Abstract
This study examines the origin and differentiation of stem-like CD8+ T cells that are essential for sustained T cell immunity in chronic viral infections and cancer and also have a key role in PD-1 directed immunotherapy1,2,3,4,5,6,7,8,9,10. These PD-1+TCF-1+TOX+ stem-like CD8+ T cells (also known as precursors of exhausted T cells8,9) have a distinct program that enables them to adapt to chronic antigen stimulation. Here, using the mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection, we find that virus-specific stem-like CD8+ T cells are generated early (day 5) during chronic infection, suggesting that this crucial fate commitment occurs irrespective of the infection outcome. Indeed, we find that nearly identical populations of stem-like CD8+ T cells were generated early during acute or chronic LCMV infection, and that antigen was essential for maintaining the stem-like phenotype. We performed reciprocal adoptive transfer experiments to determine the fate of these early stem-like CD8+ T cells after viral clearance versus persistence. After transfer of day 5 stem-like CD8+ T cells from chronically infected mice into acutely infected mice, these cells downregulated canonical markers of the chronic stem-like CD8+ T cells and expressed markers (CD127 and CD62L) associated with central memory CD8+ T cells. Reciprocally, when day 5 stem-like cells from acutely infected mice were transferred into chronically infected mice, these CD8+ T cells functioned like chronic resource cells and responded effectively to PD-1 therapy. These findings highlight the ability of these early PD-1+TCF-1+TOX+ stem-like CD8+ T cells to adapt their differentiation trajectory to either an acute or a chronic viral infection. Importantly, our study shows that the host is prepared a priori to deal with a potential chronic infection.
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Data availability
Raw RNA-seq data have been uploaded to the NCBI Gene Expression Omnibus under identifier GSE284012. Raw scRNA-seq data have been uploaded to the NCBI Gene Expression Omnibus under identifier GSE284013. Raw data for ATAC–seq analysis have been uploaded to the NCBI Gene Expression Omnibus under identifier GSE269785. Previously published datasets GSE84105 (ref. 1), GSEA datasets HALLMARK_MITOTIC_SPINDLE and HALLMARK_G2M_CHECKPOINT41 were also used for analysis in this study. Source data are provided with this paper.
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Acknowledgements
This work was supported by National Institute of Health grants R01AI030048 (R.A.) and R01AI165706 (B.B.A.-Y.); HHMI Hanna Gray Fellowship GT16001 (C.B.M.); Burroughs Wellcome Fund Postdoctoral Enrichment Program 1022361 (C.B.M.); Achievement Rewards for College Scientists Standard Scholarship (A.L.G.); NIH/NIBIB Trailblazer Award R21EB027327 (H.Y.K.); NSF Graduate Research Fellowships (K.A. and E.C.C.). We thank staff at the Emory University School of Medicine’s Flow Cytometry Core (K. Fife and R. Karaffa), the Emory Integrated Cellular Imaging Core (L. Fox-Goharioon), the Emory Integrated Genomics Core (C. Scharer) and the Yerkes Nonhuman Primate Genomics Core (K. Pellegrini and S. Bosinger; NIH P51OD011132). We acknowledge the NIH/NIAID BioArt Repository for use of their publicly available graphics in our paper (https://bioart.niaid.nih.gov/bioart/279; https://bioart.niaid.nih.gov/discover?q=T%20cell).
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D.T.M., R.M.V., Y.H., M.H., D.Y.C. and R.A. designed experiments. D.T.M., R.M.V., Y.H., A.W., T.H.N., J.L., J.L.R., D.Y.C., N.P. and M.A.C. performed experiments. D.T.M., R.M.V., Y.H. and R.A. analysed experiments. E.S., D.J.M. and H.T.K. analysed scRNA-seq data. D.T.M., C.B.M., C.D.S. and A.L.G. analysed RNA-seq data. C.D.S. analysed ATAC–seq data. B.B.A.-Y., E.C.C., K.A., A.K., H.Y.K. and J.K. contributed materials. D.T.M., R.M.V. and R.A. wrote the manuscript. All of the authors contributed to writing the manuscript and providing feedback.
