Impact of Early ARV Initiation on Relative Proportions of Effector and Regulatory CD8 T Cell in Mesenteric Lymph Nodes and Peripheral Blood During Acute SIV Infection of Rhesus Macaques

Abstract

CD8 T cells are key players in the clearance of human immunodeficiency virus (HIV)-infected cells, such that CD8 T-cell dysfunction contributes to viral persistence despite antiretroviral (ARV) therapy. Mesenteric lymph nodes (MLNs) are major sites of gut mucosal immunity. While different CD8 T cell subsets such as CD8 alpha-alpha (CD8αα), CD8 alpha-beta (CD8αβ), CD8 regulatory T cells (Treg), and mucosa-associated invariant T cells (MAIT) are present in the gut and exhibit distinct functions, their dynamics remain poorly understood due to the lack of accessibility to these tissues in humans. We thus assessed CD8 T cells in MLNs versus peripheral blood in simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs) following early ARV therapy initiation. SIV infection was associated with an increase over time of both CD8αβ and CD8αα T cells in the blood and MLNs, whereas early ARV initiation significantly decreased the frequencies of CD8αα but not CD8αβ T cells in MLNs. A significant decrease in the expression of chemokine receptors CCR6 and CXCR3 by CD8 T cells, which are essential for T-cell trafficking to the inflammatory sites, was observed in chronically SIV-infected RMs. Surprisingly, while MAIT cells are increased in ARV-treated RMs, their frequencies in MLN are extremely low and were not impacted by ARV. The acute infection resulted in an early CD39⁺FoxP3⁺ CD8 Tregs increase in both compartments, which was normalized after early ARV. Frequencies of CD8 Treg cells were positively correlated with frequencies of CD4 Tregs and accordingly negatively correlated with the Th17/Treg ratio in the blood but not in MLNs. Overall, our results underscore the difference in CD8 T-cell subset dynamics in the blood and MLNs. IMPORTANCE Changes in CD8 T-cell subsets during acute SIV/HIV infections and following early ARV initiation in gut lymphoid tissues are poorly understood. Using an acute SIV infection model in rhesus macaques, we assessed the impact of early ARV, initiated 4 days postinfection, on relative proportions of CD8 T-cell subsets in MLNs compared to blood. We found that acute SIV infection and early ARV initiation differentially affect the distribution of effector CD8 T cells, CD8 MAIT cells, and CD8 Tregs in MLNs compared to blood. Overall, early ARV initiation maintains the frequency of effector CD8 T cells while reducing immunosuppressive CD39⁺ CD8 Tregs. Our study provides deeper insight into the dynamics of the CD8 T-cell compartment in gut mucosal immune surveillance during acute SIV infection and following early ARV initiation.

Keywords: CD8 T cells; CD8 regulatory T cells (CD8 Treg); GALT; early antiretroviral therapy; gut; gut mucosal immunity; human immunodeficiency virus (HIV); simian immunodeficiency virus (SIV).

FIG 1

Impact of early ARV initiation on CD8 subsets during SIV infection. (A) CD4/CD8 ratio in both whole blood and MLNs. (B) Gating strategy used in flow cytometry to define CD8αβ T cells and CD8αα T cells in both whole blood and MLNs. (C) Percentages of CD8αβ T cells in both whole blood and MLNs. (D) Dynamics of CD8αβ T cells in matched blood versus MLNs of early ARV-treated animals. (E) Percentages of CD8αα T cells in both whole blood and MLNs. (F) Dynamics of CD8αα T cells in matched blood versus MLNs of early ARV-treated animals. Statistical significance is indicated in the figures. Differences among five study groups was determined by nonparametric Mann-Whitney rank test for unpaired variables, while the Wilcoxon rank tests were used for paired variables in the early ARV-treated group. Sample size in cross-sectional analysis as follows: uninfected, n = 10 in blood and n = 2 in MLNs; early untreated, n = 13 in blood and n = 6 in MLNs; early ARV treated, n = 8 in blood and n = 6 in MLNs; early ARV interrupted, n = 5 in blood and n = 5 in MLNs; and chronic, n = 4 in blood and n = 3 in MLNs. Sample size in paired analysis, n = 5.

