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Distinct SIV-specific CD8+ T cells in the lymph node exhibit simultaneous effector and stem-like profiles and are associated with limited SIV persistence

Abstract

Human immunodeficiency virus (HIV) cure efforts are increasingly focused on harnessing CD8+ T cell functions, which requires a deeper understanding of CD8+ T cells promoting HIV control. Here we identifiy an antigen-responsive TOXhiTCF1+CD39+CD8+ T cell population with high expression of inhibitory receptors and low expression of canonical cytolytic molecules. Transcriptional analysis of simian immunodeficiency virus (SIV)-specific CD8+ T cells and proteomic analysis of purified CD8+ T cell subsets identified TOXhiTCF1+CD39+CD8+ T cells as intermediate effectors that retained stem-like features with a lineage relationship with terminal effector T cells. TOXhiTCF1+CD39+CD8+ T cells were found at higher frequency than TCF1CD39+CD8+ T cells in follicular microenvironments and were preferentially located in proximity of SIV-RNA+ cells. Their frequency was associated with reduced plasma viremia and lower SIV reservoir size. Highly similar TOXhiTCF1+CD39+CD8+ T cells were detected in lymph nodes from antiretroviral therapy-naive and antiretroviral therapy-suppressed people living with HIV, suggesting this population of CD8+ T cells contributes to limiting SIV and HIV persistence.

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Fig. 1: TOX is upregulated on CD8+ T cells after SIV infection.
Fig. 2: TCF1+CD39+CD8+ T cells expand after SIV infection and are a unique phenotypic population.
Fig. 3: LN SIV-specific TCF1+CD39+CD8+ T cells maintain a dual-effector and stem-like transcriptional profile.
Fig. 4: Frequency of TOX+CD8+ and TCF1+CD39+CD8+ TM cell is associated with reduced viral burden.
Fig. 5: TCF1+CD39+CD8+ TM cells preferentially enter the follicular environment.
Fig. 6: TCF1+CD39+CD8+ TM cells contract during long-term ART but remain associated with viral burden.
Fig. 7: HIV infection results in expansion of LN HIV-specific TCF1+CD39+CD8+ TM cells.

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Data availability

Single-cell RNA-seq data have been deposited in the NCBI GEO database under accession number GSE266576. Proteomics data have been deposited in the PRIDE database under accession number PXD050498 and can be accessed at https://www.ebi.ac.uk/pride/archive/. All other data are present in the article as individual data points, Supplementary Information or from the corresponding authors upon reasonable request.

Code availability

The custom code used in this manuscript is available from the corresponding author upon reasonable request.

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Acknowledgements

This manuscript is dedicated in loving memory of our colleague and friend T. Hoang. We thank S. Jean, S. Ehnert, C. Wallace, J. Wood and all veterinary and animal care staff at the Emory National Primate Research Center. We also thank the Emory Flow Cytometry core (K. Gill) for flow cytometry support, the Emory National Primate Research Center Genomics Core for RNA-seq support, the NIH Tetramer Core facility at Emory University for providing tetramer, K. Easley for providing statistical advice and Accelevir Diagnostics for intact proviral DNA measurements. Peptide pools of SIVmac239 Gag were provided by the HIV Reagent Program, which were contributed by the Division of AIDS at NIAID. This work was supported by UM1AI164562 (M.P.), cofunded by NIH/NHLBI/NIDDK/NINDS/NIDA/NIAID and by the NIAID, NIH under award numbers R37AI141258 (R.S.), R01AI116379 (M.P.) and R56AI150401 (M.P.). Additional support was provided by: NIH OD, ORIP, P51 OD011132 and U42 OD011023. This work was supported by the Center for AIDS Research at Emory University (Division of Intramural Research, NIAID, P30AI050409). This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract no. 75N91019D00024; Divisions of Intramural Research of the National Institute of Allergy and Infectious Diseases, NIH 1ZIAAI001029 (J.M.B.); B03-16 project was supported by funds from the Mexican Government (Programa Presupuestal P016, Anexo 13 del Decreto del Presupuesto de Egresos de la Federación). Sequencing data were acquired on an Illumina NovaSeq6000 funded by NIH S10 OD026799 (S.E.B.). C.M.B. was supported by Swedish Research Council (grant 2023-00510). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services nor does mention of trade names, commercial products or organizations imply endorsement by the US government. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Authors

Contributions

Z.S. and M.P. conceptualized the study. Z.S., L.R.M., C.D., M.B.P., M.A.C., C.M.B., T.N.H., E.A.U., M.G., K.N., S.L., A.R.R., J.H. and H.T.K. performed the investigations. Z.S., L.R.M., C.D., M.B.P., C.M.B., M.A.C., E.A.U., M.G., G.K.T., A.R.R. and H.T.K. did the formal analysis. P.M.R.E., M.G.-N., Y.A.L., S.A.-R. and G.R.-T. provided resources. Z.S., L.R.M., C.D., M.A.C., C.M.B. and H.T.K. implemented data visualization. C.D., R.S., D.A.K., G.S., J.B., S.E.B., D.E.G., M.R.B. and M.P. acquired funding to support the work. A.S.-C., J.B., S.E.B., D.E.G., M.R.B., H.T.K. and M.P. supervised the work. Z.S., L.R.M. and M.P. wrote the original draft. Z.S., C.D., L.R.M., H.T.K., M.R.B. and M.P. reviewed and edited the final manuscript.

