Abstract
During chronic viral infection, CD8+ T cells develop into three major phenotypically and functionally distinct subsets: Ly108+TCF-1+ progenitors, Ly108−CX3CR1− terminally exhausted cells and the recently identified CX3CR1+ cytotoxic effector cells. Nevertheless, how CX3CR1+ effector cell differentiation is transcriptionally and epigenetically regulated remains elusive. Here, we identify distinct gene regulatory networks and epigenetic landscapes underpinning the formation of these subsets. Notably, our data demonstrate that CX3CR1+ effector cells bear a striking similarity to short-lived effector cells during acute infection. Genetic deletion of Tbx21 significantly diminished formation of the CX3CR1+ subset. Importantly, we further identify a previously unappreciated role for the transcription factor BATF in maintaining a permissive chromatin structure that allows the transition from TCF-1+ progenitors to CX3CR1+ effector cells. BATF directly bound to regulatory regions near Tbx21 and Klf2, modulating their enhancer accessibility to facilitate the transition. These mechanistic insights can potentially be harnessed to overcome T cell exhaustion during chronic infection and cancer.
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Data availability
Both raw data files and processed data files from ATAC-seq and CUT&Tag-seq experiments were deposited in the GEO database with accession codes GSE149752, GSE149796 and GSE149810. Source data are provided with this paper.
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Acknowledgements
This work is supported by NIH grants AI125741 (W.C.), AI148403 (W.C.), AI153537 (R.A.Z.), DK108557 (D.M.S.) and DK127526 (M.Y.K.); by an American Cancer Society Research Scholar grant (W.C.); and by an Advancing a Healthier Wisconsin Endowment grant (W.C.). R.A.Z. is supported by a Cancer Research Institute Irvington fellowship. D.M.S. and M.Y.K. are members of the Medical Scientist Training Program at the MCW, which is partially supported by a training grant from the NIGMS (T32-GM080202). This research was completed in part with computational resources and technical support provided by the Research Computing Center at the MCW. We thank S. Henikoff for providing the 3×Flag-pA-Tn5-Fl plasmid and N. Zhu for providing the protein A–Tn5 fusion protein.
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Y.C., R.A.Z., X.W., D.M.S., J.S. and W.C. designed and performed experiments and analyzed data. M.Y.K., S.Z. and R.B. helped with sequencing data analysis and visualization. E.J.T. provided helpful insights and contributed a key biological resource (Batffl/fl mice). Y.C. and W.C. wrote the manuscript. W.C. supervised the study.
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Peer review information Nature Immunology thanks Stephen Turner, Daniel Utzschneider and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Extended data
Extended Data Fig. 1 SCENIC analysis revealed distinct transcriptional regulatory circuits for CD8+ T cell subsets during chronic viral infection.
a, Dot plot showing expression of signature genes for the TPRO cells, TEFF cells, and TEXH cells. b,c,d, t-SNE projections showing binary regulon activity of cell-specific regulons for the TPRO, TEXH and TEFF subsets.
Extended Data Fig. 2 Unsupervised clustering analysis identified three major cell populations in the integrated dataset.
a, Heatmap showing the top 15 differentially expressed genes for each cluster as defined in Fig. 2b. Columns and rows correspond to cells and genes, respectively. Cells from the same cluster are grouped together. The color scale representing Z-Score that is generated from log2 read counts. b,c,d,e,h, Showing the comparison of CD8 T cells from day 9 post-acute infection and day 30 post-chronic infection. Cells are gated on CD8+CD44+ GP33-41+ cells. b, left. Summary data showing the expression of surface makers for SLECs and TEFF cells. b, right. Summary data showing the expression of surface makers for MPECs and TPRO cells. c, Summary data showing the expression of GzmB, proportion of CD8+ T cell degranulating (CD107a+) and producing IFN-γ, and proportion of CD8+ T cell producing TNF and IFN-γ upon ex vivo stimulation with GP33-41 peptide. d,h, Summary data showing the expression of transcription factors and inhibitory receptors. e, Summary data showing the relative cytotoxicity of SLECs and TEFF cells against peptide-pulsed target cells. f, Heatmap showing binary regulon activities of CD8 T cells from day 9 post-acute infection and day 30 post-chronic infection. g, t-SNE projection depicting clustering of cells by regulon activity. b, 10 mice for acute infection and 10 mice for chronic infection. c,d,h, 5 mice for acute infection and 5 mice for chronic infection. e, 10 mice for acute infection and 7 mice for chronic infection. b-h, Data pooled from 2 independent experiments. Data are expressed as mean ± s.e.m. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. b, Unpaired t-test with two-stage step-up method of Benjamini, Krieger, and Yekutieli. c,d,e,h, Ordinary one-way ANOVA.
