Arid1a-dependent canonical BAF complex suppresses inflammatory programs to drive efficient Germinal Center B cell responses

doi:10.21203/rs.3.rs-3871185/v1

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Arid1a-dependent canonical BAF complex suppresses inflammatory programs to drive efficient Germinal Center B cell responses

https://doi.org/10.21203/rs.3.rs-3871185/v1

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Differentiating B cells in germinal centers (GC) require tightly coordinated transcriptional and epigenetic transitions to generate efficient humoral immune responses. The mammalian Brg1/Brm-associated factor (BAF) complexes are major regulators of nucleosomal remodeling, crucial for cellular differentiation and development, and are commonly mutated in several cancers, including GC-derived B cell lymphomas. However, the specific roles of distinct BAF complexes in GC B cell biology and generation of functional humoral immune responses are not well understood. Here, we show that the A–T Rich Interaction Domain 1a (Arid1a) containing canonical BAF (cBAF) complex is required for maintenance of GCs and therefore high affinity antibody responses. While Arid1a-deficient B cells undergo activation to initiate GC responses, they fail to sustain the GC program resulting in premature GC collapse. We discovered that Arid1a-dependent cBAF activity establishes permissive chromatin landscapes during B cell activation and is concomitantly required to suppress inflammatory gene programs to maintain transcriptional fidelity in early GC B cells. Interestingly, the inflammatory signatures instigated by Arid1a deficiency in early GC B cells recruited neutrophils and inflammatory monocytes and eventually disrupted GC homeostasis. Dampening of inflammatory cues with anti-inflammatory glucocorticoid receptor signaling rescued GC B cell differentiation of Arid1a-deficient B cells, thus highlighting a critical role of inflammation in impeding GC responses. In sum, our work identifies essential functions of Arid1a-dependent BAF activity in promoting efficient GC responses. These findings further support an emerging paradigm in which unrestrained inflammation limits GC-derived humoral responses, as reported in the context of severe bacterial and viral infections.

Immunology

B cells

Germinal Center

Epigenetics

BAF complex

Inflammation

The authors declare no competing interests.

