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The transcription factor Runx3 guards cytotoxic CD8+ effector T cells against deviation towards follicular helper T cell lineage

Abstract

Activated CD8+ T cells differentiate into cytotoxic effector (TEFF) cells that eliminate target cells. How TEFF cell identity is established and maintained is not fully understood. We found that Runx3 deficiency limited clonal expansion and impaired upregulation of cytotoxic molecules in TEFF cells. Runx3-deficient CD8+ TEFF cells aberrantly upregulated genes characteristic of follicular helper T (TFH) cell lineage, including Bcl6, Tcf7 and Cxcr5. Mechanistically, the Runx3-CBFβ transcription factor complex deployed H3K27me3 to Bcl6 and Tcf7 genes to suppress the TFH program. Ablating Tcf7 in Runx3-deficient CD8+ TEFF cells prevented the upregulation of TFH genes and ameliorated their defective induction of cytotoxic genes. As such, Runx3-mediated Tcf7 repression coordinately enforced acquisition of cytotoxic functions and protected the cytotoxic lineage integrity by preventing TFH-lineage deviation.

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Figure 1: Runx3 is essential for clonal expansion and cytotoxic functions in CD8+ TEFF cells.
Figure 2: Runx3 activates the cytotoxic program in CD8+ TEFF cells.
Figure 3: Runx3 represses TFH-associated genes in CD8+ TEFF cells.
Figure 4: Runx3-deficient CD8+ TEFF cells provide B cell help.
Figure 5: The Runx-CBF complex deploys H3K27me3 to repress TFH genes during CD8+ TEFF cell differentiation.
Figure 6: The Runx-CBF complex acts on promoters and enhancers to activate the cytotoxic program during CD8+ TEFF cell differentiation.
Figure 7: Runx3 represses Tcf1 expression to prevent activation of the TFH program and to assist activation of the cytotoxic program in CD8+ TEFF cells.

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Acknowledgements

We thank J. Fishbaugh, H. Vignes and M. Shey (University of Iowa Flow Cytometry Core Facility) for cell sorting, J. Bair and E. Snir (Genomics Division, Iowa Institute of Human Genetics) for ChIP-Seq, J. Shao (University of Iowa Central Microscopy Research Facility) for assistance with immunofluorescence staining, I. Antoshechkin (California Institute of Technology) for RNA-Seq, S. Crotty (La Jolla Institute) for sharing the protein immunization protocol, T.J. Waldschmidt for helpful discussions on B cell responses, W. Chen (Beijing Institute of Genomics) for help with RNA-Seq data analysis, and Y. Groner (Weizmann Institute of Science, Israel) for sharing high-throughput data on Runx3. This study was supported by grants from the US National Institutes of Health (NIH; AI112579 and AI115149 to H.-H.X., AI119160 to H.-H.X. and V.P.B., AI042767 to J.T.H., AI114543 and GM113961 to V.P.B., AI121080 to H.-H.X. and W.P., and AI113806 to W.P.) and the US Department of Veteran Affairs (I01 BX002903 to H.-H.X.). The flow cytometry core facility at the University of Iowa is supported by the Carver College of Medicine, Holden Comprehensive Cancer Center, the Iowa City Veteran's Administration Medical Center, and by grants from the National Cancer Institute (P30CA086862) and the National Center for Research Resources of the NIH (S10 OD016199). M.D.M. is supported by a T32 Post-doctoral Training Grant (4T32AI007260-30). J.A.G. is a recipient of the Ballard and Seashore Dissertation Fellowship.

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Contributions

Q.S. performed most of the experiments with the help of S.X., F.L., S.M.H., J.A.G., S.P.K., N.V.B.-B., Y.S. and M.D.M. Z.Z. analyzed the high-throughput data under the supervision of W.P. S.M.V., I.T., J.T.H. and V.P.B. contributed critical reagents and provided scientific insights. H.-H.X. conceived the project and supervised the overall study.

