The mechanisms underlying the heightened protection mediated by central memory CD8+ T (TCM) cells remain unclear. Here we show that the transcription factor Tcf1 was required in resting TCM cells to generate secondary effector CD8+ T cells and to clear pathogens during recall responses. Recall stimulation of CD8+ TCM cells caused extensive reprogramming of the transcriptome and chromatin accessibility, leading to rapid induction of glycolytic enzymes, cell cycle regulators and transcriptional regulators, including Id3. This cluster of genes did not require Tcf1 in resting CD8+ TCM cells, but depended on Tcf1 for optimal induction and chromatin opening in recall-stimulated CD8+ TCM cells. Tcf1 bound extensively to these recall-induced gene loci in resting CD8+ TCM cells and mediated chromatin interactions that positioned these genes in architectural proximity with poised enhancers. Thus, Tcf1 preprogramed a transcriptional program that supported the bioenergetic and proliferative needs of CD8+ TCM cells in case of a secondary challenge.
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ChIP-seq, RNA-seq, ATAC-seq and Hi-C data in CD8+ TCM cells are deposited at GEO under accession no. GSE177064.
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We thank the University of Iowa Flow Cytometry Core facility (J. Fishbaugh, H. Vignes and G. Rasmussen) and the HMH-CDI Flow Cytometry Core facility (M. Poulus and W. Tsao) for cell sorting, B. Wagner (University of Iowa Free Radical and Radiation Biology Core) and P. Ramalingam (HMH-CDI) for assistance with the use of Seahorse Extracellular Flux Analyzer, and K. Zhao (NHLBI, NIH) for sharing the Hi-C protocol. This study is supported, in part, by grants from the National Institutes of Health (AI121080 and AI139874 to H.-H.X. and W.P.; AI112579 to H.-H.X. and C.Z.; and GM134880 and AI114543 to V.P.B.).
The authors declare no competing interests.
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Extended Data Fig. 1 Double deletion of Tcf1 and Lef1 diminished expression of cytotoxic effector molecules during primary CD8+ T cell responses.
a. Gating stratey to identify CD45.2+ donor-derived P14 CD8+ T cells in CD45.1+ wild-type recipients. b. Validating early deletion of Tcf1 protein by Gzmb-Cre by intracellular stating at 36 hrs after willd-type (WT) or Tcf7ΔGzmb P14 CD8+ T cells were labeled with cell-trace violet (CTV) and adoptively transferred into CD45.1+ recipients followed by i.v. infection with LCMV-Arm the next day. Dot plots are representative from 2 independent experiments, where values denote the numbers of cell division and redline marks signal levels with isotype control staining. c,d. Detection of cytokine production by effector CD8+ T cells in recipient spleens during primary response on day 8 after WT, Tcf7ΔGzmb or Tcf7ΔGzmbLef1ΔGzmb P14 CD8+ T cells were adoptively transferred into CD45.1+ wild-type recipients, followed by LCMV-Arm infection the next day. GP33-induced production of IFN-γ (c), TNF and IL-2 (d) was detected in CD45.2+CD8+ T cells. Representative contour plots (left) are from ≥3 experiments, with values denoting percentages of the gated population. Cumulative data (right) of the percentage of IFN-γ+ population in P14 CD8+ T cells, IFN-γ relative geometric mean fluorescent intensity (gMFI) (c), and the percentage of TNF+ or IL-2+ populations in IFN-γ+ P14 CD8+ T cells (d) are means ± s.d. e. Detection of granzyme B expression in effector CD8+ T cells by intracellular staining on day 8 p.i. Representative histographs (left) are from ≥3 experiments with the values denoting percentage of granzyme B+ cells and those in parentheses denoting gMFI of granzyme B. Cumulative data (left) are means ± s.d. For data in c–e, statistical significance was first determined with one-way ANOVA for multi-group comparisons, and as post hoc correction, Tukey’s test was used for indicated pairwise comparison. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not statistically significant.
Extended Data Fig. 2 Tcf1 is largely dispensable for induction of cytotoxic effector molecules during CD8+ TCM recall response.
