Abstract
Human immunodeficiency virus 1 (HIV-1) infection is characterized by a dynamic and persistent state of viral replication that overwhelms the host immune system in the absence of antiretroviral therapy (ART). The impact of prolonged treatment on the antiviral efficacy of HIV-1-specific CD8+ T cells has nonetheless remained unknown. Here, we used single-cell technologies to address this issue in a cohort of aging individuals infected early during the pandemic and subsequently treated with continuous ART. Our data showed that long-term ART was associated with a process of clonal succession, which effectively rejuvenated HIV-1-specific CD8+ T cell populations in the face of immune senescence. Tracking individual transcriptomes further revealed that initially dominant CD8+ T cell clonotypes displayed signatures of exhaustion and terminal differentiation, whereas newly dominant CD8+ T cell clonotypes displayed signatures of early differentiation and stemness associated with natural control of viral replication. These findings reveal a degree of immune resilience that could inform adjunctive treatments for HIV-1.
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Data availability
Flow cytometry data reported in this paper will be shared by the corresponding author upon reasonable request. All raw and processed scRNA-seq data from this study are publicly available in the Gene Expression Omnibus via accession number GSE270651. Alignments and analyses were performed using the hg38 reference genome obtained from the University of California Santa Cruz Genome Browser (http://genome.ucsc.edu).
Code availability
The scripts and code used in this study are available via GitHub at https://github.com/EoghannWhite/ImmunocoProject.
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Acknowledgements
We thank all donors for participating in this study. TCR-deficient Jurkat cells were kindly provided by T. Ueno (Kumamoto University). We also thank P. Goepfert (University of Alabama) for assistance with the identification of appropriate donors to confirm the primary findings of this study, D. Ambrozak and N. Santana Lima for assistance with flow cytometry at the Vaccine Research Center (National Institute of Allergy and Infectious Diseases, NIH), and V. Pitard and A. Zouine for assistance with flow cytometry at the Université de Bordeaux (CNRS UMS 3427, INSERM US 005). This work was funded by the Université de Bordeaux (Senior IdEx Chair) and INSERM (AgeMed Program) and by grants from the ANR (14CE16002901), the ANRS (2016-A00400-51) and the Japan Agency for Medical Research and Development (JP22fk0410052). E.W. was supported by Sidaction and Sorbonne Université. D.A.P. was supported by a Wellcome Trust Senior Investigator Award (100326/Z/12/Z). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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B.A. and V.A. conceived the study. E.W., L.P., K.S., A.R.H., D.C.R., P.R.-P., D.A.P., M.-A.A., D.C.D. and V.A. designed and/or performed experiments. E.W., L.P., A.S., K.S., B.H., D.C.R., S.D., Y.D., S.A.M., Y.S., R.T., C.K. and V.A. analyzed data. A.S., Y.D., S.L.-L., F.B., S.A.M., D.A.P., C.K. and D.C.D. provided reagents, resources and/or samples. L.A.C., D.A.P. and V.A. acquired funding. D.A.P., D.C.D. and V.A. supervised the work. E.W., L.P., D.A.P. and V.A. wrote the manuscript. All authors contributed intellectually.
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Extended data
Extended Data Fig. 1 Viral load and CD4+ T cell trajectories of PLWH from the IMMUNOCO cohort.
Viral load (dashed lines) and CD4+ T cell trajectories (solid lines) for individuals from the IMMUNOCO cohort. Red, orange and blue lines indicate the early, intermediate and late time points, respectively.
Extended Data Fig. 2 TCF-1 expression is linked to CD8+ T cell differentiation and increased in HIV-1-specific CD8+ T cells from long-term treated PLWH.