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Extended data figures and tables
Extended Data Fig. 1 Early anatomical commitment of stem-like CD8 T cells during chronic infection.
a, On days 5 and >45 after chronic infection, mice were injected with fluorescently-labelled anti-CD8α (3 μg/mouse) and euthanized 3 min later for flow cytometry analysis. b, Representative flow plots/histograms showing frequencies of GP33+TCF-1+Tim-3− (stem) and TCF-1−Tim-3+ (effect./TD) CD8+ T cells in the white pulp (CD8α i.v. antibody negative) or the red pulp (CD8α i.v. antibody positive). Numbers on flow plots are frequencies. c, Frequencies of stem and effect./TD CD8+ T cells in the white pulp (left) and red pulp. Mean ± SEM is shown. Results pooled from 2 of 3 independent experiments with n = 10 mice (day 5) and n = 10 mice (day >45). P values shown were calculated using Wilcoxon matched-pairs signed rank test. d, Representative immunofluorescent histology showing the location of stem-like (green) and effector (orange) CD8+ T cells in the spleen on days 5 and >45 after chronic infection. Dashed lines denote border between the white pulp (WP) and red pulp (RP). Stem-like cells were identified as CD8β+PD-1+TCF-1+DAPI+ cells. Effector cells were identified CD8β+PD-1+TCF-1−DAPI+ cells. e, Frequency of indicated subsets in white pulp (left) and red pulp (right) based on image analysis. (n = 3-4 mice per experiment). Summary graphs show mean ± SEM and are pooled from 2 independent experiments with n = 6 mice (day 5), and n = 7 mice (day >45). P values shown were calculated using Wilcoxon matched-pairs signed rank test.
Extended Data Fig. 2 scRNA-seq of LCMV GP33-specific CD8+ T cells on days 5 and >45 after chronic infection.
a, Gating strategy for sorting of splenic DbGP33+CD8+ T cells on days 5 (60,000 cells) and >45 (40,000 cells) for scRNA-seq. b,c, Gene set enrichment analysis enumerating enrichment of day 5 and >45 cell clusters for stem-like and effect/TD gene signatures: GSE84105, Im et al.1. Max = maximum. d,e,UMAP showing day 5 cell clusters (d) and expression of selected genes by each cluster (e). f,g, UMAP showing day >45 cell clusters (f) and expression of selected genes by each cluster (g).
Extended Data Fig. 3 RNA-seq and ATAC-seq analyses of sorted CD8 T cell subsets on days 5 and >45 after chronic infection.
a, Gating strategy for sorting of day 5 and >45 PD-1+ stem-like (CD73+ Tim-3−) and effector/terminally differentiated (CD73−Tim-3+) CD8+ T cells for RNA-seq. b, 2,500 P14 cells were transferred into congenically distinct naïve mice prior to intravenous infection with 2 × 106 LCMV clone 13. Gating strategy for sorting of day 5 and >45 LCMV GP33-specific P14 stem-like (CD73+ Tim-3−) and effector/terminally differentiated (CD73−Tim-3+) CD8+ T cells for ATAC-seq. Naïve CD8+ T cells (CD44−PD-1−) from uninfected mice were used as a control in each analysis. c,d, Genome plots showing chromatin accessibility (ATAC-seq, left) and normalized counts (RNA-seq, right) for Slamf6 (c) and Xcl1 (d) in naïve CD8 T cells from uninfected mice and day 5 vs. >day 45 stem-like and effector CD8 T cells from chronic infection. The location of the transcription start site is indicated by an arrow and a bar depicts the location of the gene body. Bar graphs for normalized counts are based on technical replicates with n = 3 per group. e, Density scatter plot comparing the log2 fold-change in ATAC-seq (x-axis) to the corresponding RNA-seq (y-axis) data for the indicated comparison. Each dot represents accessibility of a single peak and the matching gene expression of the nearest gene. Blue lines indicate data density. Linear correlation trend lines are plotted, and Pearson correlation coefficient and significance of trend are indicated. Coloured dots correspond to the data for Pdcd1, Xcl1, Tcf7, and Gzmb as indicated. f, Heatmap of the Pearson correlation coefficient between ATAC-seq and RNA-seq data for each pairwise comparison between the sample groups. RNA- and ATAC-seq results are each from 1 independent experiment.