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Correspondence to Mirko Paiardini.

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Z.S. is employed by and/or has financial interests in Merck and Co.. All other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 LN TOXhiTCF1+CD39+CD8+ TM cells express high levels of inhibitory receptors after SIV infection.

a) Expression of PD-1, TIGIT, CD101 and Ki-67 on TOX+ and TOX LN CD8+ TM cells at day 42 p.i. (n = 28 macaques). b) UMAP visualization of Phenograph clusters as in a. c) Heatmap of expression of TOX, TCF1, CD39, PD-1, TIGIT, CD101, Ki-67, GzmB and EOMES across all Phenograph clusters as in a. d) Proportion of total CD8+ TM cell population made up by individual Phenograph clusters (n = 28) as in a. e) Frequency of TCF1+CD39+ cells within LN CD8+ TM cells in 5 unique cohorts of macaques: uninfected (n = 10), day 21 p.i. (n = 7), day 35 p.i. (n = 20), day 42 p.i. (n = 28), late chronic infection (mean 17 months p.i. (n = 10). f-g) Expression of TOX, PD-1, TIGIT, CD101, Ki-67 and GzmB in TCF1+CD39, TCF1+CD39+ and TCF1CD39+ subsets of CD8+ TM cells at day 35 p.i. (n = 20) (f) and late chronic infection (n = 10) (g). P values determined by Wilcoxon matched-pairs signed rank test, Kruskal-Wallis one way ANOVA with Dunn’s multiple comparison correction (e) or two-way ANOVA with Tukey’s multiple comparisons test (f-g). Box and whiskers are displayed via Tukey method. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Extended Data Fig. 2 TCF1+CD39+CD8+ TM cells maintain a dual effector and stem-like proteomic profile.

a) Representative gating strategy for sorting of TCF1+CD39, TCF1+CD39+ and TCF1CD39+ subsets of CD8+ TM at day 42 p.i. b-c) Volcano plots displaying significantly differently expressed proteins of interest between sorted LN TCF1+CD39 and TCF1+CD39+ CD8+ TM cells (b) and between sorted LN TCF1+CD39 and TCF1+CD39+ CD8+ TM cells (c) at day 42 p.i. d) Selected proteins with significantly different expression are listed (n = 4 macaques). All statistical comparisons were performed based on two-tailed Student’s t-tests using Spectronaut and custom R scripts.

Extended Data Fig. 3 Sorting of LN SIV-specific CD8+ TM cells.

a) Sorting strategy for LN SIV-specific CM9+CD8+ TM cells for downstream scRNA-seq at day 42 p.i. b) Plots and frequencies of sorted LN SIV-specific CM9+CD8+ TM cells for all 5 macaques as in a.

Extended Data Fig. 4 Transcriptional clustering of LN SIV-specific CD8+ TM cells overlaps with established human CD8 T cell signatures.

a) Heatmap of top 20 differentially expressed genes across the 5 clusters of LN SIV-specific CM9+CD8+ TM cells at day 42 p.i. b) UMAP projection of cells matching established human CD8+ T cell signatures (Supplementary Data File 1) of cell cycling, type 1 interferon, human stem-like CD8 and human terminally differentiated (TD) CD8. c) Distribution of LN SIV-specific CM9+CD8+ TM cells across the 5 clusters for all macaques as in a. d) Scatter plot of expression levels and thresholds (dotted lines) for identifying TCF7+ENTPD1+ CD8+ TM cells as in a.

Extended Data Fig. 5 TCR analysis of expanded TCF7+ENTPD1+CD8+ TM cells demonstrates lineage relationship with terminally differentiated CD8+ TEFF cells.

a-e) UMAP projection of the top 3 clonotypes within TCF7+ENTPD1+ CD8+ TM cells and the top 5 clonotypes of TCF7+ENTPD1+ CD8+ TM cells population from each macaque and the cluster distribution frequency of those clones across all SIV-specific CM9+CD8+ TM cells at day 42 p.i. Number of total cells expressing each TCR clonotype is shown below each bar.