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A. Representative flow cytometry analysis of bone marrow (BM) B cell subsets from Arid1a fl/fl(YFP-) and CD19 Arid1a KO (YFP+) mice. Numbers represent frequencies of pre and pro B cells (pre/proB), Immature B cells, mature B cells and recirculating B cells. B. Quantification of BM B cell subsets from Arid1a fl/fl (YFP-; n=5) and CD19 Arid1a KO (YFP+; n=5). C.Immunoblot showing Arid1a expression from Arid1a fl/fl and CD19 Arid1a KO naïve B cells. Beta-actin (β-actin) is used as loading control. D-F. Quantification of spleen cellularity, B cell frequency and numbers from Arid1a fl/fl (YFP-; n=5) and CD19 Arid1a KO (YFP+; n=5). G.Representative flow cytometry plots quantifying percent apoptotic cells as cleaved Caspase 3 staining from Arid1a fl/fl (YFP-; n=3) and CD19 Arid1a KO (YFP+; n=3) naïve B cells. H. Quantification of apoptotic B cells (cleaved Caspase 3 positive) from Arid1a fl/fl and CD19 Arid1a KO mice. I. Representative flow cytometry plots quantifying percent proliferating cells with Ki67 staining from Arid1a fl/fl (YFP-; n=3) and CD19 Arid1a KO (YFP+; n=3) naïve B cells. J. Quantification of proliferation (Ki67 positive) from Arid1a fl/fl and CD19 Arid1a KO mice. Statistical significance is calculated using multiple unpaired t-tests for B and two-tailed Student’s t-test for D, E, F, H and J. Error bars represent mean ± s.e.; *p value ≤ 0.05; **p value ≤ 0.01; ***p value ≤ 0.0005; ****p value < 0.0001.
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A. Representative flow cytometry analysis of mature (AA4.1- B220+) and immature B cells (AA4.1+ B220+) subsets from splenic B cells in Arid1a fl/fl (YFP-) and CD19 Arid1a KO (YFP+) mice. B. Quantification of splenic mature and immature B cell subsets from Arid1a fl/fl (YFP-) and CD19 Arid1a KO (YFP+) mice. C and D. Quantification of splenic Follicular (FO) B cells (IgD hi, IgM low) and Marginal zone (MZ) B cells (IgD low, IgM hi) from Arid1a fl/fl (YFP-) and CD19 Arid1a KO (YFP+) mice. E. Representative flow cytometry analysis showing YFP expression in B cells (left panel) and GC B cells (right panel) from Peyer’s patches of CD19 Arid1aKO mice. F. Representative flow cytometry analysis showing YFP reporter expression in GC B cells (middle panel) and non-GC B cells (right panel) from SRBC immunized Cg1 cre Rosa26 loxp-stop-loxp YFP reporter mice. G. Representative flow cytometry analysis of splenic GC B cells gated on B cells from NP-Ova immunized Arid1a fl/fl, Cg1 Arid1a fl/+(Het) and Cg1 Arid1a KO mice at 14 days post-immunization. H. Schematic of a flow cytometry-based assay to quantify SRBC-specific IgM and IgG1 antibodies in the sera. I and J. Quantification of serum SRBC-specific IgM and IgG1 antibodies upon SRBC immunization in Ctrl (Arid1fl/fl and Cg1 Cre) and Cg1 Arid1aKO mice. Statistical significance is calculated using multiple unpaired t-tests for B and two-tailed Student’s t-test for C, D, I and J. Error bars represent mean ± s.e.; *p value ≤ 0.05; **p value ≤ 0.01; ***p value ≤ 0.0005; ****p value < 0.0001.
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A. Representative flow cytometry analysis showing YFP expression in Cg1 Arid1a KO B cells at pre- and post-stimulation (day 4) on 40LB co-cultures with IL4 (in vitro). B. Representative flow cytometry analysis showing GL7 expression in Cg1cre(controls) and Cg1 Arid1a KO B cells at day 4 of in vitro 40LB co-culture. C. Representative flow cytometry analysis of IgD and IgM expression from Arid1a fl/fl (YFP-) and CD19 Arid1a KO (YFP+) splenic B cells. The median fluorescence intensity (MFI) for IgD in quadrant1 (Q1) is shown in the plots. D. Representative flow cytometry analysis quantifying percent apoptotic cells (cleaved Caspase3) from Cg1 cre(n=4) and Cg1 Arid1a KO (n=3) B cells at day 4 of in vitro 40LB co-cultures. E. Quantification of apoptotic B cells (cleaved Caspase 3 positive) from Cg1 cre (n=4) and Cg1 Arid1a KO (n=3) on 40LB co-cultures. F. Representative flow cytometry analysis of proliferating cells (Ki67 positive) from Cg1 cre and Cg1 Arid1a KO B cells at day 4 of in vitro 40LB co-cultures. G. Quantification of proliferating B cells (Ki67 positive) from Cg1 cre (n=4) and Cg1 Arid1a KO (n=3) on 40LB co-cultures. H. Representative flow cytometry analysis quantifying percent plasma cells (CD138+) from Cg1 creand Cg1 Arid1a KO B cells at day 4 of in vitro 40LB co-cultures. I. Quantification of percent of CD138+ plasma cells from Cg1 cre (n=4) and Cg1 Arid1a KO (n=3) B cells at day 4 of in vitro 40LB co-cultures. Statistical significance is calculated using two-tailed Student’s t-test for E, G and I. Error bars represent mean ± s.e.; *p value ≤ 0.05; **p value ≤ 0.01; ***p value ≤ 0.0005; ****p value < 0.0001.
