Abstract
T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8+ T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.
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Acknowledgements
We thank S.A. Rosenberg, K. Hanada, K. Hirahara, K. Mousavi, H. Zare, V. Sartorelli, N. Van Panhuys, S. Kerkar and A. Restifo for ideas and discussion, A. Mixon and S. Farid for expertise with cell sorting, members of the NHLBI sequencing core facility for help with sequencing, L. Samsel for help with ImageStream imaging flow cytometry and G. McMullen for expertise with mouse handling. Supported by the Intramural Research Programs of the NCI and NHLBI, Wellcome Trust/Royal Society grant 105663/Z/14/Z (R.R.) and UK Biotechnology and Biological Sciences Research Council grant BB/N007794/1 (R.R. and K.O.).
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R.R., D.C. and N.P.R. wrote the manuscript and designed experiments. R.R., D.C., K.M.Q., Y.J., Z.Y., J.H.P., Y.K., Y.W., L.G. and G.F. performed experiments. P.L. and R.R. analyzed bioinformatic data. C.A.K., D.C.P., D.C.M., M.S., S.J.P., H.-Y.S., R.S., A.M., L.G., R.L.E., J.Z., K.O., J.J.O., K.I. and W.J.L. edited the manuscript.
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Supplementary Figure 1 Cell-intrinsic function of BACH2 in CD8+ T cells.
a-c, Mice were reconstituted with mixtures of congenically distinct Lin− WT and KO bone marrow cells (BM) (a) and CD44 and CD62L expression was measured on the surface of WT and KO CD4+ and CD8+ T cells 2 months following reconstitution (b). Replicate measurements (c) of the proportions of naïve (NAI; CD44+ CD62L+), central memory (CM; CD44+ CD62L+) and effector (EFF; CD44+ CD62L−) cells are shown. d, Experimental schema. Congenically distinct naïve WT Ly5.1+ and KO Thy-1.1+ OT-I TCR-transgenic CD8+ T cells were sorted from mixed BM chimeric mice reconstituted with WT and KO OT-I BM. Cells were mixed at ~1:1 ratios and transferred into naïve C57BL/6 recipient mice prior to infection with VV-OVA (2x10-6 pfu) and kinetic analysis. Numbers in gates indicate percentages. Bars and error (c) represent mean and s.e.m. **P<0.01. Data are representative of 3 independently repeated experiments.
Supplementary Figure 2 Phenotypic analysis of WT and Bach2−/– CD8+ T cells responding to viral infection.
a, Absolute number of KLRG1+ WT and KO CD8+ T cells at indicated timepoints following mixed transfer of naïve sorted WT and KO OT-I CD8+ T cells into recipient mice and infection with VV-OVA. b, Expression of CD127 on the surface of gated CD62L− KLRG1− OT-I CD8+ T cells at day 7 following infection with VV-OVA. Representative flow cytometry (left) and replicate measurements (right) are shown. c-d, Expression of CD43 (c) and CD27 (d) on the surface of WT and KO cells at day 7 post-infection. Representative flow cytometry (left) and replicate measurements (right) are shown. Bars and error (b-d) mean and s.e.m. *P<0.05; **P<0.01; ****P<0.001.
Supplementary Figure 3 Increased activation of Bach2−/– cells is antigen dose dependent and proliferation independent.
a, CD44 and CD62L expression 4 days after stimulation of naïve WT and KO OT-I CD8+ T cells with indicated concentrations of cognate peptide ligand (SL9) in the presence of congenically distinct feeder splenocytes. Numbers in gates indicate percentages. Data are representative of 2 independently repeated experiments. Naïve cells for assays were isolated by flow cytometric sorting. b, CFSE dilution and CD62L expression amongst CFSE-labelled WT and KO naïve CD8+ T cells stimulated in vitro for 3 days. c, Expression of CD69 and CD25 at day 4 following in vitro stimulation of naïve WT and KO CD8+ T cells with platebound anti-CD3 and anti-CD28 in the presence of IL-2. d, CFSE dilution and Annexin V staining on CFSE-labelled WT and KO naïve CD8+ T cells stimulated in vitro for 3 days with platebound anti-CD3 and anti-CD28 antibodies in the presence of 100 IU IL-2. Numbers in gates indicate percentages. e, Naïve WT and KO CD8+ T cells were stimulated with anti-CD3 and anti-CD28 antibodies in the presence of 100IU IL-2 and harvested for analysis at day 4. Expression of the indicated proteins was measured by SDS-PAGE and western blotting. Data are representative of 2 independently repeated experiments.
