Abstract
During chronic infection and cancer, a self-renewing CD8+ T cell subset maintains long-term immunity and is critical to the effectiveness of immunotherapy. These stem-like CD8+ T cells diverge from other CD8+ subsets early after chronic viral infection. However, pathways guarding stem-like CD8+ T cells against terminal exhaustion remain unclear. Here, we show that the gene encoding transcriptional repressor BACH2 is transcriptionally and epigenetically active in stem-like CD8+ T cells but not terminally exhausted cells early after infection. BACH2 overexpression enforced stem-like cell fate, whereas BACH2 deficiency impaired stem-like CD8+ T cell differentiation. Single-cell transcriptomic and epigenomic approaches revealed that BACH2 established the transcriptional and epigenetic programs of stem-like CD8+ T cells. In addition, BACH2 suppressed the molecular program driving terminal exhaustion through transcriptional repression and epigenetic silencing. Thus, our study reveals a new pathway that enforces commitment to stem-like CD8+ lineage and prevents an alternative terminally exhausted cell fate.
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Data availability
All data generated to support this study are available within the paper. The ATAC-seq (Figs. 1 and 7 and Extended Data Figs. 1 and 7), scRNA-seq (Figs. 3 and 6 and Extended Data Figs. 3 and 6) and RNA-seq (Figs. 4 and 6 and Extended Data Figs. 4 and 6) data have been deposited at the Gene Expression Omnibus (GEO accession no. GSE152379). H3K27ac ChIP–seq and RNA-seq data of stem-like and terminally exhausted CD8 T cells were previously published (GEO accession no. GSE119943).
Code availability
Custom code used for ATAC-seq, RNA-seq and scRNA-seq analyses is available from the corresponding author upon request.
Change history
03 March 2021
A Correction to this paper has been published: https://doi.org/10.1038/s41590-021-00906-4
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Acknowledgements
We thank T. Kupfer (CU), G. Hedlund (CU), K. Helm (CU), Y. Zhang (CU), M. Kirby (NHGRI), S. Anderson (NHGRI), J. Reilley (NIAID), R. Handon (NHGRI), L. Garrett (NHGRI), I. Ginty (NHGRI), G. Gutierrez-Cruz (NIAMS) and S. Dell’Orso (NIAMS) for their excellent technical support. This research was supported in part by an NIH grant (AG056524) to T.W.; the intramural programs of the NIAMS (to J.J.O.), NIAID (to P.L.S.), NHGRI (to P.L.S.), NINDS (to D.B.M.), National Cancer Institute (to L.G.) and NIDDK (to B.A.); an NIH Office of Dietary Supplements Research Scholar scholarship to D.C.; grants from the NIH (AI105343, AI117950, AI082630, AI112521, AI115712, AI108545 and CA210944) and Stand Up To Cancer to E.J.W.; and NIH grant (CA234842) to Z.C. E.J.W. is also supported by the Parker Institute for Cancer Immunotherapy, which supports the Cancer Immunology program at Penn Institute for Immunology.
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Contributions
T.W. and C.Y. conceived the study. T.W. and C.Y. designed the experiments. C.Y., G.L., T.W., Z.Z., Y.S., E.A.M., D.C., M.A.D., W.S. and J.C. performed the experiments. C.Y., T.W., G.L., H.-W.S., Z.C., P.L.S., J.J.O., D.B.M., L.G., B.A., E.J.W. and Y.Z. analyzed and interpreted the results. T.W. and C.Y. drafted the manuscript. J.S. designed and generated retroviral gRNA constructs targeting Prdm1 and Runx3. K.K. and T.K. provided mouse models critical to this study.
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Competing interests
E.J.W. has consulting agreements with and/or is on the scientific advisory board for Merck, Roche, Pieris, Elstar, Related Sciences and Surface Oncology. E.J.W. is a founder of Surface Oncology and Arsenal Biosciences. E.J.W. has a patent licensing agreement on the PD-1 pathway with Roche/Genentech. L.G. is inventor on a patent describing methods for the generation and isolation of stem-like memory T cells. L.G. has consulting agreements with Lyell Immunopharma and Advaxis Immunotherapies.
