Delayed differentiation of potent effector CD8+ T cells reducing viremia and reservoir seeding in acute HIV infection

Abstract

CD8⁺ T cells play a critical role in controlling HIV viremia and could be important in reducing HIV-infected cells in approaches to eradicate HIV. The simian immunodeficiency virus model provided the proof of concept for a CD8⁺ T cell-mediated reservoir clearance but showed conflicting evidence on the role of these cells to eliminate HIV-infected cells. In humans, HIV-specific CD8⁺ T cell responses have not been associated with a reduction of the HIV-infected cell pool in vivo. We studied HIV-specific CD8⁺ T cells in the RV254 cohort of individuals initiating ART in the earliest stages of acute HIV infection (AHI). We showed that the HIV-specific CD8⁺ T cells generated as early as AHI stages 1 and 2 before peak viremia are delayed in expanding and acquiring effector functions but are endowed with higher memory potential. In contrast, the fully differentiated HIV-specific CD8⁺ T cells at peak viremia in AHI stage 3 were more prone to apoptosis but were associated with a steeper viral load decrease after ART initiation. Their capacity to persist in vivo after ART initiation correlated with a lower HIV DNA reservoir. These findings demonstrate that HIV-specific CD8⁺ T cell magnitude and differentiation are delayed in the earliest stages of infection. These results also demonstrate that potent HIV-specific CD8⁺ T cells contribute to the reduction of the pool of HIV-producing cells and the HIV reservoir seeding in vivo and provide the rationale to design interventions aiming at inducing these potent responses to cure HIV infection.

Fig. 1

HIV-specific CD8⁺ T cells expansion in acute HIV-1 infection. (A) Representative dot plot of Ki-67⁺Bcl-2^lo CD8⁺ T cells from AHI week 0. (B) Percentages of Ki-67⁺Bcl-2^lo cells in CD45RA^-CD8⁺ T cells from 14 HIV- and 16 AHI 4^thG Stages 1, 20 Stage 2, and 27 stage 3 individuals. (C) Plasma viral load during the different stages of AHI week 0. (D) Correlation between plasma viral load and percentages of Ki-67⁺Bcl-2^lo CD8⁺ T cells. (E) HLA-A*1101 NEF or EBV tetramer⁺ CD8⁺ T cells in two acutely-infected individuals gated on total live peripheral blood lymphocytes (PBLs) (Top). Overlay of Ki-67⁺Bcl-2^lo expression on tetramer⁺ (Red) and total CD45RA^-CD8⁺ T cells (Blue) from the same participants (Bottom). (F) Percentage of Ki-67⁺Bcl-2^lo in HIV or EBV tetramer⁺ cells from 2 AHI stage 1, 3 stage 2, and 6 stage 3 participants. (G) HLA-A*1101 NEF or EBV tetramer⁺ CD8⁺ T cells gated on Ki-67⁺Bcl-2^lo CD8⁺ T cells. Differences between groups were analyzed by Mann-Whitney tests. Associations between two variables (p and r) were analyzed by Spearman correlations.*P< 0.05; **P< 0.01; ****P< 0.0001.

Fig. 2

Fully differentiated effector HIV-specific CD8⁺ T cells in acute HIV infection contribute to viral load decrease after ART initiation. (A) Expression of perforin and T-bet in naive CD8⁺ T cells and Ki-67⁺Bcl-2^lo CD45RA^-CD8⁺ T cells from 14 HIV- and 9 AHI stage 1, 9 stage 2, and 14 stage 3 individuals. (B) mRNA expression of granzyme B in CD38⁺HLA-DR⁺ CD8⁺ T cells from AHI stage 1/2 and 3 individuals. (C) Plasma viral load at week 2 in individuals who initiated ART in AHI stage 1-3. (D) Plasma viral load decrease between week 0 and week 2 after ART initiation in AHI stage 1-3 individuals. Differences between week 0 and week 2 were analyzed by Wilcoxon test. Slope differences are based on linear regression among stages and shown as p values. (E) Correlation between percentage of Ki-67⁺Bcl-2^lo cells in CD45RA^-CD8⁺ T cells at week 0 and plasma viral load fold change (week 0/ week 2). (F) Correlation between expression of effector molecules perforin and T-bet in Ki-67⁺Bcl-2^lo CD8⁺ T cell at week 0 and plasma viral load fold change (week 0/ week 2). Differences between groups were analyzed by Mann-Whitney tests. Associations between two variables (p and r) were analyzed by Spearman correlations. *P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001.

