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Bcl-6 is the nexus transcription factor of T follicular helper cells via repressor-of-repressor circuits

Abstract

T follicular helper (TFH) cells are a distinct type of CD4+ T cells that are essential for most antibody and B lymphocyte responses. TFH cell regulation and dysregulation is involved in a range of diseases. Bcl-6 is the lineage-defining transcription factor of TFH cells and its activity is essential for TFH cell differentiation and function. However, how Bcl-6 controls TFH biology has largely remained unclear, at least in part due to the intrinsic challenges of connecting repressors to gene upregulation in complex cell types with multiple possible differentiation fates. Multiple competing models were tested here by a series of experimental approaches to determine that Bcl-6 exhibits negative autoregulation and controls pleiotropic attributes of TFH differentiation and function, including migration, costimulation, inhibitory receptors and cytokines, via multiple repressor-of-repressor gene circuits.

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Fig. 1: TFH differentiation is not the default pathway.
Fig. 2: Bcl-6 exhibits direct negative autoregulatory feedback.
Fig. 3: A testable simple circuitry model of TFH differentiation.
Fig. 4: Bcl-6 drives CXCR5 expression via repression of Id2-E2A pathway.
Fig. 5: Integrated analysis of multiple genetic backgrounds and data types.
Fig. 6: Identification of candidate TFs.
Fig. 7: Identification of Runx2, Runx3 and GATA-3 as repressors of TFH genes, acting downstream of Bcl-6.
Fig. 8: Identification of Klf2 as a repressor acting downstream of Bcl-6 regulating major TFH genes.

Data availability

RNA-seq and ATAC–seq data were deposited to the Gene Expression Omnibus (GEO) under the GSE140187 super series. Scripts for analysis are available on Github: https://github.com/ScrippsPipkinLab/JYC_DataAnalysis. All other data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

We thank C. Kim, L. Boggeman, D. Hinz and C. Dillingham of the La Jolla Insitute for Immunology (LJI) Flow Cytometry Core Facility for cell sorting; J. Greenbaum and A. Sethi of the LJI Bioinformatics core for bioinformatics analysis; G. Seumois and J. Day of the LJI Sequencing core; and K. Jepsen of the University of California San Diego IGM sequencing core for consultation. We thank MaxCyte for generously providing the MaxCyte ATX electroporator and reagents. This work was funded by grants from the US National Institutes of Health (NIH), including the National Institute of Allergy and Infectious Diseases (NIAID) U19 AI109976 and NIAID R01 AI072543, NIH S10 RR027366 (LJI) and internal LJI institutional funds to S.C.

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Authors

Contributions

J.C. designed and performed experiments, analyzed the data and wrote the manuscript. H.D. and M.E.P analyzed ATAC–seq data. C.E.F. developed CRISPR–Cas9-mediated gene deletion experiments. J.T., M.R. and S.B. performed experiments. B.Y. performed PageRank analysis. A.W.G and M.E.P provided advice, reagents and computational analyses. S.C. supervised the project, designed experiments, analyzed data and wrote the manuscript.

Corresponding author

Correspondence to Shane Crotty.

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The authors declare no competing interests.

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Editor recognition statement 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.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 TFH differentiation is not the default pathway.