Extended Data Fig. 6 Response of TCF1+CD39+CD8+ TM cells to dual IL-10 + PD-1 mAb blockade.

a) Study schematic demonstrating dosing regimen and timing of lymph node biopsies (week -1 and week 12 post-treatment) for flow cytometry analysis placebo-treated control (n = 6) and treated (n = 9) macaques. b) Fold change of the frequency of TCF1+CD39, TCF1+CD39+ and TCF1CD39+ subsets of LN CD8+ TM cells, calculated using frequencies at week 12 post-treatment compared to baseline frequencies at week -1 (control (n = 6) and treated (n = 9)). c) Fold change of the frequency of LN Ki-67+ cells within TCF1+CD39, TCF1+CD39+ and TCF1CD39+ subsets of CD8+ TM cells (control (n = 6) and treated (n = 9)). P values determined by multiple Mann-Whitney tests with Holm-Sidak correction for multiple comparison. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Extended Data Fig. 7 TCF1+CD39+CD8+ TM cells are associated with reduced viral burden.

a-b) Association of the frequency of TOX+ (a) and TCF1+ CD39+ (b, red) cells within LN CD8+ TM cells with cell-associated SIV RNA levels in LN at day 42 p.i. (n = 26). c-h) Association of the frequency of TCF1+ CD39+ (c-e, red) and TOX+ (f-h) cells within LN CD8+ TM cells with plasma viral load (c,f), total SIV DNA (d,g) and cell associated RNA (e,h) at day 35 p.i. (n = 20). All correlations were determined using two-tailed Spearman analysis.

Extended Data Fig. 8 Expression of CXCR5 by TOX+TCF1+CD39+CD8+ TM cells is associated with viral burden.

a-c) Association of the frequency of CXCR5+ cells within LN CD8+ TM cells with plasma viral load (a), total SIV DNA (b) and cell associated RNA (c) at day 42 p.i.(a n = 27 macaques, b-c n = 25). d-f) Association of frequency of CXCR5+ cells within TCF1+CD39(d, burgundy), TCF1+CD39+ (e, red) and TCF1CD39+ (f, pink) subsets of LN CD8+ TM cells with cell-associated SIV RNA levels in sorted LN memory CD4 T cells as in a (n = 25). g) Frequency of CXCR5+ cells within LN GzmBGzmK+, GzmB+GzmK+ and GzmB+GzmK CD8+ TM cells LN memory CD8 T cells as in a (n = 27). h) Representative image of immunofluorescence staining of CD39 (green) TCF1 (blue) and TOX (red). i) Representative image of immunofluorescence analysis identifying TCF1+ and TOX+ CD8+ T cells in the B cell follicle and T cell zone. Arrows designate examples of TCF1+ TOX+ CD8+ T cells. j-k) Representative image of immunofluorescence analysis identifying SIVinfected CD4+ T cells and TCF1+TOX+CD8+ T cells in the T cell zone (j) and the dark zone of the b cell follicle (k). The perimeter of analysis is defined by the red circle. Blue arrows indicate uninfected CD4+ T cells not surrounded by TCF1+TOX+CD8+ T cells. The dotted line delimits the edge of the b cell follicle. Number of events analyzed per LN area: B cell follicle SIV+ = 175, B cell follicle SIV− = 178, T cell zone SIV+ = 501, T cell zone SIV− = 475, dark zone B cell follicle SIV+ = 217, dark zone bell cell follicle SIV- = 153. All analyses were performed on samples from animals at early chronic infection (D42 p.i.). Correlations represent Spearman analysis. P values were determined using Friedman test one-way ANOVA with Dunn’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Extended Data Fig. 9 TCF1+CD39+CD8+ TM cells express high levels of inhibitory receptors and low cytolytic markers in ART-suppressed PLWH.

a-d) Expression frequency of TOX, PD-1, TIGIT (a), Ki-67 (c), GzmB and GzmK (c-d) in TCF1+CD39, TCF1+CD39+ and TCF1CD39+ subsets of LN CD8+ TM cells in ART-suppressed PLWH (n = 10). P values determined by two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Extended Data Fig. 10 TCF1+CD39+CD8+ TM cells are not associated with viral burden in ART-naïve PLWH.

a-c) Associations of the frequency of TCF1+CD39 (burgundy), TCF1+CD39+ (red), and TCF1CD39+ (pink) subsets within LN CD8+ TM cells from ART-naïve PLWH with plasma viral load (n = 18) (a), intact reservoir size as measured by IPDA (n = 10) (b) and total HIV DNA (n = 10) (c). d-e) Associations of the frequency of TCF1+CD39 (burgundy), TCF1+CD39+ (red), and TCF1CD39+ (pink) subsets within LN CD8+ TM cells from ART-Suppressed PLWH with intact reservoir size as measured by IPDA (n = 10) (d) and total HIV DNA (n = 10) (e). Correlation evaluated by two-tailed Spearman analysis.

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Supplementary Data 1

scRNA-seq differentially expressed genes and reference human gene signature sets.

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Strongin, Z., Raymond Marchand, L., Deleage, C. et al. Distinct SIV-specific CD8+ T cells in the lymph node exhibit simultaneous effector and stem-like profiles and are associated with limited SIV persistence. Nat Immunol 25, 1245–1256 (2024). https://doi.org/10.1038/s41590-024-01875-0

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