Extended Data Fig. 4 Changes in stem-like CD8 T cells as chronic infection progresses from days 5 to >45.
a, Enrichment of day 5 and >45 GP33+CD8+ T cell clusters for cell cycle gene signature based on gene set enrichment analysis based on scRNA seq analysis. Cell cycle signature combines genes from two GSEA datasets: HALLMARK_MITOTIC_SPINDLE and HALLMARK_G2M_CHECKPOINT (ref. 41). b, Expression of Mki67 by day 5 and day >45 GP33+ cells based on scRNA-seq analysis. c, FACs analysis of splenic GP33+TCF-1+Tim-3− stem-like CD8+ T cells from days 5 and >45. Histogram (left) and summary graph (right) showing cell size based on forward scatter (FSC-A). Mean ± SEM is shown. Results pooled from 3 independent experiments with n = 10 mice (D5 stem) and n = 10 mice (>D45 stem). P values shown were calculated using two-tailed, unpaired Mann-Whitney U test. d, Enrichment for indicated gene sets by PD-1+ stem-like CD8+ T cells from day 5 versus >45 (RNA-seq). e, Bar graph showing relative expression of top 50 most significant differentially expressed genes (DEGs) from mTORC1 signalling pathway between PD-1+ day 5 and >45 stem-like CD8+ T cells (RNA-seq). f, Expression of indicated genes by naïve (CD44−PD-1−), and PD-1+ day 5 and >45 stem-like CD8+ T cells based on normalized RNA counts. Circles correspond to technical replicates with n = 3 per group. RNA- and ATAC-seq results are each from 1 independent experiment.
Extended Data Fig. 5 Identical populations of virus-specific stem-like CD8+ T cells are generated early after acute and chronic LCMV infection.
a,b, Phenotypic analysis of GP33+CD8+ T cells in the spleen on day 5 after LCMV Armstrong (acute, 2 × 106 PFU i.v.) or clone 13 (chronic, 2 × 106 PFU i.v.) infection. Flow plots are representative of 3 independent experiments (n = 4-5 recipient mice per donor per experiment). c,d, Frequency (c) and number (d) of GP33+PD-1+TCF-1+CD8+ T cells in the spleen on day 5 after acute versus chronic infection. For c and d, mean ± SEM are shown. Results are pooled from 3 independent experiments with n = 15 mice (acute) and n = 15 mice (chronic). P values shown were calculated using two-tailed, unpaired Mann-Whitney U test. e, Gating strategy used for sorting LCMV-specific GP33+ P14 stem-like (PD-1+CD73+Tim-3) and effector (PD-1+CD73−Tim-3+) CD8+ T cells from Armstrong or clone 13-infected spleens on day 5 for RNA- and ATAC-seq. f, Heatmaps showing relative expression (left) and accessibility (right) of selected genes by indicated subsets. g, Accessibility tracks (ATAC-seq, left) and normalized counts (RNA-seq, right) for selected genes. The location of the transcription start site is indicated by an arrow and a bar depicts the location of the gene body. Bar graphs for normalized counts are based on technical replicates with n = 3 per group. RNA- and ATAC-seq results are each from 1 independent experiment. h, On day 5 after acute or chronic infection, mice were injected with fluorescently-labelled anti-CD8α (3 μg/mouse) and euthanized 3 min later for flow cytometry analysis. i, Flow plots showing frequencies of GP33+TCF-1+Tim-3− (stem-like) and TCF-1−Tim-3+ (effect./TD) CD8+ T cells in the white pulp (CD8α i.v. negative) or the red pulp (CD8α i.v. positive). Numbers on flow plots are frequencies. Flow plots are representative of 1 of 2 independent experiments (n = 3-5 mice per infection group). j, Frequencies of stem-like and effect./TD CD8+ T cells in the white pulp (left) and red pulp (right). Graph shows 1 of 2 independent experiments with n = 3 mice (Arm) and n = 3 mice (Cl13).
