The transcription factor Hhex cooperates with the corepressor Tle3 to promote memory B cell development

Abstract

Memory B cells (MBCs) are essential for long-lived humoral immunity. However, the transcription factors involved in MBC differentiation are poorly defined. Here, using single-cell RNA sequencing analysis, we identified a population of germinal center (GC) B cells in the process of differentiating into MBCs. Using an inducible CRISPR–Cas9 screening approach, we identified the hematopoietically expressed homeobox protein Hhex as a transcription factor regulating MBC differentiation. The corepressor Tle3 was also identified in the screen and was found to interact with Hhex to promote MBC development. Bcl-6 directly repressed Hhex in GC B cells. Reciprocally, Hhex-deficient MBCs exhibited increased Bcl6 expression and reduced expression of the Bcl-6 target gene Bcl2. Overexpression of Bcl-2 was able to rescue MBC differentiation in Hhex-deficient cells. We also identified Ski as an Hhex-induced transcription factor involved in MBC differentiation. These findings establish an important role for Hhex–Tle3 in regulating the transcriptional circuitry governing MBC differentiation.

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Fig. 1: Identification of eight B cell clusters following viral infection using scRNA-seq.
Fig. 2: Identification of PreMem B cells using scRNA-seq.
Fig. 3: In vivo conditional Cas9 screen of transcription factors expressed by PreMem B cells.
Fig. 4: Overexpression of Hhex or Tle3 promotes MBC differentiation.
Fig. 5: Hhex promotes MBC differentiation through binding to DNA and interaction with Tle3.
Fig. 6: Ablation of Hhex in GC B cells impairs MBC differentiation.
Fig. 7: Hhex promotes the development of PreMem B cells.
Fig. 8: Hhex promotes MBC development through induction of Bcl2 and Ski.

Data availability

Raw and processed data files for the scRNA-seq and RNA-seq analyses have been deposited in the NCBI Gene Expression Omnibus under accession number GSE148805.

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Acknowledgements

We thank T. Roth and A. Marson (University of California, San Francisco) for the pTR plasmid, T. Okada for the S1pr2-Cre-ERT2 mice, J. An for expert technical assistance, J. Derisi for support and P. S. Jayaraman for helpful discussions. This work was supported by grants from the NIH (R01AI045073 and R01AI040098 to J.G.C. and T32AI07019 to B.J.L.). B.J.L. is a Howard Hughes Medical Institute Fellow of the Damon Runyon Cancer Research Foundation (DRG-2265–16). J.G.C. is an investigator of the Howard Hughes Medical Institute.

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Contributions

B.J.L. and J.G.C. conceived of and designed the experiments. B.J.L., L.D., Y.X. and S.E.V. performed the experiments. B.J.L. and J.G.C. analyzed the data. B.J.L. and J.G.C. wrote the manuscript.

Corresponding author

Correspondence to Jason G. Cyster.

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The authors declare no competing interests.

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Peer review information Peer reviewer reports are available. 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 Differential gene expression for eight B cell clusters.

a, Gating strategy for the sorted B cells used for droplet-based scRNA-seq analysis from S1pr2-ERT2creTdTomato mice at day 11 post LCMV infection. The sorted population consisted of a mixture of 90% GC B cells, 5% FO B cells, and 5% MBCs. Colored gates and labels are used to show the order of the gating scheme. Cells for scRNA-seq sort were pooled from 4 mice. b, Heatmap of each cell’s (column) expression of the top ten DEGs per cluster (rows). Select genes are labeled. Log-normalized expression scaled for each gene. Cluster name displayed below. c, Violin plots of select gene from LZ 1 (n = 2,675 cells), DZ 1 (n = 2,412 cells), DZ 2 (n = 2,017 cells), Mem (n = 1,255 cells), LZ 2 (n = 1,079 cells), DZ 3 (n = 713 cells), FO (n = 670 cells), and Myc+ clusters (n = 207 cells) with highest log-normalized expression value labeled. Violin plots are presented with floating boxes showing median (middle line) and quartiles (top and bottom). Minima and maxima are shown as the bottom and top of the violin plot. d, Enrichment score for gene signature distinguishing MBCs (far left), GC B cells (middle left), and DZ GC B cells (middle right) projected onto tSNE plots. Color scaled for each gene signature with log-normalized expression level noted. Stage of cell cycle based on gene expression projected onto tSNE plot (far right). Cells for scRNA-seq sort were pooled from 4 mice.