(a) Frequencies of tetramer+ CD8+ T cells expressing CD27 (left), HLA-DR (center) or CD45RA (right) at the early (n = 11), intermediate (n = 6) and late time points (n = 8). Bars indicate median values. Each dot represents a distinct tetramer+ population. (b) TCF-1 expression frequencies among CD8+ T cell subsets from untreated HIV-1 progressors (n = 9) and PLWH treated with long-term ART (n = 5) defined phenotypically as naive (CCR7+CD45RA+CD28+), central memory (CM, CCR7+CD45RA−CD28+), effector memory (EM, CCR7−CD45RA−CD28−/+) or effector memory with revertant expression of CD45RA (EMRA, CCR7−CD45RA+CD28−). Data are shown as standard box-and-whisker plots. (c) Representative flow cytometry plots showing tetramer staining of HIV-1-specific CD8+ T cells (top) and the expression of TCF-1 among total and tetramer+ CD8+ T cells (bottom) from an untreated HIV-1 progressor (left) and a donor treated with long-term ART (right). (d) TCF-1 expression frequencies among tetramer+ CD8+ T cells from untreated progressors (n = 9) and PLWH treated with long-term ART (n = 5). Data are shown as standard box-and-whisker plots. (a, b, d) P values were determined using a two-tailed Mann–Whitney U test.
Extended Data Fig. 3 HIV-1-specific CD8+ T cells exhibit a stem-like profile in PLWH treated with long-term ART.
(a) Representative flow cytometry plots showing KF11/HLA-B*57:01 tetramer staining of CD8+ T cells from donor PS03 at the early and late time points. Numbers indicate percentages in the drawn gates. (b) UMAP projection of KF11/HLA-B*57:01 tetramer+ CD8+ T cells from donor PS03 segregated according to time point. Each dot represents one cell. (c) Volcano plot showing differentially expressed genes between KF11/HLA-B*57:01 tetramer+ CD8+ T cells from donor PS03 at the early and late time points (red, upregulated at early; blue, upregulated at late). The x-axis represents log fold change, and the y-axis represents −log10-adjusted P values. Significance was assessed using the findMarkers function in scran, which employs a one-tailed Wilcoxon rank-sum test. Adjustments for multiple comparisons were made using the false discovery rate (FDR). (d) Heatmap representation of the top 369 differentially expressed genes in KF11/HLA-B*57:01 tetramer+ CD8+ T cells from donor PS03 between the early and late timepoints. Significance was determined using a one-tailed Wilcoxon rank-sum test. (e) Violin plots showing genes associated with exhaustion and inhibition (top), IFN signaling (middle) and stemness (bottom) in KF11/HLA-B*57:01 tetramer+ CD8+ T cells from donor PS03 at the early (red) and late time points (blue). Each violin represents the probability density at each value. Each dot represents one cell. Significance was determined using a one-tailed Wilcoxon rank-sum test (FDR-adjusted values). (f) GSEA showing the enrichment of genes associated with IFN signaling, coinhibitory molecules, terminal exhaustion, apoptosis and stabilization of p53 among early KF11/HLA-B*57:01 tetramer+ CD8+ T cells or central memory differentiation among late KF11/HLA-B*57:01 tetramer+ CD8+ T cells from donor PS03. Significance was assessed using the fgsea package, which employs an adaptive multilevel Monte Carlo scheme. The normalized enrichment score (NES) refers to the ES normalized to the mean enrichment of random samples of the same size. FDR-adjusted P values are shown. The horizontal line represents the enrichment profile, with each vertical bar corresponding to a hit (gene). The red line above the bars indicates enrichment at the early time point, and the blue line below the bars indicates enrichment at the late time point.
Extended Data Fig. 4 HIV-2-specific CD8+ T cells resemble HIV-1-specific CD8+ T cells in PLWH treated with long-term ART.
(a) Top: representative flow cytometry plots showing TL9/HLA-B*53:01 tetramer staining of CD8+ T cells from an untreated HIV-2-infected controller. Middle and bottom: representative flow cytometry plots showing the expression of CD28, CD38 and CD57 among total and TL9/HLA-B*53:01 tetramer+ CD8+ T cells from an untreated HIV-2-infected controller. Numbers indicate percentages in the drawn gates. (b) Heatmap representation of the top 532 differentially expressed genes between early and late HIV-1-specific CD8+ T cells from donor PS03 and HIV-2-specific CD8+ T cells from two untreated donors with a controller phenotype.
