Early treatment and PD1 inhibition enhance HIV-specific functionality of follicular CD8+ T cells

Abstract

People living with HIV treated during acute infection are the group for whom achieving functional cure appears most viable. Follicular CD8+ T cells could contribute to HIV reservoir clearance by accessing B cell follicles through CXCR5 expression. This study examines peripheral follicular CD8+ T cells using flow cytometry, transcriptome analyses, and functional assays in people treated during acute (n = 37) and chronic (n = 18) infection, as well as in individuals naturally controlling HIV (n = 20) and living without HIV (n = 10). Our results reveal that early, as opposed to late, treatment initiation preserves antiviral effector functions of follicular CD8+ T cells, which are further enhanced by PD1 inhibition. We also identify a correlation between follicular CD8+ T cells and intact proviral HIV DNA levels in acute, but not chronic, infection. Longitudinal transcriptomic analysis of peripheral effector cells after 48 weeks of suppressive therapy indicated traits of recent antigen exposure, suggesting potential recirculation into lymphoid tissue. These findings underscore the pivotal role of follicular CD8+ T cells in anti-HIV responses and support investigating targeted cure strategies, such as anti-PD1 therapy, especially in individuals initiating treatment during acute infection.

Keywords: AIDS/HIV; Adaptive immunity; Immunology; T cells.

Figure 1. Study design and progress-describing parameters.

(A) Overview of the study design. PLWOH, people living without HIV; LTNP, individuals naturally controlling HIV; Acute, individuals treated during acute HIV infection; Chronic, individuals treated during chronic HIV infection; Tonsils, individuals living with or without HIV and donating tonsils; Tx, treatment. Created in BioRender; https://BioRender.com/i33u513 (B and C) Longitudinal and individual CD4⁺ counts (B) and plasma viral load (C) in peripheral blood of participants analyzed by flow cytometry. The dotted line indicates the detection limit (<50 copies/mL). Time points on ART are shaded in gray.

Figure 2. Circulating CXCR5⁺CD8⁺ T cells coexpress PD1 and CD127.

(A) Frequency of CXCR5⁺CD8⁺ T cells in peripheral blood of individuals treated during acute infection (n = 37; median 52 weeks on ART). Red, follicular (CXCR5⁺); blue, nonfollicular (CXCR5^–). (B) Memory subset distribution stratified by CD45RA and CCR7 expression in CXCR5⁺CD8⁺ versus CXCR5^–CD8⁺ T cells in the same group as A. Median values were adjusted to the total, defined as 100%. P values by Wilcoxon’s matched-pairs signed-rank test. (C–E) Frequencies of CXCR5^– (blue) and CXCR5⁺ (red) CD8⁺ T cells expressing PD1 (C), CD127 (D), and coexpressing PD1/CD127 (E) in individuals treated during acute infection (n = 37; circles; median 52 weeks on ART), in individuals naturally controlling HIV (LTNPs; n = 20; diamonds), and in people LWOH (PLWOH; n = 10; squares). P values by Wilcoxon’s matched-pairs signed-rank test (within 1 group) and Kruskal-Wallis test with Dunn’s correction for multiple comparisons (between groups). Medians and IQRs are indicated. (F) Memory subset distribution stratified by CD45RA/CCR7 expression in CD127⁺PD1⁺CXCR5⁺CD8⁺ T cells of individuals treated during acute infection (n = 37; median 52 weeks on ART) and individuals naturally controlling HIV (LTNPs; n = 20). Median values were adjusted to the total, defined as 100%. TN, naive T cell; TCM, central memory T cell; TEM, effector memory T cell; TEMRA, effector memory T cells re-expressing CD45RA.

Figure 3. CXCR5⁺CD8⁺ T cell localization in tonsils and their distinct transcriptomic profile in circulation compared with CXCR5^–CD8⁺ T cells.