Related to Fig. 1. a, Schematic diagram of a mutually antagonistic relationship of Bcl-6 and Blimp-1. UCSC browser tracks from BCL-6 ChIP–seq and Blimp-1 ChIP–seq were indicated below. b, A null hypothesis of a default TFH differentiation pathway model. c, SMARTA CD4 T cells were transferred to C57BL/6 host mice, followed by immunization of the host mice with KLH-gp61 in alum only, alum + LPS, alum + Poly(I:C), or alum + cGAMP adjuvants, and analyzed 7 days later. Representative flow cytometry of TFH and non-TFH SMARTA cell subsets from draining LNs (dLNs) of KLH-gp61 immunized mice. Two independent experiments were performed; each dot represents one mouse (n = 4). Data are mean ± s.d., unpaired two-tailed Student’s t-test. d, Quantification of results from Fig. 1b. e, Schematic of the SMARTA cell transfer system used for LCMVArm infection. WT, Bcl6f/f, Prdm1f/f, and Bcl6f/fPrdm1f/f CreCD4 SMARTA CD4+ T cells were transferred to C57BL/6 host mice, followed by infection of the host mice with LCMVArm, and analyzed 7 days later. f, Representative flow cytometry of GC-TFH, TFH and non-TFH SMARTA cell subsets from spleens of LCMVArm infected mice in Extended Data Fig. 1c. Three independent experiments were performed; each dot represents one mouse (n = 4). Data are mean ± s.d., unpaired two-tailed Student’s t-test. g, Quantification of results from Extended Data Fig. 1f. h, Representative histology section to define GC, B cell zone, T cell zone, and T-B border. T-B border was defined as ±15 μm region at a boundary line of T cell zone and B cell zone. Scale bar, 200 μm. Related to Fig. 1g. i, Histology of draining LNs at d8 after KLH-gp61 immunization in Fig. 1d. Blue, TCRβ; red, GL7; green, IgD; white, CD45.1 SMARTA. SMARTA cells were presented with large dots for clarity. Scale bar, 200 μm. Related to Fig.1g.

Extended Data Fig. 2 Bcl-6 exhibits direct negative autoregulatory feedback.

Related to Fig. 2. a, Sequence homology at BPS1 locus between various species. The numbers indicate region of mouse chromosome 19 (mm10). b, Schematic diagram of self-inactivating (SIN) vectors. pQdT SIN vector were generated from original pQCXIP SIN vector by removing IRES-PuroR cassette and additional deletion of CCAAT box and TATA box. c, Schematic of the RV+ SMARTA cell transfer system used for LCMVArm infection. SMARTA CD4 T cells transduced with WT Thy1.1-RV or ΔBPS1 Thy1.1-RV were adoptively transferred to C57BL/6 host mice, followed by infection of the host mice with LCMVArm, and analyzed 7 days after infection. Related to Fig. 2a-b and Extended Data Fig. 2d-e. d, TFH and TH1 gates used for Fig. 2b and Extended Data Fig. 2e. e, Chromatogram of DNA sequencing from WT or ΔBPS1 mouse. f, Schematic of the SMARTA cell transfer system used for LCMVArm infection. WT or ΔBPS1 SMARTA CD4 T cells were transferred to C57BL/6 host mice, followed by infection of the host mice with LCMVArm, and analyzed 7 days after infection. Related to Fig. 2c-d. g, Quantification of expression of CXCR5, SLAM, and PSGL1 in Fig. 2d, gated on CXCR5hiSLAMlo TFH or CXCR5loSLAMhi TH1 cells. Two independent experiments were performed; each dot represents one mouse (n = 4). Data are mean ± s.d., unpaired two-tailed Student’s t-test. h, Genome-browser tracks depict BCL-6 or NCOR ChIP–Seq peaks at BCL6 locus from OCI-LY1 cell line. Peak calls indicated below each track.

Extended Data Fig. 3 RNA-seq of TFH and non-TFH populations from multiple genetic backgrounds.