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A. Representative flow cytometry analysis of IgG1 and IgM positive B cells from Arid1a fl/fl and CD19 Arid1a KO mice after 4 days of 40 LB co-cultures. B and C Quantification of IgM and IgG1 frequencies from day 4 of 40LB co-culture. D and E.Median fluorescence intensity (MFI) of IgM and IgG1 from day 4 of 40LB co-culture. F. Cell counts of Arid1a fl/fl and CD19 Arid1aKO B cells stimulated on 40LB co-cultures for 4 days. G and H Quantification of apoptotic (cleaved Caspase 3 positive) and proliferating B cells (Ki67 positive) respectively from Arid1a fl/fl(n=3) and Cd19 Arid1a KO (n=3) on 40LB co-cultures. I. Representative flow cytometry analysis quantifying percent plasma cells (CD138+) from Arid1a fl/fl (n=3) and Cd19 Arid1a KO (n=3) B cells at day 4 of in vitro 40LB co-cultures. J. Quantification of percent of CD138+ plasma cells from Arid1a fl/fl(n=3) and Cd19 Arid1a KO (n=3) B cells at day 4 of in vitro 40LB co-cultures.Statistical significance is calculated using two-tailed Student’s t-test for B-H and J. Error bars represent mean ± s.e.; *p value ≤ 0.05; **p value ≤ 0.01; ***p value ≤ 0.0005; ****p value < 0.0001.
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A-C. Gene set enrichment analysis (GSEA) plots for the transcriptional profile of Cg1 Arid1a KOand control B cells, using gene sets from. Pyroptosis (A), Innate immune response (B) and IL-6 production (C) pathways. Y-axis denotes enrichment score. NES, Normalized enrichment score, FDR, False discovery rate. D. Heatmap showing ATAC-Seq signals from individual replicates in differentially accessible regions identified in Cg1 Arid1a KO and control B cells. Reads per million (RPM) values in DARs are plotted in +/− 2 kb windows from the center of the peak. E. Genome browser view of RNA-seq and ATAC-seq data at Prdm1 (top); and Ccl2 and Ccl7 (bottom) loci. Asterisks denote changes in accessibility (red and black asterisks denote increased and decreased accessibility signals in Cg1 Arid1a KO cells, respectively). Please see bioinformatics analysis in methods for additional information.
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A. Representative flow cytometry analysis of early GC B cells (PNA+ CD38+) gated from splenic B cells of SRBC immunized Arid1a fl/fl and CD19 Arid1aKO mice at day 4 post-immunization. C. Quantification of PNA+ CD38+ early GC B cell frequency in spleen of Arid1a fl/fl (YFP-) and CD19 Arid1a KO(YFP+) mice at day 4 post-immunization. C. Representative flow cytometry plots for EfnB1+CD38+ early GC B cells and EfnB1+ CD38low mature GC B cells gated on splenic B cells from SRBC immunized Arid1a fl/fl (YFP-) and CD19 Arid1aKO (YFP+) mice at day 10 post-immunization. D and E.Quantification of Irf4 and Bcl6 expressing B cells from Arid1a fl/fl and CD19 Arid1a KO mice at day 4 post-SRBC immunization. F.Representative flow cytometry analysis of T follicular helper (Tfh) cells gated from CD4+ T cells of SRBC immunized Arid1a fl/fl and CD19 Arid1aKOmice at day 4 post-immunization. G. Quantification of Tfh cell frequency in spleen of Arid1a fl/fl and CD19 Arid1a KO mice at day 4 post-SRBC immunization. H. Representative flow cytometry analysis of Tfh cells gated on CD4+ T cells from SRBC immunized Arid1a fl/fl and CD19 Arid1aKO mice at day 10 post-immunization. I and J. Representative flow cytometry analysis and quantification for Tfh cells gated on CD4+ T cells from NP-Ova immunized Arid1a fl/fl and CD19 Arid1aKO mice at day 14 post-immunization. K and L. Representative flow cytometry plots and quantification for Tfh cells gated on CD4+ T cells from NP-Ova immunized Arid1a fl/fl and Cg1 Arid1aKO mice at day 14 post-immunization. Statistical significance is calculated using two-tailed Student’s t-test for B, D, E, G and J and L. Error bars represent mean ± s.e.; *p value ≤ 0.05; **p value ≤ 0.01; ***p value ≤ 0.0005; ****p value < 0.0001.
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A-B. Representative flow cytometry analysis and quantification of IgD low CD38+ memory B cell frequencies in Arid1a fl/fl and CD19 Arid1aKO mice at day 14 post-immunization with NP-Ova. C-D. Representative flow cytometry analysis and quantification of NP+ memory B cells identified gated on IgD lo CD38+ memory B cells in Arid1a fl/fl and CD19 Arid1aKO mice at day 14 post-immunization with NP-Ova. E. Quantification of IgG1+ NP+ memory B in the spleen of Arid1a fl/fl and CD19 Arid1a KO mice at day 14 post-immunization with NP-Ova. F-G. Genome browser view of RNA-seq and ATAC-seq data at Bcl6 (F) and Irf4 (G) loci for CD19 Arid1aKO and control B cells. H-K. Genome browser view of RNA-seq and ATAC-seq data showing gene expression and accessibility enrichment distribution at Ccl3, Ccl4 (H), Il1b (I), Havcr1 (J) and Igh loci (K). Asterisks denote changes in accessibility (red and black asterisks denote increased and decreased accessibility signals in CD19 Arid1a KO cells, respectively). L. Quantification of Control (Ctrl) (YFP-) and CD19 Arid1aKO (YFP+) non-GC B cells (EfnB1- CD38+) frequency for expression of Il1b (pro-form), Ifng and Il6 proteins at day 4 post-SRBC immunization. M. Gene set