Corresponding authors

Correspondence to Weiqun Peng or Hai-Hui Xue.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Specific deletion of Runx3 in mature T cells bypasses the requirement in thymic CD8+ T cell development.

a) hCD2-Cre-mediated inactivation of Runx3 has no detectable impact on T cell development. Thymocytes were isolated from Runx3−/− and littermate controls and surface-stained for CD4 and CD8 expression. Shown are total thymic cellularity (left) and numbers of CD4+ and CD8+ single positive (SP) thymocytes (right). Data are means ± S.D. from two experiments (n ≥ 7). ns, not statistically significant, by Student’s t-test.b) hCD2-Cre specifically targets floxed genes in T cells but not NK cells. Spleens and lymph nodes from hCD2-Cre+Rosa26GFP mice were isolated and surface-stained. GFP expression, which marks Cre-mediated deletion of floxed alleles, was detected in TCRβ+CD8+ T cells (right) and TCRβNK1.1+DX5+ NK cells (left). The percentage of GFP+ cells is shown in representative histograms in LNs, with similar results obtained from splenic cells (n = 2).c) hCD2-Cre effectively ablates Runx3 protein in CD8+ T cells. Surface-stained splenocytes from Runx3−/− and littermate controls were intracellularly stained with an anti-Runx3 antibody (solid lines) or isotype control (shaded areas). Data are representative from 2 experiments with similar results (n = 2).d-e) hCD2-mediated Runx3 deletion modestly diminishes CD8+ T cell pool without causing aberrant activation. Splenocytes were isolated from Runx3−/− and littermate controls and surface-stained. GFP+TCRβ+ cells were analyzed for distribution of CD4+ and CD8+ T cells, which were further analyzed for naïve (CD62L+CD44lo), effector-phenotype (CD62LCD44hi) and memory-phenotype (CD62L+CD44hi) cells. Data in e are representative from 2 experiments (n = 3). Note that inactivation of Runx3 in thymocytes causes partial derepression of the CD4 coreceptor in CD8 + T cells (Egawa et al, J. Exp. Med. 206, 51, 2009); in contrast, late deletion of Runx3 in peripheral T cells did not show this effect, consistent with the idea that the Cd4 silencing is epigenetically maintained in mature CD8 + T cells, independent of the Cd4 silencer and hence the silencer-associated protein factors (Zou et al. Nat. Genet. 29, 332, 2001).

Supplementary Figure 2 Runx3 has a predominant role in regulating CD8+ T cell responses to acute viral and bacterial infections.