Extended Data Fig. 6 Stem-like CD8+ T cells from acute and chronic infection are equally able to expand, self-renew, and differentiate into effector cells after clone 13 challenge.
a, LCMV-specific GP33+ P14 stem and effect. CD8+ T cells (CD45.1+CD45.2−) were isolated from the spleens of LCMV Armstrong or clone 13-infected mice on day 5 and transferred into naïve mice (CD45.1−CD45.2+). Recipients were then challenged i.v. with 2 × 106 PFU of LCMV Cl13. b, Gating strategy used to isolate stem-like (Slamf6+ Tim-3−) and effector (Slamf6−Tim-3+) P14 CD8 T+ cells from acute versus chronic LCMV infection on day 5 to evaluate their response to clone 13 challenge. c, FACs plots showing frequency of donor cells recovered from indicated recipient livers on day 7 after clone 13 challenge. Data are representative of 1 of 3 independent experiments. d, Numbers of donor cells recovered from indicated recipient livers on day 7 after clone 13 challenge. Mean ± SEM is shown. Results pooled from 3 independent experiments with n = 9 mice (Arm/Cl13 stem donor) and n = 9 mice (Arm/Cl13 effect. donor). P values shown were calculated using two-tailed, unpaired Mann-Whitney U test. e, FACs analysis of stem-like donor cells from acute (top) or chronic (bottom) infection pre- and on day 7 post-clone 13 challenge. Data are representative of 1 of 3 independent experiments. f, FACs analysis of effector donor cells from acute (top) or chronic (bottom) infection pre- and on day 7 post-clone 13 challenge. Data are representative of 1 of 3 independent experiments. g, Numbers of splenic stem-like (TCF-1+ GzmB−) and effector (TCF-1−GzmB+) CD8+ T cells derived from indicated effector donor cells. Results pooled from 3 independent experiments with n = 9 mice (Arm effector donor) and n = 9 mice (Cl13 effect. donor). P values shown were calculated using two-tailed, unpaired Mann-Whitney U test.
Extended Data Fig. 7 Antigen is required for the maintenance of the stem-like CD8+ T cell phenotype during acute viral infection.