Extended Data Fig. 5 HIV-1 epitope sequences are stable over time in donors from the IMMUNOCO cohort.
Viral epitope sequences from donors in the IMMUNOCO cohort at the early, intermediate and late time points compared with the reference strain HXB2. Mutations are highlighted in red.
Extended Data Fig. 6 The clonotypic composition of HIV-1-specific CD8+ T cell populations evolves in PLWH treated with long-term ART.
(a–c) TRAV, TRAJ, TRBV and TRBJ gene use and CDR3α and CDR3β amino acid sequences for FL8-specific (a) and EI8-specific clonotypes from donor PS24 (b) and KF11-specific clonotypes from donor PS03 (c) at the early/intermediate and late time points. Clonotype frequencies are indicated at each time point. Old and newly detected clonotypes are highlighted to match the color code in Fig. 5a–c.
Extended Data Fig. 7 The transcriptomic profile of HIV-1-specific CD8+ T cells evolves as a function of clonal succession in PLWH treated with long-term ART.
(a) Viral load and CD4+ and CD8+ T cell count trajectories in donor VA02 from the initiation of ART. (b) KK10/HLA-B*27:05 tetramer+ CD8+ T cell frequencies from donor VA02 and percent expression of CD28 on KK10/HLA-B*27:05 tetramer+ CD8+ T cells from the initiation of ART. (c) Volcano plot showing differentially expressed genes between KK10/HLA-B*27:05 tetramer+ CD8+ T cells from donor VA02 at the early and late time points (red, upregulated at early; blue, upregulated at late). The x-axis represents log fold change, and the y-axis represents −log10-adjusted P values. Significance was assessed using the findMarkers function in scran, which employs a one-tailed Wilcoxon rank-sum test. Adjustments for multiple comparisons were made using the false discovery rate (FDR). (d) Violin plots showing genes associated with exhaustion and inhibition (top) and stemness (bottom) in KK10/HLA-B*27:05 tetramer+ CD8+ T cells from donor VA02 at the early (red) and late time points (blue). Each violin represents the probability density at each value. Each dot represents one cell. Significance was determined using a one-tailed Wilcoxon rank-sum test (FDR-adjusted values). (e) Evolution of KK10-specific clonotypes from the initiation of ART in donor VA2. Each color represents a distinct clonotype with paired sequences for TCRα and TCRβ. (f) Violin plots showing genes associated with exhaustion and inhibition (top) and stemness (bottom) for two dominant clonotypes in donor VA2 at the late time point. Each violin represents the probability density at each value. Each dot represents one cell. Significance was determined using a a one-tailed Wilcoxon rank-sum test (FDR-adjusted values).
Extended Data Fig. 8 Jurkat cells are transduced with TCRs from old or newly dominants clonotypes to measure their avidity.
(a) Graphical representation of the experimental approach (created using BioRender.com). (b, c) Jurkat cells were transduced with CD8αβ and FL8-specific (b) or KF11-specific TCRs (c). Transduction was verified using flow cytometry to quantify the expression of CD3 and CD8. The corresponding tetramers were used to confirm specificity. Mock-transduced Jurkat cells were used as a control for nonspecific tetramer uptake.
Extended Data Fig. 9 Flow cytometric gating strategy.
Flow cytometric gating strategy for the identification and phenotypic characterization of HIV-1-specific CD8+ T cells in this study.
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White, E., Papagno, L., Samri, A. et al. Clonal succession after prolonged antiretroviral therapy rejuvenates CD8+ T cell responses against HIV-1. Nat Immunol 25, 1555–1564 (2024). https://doi.org/10.1038/s41590-024-01931-9
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DOI: https://doi.org/10.1038/s41590-024-01931-9
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