Related to Fig. 3 a, Schematic of the SMARTA cell transfer system used for RNA-seq analysis in KLH-gp61 immunization. Non-TFH (CXCR5loPSGL1hi) populations from WT, Bcl6f/f CreCd4, Prdm1f/f CreCD4, or Bcl6f/fPrdm1f/f CreCd4 SMARTA cells and TFH (CXCR5hiPSGL1lo) populations from WT or Prdm1f/f CreCD4 SMARTA cells were sorted from C57BL/6 host mice given WT, Bcl6f/f, Prdm1f/f, or Bcl6f/fPrdm1f/f CreCD4 SMARTA CD4 T cells, followed by infection of the host mice with KLH-gp61 in alum + cGAMP, and analyzed 8 days later. Naive SMARTA cells were isolated as CD44loCD62LhiCD45.1+ from uninfected mice. Representative flow cytometry of TFH and TH1 subsets from four independent experiments. b, Heatmap of gene expression of curated TFH-associated genes. Scale, row z-score. DKO, Bcl6f/fPrdm1f/f CreCD4. As a first analysis the effect of Bcl6/Prdm1 double-deficiency on the TFH biology, we assessed expression of a broad curated set of TFH-associated genes across all samples from RNA-seq gene expression profiling. Bcl6f/fPrdm1f/fCreCD4 TFH-like cells lost expression of positively TFH-associated genes in comparison to WT TFH cells or Prdm1f/fCreCD4 TFH cells. Conversely, Bcl6f/fPrdm1f/fCreCD4 TH1-like cells had a gene expression profile different from WT TH1 or Bcl6f/fCreCD4 TH1 cells. c, Principal component analysis of differential gene expression from RNA-seq of LCMVArm infection. Principal component analysis provided similar findings as (b), supporting the overall hypothesis that TFH is not a default differentiation pathway of CD4+ T cells and Bcl-6 has important activities beyond inhibition of Prdm1. d, Gene expression changes were clustered by k-means clustering analysis (k=10) of LCMVArm infection. e, Hierarchical clustering analysis of genes upregulated or downregulated in TFH cells relative to their expression in TH1 cells (1.4-fold cut off, Adj. P <0.05) of LCMVArm infection shown in Fig. 3c. Scale, row z-score. f, GSEA of BCL-6 bound genes from human tonsillar GC-TFH compared to Cluster 2 genes (left) or Cluster 3 genes (right) differentially expressed between Bcl6f/fPrdm1f/f TFH-like cells and Prdm1f/f TFH cells. NES, normalized enrichment score; FDR, false discovery rate. g, GSEA of Blimp-1 bound genes from CD8 and TH17 cells in comparison of Cluster 2 and Cluster 3 genes differentially expressed between Bcl6f/fPrdm1f/f TH1-like cells and Bcl6f/f TH1 cells.

Extended Data Fig. 4 Bcl-6 drives CXCR5 expression via repression of Id2-E2A pathway.

Related to Fig. 4 a, Quantification of GC-TFH core signature markers in SMARTA cells from dLNs of KLH-gp61 immunized mice in Fig. 4d. b, Schematic of the SMARTA cell transfer system used for LCMVArm infection. WT, Bcl6f/fPrdm1f/f, or Bcl6f/fPrdm1f/fId2f/f CreCD4 SMARTA CD4 T were transferred to C57BL/6 host mice, followed by infection of the host mice with LCMVArm, and analyzed 7 days later. Related to Fig. 4g and Extended Data Fig. 4c-g. c,e, Representative flow cytometry of TFH and GC-TFH SMARTA cell subsets from spleen of LCMVArm infected mice. Two independent experiments performed; each dot represents one mouse (n = 4). Data are mean ± s.d., unpaired two-tailed Student’s t-test. d, Quantification of expression of CXCR5 in Extended Data Fig. 4c, gated on SMARTA cells. f, Quantification of frequency of CXCR5hiPD1hi GC-TFH cells in Extended Data Fig. 4e, gated on SMARTA cells. g, Quantification of GC-TFH core signature markers in Extended Data Fig. 4e, gated on SMARTA cells.

Extended Data Fig. 5 ATAC–seq of TFH and non-TFH populations from multiple genetic backgrounds.

