Double-Negative T-Cells during Acute Human Immunodeficiency Virus and Simian Immunodeficiency Virus Infections and Following Early Antiretroviral Therapy Initiation

Abstract

HIV infection significantly affects the frequencies and functions of immunoregulatory CD3⁺CD4^-CD8^- double-negative (DN) T-cells, while the effect of early antiretroviral therapy (ART) initiation on these cells remains understudied. DN T-cell subsets were analyzed prospectively in 10 HIV+ individuals during acute infection and following early ART initiation compared to 20 HIV-uninfected controls. In this study, 21 Rhesus macaques (RMs) were SIV-infected, of which 13 were assessed during acute infection and 8 following ART initiation four days post-infection. DN T-cells and FoxP3⁺ DN Treg frequencies increased during acute HIV infection, which was not restored by ART. The expression of activation (HLA-DR/CD38), immune checkpoints (PD-1/CTLA-4), and senescence (CD28^-CD57⁺) markers by DN T-cells and DN Tregs increased during acute infection and was not normalized by ART. In SIV-infected RMs, DN T-cells remained unchanged despite infection or ART, whereas DN Treg frequencies increased during acute SIV infection and were not restored by ART. Finally, frequencies of CD39⁺ DN Tregs increased during acute HIV and SIV infections and remained elevated despite ART. Altogether, acute HIV/SIV infections significantly changed DN T-cell and DN Treg frequencies and altered their immune phenotype, while these changes were not fully normalized by early ART, suggesting persistent HIV/SIV-induced immune dysregulation despite early ART initiation.

Keywords: CD4−CD8− T-cells; acute HIV infection; acute SIV infection; double negative (DN) T-cells; early ART; regulatory T-cells (Tregs).

Figure 1

Study protocol. A total of 21 female Rhesus macaques (RMs) were infected intravenously with 20 50% animal infectious doses (AIDs) of SIVmac251 virus, and the specimens were collected in the acute phase of infection in 13 animals in the absence of ART. Eight monkeys were treated four days after the infection in a daily manner with an ART cocktail. Blood specimens were obtained from 10 SIV-uninfected animals that were used as controls. Black arrows represent the time when samples from whole blood were taken. Of note, each “D” followed by a number indicates one animal; therefore, in some cases, blood was collected from more than one animal on the same day. Nota bene: Blood samples from 3 animals were collected before and after SIV infection.

Figure 2

(A) Gating strategy used in flow cytometry to determine total CD3⁺CD4⁻CD8⁻ (double-negative, DN) T-cell (left) and DN T-cell memory subsets based on CD45RA/CD28 and CD127 expression (right) in the human study. Percentages determined in flow cytometry of total DN T-cells (B), central memory (CM, CD45RA⁻CD28⁺) (C), terminally differentiated (TD, CD45RA⁺CD28⁻) (D), effector memory (EM, CD45RA⁻CD28⁻) (E), and naïve (CD45RA⁺CD28⁺) (F) subsets within DN T-cells in the human study. (G) Percentages determined in flow cytometry of CD127⁺ DN T-cells in the human study. After the Kruskal–Wallis analysis, the differences among the three study groups were determined by a nonparametric Mann–Whitney rank test for unpaired variables (non-infected vs. acute, non-infected vs. ART-treated) and a Wilcoxon signed-rank test for paired variables (acute vs. ART-treated). Sample sizes in flow cytometry analysis: non-infected (n = 20), acute, and ART-treated (n = 10). Horizontal lines in graphs represent the median. Only statistical significances are presented (*, p < 0.05; **, p < 0.01).

Figure 3

(A) Gating strategy used in flow cytometry to determine CD73/CD39, CD38/HLA-DR, CTLA-4/PD-1, and CD57/CD28 expression within DN T-cells in the human study. Percentages determined in flow cytometry of CD73⁺ (B), CD39⁺ (C), CD39⁺CD73⁺ (D), CD38⁺ (E), HLA-DR⁺ (F), HLA-DR⁺CD38⁺ (G), PD-1⁺ (H), CTLA-4⁺ (I), CTLA-4⁺PD-1⁺ (J), and CD28⁻CD57⁺ (K) within DN T-cells in the human study. After the Kruskal–Wallis analysis, the differences among the three study groups were determined by a nonparametric Mann–Whitney rank test for unpaired variables (non-infected vs. acute, non-infected vs. ART-treated) and Wilcoxon signed-rank test for paired variables (acute vs. ART-treated). Sample sizes in flow cytometry analysis: non-infected (n = 20), acute, and ART-treated (n = 10). Horizontal line s in graphs represent the median. Only statistical significances are presented (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Figure 4

