Long-term antiretroviral therapy initiated in acute HIV infection prevents residual dysfunction of HIV-specific CD8+ T cells

Abstract

Background: Harnessing CD8⁺ T cell responses is being explored to achieve HIV remission. Although HIV-specific CD8⁺ T cells become dysfunctional without treatment, antiretroviral therapy (ART) partially restores their function. However, the extent of this recovery under long-term ART is less understood.

Methods: We analyzed the differentiation status and function of HIV-specific CD8⁺ T cells after long-term ART initiated in acute or chronic HIV infection ex vivo and upon in vitro recall.

Findings: ART initiation in any stage of acute HIV infection promoted the persistence of long-lived HIV-specific CD8⁺ T cells with high expansion (P<0·0008) and cytotoxic capacity (P=0·02) after in vitro recall, albeit at low cell number (P=0·003). This superior expansion capacity correlated with stemness (r=0·90, P=0·006), measured by TCF-1 expression, similar to functional HIV-specific CD8⁺ T cells found in spontaneous controllers. Importanly, TCF-1 expression in these cells was associated with longer time to viral rebound ranging from 13 to 48 days after ART interruption (r =0·71, P=0·03). In contrast, ART initiation in chronic HIV infection led to more differentiated HIV-specific CD8⁺ T cells lacking stemness properties and exhibiting residual dysfunction upon recall, with reduced proliferation and cytolytic activity.

Interpretation: ART initiation in acute HIV infection preserves functional HIV-specific CD8⁺ T cells, albeit at numbers too low to control viral rebound post-ART. HIV remission strategies may need to boost HIV-specific CD8⁺ T cell numbers and induce stem cell-like properties to reverse the residual dysfunction persisting on ART in people treated after acute infection prior to ART release.

Funding: U.S. National Institutes of Health and U.S. Department of Defense.

Keywords: Antiretroviral therapy; CD8 T cells; Cell differentiation; HIV; TCF-1.

Figure 1

HIV-specific CD8⁺ T cell numbers before ART initiation in AHI and CHI and under long-term ART. (a) Sequence logo for the 5 HIV CD8⁺ T cell epitopes tested with tetramers: GK10, QK10, AK9, YI9, CL9. The sequence conservation for each epitope was measured using reference datasets of circulating CRF01_AE sequences. The relative size of each AA letter at a site represents the frequency of the residue across circulating viruses. The total height indicates the information content of the position. (b) Number of HIV- and EBV-specific tetramer⁺ CD8⁺ T cells per mL of peripheral blood in individuals prior to ART initiation in AHI (UnTx AHI, n=19) or CHI (UnTx CHI, n=15). (c) Number of HIV-specific CD8⁺ T cells before (UnTx) and during (Tx) long-term ART measured longitudinally in individuals who initiated treatment in AHI or CHI and were on ART for more than 1 year. (d) Number of HIV- and EBV-specific tetramer⁺ CD8⁺ T cells in individuals who initiated treatment in AHI (n=41) or in CHI (n=27) and were on ART for more than 1 year. Differences between UnTx AHI group, UnTx CHI group, and EBV-specific CD8⁺ T cells were analyzed by a Kruskal-Wallis test. Differences between samples collected before and after ART were analyzed by a Wilcoxon test. *P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001.

Figure 2

Differentiation status and phenotype of HIV-specific CD8⁺ T cells in people during acute and chronic HIV infection before ART initiation. (a) Frequencies of naïve-like (Tna), stem cell memory (Tscm), central memory (Tcm), transitional memory (Ttm), CD45RA⁺ transitional memory (RA⁺ Ttm), effector memory (Tem), and terminally differentiated cells (Ttd) in HIV/EBV-specific CD8⁺ T cells prior to ART initiation in CHI and AHI. Chi-square permutation test was used to compare the overall frequency distribution of the CD8⁺ T cell subsets among the groups. (b-e) Percentage of Ki-67⁺Bcl-2^low cells (b) and expression of T-bet (c), TOX (d), and PD-1 (e) on HIV/EBV-specific CD8⁺ T cells. Differences between groups were analyzed by Kruskal-Wallis test. *P< 0.05; **P< 0.01; ****P< 0.0001.

Figure 3

Differentiation status and phenotype of HIV-specific memory CD8⁺ T cells in people treated from acute and chronic HIV infection. (a) Average frequencies of naive-like (Tna), stem cell memory (Tscm), central memory (Tcm), transitional memory (Ttm), CD45RA⁺ transitional memory (RA⁺ Ttm), effector memory (Tem), and terminally differentiated cells (Ttd) in HIV/EBV-specific CD8⁺ T cells from individuals who initiated treatment in AHI (n=33) or in CHI (n=13) and were on ART for more than 1 year. Chi-square permutation test was used to compare the overall frequency distribution of the CD8⁺ T cell subsets among the groups. (b, c, and d) Expression of CD127 (b), TOX (c), and PD-1 (d) on/in HIV/EBV-specific CD8⁺ T cells. Differences between groups were analyzed by a Kruskal-Wallis test. *P< 0.05; **P< 0.01; ****P< 0.0001.