Related to Fig. 5 a, Representative flow cytometry of naive SMARTA cells (CD44loCD62LhiCD45.1+) used for ATAC–seq analysis. b, Genome-browser tracks depict ATAC–seq chromatin accessibility at Cxcr5 locus. Peak calls indicated below each track. * indicates DEseq2 raw p ≤ 0.05 in comparison between WT TFH and TH1. c, Genome-browser tracks depict ATAC–seq chromatin accessibility at Tbx21 and Gata3 loci. Peak calls indicated below each track. Bcl-6 liftover peaks from human to mouse reference genome are indicated. * and ** indicate DEseq2 raw p-val ≤ 0.05 and ≤ 0.01, respectively, in comparison between WT TFH and TH1. Gene expressions from RNA-seq data of LCMVArm infected mice are graphed. DKO, Bcl6f/fPrdm1f/f CreCD4. d, Genome-browser tracks depict BCL-6 ChIP–Seq peaks at SELPLG locus. Peak calls indicated below the track. e, Representative flow cytometry and quantification of the level of Bcl-6 in RV+ TFH or non-TFH SMARTA cell subsets from spleen of LCMVArm infected mice. SMARTA CD4 T cells transduced with GFP-RV or Bcl6-RV were transferred to C57BL/6 host mice, followed by infection of the host mice with LCMVArm, and analyzed 3 days after infection. Non-TFH: GFP-RV+ and TFH: GFP-RV+ indicate the expression level of endogenous Bcl-6 in each population. Each dot represents one mouse (n = 4). Data are mean ± s.d., unpaired two-tailed Student’s t-test. f, Representative flow cytometry and quantification of GC-TFH and BGC subpopulations from spleen of LCMVArm infected mice. Bcl6f/f CreCD4 SMARTA CD4 T cells transduced with Bcl6-RV or myctagN-Bcl6-RV were transferred to Bcl6f/f CreCD4 host mice, followed by infection of the host mice with LCMVArm, and analyzed 8 days after infection. We validated that an myctagN-Bcl6-RV was functionally comparable with non-tagged Bcl6-RV (Bcl6-RV), based on myctagN-Bcl6-RV rescue of TFH differentiation and B cell help function of Bcl6f/f CreCD4 CD4 T cells in LCMV infected mice. Each dot represents one mouse (n = 4). Data are mean ± s.d.

Extended Data Fig. 6 Identification of candidate TFs.

Related to Fig. 6 a, Heatmap plots representing the frequencies of the most enriched TF motifs in regions in decreased accessibility (relatively less open in first group than second group, DEseq2 raw p-val < 0.05). Scale, motif frequencies (%). Related to Fig. 6b. b, Motif analysis of the center of TF footprint (green bar) in Fig. 6c and Extended Data Fig. 6e from ATAC–seq reads. c, Genome-browser tracks depict ATAC–seq chromatin accessibility and TF occupancy at Icos and Cd200 loci. Peak calls indicated below each track. ** indicates DEseq2 raw p-val ≤ 0.01 in comparison between Bcl6f/fPrdm1f/f CreCD4 TFH-like and Prdm1f/f CreCD4 TFH. d, Gene expression of Klf genes from RNA-seq data of LCMVArm infected mice or KLH-gp61 immunized mice. e, TF footprints derived from ATAC–seq reads over representative TF motifs within accessible ATAC–seq regions. f, Heatmap plots of relative PageRank scores. Scale, row z-score.

Extended Data Fig. 7 CRISPR–Cas9-mediated gene knockdown of SMARTA cells.