(A–C) Immunofluorescence microscopy of tonsil tissue from participants LWH: viremic (A) and aviremic participant on ART (B) and an individual naturally controlling HIV (C). VL, viral load; cp, copies. Whole tonsil tissue sections were stained, and GCs identified (white dotted circles). Inserts represent zoomed into areas of GCs (white rectangles). Orange, CXCR5; pink, CD8; green, HIV p24; blue, DAPI/nuclear counterstain. Original magnification, ×40. Scale bars: 100 μm, 10 μm (insets), and 5 μm (zoomed-in insets). See Supplemental Figure 3C for the isotype control staining. (D) Heatmap of selected differentially expressed genes (DEGs) in CXCR5⁺CD8⁺ versus CXCR5^–CD8⁺ T cells from people LWH (n = 13; max 4 weeks on ART) categorized in functional gene groups. Relative changes in gene expression between CXCR5⁺CD8⁺ and CXCR5^–CD8⁺ T cells are depicted. Normalized read counts, calculated with DESeq2, were converted into z scores and mean z scores of all analyzed individuals per gene per CXCR5⁺ and CXCR5^– cell types are plotted. Plotted DEGs were selected based on their function. Red indicates higher and blue lower expression. (E) Enriched GO terms (biological process) from significantly DEGs that are downregulated (left, blue background) and upregulated (right, red background) in CXCR5⁺CD8⁺ T cells compared with their CXCR5^– counterparts (BH-adjusted P values < 1 × 10^–4). The line length represents the ratio of the number of genes that are part of the pathway differentially expressed between cell types to the total number of genes differentially expressed between cell types. Dot size represents the absolute observed number of DEGs in CXCR5⁺CD8⁺ versus CXCR5^–CD8⁺ T cells per pathway. Line and dot color code is according to the adjusted P value.

Figure 4. Circulating HIV-specific CXCR5⁺CD8⁺ T cells show a classical follicular, non–tissue-resident-like transcriptomic profile.

(A) Volcano plot depicting upregulated (red) and downregulated (blue) DEGs in HIV-specific CXCR5⁺CD8⁺ versus CXCR5^–CD8⁺ T cells. Cutoffs for differential expression: adjusted P value < 0.05 and absolute log₂(fold-change) > 1. Selected immune-related genes are highlighted. (B) Number of genes significantly differentially regulated in bulk and HIV-specific CXCR5⁺CD8⁺ versus CXCR5^–CD8⁺ T cells, with overlapping DEGs in red. (C) Heatmap of selected DEGs in HIV-specific CXCR5⁺CD8⁺ versus CXCR5^–CD8⁺ T cells categorized in functional groups. Relative changes in gene expression between tetramer-binding CXCR5⁺CD8⁺ and CXCR5^–CD8⁺ T cells are depicted. Normalized read counts, calculated with DESeq2, were converted into z scores and mean z scores per gene per HIV-specific CXCR5⁺ and CXCR5^– cell types are plotted. Plotted DEGs were selected based on their function. Red represents higher and blue lower gene expression. (D) GSEA shows exhaustion signature (70) enrichment in HIV-specific CXCR5^–CD8⁺ but not CXCR5⁺CD8⁺ T cells. (A–D) CXCR5⁺CD8⁺ T cells were sorted from people LWH (n = 13; max 4 weeks on ART). NES, normalized enrichment score.

Figure 5. Circulating CXCR5⁺CD8⁺ T cells exhibit HIV-specific effector functions upon antigen stimulation.

(A–D) Frequencies of CXCR5^–CD8⁺ (blue) and CXCR5⁺CD8⁺ (red) T cells coexpressing granzyme B and perforin ex vivo (A), and CD107a (B), IL-10 (C), or IL-21 (D) after HIV gag peptide stimulation in individuals treated during acute infection (n = 37; circles; median 52 weeks on ART) and individuals naturally controlling HIV (LTNPs; n = 20; diamonds). P values by Wilcoxon’s matched-pairs signed-rank test. Medians and IQRs are indicated. (E) HIV p24 concentration in culture supernatants of PHA-activated CD4⁺ T cells from 3 viremic individuals without (black) or with autologous, unstimulated CXCR5^–CD8⁺ (blue) or CXCR5⁺CD8⁺ (red) T cells (effector/target ratio 1:1) (Supplemental Table 2).

Figure 6. Circulating CXCR5⁺CD8⁺ T cells inversely correlate with intact proviral HIV DNA after 48 weeks of ART in individuals treated during acute infection.

