultured in the presence of concanavalin A (CON-A) (polyclonal positive control), Ova (OVA-PEP) (negative control), tetanus toxoid (T.T.) (antigen-specific positive control), and eight Env or Gag peptide pools in the presence of brefeldin A. Cells were surface stained for CD4 and then fixed/permeabilized and stained to detect IFN-γ. The frequency of IFN-γ-producing CD4⁺ T cells was determined by flow cytometry. Shown are data representative of 12 animals of each species. Similar data were obtained for CD8⁺ T-cell responses (data not shown).