Related to Fig. 7 a, Schematic of the CRISPR/Cas9-mediated gene knockdown of SMARTA cell system. Details are in the Method section. b, Representative flow cytometry of RNP transfection from more than four independent experiment; each dot represents one RNP+ group (n = 9). Data are mean ± s.d. Quantification of cell viability and transfection efficiency of RNP+ and RNP- (electroporation without RNP) SMARTA cells. c, Frequency of crRNA+ CD45.1+ SMARTA cells among total CD4 T cells from spleen of LCMVArm infected mice in Fig. 7a. Two independent experiments were performed; each dot represents one crRNA+ group (n = 4). Data are mean ± s.d., unpaired two-tailed Student’s t-test. d,e,f,g, WT or Bcl6f/fPrdm1f/f CreCD4 SMARTA CD4 T cells transfected with crCd8, crGata3, crRunx2, crRunx3, and crKlf2 were transferred to C57BL/6 host mice, followed by infection with LCMVArm (e,f,g) or immunization with KLH-gp61 (d) of the host mice, and analyzed 6-7 days later. crRNA+ SMARTA cells were FACS sorted from spleens (LCMVArm infection) or dLN (KLH-gp61 immunization) of host mice. Gene knockdown efficiencies were measured by flow cytometry (d), mRNA qPCR (e), or Western blot analysis (f,g). shRunx3-RV+ SMARTA cells and pMIG-Klf2-RV+ SMARTA cells were used as positive controls. Two independent experiments were performed; Each dot represents one mouse (n = 5). Data are mean ± s.d., unpaired two-tailed Student’s t-test. Details are in the Method section. h, Schematic of the CRISPR/Cas9-mediated gene knockdown of SMARTA cell system used for testing Gata3 in LCMVArm infection. WT or Bcl6f/fPrdm1f/f CreCD4 SMARTA CD4 T cells transfected with crCd8 or crGata3 were transferred to C57BL/6 host mice, followed by infection of the host mice with LCMVArm, and analyzed 6-7 days later. Related to Fig. 7c and Extended Data Fig. 7i,l,m. i,j,k, Frequency of crRNA+ CD45.1+ SMARTA cells among total CD4 T cells from spleen of LCMVArm infected mice in Fig. 7d and Extended Data Fig. 7h. Two (i) or three (j,k) independent experiments were performed; each dot represents one mouse (n = 4). Data are mean ± s.d., unpaired two-tailed Student’s t-test. l, Representative flow cytometry of TFH SMARTA cell subsets from spleen of LCMVArm infected mice in Extended Data Fig. 7h. Two independent experiments were performed; Each dot represents one mouse (n = 4). Data are mean ± s.d., unpaired two-tailed Student’s t-test. m, Quantification of expression of PSGL1 in Extended Data Fig. 7h, gated on SMARTA cells. CD44lo naive CD4 T cells were used as a negative control.

Extended Data Fig. 8 Identification of Runx2, Runx3 and Gata3 as repressors of TFH genes, acting downstream of Bcl-6.

Related to Fig. 7 a, Schematic of the RV+ SMARTA cell transfer system used for LCMVArm infection. WT SMARTA CD4 T cells transduced with pMIG (GFP-RV+), pMIG-Runx3myc (Runx3-RV+), or pMIG-Runx2myc (Runx2-RV+) were transferred to C57BL/6 host mice, followed by infection of the host mice with LCMVArm, and analyzed 7 days after infection. Related to Fig. 7i-j and Extended Data Fig. 7o-u. b, Total GFP+ RV+ SMARTA cells [RV+ high] were FACS sorted from in vitro culture. Whole cell lysates were analyzed by immunoblotting using anti-myc tag and anti-GAPDH antibodies. Relative Runx expressions are indicated. c, SMARTA CD4 T cells transduced with pMIG (GFP-RV+), pMIG-Runx3myc (Runx3-RV+ [High]), or pMIG-Runx2myc (Runx2-RV+ [High]) were transferred to C57BL/6 host mice, followed by infection of the host mice with LCMVArm, and analyzed 7 days later. Frequency of CD45.1+ SMARTA cells among total CD4 T cells from spleen of LCMVArm infected mice. Two independent experiments were performed; each dot represents one mouse (n = 4). Data are mean ± s.d., unpaired two-tailed Student’s t-test. d, Representative flow cytometry of TFH RV+ [High] SMARTA cell subsets from spleen of LCMVArm infected mice. Quantification of results from left panels. e, Bottom 10% [Low], bottom 10-30% [Med] and remaining 30-100% [High] GFP+ Runx3-RV+ SMARTA cells, and total GFP+ RV+ SMARTA cells were FACS sorted from in vitro culture. Whole cell lysates were analyzed by immunoblotting using anti-Runx3 and anti-GAPDH antibodies. Relative Runx expressions are indicated. f, Bottom 10% GFP+ Runx3-RV+ (Runx3-RV+ [Low]), bottom 30% GFP+ Runx2-RV+ (Runx2-RV+ [Med]), and total GFP+ RV+ (GFP-RV+) SMARTA cells were FACS sorted from in vitro culture. Whole cell lysates were analyzed by immunoblotting using anti-myc tag and anti-GAPDH antibodies. Relative Runx expressions are indicated. g, SMARTA CD4 T cells transduced with pMIG (GFP-RV+), pMIG-Runx3myc (Runx3-RV+ [Low]), or pMIG-Runx2myc (Runx2-RV+ [Med]) were transferred to C57BL/6 host mice, followed by infection of the host mice with LCMVArm, and analyzed 7 days later. Frequency of CD45.1+ SMARTA cells among total CD4 T cells from spleen of LCMVArm infected mice. Two independent experiments were performed; each dot represents one mouse (n = 5). Data are mean ± s.d., unpaired two-tailed Student’s t-test. h, Representative flow cytometry of TFH Runx3-RV+ [Low] and Runx2-RV+ [Med] SMARTA cell subsets from spleen of LCMVArm infected mice. Quantification of results from left panels. h, Representative flow cytometry of TFH Runx3-RV+ [Low] and Runx2-RV+ [Med] SMARTA cell subsets from spleen of LCMVArm infected mice. Quantification of results from left panels.

