HIV Productively Infects Highly Differentiated and Exhausted CD4+ T Cells During AIDS

Abstract

Background: Throughout HIV infection, productively infected cells generate billions of viral particles and are thus responsible for body-wide HIV dissemination, but their phenotype during AIDS is unknown. As AIDS is associated with immunological changes, analyzing the phenotype of productively infected cells can help understand HIV production during this terminal stage.

Methods: Blood samples from 15 untreated viremic participants (recent infection, n=5; long-term infection, n=5; active opportunistic AIDS-defining disease, n=5) and 5 participants virologically controlled on antiretroviral therapy (ART) enrolled in the Analysis of the Persistence, Reservoir and HIV Latency (APRIL) study (NCT05752318) were analyzed. Cells expressing the capsid protein p24 (p24+ cells) after 18 hours of resting or 24 hours of stimulation (HIV-Flow) revealed productively infected cells from viremic participants or translation-competent reservoir cells from treated participants, respectively.

Results: The frequency of productively infected cells tended to be higher during AIDS in comparison with recent and long-term infections (median, 340, 72, and 32/million CD4+ T cells, respectively) and correlated with the plasma viral load at all stages of infection. Altogether, these cells were more frequently CD4^low, HLA-ABC^low, CD45RA-, Ki67+, PD-1+, with a non-negligible contribution from pTfh (CXCR5+PD-1+) cells, and were not significantly enriched in HIV coreceptors CCR5 nor CXCR4 expression. The comparison markers expression between stages showed that productively infected cells during AIDS were enriched in memory and exhausted cells. In contrast, the frequencies of infected pTfh were lower during AIDS compared to non-AIDS stages. A UMAP analysis revealed that total CD4+ T cells were grouped in 7 clusters and that productive p24+ cells were skewed to given clusters throughout the course of infection. Overall, the preferential targets of HIV during the latest stages seemed to be more frequently highly differentiated (memory, T_TD-like) and exhausted cells and less frequently pTfh-like cells. In contrast, translation-competent reservoir cells were less frequent (5/million CD4+ T cells) and expressed more frequently HLA-ABC and less frequently PD-1.

Conclusions: In long-term infection and AIDS, productively infected cells were differentiated and exhausted. This could indicate that cells with these given features are responsible for HIV production and dissemination in an immune dysfunction environment occurring during the last stages of infection.

Keywords: AIDS; exhaustion; phenotype; productively infected cells; recent infection.

Figure 1.

The phenotype of productively infected p24+ cells differs from p24-cells and changes over time. A) Representative dot plot showing the gating strategy for identification of productively infected p24+ cells (orange gate) and p24-cells (grey gate). B) Frequency of p24+ cells per group (median). C-D) Correlations between p24+ cells frequencies and plasma viremia (C) or CD4+ T-cell count (D). The regression line was plotted with 95% CI, and correlation was assessed (Spearman). E) Frequencies of p24+ and p24-cells expressing each marker or combination of markers (CD4^low, HLA-ABC^low, CD45RA-, CCR5+, CXCR4+, Ki67+, PD-1+, TIGIT+, CXCR5+, peripheral T follicular helpers [pTfh] cells [CXCR5+PD-1+]) are depicted for each participant (n=15) with median. Significant changes are highlighted with the P value (Wilcoxon). F) The frequency of p24+ (orange lines) and p24-(grey lines) cells expressing each marker or combination of markers is depicted for each stage of infection. Median values are plotted with range. Significant changes are highlighted with the P value of the differing point (Mann-Whitney). G) Ratio of frequencies of cells (p24+/p24-) expressing each marker or combination of markers according to infection stage are depicted for each participant (n=15) with a median. Significant changes are highlighted (Mann-Whitney).

Figure 2.

Productively infected p24+ cells are skewed to given clusters throughout the course of infection. A) p24+ cells and a fraction of p24-cells phenotypic data were integrated in a UMAP analysis and generated 7 cell clusters. B) Dot plots of the UMAP analysis of all cells (in grey) as shown in panel A, on which the p24- and p24+ cells by stage of infection are overlaid in colors. Numbers of samples analyzed are indicated at the top of the dot plots, and the number of cells is indicated at the bottom. C) The frequency of each of these clusters among p24+ and p24-cells is depicted in bar graphs according to the stage of infection. Numbers of samples analyzed are as in B. The table shows the name of the identified clusters hypothesized according to their phenotype and the detailed marker expression frequencies for each cell cluster (%). D) Frequencies of p24+ and p24-cells included in each cluster are depicted for each participant (n=15) with a median. Significant changes are highlighted with the P value (Wilcoxon). E) The frequency of p24+ (orange lines) and p24-(grey lines) cells included in each cluster is depicted for each stage of infection. Median values are plotted with range. Changes close to significance are highlighted with the P value of the trending point (Mann-Whitney).

Figure 3.

The phenotype of translation-competent latently infected cells differs from productively infected cells. A) Frequency of p24+ cells per group (median). Productively infected cells were more frequent than translation-competent latently infected cells (Mann-Whitney). B) Correlation between p24+ cells frequencies and CD4+ T-cell count. The regression line was plotted with 95% CI, and correlation was assessed (Spearman). C) Frequencies of p24+ and p24-cells expressing each marker or combination of markers (HLA-ABC^low, CD45RA-, Ki67+, PD-1+) are depicted for each participant (n=20) with median. Significant differs between viremic untreated and ART-treated participants are highlighted with the P value (Wilcoxon). D) Ratio of frequencies of cells (p24+/p24-) expressing each marker or combination of markers according to infection stage are depicted for each participant (n=20) with a median. Significant or close to significance changes are highlighted with P value (Mann-Whitney).