FIG 2

Effect of early ARV initiation on the expression of migration markers CCR6 and CXCR3 by memory CD8 T cells. (A) Gating strategy used in flow cytometry to define CCR6⁺ memory CD8 T cells in both whole blood and MLNs. (B) Percentages of CCR6⁺ memory CD8 T cells in both whole blood and MLNs. (C) Dynamics of CCR6⁺ memory CD8 T cells in matched blood versus MLNs of early ARV-treated animals. (D) Gating strategy used in flow cytometry to define CXCR3⁺ memory CD8 T cells in both whole blood and MLNs. (E) Percentages of CXCR3⁺ memory CD8 T cells in both whole blood and MLNs. (F) Dynamics of CXCR3⁺ memory CD8 T cells in matched blood versus MLNs of early ARV-treated animals. Statistical significance is indicated in the figures. Differences among five study groups were determined by nonparametric Mann-Whitney rank test for unpaired variables, while the Wilcoxon rank tests were used for paired variables in the early ARV-treated group. Sample size in cross-sectional analysis as follows: uninfected, n = 10 in blood and n = 2 in MLNs; early untreated, n = 13 in blood and n = 6 in MLNs; early ARV treated, n = 8 in blood and n = 6 in MLNs; early ARV interrupted, n = 5 in blood and n = 5 in MLNs; and chronic, n = 4 in blood and n = 3 in MLNs. Sample size in paired analysis, n = 5.

FIG 3

Effect of early ARV initiation on CD8 MAIT cells. (A) Gating strategy used in flow cytometry to define CD8 MAIT cells in both whole blood and MLNs. (B) Percentages of CD161⁺TCRVa7.2⁺ CD8 MAIT cells in both whole blood and MLNs. (C) Dynamics of CD161⁺ TCRVα7.2⁺ CD8 MAIT cells in matched blood versus MLNs of early ARV-treated animals. Statistical significance is indicated in the figures. Differences among five study groups was determined by nonparametric Mann-Whitney rank test for unpaired variables, while the Wilcoxon rank tests were used for paired variables in the early ARV-treated group. Sample size in cross-sectional analysis as follows: uninfected, n = 10 in blood and n = 2 in MLNs; early untreated, n = 13 in blood and n = 6 in MLNs; early ARV treated, n = 8 in blood and n = 6 in MLNs; early ARV interrupted, n = 5 in blood and n = 5 in MLNs; and chronic, n = 4 in blood and n = 3 in MLNs. Sample size in paired analysis, n = 5.

FIG 4

Effect of early ARV on total CD8 Tregs and CD39⁺ CD8 Tregs. (A) Gating strategy used in flow cytometry to define CD8 Tregs (FoxP3⁺ CD8 T cells) and CD39⁺ CD8 Tregs (CD8⁺ FoxP3⁺ CD39⁺) in both whole blood and MLNs. (B) Percentages of total CD8 Tregs in both whole blood and MLNs. (C) Dynamics of total CD8 Tregs in matched blood versus MLNs of early ARV-treated animals. (D) Percentages of CD39⁺ CD8 Tregs in both whole blood and MLNs. (E) Dynamics of CD39⁺ CD8 Tregs in matched blood versus MLNs of early ARV-treated animals. Statistical significance is indicated in the figures. Differences among five study groups was determined by nonparametric Mann-Whitney rank test for unpaired variables, while the Wilcoxon rank tests were used for paired variables in the early ARV-treated group. Sample size in cross-sectional analysis as follows: uninfected, n = 10 in blood and n = 2 in MLNs; early untreated, n = 13 in blood and n = 6 in MLNs; early ARV treated, n = 8 in blood and n = 6 in MLNs; early ARV interrupted, n = 5 in blood and n = 5 in MLNs; and chronic, n = 4 in blood and n = 3 in MLNs. Sample size in paired analysis, n = 5.

FIG 5

Correlation between CD8 Tregs and CD4 Tregs (A) and memory CCR6⁺ Th17/Treg ratio (B) in both blood and MLNs. (C) Correlation between CD39⁺ CD8 Tregs and memory CCR6⁺ Th17/Treg ratio in both blood and MLNs. Correlations were done with the following sample size: uninfected, n = 10 in blood and n = 2 in MLNs; early untreated, n = 13 in blood and n = 6 in MLNs; early ARV treated, n = 8 in blood and n = 6 in MLNs; early ARV interrupted, n = 5 in blood and n = 5 in MLNs; and chronic, n = 4 in blood and n = 3 in MLNs. P values were obtained following the Spearman correlation coefficient test.

FIG 6

Study protocol. A total of 32 female RMs were enrolled in this study. A total of 25 animals were infected intravenously with 20 50% animal infectious doses of SIVmac251 virus, and the specimens were collected in both acute (early untreated) and chronic phases of infection. Nine monkeys were treated 4 days after the infection in a daily manner with an ARV cocktail. After 8 weeks of ARV treatment, the therapy was interrupted in five RMs. Two groups of nontreated animals in both acute (early untreated, n = 13) and chronic (n = 4) phases were also assessed. Blood specimens were obtained from 10 animals 3 days before the SIVmac251 infection to assess the uninfected baseline status. In addition, MLN specimens from two uninfected animals were also assessed to establish a baseline status in the MLNs. In the figure, black arrows represent the time point where samples from whole blood and/or mesenteric lymph nodes (MLNs) were taken.*, samples from MLNs were taken upon euthanasia; **, for one animal, only sample from MLNs was taken.