Mice were infected intravenously with 2 × 106 PFU of LCMV Armstrong (acute). a, Viral titre kinetics (blue curve) and numbers of LCMV-specific GP33+CD8+ T cells in the spleen over time. Viral titre data shown are from one independent experiment with n = 5 mice per time point and are consistent with historical data from our lab. Black line shows number of GP33+CD8+ T cells at indicated time points. Data pooled from 3 independent experiments with n = 15 mice (day 5), n = 12 mice (day 8), and n = 12 mice (day 14). Mean ± SEM are shown. b, Frequency of GP33+PD-1+TOX+TCF-1+ stem-like CD8+ T cells in the spleen at indicated times post- acute infection. Results pooled from 3 independent experiments with n = 12 mice (D5), n = 9 mice (D8), n = 9 mice (D14). Mean ± SEM is shown. P values shown were calculated using two-tailed, unpaired Mann-Whitney U test. c, Expression of indicated markers on GP33+TCF-1+CD8+ T cells at indicated times after acute LCMV infection. Numbers in flow plots indicate frequency. Flow plots are representative of 1 of 3 independent experiments (n = 3-5 mice per time point per experiment). d, On day 5 after acute infection, mice were injected i.v. with 200 μg of GP33 peptide on days 5, 7, and 10. After clearance of infection (day 14), the phenotype of GP33+CD8+ T cells was examined in acutely infected mice that were untreated or injected with peptide. e, Flow plots showing frequencies of GP33+CD44+CD8+ T cells in the spleen in the two groups of mice. Data are representative of 1 of 3 independent experiments (n = 3-5 mice per group). f, Frequency of GP33+CD8+ T cells in the spleen. Results pooled from 4 independent experiments with n = 13 mice (day 5), n = 18 mice (day 14, no pep.), n = 12 mice (day 14, GP33 pep.). Mean ± SEM is shown. P values shown were calculated using two-tailed, unpaired Mann-Whitney U test. g, Expression of indicated markers on GP33+TCF-1+CD8+ T cells. Data are representative of 1 of 4 independent experiments (n = 3-5 mice per group). h, Frequency of GP33+PD-1+TCF-1+CD8+ T cells in the spleen for indicated groups of mice. Results pooled from 4 independent experiments with n = 13 mice (day 5), n = 18 mice (day 14, no pep.), n = 12 mice (day 14, GP33 pep.). Mean ± SEM is shown. P values shown were calculated using two-tailed, unpaired Mann-Whitney U test.
Extended Data Fig. 8 Antigen is required for the maintenance of the stem-like CD8+ T cell phenotype during chronic viral infection.
a, 2,000 P14 CD8+ T cells were transferred into congenically distinct naïve mice. Recipients were then infected with either wild-type LCMV clone 13 alone (WT Cl13, black) or mixed 1:2 with a mutant GP33-deficient clone 13 strain (WT/Mut, red). b, FACs analysis of P14s or endogenous LCMV-specific DbGP276+CD8+ T cells. Flow plots are representative of 1 of 2 independent experiments (WT Cl13 n = 5-10 mice, WT/Mut Cl13 n = 4-5 mice). c, Nur77-GFP reporter mice were infected with either acute or chronic LCMV infection. d, Frequencies of Nur77-GFP+ cells among indicated GP33+ subsets. Results pooled from 2 independent experiments with n = 8 mice (chronic LCMV, >D45) and n = 4 mice (Acute LCMV, >D60) with mean ± SEM is shown.
Extended Data Fig. 9 Adoptive transfer of stem-like and effector CD8+ T cells from day 5 LCMV clone 13 (chronic) mice into day 5 LCMV Armstrong (acute) infected mice.
Same experimental setup as shown in Fig. 5a. a, Frequency of stem-like and effector donor cells recovered from the spleens of LCMV Armstrong-infected recipients on days 8/15 post-transfer. Data representative of 1 of 3 independent experiments. b, Expression of CD127, KLRG1, CD62L and TCF-1 on stem-like donor cells in the spleen pre- (day 5 p.i., chronic) and post-transfer (days 8/15 post-transfer, acute). Data representative of 1 of 3 independent experiments. c, Frequencies of KLRG1+CD127− (left) and KLRG1−CD127+ (right) P14 CD8 T cells derived from stem donor cells post-transfer on days 8/15 post-transfer. Results pooled from 3 independent experiments with n = 10 mice (stem donor recipients) with mean ± SEM is shown. P values shown were calculated using two-tailed, unpaired Mann-Whitney U test. d, Expression of CD127, KLRG1, CD62L and TCF-1 on effector donor cells in the spleen pre- (day 5 p.i., chronic) and post-transfer (days 8/15 post-transfer, acute). e, Frequencies of KLRG1+CD127− (left) and KLRG1−CD127+ (right) P14 CD8 T cells derived from effector donor cells post-transfer on days 8/15 post-transfer. Results pooled from 3 independent experiments with n = 16 mice (effect. donor recipients). Mean ± SEM is shown. P values shown were calculated using two-tailed, unpaired Mann-Whitney U test.