a-b) CBFβ and Runx3 deficiency causes remarkably similar impairment of CD8 + T cell responses to acute viral infection. a) hCD2-Cre-mediated deletion of CBFβ protein in CD8+ T cells. hCD2-Cre+Rosa26GFPCbfbfl/fl (called Cbfb−/−) mice were generated. GFP+CD8+ T cells were sorted from the spleens of control P14, Cbfb−/− P14 or Runx3−/− P14 TCR transgenic mice, and immunoblotted for CBFβ or β-actin.b) CBFβ deficiency profoundly impaired magnitude and functionality of CD8+ T cell responses to acute viral infection. Cbfb−/− or littermate controls were infected with LCMV-Arm. On 8 dpi, splenocytes were harvested, and the numbers of total (top) or antigen-specific Thy1.2+CD8+ effector T cells were determined by either GP33 tetramer (middle) or GP33 peptide-stimulated IFN-γ production (bottom). Data are means ± S.D. from 3 independent experiments (n = 5). *, p<0.05; **, p<0.01; ***, p<0.001 (Student’s t-test). Note that data above (CBFβ deficiency) and those in Fig. 1a (Runx3 deficiency) showed remarkable similarity in profoundly impairing CD8 + T cell responses. These data suggest that the Runx3-CBFβ complex has a predominant role in regulating CD8 + effector differentiation and may not strongly involve Runx1-CBFb. c-e) Runx3 deficiency profoundly affects magnitude and functionality of CD8+ T cell responses to bacterial infection. Runx3−/− and littermate controls were infected with attenuated Listeria monocytogenes expressing both the Ovalbumin 257-264 (OVA257) and GP33 epitopes (LM-OVA-GP33), and seven days later, total, OVA-specific and GP33-specific CD8+ T cells were characterized.c) Runx3 deficiency diminishes CD8+ effector T cell expansion. Splenocytes were collected from the infected mice and surface-stained. The frequency of Thy1.2+CD8+ T cells is shown in representative contour plots (left), and cumulative data on the numbers of total CD8+ T cells are summarized in bar graphs (right).d) Runx3 deficiency diminishes expansion of OVA- or GP33-specific effector CD8+ T cells. GFP+Thy1.2+CD8+ splenic T cells were stained with OVA- (top) or GP33-specific (bottom) tetramers. The frequency of OVA- or GP33-specific CD8+ T cells is shown in representative contour plots (left), and cumulative data on the numbers of antigen-specific CD8+ T cells are in bar graphs (right).e) Runx3 deficiency impairs IFN-g production stimulated by OVA or GP33 peptide. Splenocytes from infected mice were stimulated with OVA (top) or GP33 peptide (bottom), and 5 hrs later, Thy1.2+CD8+ T cells were intracellularly stained for IFN-γ. The frequency of IFN-g-producing CD8+ T cells is shown in representative contour plots (left), and cumulative data on the numbers of IFN-γ+CD8+ T cells are in bar graphs (right). Data are from two experiments measuring four replicates. *, p<0.05; **, p<0.01; and ***, p<0.001 by Student’s t-test.In all panels, the fold changes between Cbfb−/− and littermate controls and those between Runx3−/− and controls are also marked.

Supplementary Figure 3 Runx3 controls survival and a broad transcriptional program of CD8+ effector T cells

a) Runx3 supports the survival of CD8+ effector T cells. Runx3−/− or control P14 CD8+ T cells were adoptively transferred into congenic recipients followed by LCMV-Arm infection. On days 4, 6 and 8 post-infection, splenocytes were harvested, and caspase-3/7 activation was determined in donor-derived CD45.2+ CD8+ effector T cells. Representative data are from two experiments, and cumulative data are summarized in bar graphs (n = 5). ***, p<0.001 (Student’s t-test).b-c) Analysis of Runx3-deficient CD8+ effector T cells in vivo reveals key differences from what has been observed in vitro. Conditionally targeted Runx3−/− or control CD8+ effector T cells were isolated from LCMV-infected recipients on 4 dpi and used for RNA-Seq analysis. Differentially expressed genes were compared with those identified in Ref. 22, where germline-targeted Runx3-deficient CD8+ T cells were activated in vitro with anti-CD3 and anti-CD28 in the presence of IL-2, and the resulting cells were analyzed with microarray approach. Venn diagrams show the overlap of downregulated (b) or upregulated (c) genes identified between the two sets of transcriptomic data. The commonly identified genes are listed in alphabetically order.

Supplementary Figure 4 Runx3 restrains activation of the TFH programs in CD8+ effector T cells by directly suppressing Tcf1