Figure 4

Schematic of the proliferation and cytotoxicity assay of recalled memory HIV-specific CD8⁺ T cells. CellTrace dye labeled PBMCs were stimulated with cognate HIV epitope peptides. Tetramer⁺ HIV-specific CD8⁺ T cell phenotype and cell number were analyzed by flow cytometry after 6 and 12 days of stimulation. Autologous PBMCs were concurrently expanded with PHA and rhIL-2 for 12 days in the presence of ARVs. CD4⁺ T cells were purified from the PHA expanded PBMCs by magnetic negative selection. Autologous CD4⁺ T cells were labeled with low levels of CSFE and pulsed with HIV peptide or labeled with high levels of CFSE and not loaded with peptide. Cytotoxic capacity of CD8⁺ T cells was measured by co-culturing the peptide stimulated CD8⁺ T cells at day 13 post-stimulation at an E/T ratio of 2 with the peptide-pulsed and non-pulsed CFSE labeled autologous CD4⁺ T cells mixed 50/50 as targets.

Figure 5

Magnitude of recalled memory CD8⁺ T cells from people treated in AHI and CHI. (a) Number of HIV- or EBV-specific CD8⁺ T cells in culture 12 days after peptide stimulation. Thirty-three individuals treated from AHI and 27 individuals treated from CHI were analyzed. (b) Fold expansion of HIV- or EBV-specific CD8⁺ T cells from day 0 (ex vivo) to day 12 after peptide stimulation. (c) Correlation between the fold expansion of HIV-specific CD8⁺ T cells and combined ex vivo frequency of Ttm, Tem, and Ttd in HIV-specific CD8⁺ T cells of people treated from CHI. (d) Correlation between the fold expansion of HIV-specific CD8⁺ T cells and expression of T-bet in day 6 recalled HIV-specific CD8⁺ T cells of people treated from AHI. Differences among groups were analyzed by Kruskal-Wallis test (**P< 0.01; ***P< 0.001; ****P< 0.0001). Correlations were analyzed by Spearman correlation with the Benjamini-Hochberg procedure for multiple comparisons (FDR < 0.1).

Figure 6

Effector function of recalled memory CD8⁺ T cells from people treated in AHI and CHI. (a) Expression of Granzyme B in ex vivo HIV/EBV-specific CD8⁺ T cells from people treated from AHI and CHI. (b, c, and f) Expression of T-bet (b), perforin (c), and PD-1 (f) in/on HIV/EBV-specific CD8⁺ T cells 12 days after stimulation. (d) Cytotoxic capacity of day 13 recalled HIV Nef GK10- or EBV- specific CD8⁺ T cells cultured with autologous CD4⁺ T cells at an effector/ target ratio of 2. (e) Expression level of PD-1 on day 12 recalled HIV/EBV-specific CD8⁺ T cells from individuals treated from AHI and CHI. Differences among groups were analyzed by Kruskal-Wallis test (*P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001). Correlations were analyzed by Spearman correlation with the Benjamini-Hochberg procedure for multiple comparisons (FDR < 0.1).

Figure 7

Initiation of ART in AHI prevents loss of TCF-1 expression and the expression supports expansion of functional memory HIV-specific CD8⁺ T cells and associates with time to viral rebound after ATI. (a) Expression of TCF-1 in HIV/EBV-specific CD8⁺ T cells from people before ART initiation in AHI or CHI. (b) Expression of TCF-1 in HIV-specific CD8⁺ T cells before and during long-term ART initiated in AHI or CHI. (c) Expression of TCF-1 in HIV/EBV-specific CD8⁺ T cells from people treated from AHI or CHI. (d) Correlation between the fold expansion of HIV-specific CD8⁺ T cells and ex vivo expression of TCF-1 in HIV-specific CD8⁺ T cells from people who initiated treatment in AHI and subsequently underwent ATI. (e) Correlation between days to viral load rebound (VL>20 copies/mL) after ATI and expression of TCF-1 in HIV-specific CD8⁺ T cells 12 days after in vitro recall. Differences between samples collected before and after ART were analyzed by a Wilcoxon test. Differences between groups were analyzed by a Kruskal-Wallis test. Correlations were analyzed by Spearman correlation with the Benjamini-Hochberg procedure for multiple comparisons (FDR < 0.1). *P< 0.05; ***P< 0.001.