Extended Data Fig. 10 Early stem-like CD8+ T cells from acute viral infection become chronic resource CD8+ T cells after transfer into chronically infected mice.
a, 2 × 103TCF7-YFP reporter P14 CD8+ T cells (CD45.1−CD45.2+) were transferred into congenically distinct naïve mice (CD45.1+CD45.2−) prior to intravenous infection with 2 × 106 PFU of LCMV Armstrong (acute). On day 5, TCF7-YFP+Tim-3− (stem) andTCF7-YFP−Tim-3+ (effect.) P14 CD8+ T cells (CD45.1−CD45.2+) were FACS sorted from the spleen and 1 × 106 cells of each sorted subset were transferred intravenously into separate groups of congenically distinct, day 5 LCMV clone 13 (chronic) infected mice (CD45.1+CD45.2−). The recovery and phenotype of donor cells were analysed 8 days after transfer. b, Flow plots showing stem and effector subsets pre- and post-sort with purity indicated. c, FACs analysis showing frequencies of stem-like and effector P14 donor cells recovered from indicated tissues. Flow plots represent 1 independent experiment. d, Numbers of stem-like and effector P14 donor cells recovered from indicated tissues. Data are from 1 independent experiment with n = 3 mice (stem donor recipients) and n = 4 mice (effector donor recipients). e, Expression of indicated markers on stem-like donor P14 cells pre- (spleen, day 5 p.i., acute) and day 8 post-transfer (spleen, liver and lung, chronic). Arrow indicates differentiation trajectory of donor Tim-3− stem-like cells into Tim-3+ effector cells. f, Expression of indicated markers on effector donor P14 cells pre-transfer (spleen, day 5 p.i., acute, top panel) and day 8 post-transfer (spleen, chronic, bottom panel). Flow plots represent 1 independent experiment.
Extended Data Fig. 11 Longitudinal tracking of day 5 stem-like and effector CD8+ T cells from acute viral infection following transfer into chronically infected mice.
a, 2 × 103 TCF7-YFP reporter P14 CD8+ T cells (CD45.1−CD45.2+) were transferred into congenically distinct naïve mice (CD45.1+CD45.2−) before i.v. infection with 2 × 106 PFU of LCMV Armstrong (acute). On day 5 p.i. stem-like (TCF7-YFP+ Tim-3−) and effector (TCF7-YFP−Tim-3+ (effect.) P14 T cells were isolated from the spleen via FACS and transferred into separate groups of day 5 LCMV clone 13-infected recipients (CD45.1+CD45.2−), which had been transiently depleted of CD4+ T cells before infection. b, Flow plots showing stem and effector subsets pre- and post-sort with purity indicated. c, Frequencies of stem-like and effector P14 donor cells recovered from the spleens, livers, and lungs, of chronic LCMV recipient mice at indicated times post-transfer. Data represent one independent experiment. d, Expression of TCF-1 and Tim-3 on stem-like P14 donor cells pre-transfer (spleen, day 5 p.i., acute) and at indicated times post-transfer in the livers and lungs of chronically infected recipients. Data represent one independent experiment. e, UMAP plot showing scRNA-seq analysis of stem donor P14 cells on day 15 post-transfer (right) and endogenous GP33+CD8+ T cells from stem donor P14 recipient mice (left). UMAPs represent 1 independent experiment.
Supplementary information
Supplementary Table 1
Detailed information for the antibodies used in flow cytometry and immunohistochemistry.
Supplementary Table 2
Specific Gene Ontology pathways and identifiers used for analysis in Fig. 2d,e.
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McManus, D.T., Valanparambil, R.M., Medina, C.B. et al. An early precursor CD8+ T cell that adapts to acute or chronic viral infection. Nature 640, 772–781 (2025). https://doi.org/10.1038/s41586-024-08562-y
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DOI: https://doi.org/10.1038/s41586-024-08562-y
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