a. Detecting cell viability of early effector CD8+ T cells in secondary recipients at 60 hrs after CD45.2+ wild-type or Tcf7ΔGzmb CD62L+CD8+ TCM cells (sorted from the primary recipients on day ≥30–35 p.i., as generated in Fig. 1g) were transferred into CD45.1+ wild-type recipients, which were infected with LCMV-Arm the next day. AnnexinV–7-AAD– viable cells were detected in CTV+CD45.2+CD8+ T cells, with representative contour plots (left) from 2 independent experiments, and cumulative data (right) as means ± s.d. b, c. Detection of cytokine production in secondary effector CD8+ T cells on day 8 after CD45.2+ wild-type or Tcf7ΔGzmb CD62L+CD8+ TCM cells (sorted from the primary recipients on day ≥30–35 p.i., as generated in Fig. 1g) were transferred into CD45.1+ wild-type recipients, which were infected with LCMV-Arm the next day. GP33-induced production of IFN-γ (b), TNF and IL-2 (c) was detected in CD45.2+CD8+ T cells. Representative contour plots (left) are from 2 independent experiments, with values denoting percentages of the gated population. Cumulative data (right) of the percentage of IFN-γ+ population in P14 CD8+ T cells, IFN-γ relative gMFI (b), and the percentage of TNF+ or IL-2+ populations in IFN-γ+ P14 CD8+ T cells (c) are means ± s.d. d. Detection of granzyme B expression in secondary effector CD8+ T cells by intracellular staining on day 8 p.i. Representative histographs (left) are from 2 independent experiments, with the values denoting percentage of granzyme B+ cells and those in parentheses denoting gMFI of granzyme B. Cumulative data (left) are means ± s.d. ns, not statistically significant; **, p < 0.01 as determined with two-tailed Student’s t-test.
Extended Data Fig. 3 Tcf1 critically regulates transcriptomic changes in CD8+ TCM cells at resting state and after recall stimulation.
a. Principal component analysis (PCA) of the transcriptomes of wild-type or Tcf7ΔGzmb CD8+ TCM cells at resting state and after 24-hr GP33 stimulation, where log2(RPKM + 1) for each gene in different samples were used as the input data. b,d,f. Heatmaps of select genes involved in ‘mitochondria’ and ‘oxidative phosphorylation’ from Cluster 3 + 4a (b), genes in Cluster 1 (d) and Cluster 2 (f), with the color represented row-normalized RPM for the genes across different samples. c,e. Functional annotation of Cluster 1 (c) and Cluster 2 (e) genes, as defined in Fig. 3e, using DAVID Bioinformatic Resources, with select top gene ontology (GO) terms shown in dot plots and statistical significance denoted with a color scale.
Extended Data Fig. 4 Tcf1 critically regulates chromatin accessibility changes in CD8+ TCM cells at resting state and after recall stimulation.
a. Principal component analysis (PCA) of the ChrAcc profiles of wild-type or Tcf7ΔGzmb CD8+ TCM cells at resting state and after 24-hr GP33 stimulation, where normalized ATAC-seq signal on merged peaks were used as input data. b. Functional annotation of recall-‘closed’ chromatin sites in wild-type CD8+ TCM cells (the 1,484 sites in Fig. 4d) using GREAT analysis, with select top GO terms shown in dot plots and statistical significance denoted with a color scale. c,d. Motif analysis of recall-‘opened’ 17,763 (c) and –‘closed’ 1,484 ChrAcc sites (d) in GP33-stimulated WT CD8+ TCM cells, as defined in Fig. 4d. Listed are the top transcription factor or hybrid motifs and corresponding logos, along with statistical significance and frequency of occurrence at the target ChrAcc sites.
a. Scatter-plots showing reproducibility of two biological replicates of Hi-C libraries from resting wild-type or Tcf7ΔGzmb CD8+ TCM cells. The x- and y-axis values for each data point (marked with a dot) represent the interaction scores of an anchor in replicate 1 (Rep1) and replicate 2 (Rep2), respectively, and the R values denote Pearson correlation coefficient. b. Functional annotation of Clusters 3 + 4a genes associated with Tcf1-dependent ChrInt using DAVID, with selected gene ontology (GO) terms shown in dot plots and statistical significance denoted with a color scale. c. Proposed model for Tcf1-mediated pre-programming of responsiveness of CD8+ TCM cells to recall stimulation. Left, CD8+ TCM cells at resting state express Tcf1 and basal expression of secondary effector CD8+ T cell-associated genes, including transcription regulators such as Id3 and glycolytic enzymes. In this context, the presence of Tcf1 mediates chromatin interactions that engage the effector gene promoters with distal regulatory regions (top row) or span the effector gene loci (bottom row). The Tcf1-dependent chromatin interactions may thus bring the effector gene promotors and the poised enhancer elements (blue flags) into spatial proximity, which constitutes a ‘preprogrammed’ state predisposed for transcriptional activation. Right, recall-stimulated CD8+ TCM cells downregulate Tcf1, but TCR-mobilized transcription factors, such as those in the AP-1, NFAT and NF-κB families, increase chromatin accessibility of the poised regulatory elements to become active enhancers (red flags), and hence induce the poised effector genes with higher efficacy. The induced Id3 can further boost activation of glycolytic genes in recall-stimulated CD8+ TCM cells to ensure bioenergetic supplies.