enrichment analysis (GSEA) plots for the transcriptional profile of Cg1 Arid1a KO and control B cells, using gene sets from. Acute inflammatory response (top), Lymphocyte Chemotaxis (middle) and Monocyte Chemotaxis (bottom) pathways in transcription profile of CD19 Arid1aKO and control early GC B cells. Y-axis denotes enrichment score. NES, Normalized enrichment score, FDR, False discovery rate. N. Heatmap showing enrichment of ATAC-seq signal (average of 2 replicates) in CD19 Arid1aKO and control early GC B cells. Reads per million (RPM) values in DARs are plotted in +/− 2 kb windows from the center of the peak. Statistical significance for B, E, D and L is calculated using two-tailed Student’s t-test. Error bars represent mean ± s.e.; *p value ≤ 0.05; **p value ≤ 0.01; ***p value ≤ 0.0005; ****p value < 0.0001. Please see bioinformatics analysis in methods for additional information.
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A. Representative flow cytometry analysis of splenic CD11b+ myeloid cells in Arid1a fl/fl and Cg1 Arid1aKO mice at day 14 post-immunization with NP-Ova. B.Quantification of CD11b+ myeloid cells frequency in spleen of Arid1a fl/fl and Cg1 Arid1a KO mice at day 14 post-immunization with NP-Ova. C. Representative flow cytometry plots showing neutrophils (Ly6G+Ly6C low) and inflammatory monocytes (Ly6G-Ly6C hi) in Arid1a fl/fl and CD19 Arid1aKO mice at day 10 post-immunization with SRBCs. D. Quantification (absolute numbers) of splenic neutrophils (Ly6G+Ly6C low) and inflammatory monocytes (Ly6G-Ly6C hi) from Arid1a fl/fl and CD19 Arid1aKO mice at day 4 post-immunization with SRBC. E. Representative immunohistochemistry images for PNA, CD11b, Gr1 and CD3e staining on formalin fixed spleen serial sections (displaying the same follicle) from Arid1a fl/fl and CD19 Arid1a KO mice at day 14 post-immunization with NP-Ova. Statistical significance is calculated using two-tailed Student’s t-test for B and multiple unpaired t-tests for D. Error bars represent mean ± s.e.; *p value ≤ 0.05; **p value ≤ 0.01; ***p value ≤ 0.0005; ****p value < 0.0001.
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A. Representative flow cytometry analysis depicting the distribution of CD45.2 B cells (red boxes) and CD45.1 B cells (dark grey boxes) from SRBC immunized Arid1a fl/fl and Cg1 Arid1aKO chimeras at day 11 post-immunization. The upper panel shows representative data from DMSO treated mice and lower panel corresponds to Dexa treatment. B. Quantification of splenic CD45.2 B cell frequency normalized to CD45.1 B cells from Arid1a fl/fl and Cg1 Arid1aKO chimeras at day 11 post-immunization treated with DMSO or Dexa. C. Quantification of splenic CD45.1.2 GC B cell frequency normalized to CD45.1 GC B cells from Arid1a fl/fl and Cg1 Arid1aKO chimeras at day 11 post-immunization treated with DMSO or Dexa. For B. and C. The frequency of the respective subsets was calculated from the live cell gate to account for differences in engraftment. D. Representative flow cytometry analysis of GC B cells from SRBC immunized wild type (WT) mice treated with DMSO or Dexa. Numbers represent frequency of GC B cells at day 11 post-SRBC immunizations and treatment with DMSO or Dexa at days 3, 7 and 9. E. Quantification of GC B cell frequency in spleen of DMSO and Dexa treated SRBC immunized WT mice. F.Quantification of splenocyte numbers in Arid1a fl/fl and Cg1 Arid1aKOchimeras at day 11 post-SRBC immunization with DMSO or Dexa treatment. Arid1a fl/fl and Cg1 Arid1aKO chimeras treated with DMSO are open bars and Dexa treated groups are represented with light red filled bars. G and H.Quantification of frequencies and numbers of Tfh cells gated on CD45.1 CD4+ cells in Arid1a fl/fl and Cg1 Arid1aKO chimeras at day 11 post-SRBC immunization. I and J. Quantification of frequencies and numbers of CD11b expressing myeloid cells gated on CD45.1 cells in Arid1a fl/fl and Cg1 Arid1aKO chimeras at day 11 post-SRBC immunization. Arid1a fl/fl and Cg1 Arid1aKO chimeras treated with DMSO are open bars and Dexa treated groups are represented with light red filled bars. K and L. Quantification of frequencies and numbers of Ly6G- Ly6Chi monocytes gated on CD45.1+ CD11b expressing myeloid cells in Arid1a fl/fl and Cg1 Arid1aKOchimeras at day 11 post-SRBC immunization. M and N. Quantification of frequencies and numbers of Ly6G+ Ly6Clo neutrophils gated on CD45.1+ CD11b expressing myeloid cells in Arid1a fl/fl and Cg1 Arid1aKOchimeras at day 11 post-SRBC immunization. Statistical significance is calculated using two-tailed Student’s t-test for D and 2-way ANOVA with multiple comparisons for B and E-M. Error bars represent mean ± s.e.; *p value ≤ 0.05; **p value ≤ 0.01; ***p value ≤ 0.0005; ****p value < 0.0001.

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Arid1a-dependent canonical BAF complex suppresses inflammatory programs to drive efficient Germinal Center B cell responses

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