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Peer review information Nature Immunology thanks Stephen Jameson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. 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 Stem-like and terminally exhausted CD8+ T cells exhibited distinct open chromatin landscapes.
a, Experimental setup has been described in Fig. 1a. Gating strategy for sorting stem-like (Ly108hiTIM-3lo) and terminally exhausted (Ly108loTIM-3hi) P14 cells on day 7 p.i.. b, Venn diagram illustrating the numbers of ATAC-Seq peaks in stem-like and terminally exhausted CD8+ T cells. c, Percentages of common and variable ATAC-Seq peaks, as defined in Extended Data Fig. 1b, annotated to Intergenic, Intronic, and Promoter-TSS regions. Filled bars represent common peaks; unfilled bars represent variable peaks. P values were determined by the Chi-squared test. d, Volcano plots of differentially accessible chromatin regions between stem-like and terminally exhausted CD8+ T cells are shown. The x axis represents log2 fold change of chromatin accessibility; the y axis represents -log10FDR. The horizontal dash line indicates FDR = 0.1; vertical dash lines indicate fold change of +/− 1.2. Dots represent peaks associated with significantly upregulated genes (fold change >1.5, FDR < 0.05) in stem-like (red) or terminally exhausted (green) CD8+ T cells, and non-significantly regulated genes (grey). FDR is calculated by edgeR. ATAC-Seq data include n = 2 independent experiments per group. e, The transcript abundance of Bach2 and Prdm1 in stem-like and terminally exhausted P14 CD8+ T cells harvested on day 7 (left, n = 2 independent samples) and day 20 (right, n = 3 independent samples) after LCMV clone 13 infection are shown in bar graphs. FDR was determined using edgeR. f, Cas9; P14 CD8+ T cells transduced with control or Prdm1 gRNA constructs were adoptively transferred into C57BL/6 mice that were subsequently infected with LCMV clone 13. Splenic P14 cells were analyzed on day 7 p.i.. The protein expression of BACH2 in control or Prdm1 gRNA transduced P14 CD8+ T cells are shown in representative FACS plot (left) and bar graph (right). g. A Venn diagram shows transcription factors (TFs) upregulated in stem-like compared to terminally exhausted CD8+ T cells (red) and TFs with motif enriched in the differentially accessible chromatin regions between stem-like and terminally exhausted CD8+ T cells (blue). Statistical significance in f was calculated with a two-sided Student’s t-test. ****P < 0.0001.
Extended Data Fig. 2 BACH2 overexpression enhances stem-like CD8+ T cell differentiation during chronic LCMV infection.
a, Representative FACS plots of TCF-1 and TIM-3 expression in pMIG and BACH2 OE P14 CD8+ T cells on day 28 p.i. b, Left panel: representative FACS plots of pMIG and BACH2 OE P14 cells in CD8+ T cells on day 14 and day 28 p.i.. Right panel: fold changes in the numbers of BACH2 OE P14 cells relative to the numbers of pMIG P14 cells on day 14 and day 28 p.i.. n = 5 mice/group. c-e, FACS analyses of PD-1 (c, n = 5 mice/group), TIM-3 (d, n = 5 mice/group), TIGIT (e, n = 5 mice/group) expression in pMIG and BACH2 OE P14 CD8+ T cells on day 14 p.i.. f-h, FACS analyses of PD-1 (f, n = 5 mice/group), TIM-3 (g, n = 5 mice/group), TIGIT (h, n = 5 mice/group) expression in pMIG and BACH2 OE P14 CD8+ T cells on day 28 p.i.. i,j, FACS analyses of KLRG1 expression in pMIG and BACH2 OE P14 CD8+ T cells on day 14 (i, n = 5 mice/group) and day 28 (j, n = 5 mice/group) p.i.. k,l, FACS analyses of EOMES expression in pMIG and BACH2 OE P14 CD8+ T cells on day 14 (k, n = 5 mice/group) and day 28 (l, n = 5 mice/group) p.i.. m-o, FACS analyses of granzyme B (m, n = 5 mice/group), IFNγ (n, n = 5 mice/group), TNFα (o, n = 5 mice/group) expression in pMIG and BACH2 OE P14 CD8+ T cells on day 7 p.i.. p, Numbers of pMIG (n = 4 mice) and BACH2 OE (n = 5 mice) P14 CD8+ T cells in the liver (left) and the ratio of P14 cells in the liver versus P14 cells in the spleen in pMIG (n = 4 mice) and BACH2 OE (n = 5 mice) groups (right) on day 7 p.i.. q, Numbers of pMIG (n = 4 mice) and BACH2 OE (n = 5 mice) P14 CD8+ T cells in the lung (left) and the ratio of P14 cells in the lung versus P14 cells in spleen in pMIG (n = 4 mice) and BACH2 OE (n = 5 mice) groups (right) on day 7 p.i.. r,s, Representative FACS plots (left) and bar graph (right) showing the percentage of stem-like (TCF-1hiTIM-3lo) pMIG (n = 4 mice) and BACH2 OE (n = 5 mice) P14 CD8+ T cells in the liver (r) and lung (s). Data are representative of at least two independent experiments. Circles represent individual mice. Bar graphs represent mean ± s.d.. Statistical significance was calculated with a two-sided Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Extended Data Fig. 3 ScRNA-Seq analyses of pMIG and BACH2 OE P14 cells.