Extended Data Fig. 9 Identification of Klf2 as a repressor, acting downstream of Bcl-6 regulating major of TFH genes.

Related to Fig. 8 a-c, WT or Bcl6f/fPrdm1f/f CreCd4 SMARTA CD4 T cells transfected with crCd8 or crKlf2 were transferred to C57BL/6 host mice, followed by infection of the host mice with LCMVArm, and analyzed 6-7 days later (n=4-5 mice per group). (a) Frequency of CD45.1+ SMARTA cells among total CD4 T cells from spleen of LCMVArm infected mice. (b) Representative flow cytometry of gp66-restimulated IL-21+ SMARTA cells. Quantification of results from left panels. (c) Quantification of expression of Tcf-1, T-bet, GATA-3, and Maf, gated on SMARTA cells. Three independent experiments were performed; each dot represents one mouse (a, n = 4; b-c, n = 5). Data are mean ± s.d., unpaired two-tailed Student’s t-test. Related to Fig. 8a-c. d, Schematic of the CRISPR/Cas9-mediated gene knockdown of SMARTA cell system used for testing Klf2 in KLH-gp61 immunization. WT or Bcl6f/fPrdm1f/f CreCD4 SMARTA CD4 T cells transfected with crCd8 or crKlf2 were transferred to C57BL/6 host mice, followed by immunization of the host mice with KLH-gp61 and alum + cGAMP, and analyzed 8 days later. Related to Fig. 8d-f. e,g, Genome-browser tracks depict ATAC–seq chromatin accessibility and TF occupancy. Peak calls indicated below each track. * and ** indicate DEseq2 raw p-val ≤ 0.05 and ≤ 0.01, respectively, in comparison between Bcl6f/fPrdm1f/f CreCD4 TFH-like and Prdm1f/f CreCD4 TFH. Gene expression from RNA-seq data of LCMVArm infected mice and KLH-gp61 immunized mice are graphed. f, Genome-browser tracks depict Tcf1 ChIP–Seq peaks at Pdcd1 and IL6ra loci from murine TFH cells. h, Gene expression of Tox and Tox2 from RNA-seq data of LCMVArm infected mice or KLH-gp61 immunized mice. i, Representative flow cytometry of TFH cells, gated on SMARTA cells from spleens of LCMVArm infected mice (n=4 mice per group) in Fig. 8a. Quantification of results from left panels.

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Choi, J., Diao, H., Faliti, C.E. et al. Bcl-6 is the nexus transcription factor of T follicular helper cells via repressor-of-repressor circuits. Nat Immunol 21, 777–789 (2020). https://doi.org/10.1038/s41590-020-0706-5

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