Extended Data Fig. 2 Analysis of memory cluster cells.

a, Number of cells per cluster (left) and DEGs per cluster (right) following subclustering analysis of Mem cluster. b, Enrichment score for gene signature distinguishing CD80PDL2 MBCs (left) and CD80+PDL2+ MBCs (right) projected onto tSNE plots of Mem cluster (n = 1,255 cells). Color scaled for each gene signature with highest log-normalized expression level noted. Cells for scRNA-seq sort were pooled from 4 mice. c, Violin plots of Sell, Cd44, and Il21r expression in cells from PreMem (n = 106 cells), Mem α (n = 482 cells), and Mem β clusters (n = 677 cells) with log-normalized expression value labeled. Violin plots are presented with floating boxes showing median (middle line) and quartiles (top and bottom). Minima and maxima are shown as the bottom and top of the violin plot. d, Representative FACS plots of CD44 and CD62L expression at day 11 post LCMV infection on MBCs (B220+IgDloGL7CD38+S1pr2-Tomato+) showing example gating scheme for identification of CD44+CD62L+ and CD44+/-CD62L subsets. Data are representative of 4 independent experiments with at least 2 mice per group.

Extended Data Fig. 3 Transcription factor expression in mouse and human memory B cells.

a, Heatmap of each cell’s (column) expression of select TFs per cluster (rows). Log-normalized expression scaled for each gene. Cluster name displayed below. b, Heatmap of TF expression from bulk RNA-seq of splenic GC B cells, PreMem B cells, and MBCs at day 11 post LCMV infection. c, TF expression in human naïve, GC, and MBCs as determined using datasets compiled by Genevestigator. 3 samples were included in each dataset. Level of expression is a unitless value on a log2 scale computed following normalization and scaling of raw data in order to allow comparison of expression of given gene between samples. Statistical analyses were performed using the two-sided Holm-Sidak multiple comparisons test (****, p < 0.0001). Scatter plots indicate mean (middle line) with error bars indicating standard error mean. d, Representative FACS plots of CXCR3+ cells (left) among Thy1.1+BFP+ MBCs (B220+IgDloGL7CD38+GFP+) at day 30 post LCMV infection in mice containing cells transduced with sgRNAs targeting a control nonexpressed gene and CXCR3. Percentage of CXCR3+ cells among Thy1.1+, BFP+, Thy1.1+BFP+, and Thy1.1-BFP MBCs (right). Data are pooled from 4 independent experiments with at least 4 mice per group. Scatter plots indicate mean (middle line) with error bars indicating standard error mean. e, Representative FACS plots of CD38+ cells (left) among Thy1.1+BFP+ B220+IgDloGFP+ cells at day 30 post LCMV infection in mice containing cells transduced with sgRNAs targeting a control nonexpressed gene and CD38. Percentage of CD38+ cells among Thy1.1+, BFP+, Thy1.1+BFP+, and Thy1.1-BFP B220+IgDloGFP+ (right). Data are pooled from 4 independent experiments with at least 4 mice per group. Scatter plots indicate mean (middle line) with error bars indicating standard error mean. f, Ratio of MBCs to GC B cells (B220+IgDloGL7+CD95+GFP+) among sgRNA 1-Thy1.1+sgRNA 2-BFP+ B cells in the experiment described in Fig. 3c. Data are pooled from 6 independent experiments with at least 3 mice per group. Statistical analyses were performed using the ordinary one-way ANOVA with two-sided Dunnett multiple comparison testing. Scatter plots indicate mean (middle line) with error bars indicating standard error mean.