Extended Data Fig. 6 Integrative analysis of Tcf1 occupancy and chromatin accessibility at recall-induced genes in CD8+ TCM cells.
Sequencing tracks at select glycolytic genes (a) and Irf8 (b) as displayed on the UCSC genome browser, with gene structure and transcription orientation marked on the top. The tracks included Tcf1 ChIP-seq in WT CD8+ TCM cells and Tcf1-deficient naïve CD8+ T cells, ATAC-seq in resting WT and Tcf7ΔGzmb, and GP33-stimulated WT and Tcf7ΔGzmb CD8+ TCM cells, where black bars on the top denoted high-confidence Tcf1-binding sites and those at the bottom denoted WT-prepotent (that is, Tcf1-dependent, recall-‘opened’) ChrAcc sites with corresponding regions highlighted with yellow vertical bars.
Extended Data Fig. 7 Effect of ectopic expression of select factors on recall responses by CD8+ TCM cells.
a–b. Analysis of cell surface markers (a) and Id3 expression (b) in CD8+ TCM cells at resting state or after 24-h GP33 stimulation. Note that recall-induced CD25 and CD69 proteins (right panel in a) better demarcated CD8+ TCM cell activation than recall-repressed CD62L and IL-7Rα proteins (left panels in a). Sell and Il7r genes were greatly diminished in WT CD8+ TCM cells after 24-hr GP33 stimulation, as detected among the 2,998 genes in Fig. 3b, but their encoded proteins, CD62L and IL-7Rα, remained highly expressed at this time point (left panels), likely due to longer protein half-lives. Data are representative from 3 independent experiments with similar results. c. Experimental design for ‘rescue’ studies by gene delivery into BM progenitors. mCherry- or hNGFR-expressing retroviruses were used for gene delivery into CD45.2+ BM progenitors, which were transplanted into irradiatied CD45.1+ wild-type hosts to generate BM chimeras. mCherry+ (hNGFR+) naïve CD8+ T cells were then sorted from the BM chimeras to generate TCM cells, followed by in vivo and ex vivo analyses. d. Analysis of cell surface markers in mCherry+ (hNGFR+) CD45.2+CD8+ T cells in the spleens of the BM chimeras reconstituted with BM progenitors infected with mCherry (hNGFR)-expressing retroviruses, where the frequency of CD62L+CD44med-lo naïve phenotype cells is shown in representative contour plots from 2 independent experiments. e. Detection of glycolytic gene expression in mCherry+hNGFR+CD8+ TCM cells with RT-PCR at resting state, where the transcripts of each gene were first normalized to Hprt in each cell type, and further normalized to resting WT CD8+ TCM cells to calculate the relative expression of the gene in all cell types. Data are mean ± s.d. (n = 4) from 2 independent experiments. f. Detection of CD62L+CD8+ TCM cell formation in recipient spleens at ≥30 days after CD45.2+ mCherry+ (hNGFR+) naïve P14 CD8+ T cells were sort-purified from the BM chimeras, transferred into CD45.1+ wild-type recipients followed by LCMV infection the next day. The frequency of CD62L+ CD8+ TCM cells is shown in representative contour plots (left) from 2 independent experiments, and cumulative data on CD8+ TCM cell frequency (right) are means ± s.d. g. Experimental design for ‘rescue’ studies by gene delivery into CD8+ T cells primed in vivo. WT or Tcf7ΔGzmb P14 transgenic mice were i.v. infected with LCMV-Arm to prime P14 CD8+ T cells, which were then infected with mCherry-expressing retroviruses. The retrovirus-transduced cells were transferred into primary recipients to generated CD8+ TCM cells, and the mCherry+ CD8+ TCM cells were then sort-purified for in vivo analyses in secondary Tcra–/– recipients. h. Enumerating CD45.2+mCherry+CD8+ TCM cells in the recipient spleens at ≥day 25 after EV- or Id3-mCherry retrovirus-transduced WT or Tcf7ΔGzmb P14 CD8+ T cells were transferred into CD45.1+ wild-type recipients, followed by LCMV-Arm infection the next day. Data are means ± s.d. from 2 independent experiments. Statistical significance for multiple group comparisons in e, f, and h was first determined with one-way ANOVA for multi-group comparison, and Tukey’s test was used as post hoc correction for indicated pairwise comparisons. ns, not statistically significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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Shan, Q., Hu, S.S., Zhu, S. et al. Tcf1 preprograms the mobilization of glycolysis in central memory CD8+ T cells during recall responses. Nat Immunol 23, 386–398 (2022). https://doi.org/10.1038/s41590-022-01131-3
Nature Immunology (2022)