FoxP3+ CD8 T-cells in acute HIV infection and following early antiretroviral therapy initiation

Abstract

Objectives: Besides CD4 regulatory T-cells (Tregs), immunosuppressor FoxP3⁺ CD8 T-cells are emerging as an important subset of Tregs, which contribute to immune dysfunction and disease progression in HIV infection. However, FoxP3⁺ CD8 T-cell dynamics in acute HIV infection and following early antiretroviral therapy (ART) initiation remain understudied.

Methods: Subsets of FoxP3⁺ CD8 T-cells were characterized both prospectively and cross-sectionally in PBMCs from untreated acute (n=26) and chronic (n=10) HIV-infected individuals, early ART-treated in acute infection (n=10, median of ART initiation: 5.5 months post-infection), ART-treated in chronic infection (n=10), elite controllers (n=18), and HIV-uninfected controls (n=21).

Results: Acute and chronic infection were associated with increased total, effector memory, and terminally differentiated FoxP3⁺ CD8 T-cells, while early ART normalized only the frequencies of total FoxP3⁺ CD8 T-cells. We observed an increase in FoxP3⁺ CD8 T-cell immune activation (HLADR⁺/CD38⁺), senescence (CD57⁺/CD28^-), and PD-1 expression during acute and chronic infection, which were not normalized by early ART. FoxP3⁺ CD8 T-cells in untreated participants expressed higher levels of immunosuppressive LAP(TGF-β1) and CD39 than uninfected controls, whereas early ART did not affect their expression. The expression of gut-homing markers CCR9 and Integrin-β7 by total FoxP3⁺ CD8 T-cells and CD39⁺ and LAP(TGF-β1)⁺ FoxP3⁺ CD8 T-cells increased in untreated individuals and remained higher than in uninfected controls despite early ART. Elite controllers share most of the FoxP3⁺ CD8 T-cell characteristics in uninfected individuals.

Conclusions: Although early ART normalized total FoxP3⁺ CD8 T-cells frequencies, it did not affect the persistent elevation of the gut-homing potential of CD39⁺ and LAP(TGF-β1)⁺ FoxP3⁺ CD8 T-cell, which may contribute to immune dysfunction.

Keywords: CD39; CD8 regulatory T cells (CD8 Tregs); FoxP3; TGF-β1; acute HIV infection; early antiretroviral therapy (ART).

Figure 1

Effect of early ART initiation on total FoxP3⁺ CD8 T-cells frequencies and memory subsets. (A) Gating strategies used in flow cytometry to define total FoxP3 ⁺ CD8 T-cells and FoxP3 ⁺ CD8 T-cells memory subsets within CD8 T-cells. (B) Percentages of total FoxP3 ⁺ CD8 T-cells. Frequencies of (C) naïve (CD45RA⁺CD28⁺), (D) central memory (CM, CD45RA^-CD28⁺) (E) effector memory (EM, CD45RA^-CD28^-), and (F) terminally differentiated (TD, CD45RA⁺CD28^-) FoxP3 ⁺ CD8 T-cells subsets. Statistical significance is indicated in the figures as follow: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. 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 longitudinal study. Sample size in cross-sectional analysis: non-infected n=20, Acute n=26, Chronic ART- n=10, Chronic ART+ n=11, EC n=18. Sample size in longitudinal analysis: non-infected n=20, ART- n=10, ART+ n=10.

Figure 2

Effect of early ART initiation on FoxP3⁺ CD8 T-cell immune activation and senescence. (A) Gating strategies used in flow cytometry to define activated FoxP3 ⁺ CD8 T-cells (CD8⁺FoxP3⁺HLA-DR⁺CD38⁺). (B) Gating strategies used in flow cytometry to define immunosenescent FoxP3 ⁺ CD8 T-cells (CD8⁺FoxP3⁺CD28^-CD57⁺). Frequencies of CD8⁺FoxP3⁺HLA-DR⁺CD38⁺ (C), and CD8⁺FoxP3⁺CD28^-CD57⁺ (D) within CD8 T-cells. Statistical significance is indicated in the figures as follow: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. 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 longitudinal study. Sample size in cross-sectional analysis: non-infected n=20, Acute n=26, Chronic ART- n=10, Chronic ART+ n=11, EC n=18. Sample size in longitudinal analysis: non-infected n=20, ART- n=10, ART+ n=10.

