The RNA-binding protein hnRNP F is required for the germinal center B cell response

Figures

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  Fig. 1. hnRNP F-deficient mice have normal B cell development. a, b Flow cytometric analysis of B cells in the BM. The frequencies (a) and numbers (b) of various B cell subsets in the BM of WT and Hnrnpf KO mice were analyzed and enumerated using Hardy’s gating strategy (n = 6 for WT group and n = 5 for Hnrnpf KO group). c, d Flow cytometric analysis of B cells in the spleen. The frequencies (c) and numbers (d) of different splenic B cell subsets in WT and Hnrnpf KO mice were examined and calculated (n = 6 for WT group and n = 5 for Hnrnpf KO group). Data are representative of five independent experiments (a and c). Each symbol represents an individual mouse (b and d). An unpaired two-tailed student’s t-test was used for the statistical analysis (b and d). Data are presented as mean values ± SD (b and d). Source data are provided as a Source Data file.

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  Fig. 2. hnRNPF is required for TD antibody immune response. a–d ELISA of determining antibody titers of NP-specific IgM (a), IgG1 (b), IgG2b (c), and IgG3 (d) in the serum of WT and Hnrnpf KO mice at various time points post-immunization (n = 6 for WT group and n = 7 for Hnrnpf KO group). e ELISPOT assay of NP-specific IgM and IgG1 antibody-secreting cells in the spleen and BM of WT and Hnrnpf KO mice at day 10 post-immunization (n = 6 per group). f Enumeration of antibody-secreting cells in the spleen and BM as determined in e. Each symbol represents an individual mouse analyzed. g–i Ratios of NP-specific IgG1 (g), IgG2b (h), and IgG3 (i) antibody titers as determined with NP2-BSA as the coating antigens versus those with NP20-BSA as the coating antigens in the sera of WT and Hnrnpf KO mice at the various time points post-immunization (n = 6 for WT group and n = 7 for Hnrnpf KO group). Data are from one single experiment (a–d and g–i) or representative of six independent experiments (e). Two-way ANOVA with Sidak’s multiple comparisons (a–d and g–i) or an unpaired two-tailed student’s t-test (f) was used for the statistical analysis. Data are presented as mean values ± SD (a–d and f–i).…

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  Fig. 3. hnRNP F is required for IgG1 CSR. a ELISA of determining antibody titers of total IgM, IgG1, IgG2b, IgG3, and IgA in sera of unimmunized WT (n = 8) and Hnrnpf KO (n = 8) mice. Data are from one single experiment. b, c Flow cytometry measuring CSR to IgG3 or IgG1 in activated B cells with indicated stimulation (n = 7 per group). Data are representative of four independent experiments (b). An unpaired two-tailed student’s t-test was used for the statistical analysis (a and c). Data are presented as mean values ± SD (a and c). Source data are provided as a Source Data file.

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  Fig. 4. hnRNP F is essential for GC B cell formation. a, b Flow cytometric analysis of the frequencies (a) and numbers (b) of total GC B cells in the spleen of WT and Hnrnpf KO mice (n = 6 per group) at day 10 post-immunization. c, d Flow cytometric analysis of the NP-specific IgG1+ B cells in the spleen of WT and Hnrnpf KO mice (n = 6 per group) at day 10 post-immunization. NP-specific IgG1+ B cells were determined by IgG1 and NIP+ BCR expression on the B220+Dump (CD138, IgM, CD3, IgD, Gr1)- cells (c). The frequencies and numbers of NP-specific IgG1+ B cells (d) were calculated accordingly. e–g Flow cytometric analysis of early GC B cell formation in the spleen of WT and Hnrnpf KO mice (n = 5 per group) at day 5 post-immunization. B220+GL-7+IgD-CD38- GC B cells were determined by flow cytometry (e), and their frequencies (f) and numbers (g) were summarized. h–j Flow cytometric analysis of iGB cells induced by anti-CD40, IL4, and IL21 for 4 days. B220+GL-7+Fas+CD38- iGC B cells were determined by flow cytometry (h), and their frequencies (i) and the Hnrnpf deletion efficiency (j) were summarized (n = 7 per group in (i) and n = 4 per group in j). k Analysis of proliferation of B cells…