a, Violin plots illustrating the mRNA amounts of differentially expressed genes between pMIG (iris blue) and BACH2 OE (red) P14 cells on day 7 p.i.. b, Left panel: UMAP projection of P14 cells colored by cell-cycle phases (G1: red; S: blue; G2/M: green). Middle panel: percentages of cells in different cell-cycle phases from each cluster defined in Fig. 3c. Right panel: percentages of cells in cluster 0 pMIG P14 cells or cluster 0 BACH2 OE P14 cells that are in different cell-cycle phases. c, Volcano plots showing differentially expressed genes between cluster 0 pMIG P14 cells and cluster 0 BACH2 OE P14 cells.
Extended Data Fig. 4 RNA-Seq analyses of pMIG and BACH2 OE stem-like P14 CD8+ T cells.
a, Correlation heatmap of RNA-Seq data from independent samples (n = 3/group) of pMIG and BACH2 OE stem-like (Ly108hiTIM-3lo) P14 cells in Fig. 4a. b, Significantly enriched pathways in Fig. 4c determined by GSEA. c,d, FACS analyses of phospho-ribosomal protein S6 (c) and phospho-AKT (d) in pMIG (n = 5 mice) and BACH2 OE (n = 4 mice) P14 cells on day 7 p.i.. e, Percentages of dead (Annexin V+ Aqua Live/Dead+) cells among pMIG (n = 4 mice) and BACH2 OE (n = 5 mice) P14 cells on day 7 p.i.. Data in c-e are representative of at least two independent experiments. Circles represent individual mice. Bar graphs represent mean ± s.d.. Statistical significance in c-e was calculated with a two-sided Student’s t-test. **P < 0.01, ***P < 0.001.
Extended Data Fig. 5 BACH2 deficiency impairs the differentiation of stem-like CD8+ T cells and persistence of antiviral CD8+ T cells during chronic LCMV infection.
Bach2loxP/loxP; Cre/ERT2 CD8+ T cells were transduced with P14 TCR and adoptively transferred to B6 CD45.1 mice. Recipients were treated with vehicle (control) or tamoxifen (Bach2 iKO) and infected with LCMV clone 13. a-f, Splenocytes were analyzed on day 7 post-infection. a, Representative FACS plots (left) and bar graph (right) showing the percentage of stem-like (TCF-1hiTIM-3lo) control (n = 5 mice) and Bach2 iKO (n = 4 mice) P14 cells. b,c, Numbers of stem-like (TCF-1hi) (b) and terminally exhausted (TCF-1lo) (c) control (n = 5 mice) and Bach2 iKO (n = 4 mice) P14 cells. d-f, FACS analysis of granzyme B (d), IFNγ (e), TNFα (f) in control (n = 5 mice) and Bach2 iKO (n = 4 mice) P14 cells. g-i, Splenocytes were analyzed two-week post-infection. g, Representative FACS plots (left) and bar graph (right) showing the percentage of stem-like (TCF-1hiTIM-3lo) control (n = 5 mice) and Bach2 iKO (n = 4 mice) P14 cells. h,i, Numbers of stem-like (TCF-1hi) (h) and terminally exhausted (TCF-1lo) (i) control (n = 5 mice) and Bach2 iKO (n = 4 mice) P14 cells. j-l, Splenocytes were analyzed one-month post-infection. j, Bar graph showing the percentage of stem-like control (n = 5 mice) and Bach2 iKO (n = 5 mice) P14 cells. k,l, Numbers of stem-like (k) and terminally exhausted (l) control (n = 5 mice) and Bach2 iKO (n = 5 mice) P14 cells. Data are representative of at least two independent experiments. Circles represent individual mice. Bar graphs represent mean ± s.d.. Statistical significance was calculated with a two-sided Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Extended Data Fig. 6 Transcriptome analysis of control and Bach2 gRNA transduced P14 cells.