a) Runx3 deficiency activates the TFH program in CD8+ effector T cells elicited by bacterial infection in a Tcf1-dependent manner. CD45.2+ control, Runx3−/−, Tcf7−/−, Runx3−/−Tcf7−/− naïve P14 CD8+ T cells were adoptively transferred into CD45.1+ congenic mice and then infected with LM-GP33. On 7 dpi, CD45.2+ CD8+ effectors in the recipient spleens were enumerated and surface-stained for CXCR5, SLAM, and ICOS expression. Representative contour plots (left) and cumulative numbers of CD8+ effector T cells (right) are from 2 independent experiments (n = 4-5).b) Heatmap of TFH-associated genes enriched in Runx3−/− CD8+ effector T cells. Our previous study compared TFH vs TH1 cells generated in response to viral infection and identified 350 genes that were expressed ≥ 2 fold higher in TFH than TH1 cells (Ref. 23). GSEA revealed that 151 genes in this gene set were enriched in Runx3−/− CD8+ effector T cells, and their relative expression is shown in a heatmap (generated from GSEA).c) Runx3 deficiency activates the TFH program in CD8+ effector T cells elicited by chronic viral infection in a Tcf1-dependent manner. CD45.2+ control, Runx3−/−, Tcf7−/−, Runx3−/−Tcf7−/− naïve P14 CD8+ T cells were adoptively transferred into CD45.1+ congenic mice and then infected with LCMV-Cl13. On 8 dpi, CD45.2+ CD8+ effector T cells in the recipient spleens were enumerated and surface-stained for CXCR5, SLAM, and ICOS expression. Representative contour plots (left) and cumulative numbers of CD8+ effector T cells (right) are from 2 independent experiments (n = 4-5). ns, not statistically significant; **, p<0.01; ***, p<0.001 for indicated comparisons (one-ANOVA followed by Bonferroni’s test).d) Localization of CD8+ effector T cells generated in response to chronic viral infection. On day 8 after LCMV-Cl13 infection, spleen sections were subjected to immunofluorescence staining for B220 (red) and congenically marked P14 (CD45.2+, green) cells. White dotted lines denote the boundary of B cell follicles and T cell zone. Scale bar, 50 μm. Lower panels are a 9-fold enlargement of highlighted area outlined in the main image. For quantification, the frequency of CD45.2+ cells in B cell follicles among all CD45.2+ cells in the scanned image was determined. Each dot in the dot plot denotes one recipient mouse. Data are from 2 independent experiments. **, p<0.01 by Student’s t-test.e) Runx3-deficient CD8+ effector T cells help antibody production upon protein immunization. CD45.2+ control or Runx3−/− P14 CD8+ T cells (1 × 105 each) were sort-purified and adoptively transferred into CD45.1+Bcl6−/− recipient mice, followed by immunization with KLH-GP33. Bcl6−/− mice that were not transferred with P14 cells but immunized were used as negative controls. On day 21 post-immunization, sera were collected and measured for KLH-specific total IgG at indicated dilutions using ELISA. ns, not statistically significant; *, p<0.05; **, p<0.01 for indicated comparisons (one-ANOVA followed by Bonferroni’s test). Note that ablating Tcf1 improved clonal expansion of Runx3 −/− CD8 + effector T cells in response to bacterial infection ( a ), similar to what was observed in acute viral infection ( Fig. 7c ). This beneficial effect was, however, not observed in the context of chronic viral infection ( e ) or protein immunization ( Supplementary Fig. S7a ). This is consistent with a requirement for Tcf1 in supporting the self-renewing CXCR5 + subset of exhausted CD8 + T cells in chronic infection (Ref. 27, 28), and also suggests that antigen persistence may program activated CD8 + T cells differently. f) Direct comparison of key TFH molecules between the TFH-like Runx3-deficient CD8+ T cells and WT CD4+ TFH cells. CD45.2+ WT SMARTA CD4+ and Runx3−/− P14 CD8+ T cells (1 × 105 each) were both adoptively transferred into CD45.1+ recipients, following by immunization with both GP33-KLH and GP61-KLH. Another cohort of recipients of both cell types were immunized with either GP33-KLH or GP61-KLH alone to confirm that SMARTA CD4+ T cells were only activated by GP61-KLH and Runx3−/− P14 CD8+ T cells were only activated by GP33-KLH (not shown). On day 7 post-immunization, LN cells were analyzed for TFH molecules by surface and intracellular staining. Cumulative data of indicated MFIs in all CXCR5+ cells are shown as means ± s.d. in bar graphs (n = 3). Source data

Supplementary Figure 5 Characterization of genome-wide CBFβ occupancy in CD8+ effector T cells reveals a role of Runx3-CBFβ in deploying H3K27me3 mark to repress its target genes