(A) Intact proviral HIV DNA levels in CD4⁺ T cells from individuals with acute (TopHIVFUTURE; n = 21, green circles) and chronic HIV infection (n = 4, purple triangles) before and during ART. P values by Wilcoxon’s matched-pairs signed-rank test. (B) Negative correlation between CXCR5⁺CD8⁺ T cell frequency prior to ART and intact proviral HIV DNA after 48 weeks of ART in individuals treated during acute infection (TopHIVFUTURE; n = 25). Spearman’s rank correlation was performed. (C) Spearman’s rank correlation between TIM3⁺CXCR5⁺CD8⁺ T cell frequency and intact proviral HIV DNA in individuals treated during acute infection (TopHIVFUTURE; n = 22) prior to ART. (D–F) Longitudinal analysis of PD1 (D), TIM3 (E), and CD127/PD1 (F) (co)expression on CXCR5^–CD8⁺ (blue) and CXCR5⁺CD8⁺ (red) T cells in individuals treated during acute infection (TopHIVFUTURE; n = 27). P values by Friedman’s test with Dunn’s correction for multiple comparisons.

Figure 7. ART initiation during acute HIV infection preserves HIV-specific effector functionality in CXCR5⁺CD8⁺ T cells.

(A) Frequencies of cytotoxic (granzyme B⁺perforin⁺) CXCR5⁺CD8⁺ T cells in individuals with acute (TopHIVFUTURE: n = 27; circles) and chronic (n = 10; triangles) HIV infection prior and during ART as well as in individuals naturally controlling HIV (LTNPs; n = 20; diamonds) ex vivo. P values by Kruskal-Wallis test with Dunn’s correction for multiple comparisons. (B) Correlation between HIV-specific CD107a expression on CXCR5⁺CD8⁺ T cells responding to overnight stimulation with HIV gag peptide pool at baseline and intact proviral HIV DNA after 48 weeks of ART in individuals treated during acute infection (TopHIVFUTURE; n = 25). Spearman’s rank correlation was performed. (C) Heatmap of genes significantly differentially regulated in individuals treated during acute HIV infection (n = 4; week 4 [w4] versus week 48 [w48] after ART initiation) in HIV-specific CXCR5⁺ and CXCR5^– cells as well as tetramer-negative CXCR5⁺CD8⁺ T cells categorized in functional groups. Relative changes in gene expression between w4 and w48 on ART depicted for each cell type. Normalized read counts, calculated with DESeq2, were converted into z scores and mean z scores per gene significantly differentially expressed across the time points are plotted. Plotted DEGs were selected based on their function. Red indicates higher and blue lower expression. (D) Enriched GO terms (biological process) for significantly downregulated (left) and upregulated (right) genes in HIV-specific CXCR5⁺CD8⁺ and CXCR5^–CD8⁺ T cells w4 versus w48 after ART initiation (BH-adjusted P values < 0.01). The line length represents the ratio of the number of observed DEGs per pathway to the total number of DEGs, and dot size represents the absolute observed number of DEGs in CXCR5⁺CD8⁺ versus CXCR5^–CD8⁺ T cells per pathway. Line and dot color code is according to the adjusted P value.

Figure 8. CXCR5⁺CD8⁺ T cells of individuals treated during acute infection proliferate and upregulate HIV-specific effector functions after in vitro stimulation and PD1 blockade.

PBMCs were stimulated with HIV gag peptide pool for 20 hours (day 1) or 68 hours (day 3) with PD1 blockade (nivolumab; pink) or isotype control (gray). (A) PD1 expression on CXCR5^–CD8⁺ and CXCR5⁺CD8⁺ T cell subsets (day 1 [d1] and d3 merged). (B–H) Frequency of proliferation and effector function markers coexpressed with CXCR5 on CD8⁺ T cells, as measured by intracellular cytokine staining assays. Left: Individuals treated during acute infection (12 weeks on ART; n = 9). Right: Individuals with chronic infection (Tx-naive; n = 3 LTNP and n = 3 viremic). The following markers are depicted: Ki-67 (B), CD107a (C), granzyme B/perforin (D), IL-2 (E), IFN-γ (F), IL-21 (G), and TNF (H). Mean values are shown and P values were calculated with Wilcoxon’s matched-pairs signed-rank test.