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  Fig. 5. hnRNP F controls Cd40 AS. a Scatter plots of inclusion level difference between WT and Hnrnpf KO FOB cells. Each dot represents a significantly altered splicing event (|inclusion level difference | > 10%, FDR < 0.05). b A list of top ten genes with the highest aberrant AS events. c Relative expression of Hnrnpf and Cd40 mRNA in FOB cells based on the RNA-seq data (n = 2 per group). Data are from one single experiment. FDR was calculated by DESeq2 Bioconductor package based on the negative binomial distribution and and Benjamini–Hochberg method. d Sashimi plot of changes in AS of Cd40 exon 6-8 in WT and Hnrnpf KO FOB cells. The full-length version and different variants of Cd40 were shown. e The comparison of inclusion levels of indicated Cd40 AS events between WT and Hnrnpf KO FOB cells. FDR was calculated by rMATS. Likelihood-ratio test was used to calculate p value of the Inclusion Level between two groups. Benjamini Hochberg algorithm was used to correct p value to get FDR value (n = 2 per group). Data are from one single experiment. f Schematics of various isoforms of Cd40 mRNA generated by AS. The red lines above the exon indicate exon junctions, and the red line in CD40 V3…

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  Fig. 6. hnRNP F regulates GC B cell formation by promoting signalling-competent CD40 expression. a Flow cytometric analysis of the cell surface expression of CD40 on WT and Hnrnpf KO FOB cells (n = 3 per group). b Flow cytometric analysis of the cell surface expression of CD40 on WT and Hnrnpf KO GC (B220+CD38-FAS+) and non-GC (B220+CD38+FAS-) B cells. c Knockout efficiency of hnRNP F in Hnrnpf KO GCB cells with high and low CD40 expression using qRT-PCR (n = 4 for WT group and n = 3 for Hnrnpf KO group). d Flow cytometry analysis of the cell surface expression of CD40 on 293 T cells transfected with different CD40 variants. e, f Immunoblotting of phosphorylation of IKKα/IKKβ (e) and IκBα (f) in WT and Hnrnpf KO splenic B cells stimulated by 1 μg/ml anti-CD40 antibody. Data are from one single experiment (e and f). g The experimental flow of mixed BM chimeric mice study. h Flow cytometric analysis of the cell surface expression of CD40 on splenic B cells from different chimeric mice as indicated at day 10 after immunization with NP-CGG (n = 2 per group). i Flow cytometric analysis of GC B cells in the spleen of chimeric mice at day 10 after immunization (n = 4 for WT group and n = 6 for…

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  Fig. 7. hnRNP F directly binds to G-tracts of Cd40 pre-mRNA to modulate exon inclusion and intron removal. a RIP-qPCR analysis of the enrichment of Cd40 gene bound to hnRNP F in WT splenic B cells (n = 2). Data are from one single experiment. An unpaired two-tailed Welch’s t test was used for statistical analysis. Data are presented as mean values ± SD. b Illustration of Cd40 minigenes. Filled boxes: alternatively spliced exons. Empty boxes: constitutively spliced exons. The mutated bases were highlighted in red. c, d RT-PCR analysis of Cd40 variants, including exon 6 skipping (c) and intron 7 retention (d), using primer P1 and P2 in 3T3-L1 cells transfected with WT or mutated Cd40 minigenes as indicated. CD40 V1 was used as a positive control. The ratios of CD40 V2 to V1 and V3 to V1 were shown below the gel images. e–h RNA pull-down assays using biotin-labeled RNAs. Biotinylated RNAs, containing WT or mutated intron 6 (e) or 7 (g) sequences of the Cd40 pre-mRNA were prepared. The red letters indicate the mutant bases. RNAs with the reverse sequences were included as controls. The precipitated hnRNP F proteins were examined by immunoblotting (f and h). i, j RNA EMSA detection of hnRNP F…

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  Fig. 8. hnRNP A1 and A2B1 bind directly to Cd40 pre-mRNA and regulate its AS. a Cd40 pre-mRNA-binding proteins pulled down by the sequence corresponding its exon 6 and the flanking intronic regions. The pulled-down proteins were analyzed by mass spectrometry. Top proteins with a fold change of LFQ intensity of more than 5 and MS/MS count with sense RNA of more than 15 were shown. Data are from one single experiment. b Immunoblotting of hnRNP F, A1, and A2B1 proteins in the pull-down lysates using biotinylated sense and antisense RNAs as shown in a. Data are representative from two independent experiments. c RT-PCR analysis of Cd40 variants using primers P1 and P2 in B cells retrovirally transduced with control or hnRNP A1-overexpressing vectors. d Immunoblotting of hnRNP A1 or A2B1 in the splenic B cells from the WT and different knockout mice. e RT-PCR analysis of Cd40 variants using primers P1 and P2 in the FOB cells from the WT and different knockout mice. f, g Flow cytometric (f) and statistical analysis (g) of the cell surface expression of CD40 expression in FOB cells from WT and DKO mice. Data are pooled from three independent experiments in which the pairing samples were the mice…