WT naïve P14 CD8+ T cells were adoptively transferred into congenic mice and then infected with LCMV-Arm. On 8 dpi, KLRG1hiIL-7Rαlo CD8+ effector T cells were sort-purified and used in CBFβ ChIP-Seq. Naïve CD8+ T cells from Cbfb−/− mice were used as a negative control. CBFβ binding peaks were identified using a setting of 4-fold enrichment (over the negative control), p<1×10–5, and FDR < 0.05.a) Distribution of CBFβ binding peaks in CD8+ effector T cells. Promoter region is defined as – 1 kb to + 1 kb flanking transcription start site (TSS); gene body refers to + 1 kb downstream of TSS to transcription end site (TES), and the remainder is considered as intergenic region.b) Comparison of CBFβ and Runx3 binding peaks in Runx3−/− CD8+ effector T cells. Runx3 ChIP-Seq data were retrieved from Ref. 22, and Runx3 binding peaks were re-analyzed using the same criteria used in analysis of CBFβ ChIP-Seq data. This re-analysis identified 7,907 high-confidence Runx3 binding peaks in the in vitro activated CD8+ T cells. Note that about 40% of the Runx3 peaks identified overlapped with the CBFβ peaks. The unique Runx3 and CBFβ peaks are likely due to differences in CD8 + T cell activation conditions and/or antibody avidity, or due to additional co-factors interacting with Runx3 or CBFβ. c) De novo motif analysis of CBFβ binding peaks in CD8+ effector T cells. CBFβ binding peaks were divided into two groups, 1) overlapping with the promoter regions and 2) overlapping with enhancers (defined by “H3K4me1hi + H3K4me3neg/lo”) within 5 kb of TSS. The motifs in each group were analyzed. Shown are motif logos, statistical significance, and corresponding transcription factor (TF) family. Note that SP1 and Ets motifs were also enriched in CBFβ binding peaks, suggesting possible cooperative roles of the Runx3-CBFβ complex with SP1 and Ets factors in gene regulation. d) Histone modification profiles of CBFβ peaks that are associated with upregulated genes in Runx3−/− CD8+ effector T cells. CBFβ peaks were identified within the “–5 kb to TES” regions of 422 upregulated genes in Runx3−/− CD8+ effector T cells (overlap) or at the rest of the genome outside this region (non-overlap). The profiles of H3K27me3 (left) and H3K4me3 (right) were analyzed in each group of CBFβ peaks in both WT and Runx3−/− CD8+ effector T cells.e) ChIP-Seq tracks of CBFβ binding and histone modifications at the Bcl2l11 gene. The gene structure and transcriptional orientation are shown on the top along with a genomic scale. CBFβ ChIP-Seq tracks in CD8+ effector T cells (raw data) are displayed next, and the vertical bars on top of the track denote the MACS-called high-confidence CBFβ peaks. Shown at the bottom are the histone mark ChIP-Seq tracks (island-filtered) in WT or Runx3−/− CD8+ effector T cells. A green rectangle marks the TSS, and an orange rectangle highlights the widespread changes in H3K27me3 signals in Runx3−/− CD8+ effector T cells.

Supplementary Figure 6 Runx3 acts on promoters and active enhancers to activate the cytotoxic program in CD8+ effector T cells