Figure 3

Effect of early ART initiation on FoxP3⁺ CD8 T-cells subsets with known immunosuppressive functions. Gating strategies used in flow cytometry to define FoxP3 ⁺ CD8 T-cells expressing PD-1/CTLA-4 (A) and CD39/LAP(TGF-β1) (B). Frequencies of CD8⁺FoxP3⁺PD-1⁺ (C), CD8⁺FoxP3⁺CTLA-4⁺ (D), CD8⁺FoxP3⁺CD39⁺ (E), CD8⁺FoxP3⁺LAP(TGF-β1)⁺ (F), and CD8⁺FoxP3⁺CD39⁺LAP(TGF-β1)⁺ (G) within CD8 T-cells. Statistical significance is indicated in the figures as follow: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. 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 longitudinal study. Sample size in cross-sectional analysis: non-infected n=20, Acute n=26, Chronic ART- n=10, Chronic ART+ n=11, EC n=18. Sample size in longitudinal analysis: non-infected n=20, ART- n=10, ART+ n=10.

Figure 4

Effect of early ART initiation on FoxP3⁺ CD8 T-cells potential migration. (A) Gating strategies used in flow cytometry to define CD8⁺FoxP3⁺ T-cells -expressing CCR4, CCR5, CCR6, CXCR3, Integrin-β7 and CCR9. Frequencies of CD8⁺FoxP3⁺CCR4⁺ (B), CD8⁺FoxP3⁺CCR5⁺ (C), CD8⁺FoxP3⁺CCR6⁺ (D), CD8⁺FoxP3⁺CXCR3⁺ (E), CD8⁺FoxP3⁺Integrin-β7⁺ (F), and CD8⁺FoxP3⁺CCR9⁺ (G) within CD8 T-cells. Statistical significance is indicated in the figures as follow: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. 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 longitudinal study. Sample size in cross-sectional analysis: non-infected n=20, Acute n=26, Chronic ART- n=10, Chronic ART+ n=11, EC n=18. Sample size in longitudinal analysis: non-infected n=20, ART- n=10, ART+ n=10.

Figure 5

Effect of early ART initiation on the migratory potential towards the gut of FoxP3⁺ CD8 T-cells subsets with known immunosuppressive functions. (A) Gating strategies used in flow cytometry to define CCR9⁺CD39⁺, CCR9⁺LAP(TGF-β1)⁺, Integrin-β7⁺CD39⁺, and Integrin-β7⁺LAP(TGF-β1)⁺ CD8⁺FoxP3⁺ T-cells. Frequencies of CD8⁺FoxP3⁺CCR9⁺CD39⁺ (B), CD8⁺FoxP3⁺CCR9⁺LAP(TGF-β1)⁺ (C), CD8⁺FoxP3⁺Integrin-β7⁺CD39⁺ (D), and CD8⁺FoxP3⁺Integrin-β7⁺LAP(TGF-β1)⁺ (E) within CD8 T-cells. Statistical significance is indicated in the figures as follow: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. 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 longitudinal study. Sample size in cross-sectional analysis: non-infected n=20, Acute n=26, Chronic ART- n=10, Chronic ART+ n=11, EC n=18. Sample size in longitudinal analysis: non-infected n=20, ART- n=10, ART+ n=10.

Figure 6

Effect of early ART initiation on CD8⁺CD28^-PD-1⁺ and CD8⁺CD28^-CD39⁺ T-cells. Gating strategies used in flow cytometry to define CD8⁺CD28^-PD-1⁺ and CD8⁺CD28^-CD39⁺ (A). Frequencies of CD8⁺CD28^-PD-1⁺ (B) and CD8⁺CD28^-CD39⁺ (C) within CD8 T-cells. Statistical significance is indicated in the figures as follow: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. 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 longitudinal study. Sample size in cross-sectional analysis: non-infected n=20, Acute n=26, Chronic ART- n=10, Chronic ART+ n=11, EC n=18. Sample size in longitudinal analysis: non-infected n=20, ART- n=10, ART+ n=10.