Supplementary Figure 4 Analysis of global transcriptional differences between WT and Bach2−/– CD8+ T cells at day 7 after infection.
a, Hierarchical clustering analysis of differentially expressed genes (p<0.05; log2 fold-change>1) between WT and KO OT-I cells isolated ex vivo 7 days following VV-OVA infection. Color scale shows FPKM values normalized to row maxima as indicated. b, Geneset enrichment analysis of the indicated geneset in global transcriptional differences between KO and WT cells. Statistical significance was tested using a weighted Kolmogorov–Smirnov test. FPKM values from 3 replicate RNA-Seq measurements per experimental condition are shown. c, Hierarchical clustering analysis of differentially expressed genes (p<0.05; log2 fold-change>1) between fractionated WT and KO CD62L− KLRG1− effector CD8+ T cells isolated ex vivo at day 7 following VV-OVA infection. Color scale shows average FPKM values normalized to row maxima as indicated. FPKM values from 2 replicate RNA-Seq measurements per experimental condition are shown.
Supplementary Figure 5 Analysis of JunD and BACH2 binding in d5 in vitro–activated CD8+ T cells.
a, Naïve CD8+ T cells were isolated by flow cytometric sorting from spleens of wildtype animals and stimulated with plate-bound anti-CD3 and anti-CD28 antibodies in the presence of IL-2 for 2 days followed by 3 further days of culture to generate d5 in vitro activated CD8+ T cells. Protein expression was measured at indicated timepoints following stimulation using SDS-PAGE and immunoblotting. Quantification of BACH2 abundance normalized to β-actin is shown. BACH2 expression in day 5 in vitro activated CD8+ T cells is ~25% of that in naïve cells. b, Histogram of JunD binding, centered around JunD peaks, in WT and Bach2 KO CD8+ T cells at sites bound exclusively by JunD (left histogram) or sites at which JunD and BACH2 binding sites colocalize (right histogram). A significant increase in JunD binding is observed in Bach2-deficient cells only at sites where BACH2 and JunD binding sites colocalize. c-d, Analysis of exclusive JunD binding in Bach2-deficient cells. Pie chart showing JunD binding sites in Bach2-deficient cells (c). A majority of sites (blue) were found to be shared with WT cells, while a minority of sites (red) were only present in Bach2 KO cells. Alignments of JunD binding in WT and Bach2 KO cells at loci where exclusive JunD binding in Bach2 KO cells is detected (d). Arrows indicate loci at which JunD peaks only pass a low significance threshold for peak calling in Bach2-deficient cells (p<1x10-3; Binomial test).
Supplementary Figure 6 Effect of BACH2 on chromatin accessibility and occupancy of Jun family AP-1 factors.
a, Differences in JunD binding in WT and KO d5 in vitro activated CD8+ T cells do not correlate with differences in chromatin accessibility. Scatterplot comparing differences in JunD binding (y-axis) with differences in ATAC-Seq signal around BACH2 binding sites in WT and Bach2−/– d5 in vitro activated CD8+ T cells. Statistical significance was evaluated using the two-sample Kolmogorov-Smirnov test. b, Effect of BACH2 on binding of c-Jun, JunB and JunD. Enrichment of BACH2, c-Jun, JunB and JunD at indicated loci in d5 in vitro activated KO and WT CD8+ T cells relative to input. Enrichment values are normalized to WT cells. Bars and error (b) represent mean and s.e.m. of replicate measurements. *P<0.05; **P<0.01; ***P<0.005; ****P<0.001 (Student’s t-test).
Supplementary Figure 7 Expression of Bach2 mRNA and the encoded protein within CD8+ T cells isolated ex vivo after viral infection.
a-b, Naïve CD45.2+ OT-1 CD8+ T cells were isolated by flow cytometric sorting and pelleted for analysis or transferred into congenically distinct CD45.1+ hosts. Recipient mice were infected with rVV-OVA and Ly5.2+ CD8+ T cells were sorted by flow cytometric sorting at the indicated timepoints following infection. Bach2 mRNA was measured in isolated cells relative to Actb (a) or the abundance of BACH2 and β-actin was assessed in whole cell lysates by western blot (b). Data are representative of two independently repeated experiments (a). Bars and error (a) represent mean and s.e.m. **P<0.01; ***P<0.005; ****P<0.001.
Supplementary Figure 8 Suppression of effector differentiation by pharmacological inhibition of Akt is partially dependent on BACH2.
a-b, Naïve CD44− CD62L+ WT and KO CD8+ T cells (a) were stimulated in the presence of 100IU rhIL-2 in the presence or absence of 1μM AKTi. Expression of IFN-γ and TNF was measured 4 days after stimulation by intracellular cytokine staining. Numbers in gates indicate percentages. Bars and error represent mean and s.e.m. ***P<0.005; ****P<0.001. Data (a-b) are representative of 2 independently repeated experiments.
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Roychoudhuri, R., Clever, D., Li, P. et al. BACH2 regulates CD8+ T cell differentiation by controlling access of AP-1 factors to enhancers. Nat Immunol 17, 851–860 (2016). https://doi.org/10.1038/ni.3441
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DOI: https://doi.org/10.1038/ni.3441
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