Extended Data Fig. 4 Hhex promotes memory B cell differentiation following TH1-type and TH2-type immunization.

a, Ratio of transduced (Thy1.1+) splenic MBCs (B220+IgDloGL7CD38+CD95+CD73+) to GC B cells (B220+IgDloGL7+CD95+) in Hhex-overexpressing bone marrow chimeras at day 15, 30, and 60 post LCMV infection. Data are pooled from 8 independent experiments with at least 4 mice per group. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. Scatter plots indicate mean (middle line) with error bars indicating standard error mean. b, Representative FACS plots of the percentage of transduced (Thy1.1+) cells among splenic FO (B220+IgDhiGL7CD38+CD95), GC (B220+IgDloGL7+CD95+NP+), and MBCs (B220+IgDloGL7CD38+NP+) at day 30 post NP-CGG in alum immunization in empty vector and Hhex-overexpressing bone marrow chimeras. Data are representative of 2 independent experiments with at least 4 mice per group. c, Ratio of transduced GC to FO B cells (left), MBCs to FO B cells (middle), and MBCs to GC B cells (right). Data are representative of 2 independent experiments with at least 4 mice per group. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. Scatter plots indicate mean (middle line) with error bars indicating standard error mean.

Extended Data Fig. 5 Ski and Klf2 promote memory B cell differentiation.

a, Representative FACS plots of the percentage of transduced (Thy1.1+) cells among splenic FO (B220+IgDhiGL7CD38+CD95), GC (B220+IgDloGL7+CD95+), and MBCs (B220+IgDloGL7CD38+CD95+CD73+) at day 30 post LCMV infection in Ski and Klf2-overexpressing bone marrow chimeras. Data are representative of 4 independent experiments with at least 3 mice per group. b, Ratio of transduced GC to FO B cells (top), MBCs to FO B cells (bottom left), and MBCs to GC B cells (bottom right). Vector used to transduce cells listed below. Data are pooled from 4 independent experiments with at least 3 mice per group. Statistical analyses were performed using the unpaired two-tailed Student’s t-test for the GC/FO and Mem/FO comparisons (****, p < 0.0001). Statistical analyses were performed using the ordinary one-way ANOVA with two-sided Dunnett multiple comparison testing for the Mem/GC comparisons (*****, p < 0.0001). Scatter plots indicate mean (middle line) with error bars indicating standard error mean. c, Coomassie staining of gel loaded with GST (lane 1), GST-Hhex (lane 2), and GST-Hhex F33E (lane 3) fusion proteins. Arrow indicates full-length fusion protein. Data are from 1 independent experiment. d, Interaction of in vitro translated and transcribed Tle3 with glutathione beads coated with GST, GST-Hhex, or GST-Hhex F33E as quantified using a radioactive ligand binding assay. Counts per minute (cpm) are normalized based on the count of the input Tle3 used in the binding assay. Data are pooled from 2 independent experiments with 4 samples per group. Statistical analyses were performed using the ordinary one-way ANOVA with two-sided Dunnett multiple comparison testing (*****, p < 0.0001). Scatter plots indicate mean (middle line) with error bars indicating standard error mean. e, Interaction of in vitro translated and transcribed Nkx6 (left) and Rfx6 (right) with glutathione beads coated with GST, GST-Hhex, or GST-Hhex F33E as quantified using a radioactive ligand binding assay. Counts per minute (cpm) are normalized based on the count of the input Nkx6 or Rfx6 used in the binding assay. Data are from 1 independent experiment with 2 samples per group.

Extended Data Fig. 6 Ablation of Hhex in GC B cells impairs memory B cell differentiation following TH2-type immunization.