Extended Data Fig. 3 Distinct H3K4me3 and H3K27me3 patterns associated with gene expression profiles of the three CD8+ T cell subsets.
a, Sorting strategy and sort purity of GP33-41 tetramer+ or GP276-286 tetramer+ virus-specific CD8+ T cell subsets that were sorted from C57BL/6 mice at 3-5 weeks post LCMV Cl13 infection. b,c, Heatmap showing gene promoter regions that exhibited differential H3K4me3 enrichment between TEFF cells and TEXH cells. Scale bar representing Z-Score that is generated from log2 RPKM. Bar plot showing the top pathways correlated with these genes by gene ontology (GO) analysis. d, Genome track view of representative gene loci showing H3K4me3 (green, above line) or H3K27me3 (red, below line) peaks. CUT & Tag-seq data are from two independent replicates. Each replicate was pooled together from 2-3 mice.
Extended Data Fig. 4 Distinct enhancer repertoires regulate transcriptional programs of the three CD8+ T cell subsets.
Venn diagram showing overlap of all chromatin-accessible regions (ChARs) detected by ATAC-seq. b, Bar plot showing the distance of ChARs to TSS. Left are ChARs with differential accessibility among the three CD8+ T cells subsets. Right are ChARs shared by the three subsets. c, Heatmap showing MSigDB pathway enrichment signatures in six enhancer peak sets as shown in Fig. 5c (Top). d, MA plot showing M value vs A value of the merged set of TEFF cell and TEXH cell enhancer peaks after normalization. Top 5,000 peaks are highlighted for TEFF cells (cyan) and TEXH cells (red). Dot plot showing the top 10 TFs whose motifs were significantly enriched in TEFF cell-specific enhancers compared to TEXH cell-specific enhancers.
Extended Data Fig. 5 Active and suppressive states of enhancer regions regulate gene expression in the three CD8 T cell subsets.
a, Genome track view of the gene loci showing ATAC-seq, H3K27ac, and H3K27me3 peaks in TPRO, TEXH and TEFF subsets, TFs with predictive binding sites at the enhancers (green shadow) were listed.
Extended Data Fig. 6 BATF is required for TPRO progenitor cell differentiation into TEFF cells.
a, Summary data showing viral titers in the serum of experimental mice on day 8 p.i. and day 30 p.i. b, c, Summary data showing the relative expression of KLRG1 and TCF-1 in three virus-specific CD8+ T cell subsets. d, Experimental design. e, g, Representative flow plots and summary data showing the proportion of three antigen-specific CD8+ T cell subsets in WT and BATF deficient cells on day 28 p.i. with LCMV Cl13. f, Summary data showing the expression of BATF in WT and BATF deficient cells. a, Day 8 data was collected from 4 WT and 6 BATF-HET mice. Day 21 data was collected from 4 WT and 5 BATF-HET mice. b,c, Data was collected from 4 WT and 4 BATF-HET mice. f,g, Data was collected from 8 Batf flox/ flox CreERT2 mT/mG mice. c-j, Data pooled from 2 independent experiments. Data are expressed as mean ± s.e.m. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. a, Two-tailed unpaired t-test. b,c, Unpaired t-test with Holm-Šídák method. f, Two-tailed paired t-test. g, Paired t-test with two-stage step-up method of Benjamini, Krieger, and Yekutieli.
Extended Data Fig. 7 BATF regulates chromatin accessibility of CD8+ T cells during chronic infection.
Gzmb-Cre+;Batf+/+;RosamT/mG (WT), Gzmb-Cre+;Batffl/+;RosamT/mG (BATF-HET), and Gzmb-Cre+;Batffl/fl;RosamT/mG mice (BATF-KO) were used for this experiment. At 4 weeks post-LCMV Cl13 infection, CD8+CD44+GFP+ cells, which represent polyclonal LCMV-specific CD8+ T cells, were FACS sorted to perform ATAC-seq experiments. a, Venn diagram showing overlapping and unique enhancer regions in WT, BATF-HET, and BATF-KO CD8+ T cells b, Heatmap showing enhancer regions with differential accessibility. Scale bar representing Z-Score that is generated from log2 FPKM. Each replicate was an individual mouse.
Extended Data Fig. 8 Genome track view of Pdcd1, Tcf7, Id3, Cxcr5, Cx3cr1, and Irf8.
a,b, Genome track view of the gene loci showing ATAC-seq, H3K27ac, and BATF CUT&Tag peaks in TPRO, TEXH and TEFF subsets.
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Chen, Y., Zander, R.A., Wu, X. et al. BATF regulates progenitor to cytolytic effector CD8+ T cell transition during chronic viral infection. Nat Immunol 22, 996–1007 (2021). https://doi.org/10.1038/s41590-021-00965-7
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DOI: https://doi.org/10.1038/s41590-021-00965-7
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