a-c) ChIP-Seq tracks of CBFβ binding and histone modifications at the Ifng (a), Fasl (b), and Gzma (c) genes. The gene structure and transcriptional orientation are shown on the top along with a genomic scale. CBFβ ChIP-Seq tracks in CD8+ effector T cells (raw data) are displayed next, and the vertical bars on top of the track denote the MACS-called high-confidence CBFβ peaks. Histone mark ChIP-Seq tracks (island-filtered) in WT or Runx3−/− CD8+ effector T cells are shown in the bottom tracks, where blue and red bars above the H3K4me1 tracks denote active enhancers in WT or Runx3−/− CD8+ effector T cells, respectively.Green open rectangles mark the TSS. The cyan or purple filled rectangles highlight active enhancers in WT CD8+ effector T cells, with cyan-marked ones remaining active or purple-marked ones lost enhancer activity in Runx3−/− CD8+ effector T cells.Note that Ifng was not identified as a differentially expressed gene on RNA-Seq. The Runx-CBF complex nonetheless binds to its promoter and active enhancers in WT CD8+ effector T cells ( a ). This is consistent with impaired IFN-γ production in Runx3−/− CD8+ effector T cells observed on 8 dpi ( Fig. 1d ).d) Clustering analysis of CBFβ-bound active enhancers in WT and Runx3−/− CD8+ effector T cells. Active enhancers that overlap with CBFβ binding peaks within the +/– 50 kb region flanking the gene body of Runx/CBF-activated genes were identified and assessed for the presence (colored) or absence (no color) of indicated histone marks, and clustered for consensus patterns. Genes (more than 1 in some cases) associated with each enhancer are marked. Note that whereas most of active enhancers in CD8 + effector T cells remained active in Runx3 −/− CD8 + effector T cells, some (in 3 clusters to the left) lost activity, due to loss of H3K27Ac and/or gain of H3K27me3. The presence of both H3K27 histone marks is likely because of the heterogeneity of Runx3 −/− CD8 + effector T cells, which for example, contain both CXCR5 + and CXCR5 subsets.

Supplementary Figure 7 Runx3 functions as a ‘built-in’ pathway to prevent activation of the TFH program in CD8+ effector T cells

a) Ablating Tcf1 diminishes activation of TFH program in response to protein immunization. CD45.2+ control, Runx3−/−, Tcf7−/−, Runx3−/−Tcf7−/− naïve P14 CD8+ T cells were adoptively transferred into CD45.1+ congenic mice and then immunized with KLH-GP33. On 7 dpi, CD45.2+ CD8+ effector T cells in the recipient spleens were enumerated and surface-stained for CXCR5, SLAM, and ICOS expression. Representative contour plots (left) and cumulative number of CD8+ effector T cells (right) are from 2 independent experiments (n = 4-5). ns, not statistically significant; ***, p<0.001 for indicated comparisons (one-ANOVA followed by Bonferroni’s test).b) Schematics showing intricate interplay between Runx3 and other key transcription factors in programming CD8+ effector T cell differentiation. TCR stimulation leads to CD8+ T cell activation, which may prime the T-bet, Eomes, and Blimp-1 gene loci (dotted lines) for further induction by cytokine signals such as IL-12, type I interferons, and IL-2 depending on the infection types. Runx3, on the other hand, is an inherited factor from CD8+ T cell lineage choice and further maturation in the thymus, but is only minimally induced by TCR or cytokines. This work identifies Runx3 as a distinct pathway that may cooperate with T-bet and Eomes and partly upstream of Blimp-1 to promote CD8+ effector T cell differentiation, including clonal expansion and acquisition of cytotoxic effector molecules.The cytotoxic lineage integrity of CD8+ effector T cells is also guarded by key transcription factors. T-bet, in combination with Eomes or Blimp, are induced to prevent deviation to TH17 lineage. Runx3 is ‘hardwired’ in CD8+ T cells to represses activation of the TFH program, utilizing a Tcf1-dependent mechanism. Importantly, Runx3-mediated repression of Tcf1 is also important for optimal clonal expansion and full activation of the cytotoxic program in CD8+ effector T cells.Arrows denote positive regulation, and lines ending in bars denote negative regulation. Dotted lines, presumed regulation. *, Blimp-1 cooperates with T-bet to repress the TH17 program in CD8+ effector T cells, because this effect is only evident when Blimp-1 and T-bet expression are both abrogated. Source data

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Runx/CBF direct target genes in CD8+ effector T cells (XLSX 52 kb)

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Shan, Q., Zeng, Z., Xing, S. et al. The transcription factor Runx3 guards cytotoxic CD8+ effector T cells against deviation towards follicular helper T cell lineage. Nat Immunol 18, 931–939 (2017). https://doi.org/10.1038/ni.3773

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