a, Representative FACS plots of the percentage of S1pr2-Tomato+ cells among splenic B220+IgDloGL7CD38+NP+ cells in Hhex Het and KO mice at day 30 post NP-CGG in alum immunization. Data are representative of 2 independent experiments with 3 mice per group. b, Percentage of B cells that are GC B cells (B220+IgDloGL7+CD95+NP+S1pr2-Tomato+) and MBCs (B220+IgDloGL7CD38+NP+S1pr2-Tomato+) in Hhex Het and KO mice at day 30 post NP-CGG in alum immunization. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. Scatter plots indicate mean (middle line) with error bars indicating standard error mean. c, Ratio of NP+ MBCs to NP+ GC B cells in Hhex Het and KO mice at day 30 post NP-CGG in alum immunization. Data are pooled from 2 independent experiments with 3 mice per group. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. Scatter plots indicate mean (middle line) with error bars indicating standard error mean. d, Representative FACS plots of CD44 and CD62L expression on MBCs (B220+IgDloGL7CD38+S1pr2-Tomato+) at day 12 post NP-CGG in alum immunization. Data are representative of 2 independent experiments with 3 mice per group. e, Percentage of CD44CD62L (left), CD44+CD62L (middle), and CD44+CD62L+ (right) MBCs at day 12 post NP-CGG in alum immunization. Data are pooled from 2 independent experiments with 3 mice per group. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. Scatter plots indicate mean (middle line) with error bars indicating standard error mean. f, Frequency of GC B cells (B220+IgDloGL7+CD95+S1pr2-Tomato+) and MBCs (B220+IgDloGL7CD38+S1pr2-Tomato+) in Hhex Het and KO mice at day 12 post NP-CGG in alum immunization that have W33L mutation in the canonical NP-responding gene VH186.2. Data are pooled from 2 independent experiments with 3 mice per group. g, Representative FACS plot (left) and percentage (right) of CD138+ cells that are CD45.2+S1pr2-Tomato+ at day 5 post challenge with NP-CGG in alum. Equivalent numbers of splenic CD45.2+ T and B cells from Hhex Het and KO NP-CGG in alum immune mice were transferred to naïve CD45.1+ recipients one day prior to challenge. Data are pooled from 2 independent experiments with 3 mice per group. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. Scatter plots indicate mean (middle line) with error bars indicating standard error mean. h, Percentage of CD138+ cells (left) and GC B cells (right) that were CD45.2+S1pr2-Tomato+ at day 5 post challenge with NP-CGG in alum when normalized to the percentage of MBCs present in the transferred cells. Data are pooled from 2 independent experiments with 3 mice per group. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. Scatter plots indicate mean (middle line) with error bars indicating standard error mean.

Extended Data Fig. 7 Memory B cells develop early following LCMV infection.

a, Gating scheme for PreMem B cells at day 11 post LCMV infection. b, Representative FACS plots of S1pr2-Tomato expression in B220+IgDloGL7CD38+ cells at day 60 post LCMV infection in S1pr2-ERT2creTdTomato mice treated with tamoxifen beginning at day 4, 11, 16, or 30 p.i. Data are representative of 3 independent experiments with at least 4 mice per group. c, Percentage of S1pr2-Tomato+ GC (left) and MBCs (right) at day 60 post LCMV infection in S1pr2-ERT2creTdTomato mice treated with tamoxifen beginning at day 4, 11, 16, or 30 p.i. Data are representative of 3 independent experiments with at least 4 mice per group. Statistical analyses were performed using the ordinary one-way ANOVA with two-sided Dunnett multiple comparison testing (****, p < 0.0001). Scatter plots indicate mean (middle line) with error bars indicating standard error mean.

Extended Data Fig. 8 Expression of differentially expressed genes in MBCs.

a, Heatmap of select genes from RNA-seq analysis of MBCs (B220+IgDloGL7CD38+S1pr2-Tomato+) from Hhexflox/+ (Het) and Hhexflox/flox (KO) S1pr2-ERT2-cre-TdTomato mice and GC B cells (B220+IgDloGL7+CD95+Ephrinb1+S1pr2Venus/+)16 at day 11 post LCMV infection. Data are from 3 independent experiments with at least 3 mice per experiment pooled for each sample. b, Expression of Bcl6 (left), S1pr2 (middle), and Bcl2 (right) in Hhex Het and KO GC B cells (B220+IgDloGL7+CD95+S1pr2-Tomato+) at day 12 post LCMV infection. Data are from 1 independent experiments with 4 mice. c, Expression of Bcl6, Hhex, Tle3, and Bcl2 projected onto tSNE plot of Mem cluster (n = 1,255 cells). Color scaled for each gene with log-normalized expression level noted. Cells for scRNA-seq sort were pooled from 4 mice.

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Laidlaw, B.J., Duan, L., Xu, Y. et al. The transcription factor Hhex cooperates with the corepressor Tle3 to promote memory B cell development. Nat Immunol 21, 1082–1093 (2020). https://doi.org/10.1038/s41590-020-0713-6

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