Fig. 3

Loss of cytokine secretion and survival potential of HIV-specific CD8⁺ T cells during acute HIV infection. (A) Expression of CD127 on CD38⁺HLA-DR⁺CD45RA^-CD8⁺ T cells from 12 AHI stage 1, 13 stage 2, and 14 stage 3 individuals. (B) mRNA expression of IL7R, and STAT5A in CD38⁺HLA-DR⁺ cells. (C) IFN-γ, TNF-α, and IL-2 production by CD38⁺HLA-DR⁺ cells from 9 AHI stages 1, 10 Stage 2, and 11 stage 3 individuals after anti-CD3 and CD28 Ab stimulation. (D) Correlation between plasma viral load and percentages of IL-2⁺ secreting cells within CD38⁺HLA-DR⁺ cells. (E) Percentage of live non-apoptotic cell recovery of CD38⁺HLA-DR⁺ effector CD8⁺ T cells from AHI stage 1-3 individuals based on their absolute cell numbers after 24hrs in vitro. (F) Percentages of apoptotic Annexin V^hi cells in ex vivo CD38⁺HLA-DR⁺ cells. (G) Correlation between plasma viral load and percentages of Annexin V^high cells in CD38⁺HLA-DR⁺ CD8⁺ T cells. (H) Correlation between ex vivo apoptotic CD38⁺HLA-DR⁺ cell percentage and staining intensity of MitoTracker Orange CM-H₂TMRos (Mitochondria membrane potential activity, Left) or CellRox Deep Red (total Ros level, Right) from 8 AHI stages 1, 8 Stage 2, and 8 stage 3 individuals. Differences between groups were analyzed by Mann-Whitney tests. Associations between two variables (p and r) were analyzed by Spearman correlations.*P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001.

Fig. 4

Differential fate of HIV-specific CD8⁺ T cell 2 weeks after ART initiation in acute HIV infection. (A) Tetramer⁺ HLA-A*1101 NEF-specific CD8⁺ T cells in total live PBLs at week 0 and 2 weeks after ART initiation. (B) Tetramer⁺ HIV-specific CD8⁺ T cell absolute number /μl based on total CD8 T cell count for AHI stage 1-3 individuals. ND: Not Detected. (C) Percentage of Ki-67⁺Bcl-2^lo cells in HIV-specific or EBV-specific tetramer⁺ CD8⁺ T cells from 7 AHI stage 1, 5 stage 2, and 11 stage 3 individuals at week 0 and week 2 on ART. (D) Percentage of Ki-67⁺ cells in CD8⁺ T cells from 14 stage 1, 20 stage 2, and 34 stage 3 individuals at week 0 and week 2. Differences between week 0 and week 2 were analyzed by Wilcoxon test. Slope differences are based on linear regression among stages and shown as p values. (E) Perforin expression in Ki-67⁺ and Naïve CD8⁺ T cells at week 0 and week 2. Differences between groups were analyzed by Mann-Whitney tests (*P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001).

Fig. 5

HIV-specific CD8⁺ T cells persisting 2 weeks after ART initiation contribute to limited seeding and persistence of the HIV reservoir. (A) Correlation between percentage of Ki-67⁺CD8⁺ T cells from 34 stage 3 individuals and plasma viral load at week 2. Open circle represents the detection limit. (B) Total HIV DNA copies/ 10⁶ CD4⁺ T cells among AHI stage 1-3 individuals at week 2. For samples in which no positive cells were detected, the limit of detection based on cell input is plotted as an open symbol. (C) Correlation between percentage of Ki-67⁺ cells in total CD8⁺ T cells from 20 stage 2 individuals at week 2 and Total HIV DNA copies/ 10⁶ CD4⁺ T cells at week 24. (D) Correlation between percentage of Ki-67⁺CD8⁺ T cells at week 2 and Total HIV DNA copies/ 10⁶ CD4⁺ T cells at week 24, week 36, week 48, week 72, and week 96 in AHI stage 3 individuals. (E) Correlation between percentage of Ki-67⁺CD4⁺ T cells at week 2 and Total HIV DNA copies/ 10⁶ CD4⁺ T cells at week 24. Differences between groups were analyzed by Mann-Whitney tests. Associations between two variables (p and r) were analyzed by Spearman correlations. **P< 0.01; ****P< 0.0001.