(A) Gating strategy used in flow cytometry to determine total FoxP3⁺ DN T-cells (DN Tregs) (left) in the human study. (B) Percentages determined in flow cytometry of total DN Tregs within DN T-cells in the human study. (C) Gating strategy used in flow cytometry to determine DN Treg memory subsets based on CD45RA/CD28, CD73/CD39, CD38/HLA-DR, CTLA-4/PD-1, and CD57/CD28 expression within DN Tregs in the human study. Percentages determined in flow cytometry of naïve (CD45RA⁺CD28⁺) (D), terminally differentiated (TD, CD45RA⁺CD28⁻) (E), effector memory (EM, CD45RA⁻CD28⁻) (F), central memory (CM, CD45RA⁻CD28⁺) (G), CD73⁺FoxP3⁺ (H), CD39⁺FoxP3⁺ (I), CD39⁺CD73⁺FoxP3⁺ (J), CD38⁺FoxP3⁺ (K), HLA-DR⁺FoxP3⁺ (L), HLA-DR⁺CD38⁺FoxP3⁺ (M), PD-1⁺FoxP3⁺ (N), CTLA-4⁺FoxP3⁺ (O), CTLA-4⁺PD-1⁺FoxP3⁺ (P), and CD28⁻CD57⁺FoxP3⁺ (Q) DN T-cells in the human study. After the Kruskal–Wallis analysis, the differences among the three study groups were determined by a nonparametric Mann–Whitney rank test for unpaired variables (non-infected vs. acute, non-infected vs. ART-treated) and Wilcoxon signed-rank test for paired variables (acute vs. ART-treated). Sample sizes in flow cytometry analysis: non-infected (n = 20), acute, and ART-treated (n = 10). Horizontal line in graphs represent the median. Only statistical significances are presented (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Figure 5

(A) Gating strategy used in flow cytometry to determine CCR6, CCR9, and CXCR3 expression within DN T-cells in the human study. Percentages determined in flow cytometry of CCR6⁺ (B), CCR9⁺ (C), and CXCR3⁺ (D) within DN T-cells in the human study. (E) Gating strategy used in flow cytometry to determine CCR6, CCR9, and CXCR3 expression within DN Tregs in the human study. Percentages determined in flow cytometry of CCR6⁺FoxP3⁺ (F), CCR9⁺FoxP3⁺ (G), and CXCR3⁺FoxP3⁺ (H) within DN T-cells in the human study. After the Kruskal–Wallis analysis, the differences among the three study groups were determined by a nonparametric Mann–Whitney rank test for unpaired variables (non-infected vs. acute, non-infected vs. ART-treated) and Wilcoxon signed-rank test for paired variables (acute vs. ART-treated). Sample sizes in flow cytometry analysis: non-infected (n = 20), acute, and ART-treated (n = 10). Horizontal lines in graphs represent the median. Only statistical significances are presented (*, p < 0.05; **, p < 0.01).

Figure 6

(A) Gating strategy used in flow cytometry to determine total CD3⁺CD4⁻CD8⁻ (double-negative, DN) T-cells, total FoxP3⁺ DN T-cells (DN Tregs), CD127⁺, CD37/CD73, and HLA-DR⁺ DN T-cells in Rhesus macaques. Percentages determined in flow cytometry of total DN T-cells (B), total DN Tregs (C), CD127⁺ (D), CD73⁺ (E), CD39⁺ (F), and HLA-DR⁺ (G) within DN T-cells in Rhesus macaques. (H) Gating strategy used in flow cytometry to determine CD39/CD73 within DN Tregs in Rhesus macaques. Percentages determined in flow cytometry of CD39⁺FoxP3⁺ (I) and CD73⁺FoxP3⁺ (J) within DN T-cells in Rhesus macaques. After the Kruskal–Wallis analysis, the differences among the three study groups were determined by a nonparametric Mann–Whitney rank test for unpaired variables (non-infected vs. acute, non-infected vs. early ART-treated, and acute vs. early ART-treated) in Rhesus macaques. Sample sizes in flow cytometry analysis in Rhesus macaques: non-infected (n = 10), acute (n = 13), and early ART-treated (n = 8). Horizontal lines in graphs represent the median. Only statistical significances are presented (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).