a, Violin plots illustrating the mRNA amounts of differentially expressed genes between control (iris blue) and Bach2 gRNA (purple) transduced P14 cells on day 7 p.i.. Each dot represents one cell. b, Correlation between WT GP33 tetramer+ stem-like CD8+ T cells and control stem-like P14 CD8+ T cells (left) or between Bach2 KO GP33 tetramer+ stem-like CD8+ T cells and Bach2 gRNA stem-like P14 CD8+ T cells (right). 3 independent samples per condition. c, GSEA of RNA-Seq data from WT and Bach2 KO GP33 tetramer+ stem-like CD8+ T cells shows the enrichment of gene sets containing genes upregulated (left) or downregulated (right) in Bach2 gRNA transduced stem-like P14 cells relative to control stem-like P14 cells. d, Significantly enriched pathways in Fig. 6h determined by GSEA.
Extended Data Fig. 7 The molecular program downstream of BACH2.
a, Experimental setup has been described in Fig. 7a. A pie chart illustrates the genomic distribution of differentially accessible (DA) regions between pMIG and BACH2 OE stem-like P14 cells. b, Experimental setup has been described in Fig. 7f. A pie chart illustrates the genomic distribution of DA regions between control and Bach2 gRNA transduced stem-like P14 cells. c,d, Cas9; P14 CD8+ T cells co-transduced with pMKO GFP vector expressing Bach2 gRNA and SL21 VEX vector expressing Runx3 gRNA were adoptively transferred into C57BL/6 mice that were subsequently infected with LCMV clone 13 (n = 4 mice). Splenic P14 cells were analyzed on day 7 p.i.. c, Representative FACS plots of Ly108 and TIM-3 expression on GFP-VEX-, GFP+VEX-, GFP-VEX+, and GFP+VEX+ P14 CD8+ T cells. d, Percentage of stem-like (Ly108hiTIM-3lo) cells within GFP-VEX-, GFP+VEX-, GFP-VEX+, and GFP+VEX+ P14 CD8+ T cells. e-g, Cas9; P14 CD8+ T cells co-transduced with pMKO GFP vector expressing Bach2 gRNA and SL21 VEX vector expressing Prdm1 gRNA were adoptively transferred into C57BL/6 mice that were then infected with LCMV clone 13 (n = 5 mice). Splenic P14 cells were analyzed on day 7 p.i.. Representative FACS plots of Ly108 and TIM-3 expression on GFP-VEX-, GFP+VEX-, GFP-VEX+, and GFP+VEX+ P14 CD8+ T cells (e) and percentage of stem-like (Ly108hiTIM-3lo) cells within GFP-VEX-, GFP+VEX-, GFP-VEX+, and GFP+VEX+ P14 cells (f) are shown. g, CD62L expression on GFP-VEX-, GFP+VEX-, GFP-VEX+, and GFP+VEX+ P14 cells. Data in c-g are representative of at least two independent experiments. Circles represent individual mice. Lines in d,f,g connect data points from the same individual mice. Statistical significance in d,f,g was calculated with a two-sided Student’s paired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
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Yao, C., Lou, G., Sun, HW. et al. BACH2 enforces the transcriptional and epigenetic programs of stem-like CD8+ T cells. Nat Immunol 22, 370–380 (2021). https://doi.org/10.1038/s41590-021-00868-7
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DOI: https://doi.org/10.1038/s41590-021-00868-7
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