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The RNA-binding protein PTBP1 is necessary for B cell selection in germinal centers

Nature Immunology volume 19, pages 267–278 (2018)Cite this article

Abstract

Antibody affinity maturation occurs in germinal centers (GCs), where B cells cycle between the light zone (LZ) and the dark zone. In the LZ, GC B cells bearing immunoglobulins with the highest affinity for antigen receive positive selection signals from helper T cells, which promotes their rapid proliferation. Here we found that the RNA-binding protein PTBP1 was needed for the progression of GC B cells through late S phase of the cell cycle and for affinity maturation. PTBP1 was required for proper expression of the c-MYC-dependent gene program induced in GC B cells receiving T cell help and directly regulated the alternative splicing and abundance of transcripts that are increased during positive selection to promote proliferation.

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Fig. 1: PTBP1 expression is increased in positively selected GC B cells.
Fig. 2: PTBP1 is necessary for GC B cell responses.
Fig. 3: PTBP2 is partially redundant with PTBP1 in GC B cells.
Fig. 4: PTBP1 regulates mRNA abundance and AS in GC B cells.
Fig. 5: The positive-selection gene-expression program is reduced after deletion of Ptbp1.
Fig. 6: Late S-phase progression is impaired in the absence of PTBP1.
Fig. 7: PTBP1 regulates the abundance of Tyms mRNA through control of AS.
Fig. 8: PTBP1 regulates AS of Pkm.

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Acknowledgements

We thank D. L. Black (Brain Research Institute, University of California) for Ptbp2fl/fl mice; M. Reth (Max Planck Institute of Immunobiology and Epigenetics) for Cd79aCre mice; M. Busslinger (Research Institute of Molecular Pathology) for AicdaTg-Cre mice; B. P. Sleckman (Weill Cornell Medicine Pathology & Laboratory Medicine) for MycGFP/GFP mice; F. W. Alt (Department of Genetics, Harvard Medical School) for Rag2–/– mice; J. Ule, N. Haberman and T. Curk for help with iCLIP data; G. Butcher for assistance in generating monoclonal antibodies to PTBP3; K. Bates, D. Sanger, A. Davis, the Biological Support Unit, Flow Cytometry and Bioinformatics Facilities for technical assistance; and M. Spivakov and members of the Turner and Smith laboratories for helpful discussions. Supported by The Biotechnology and Biological Sciences Research Council (BB/J004472/1 and BB/J00152X/1 to M.T.), the Wellcome Trust (200823/Z/16/Z to M.T.) and Bloodwise (14022 to M.T.).

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Authors and Affiliations

  1. Laboratory of Lymphocyte Signaling and Development, The Babraham Institute, Cambridge, UK

    Elisa Monzón-Casanova, Michael Screen, Manuel D. Díaz-Muñoz, Richard M. R. Coulson, Sarah E. Bell, Greta Lamers & Martin Turner

  2. Department of Biochemistry, University of Cambridge, Cambridge, UK

    Elisa Monzón-Casanova & Christopher W. J. Smith

  3. Molecular Diabetology, University Hospital and Faculty of Medicine, Technische Universität Dresden, Dresden, Germany

    Michele Solimena

  4. Paul Langerhans Institute Dresden of the Helmholtz Center Munich at University Hospital and Faculty of Medicine, Technische Universität Dresden, Dresden, Germany

    Michele Solimena

  5. Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany

    Michele Solimena

  6. German Center for Diabetes Research (DZD e.V.), Neuherberg, Germany

    Michele Solimena

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  1. Elisa Monzón-Casanova
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Contributions

E.M.-C., C.W.J.S. and M.T. designed experiments; E.M.-C., M. Screen, M.D.D.-M., S.E.B. and G.L. performed experiments and analyzed data; E.M.-C. carried out computational analyses; R.M.R.C. ran DaPars; M. Solimena provided Ptbp1tm1Msol mice and antibody to PTBP2; and E.M.-C. and M.T. wrote the manuscript with input from the co-authors.

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Correspondence to Martin Turner.

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Integrated supplementary information

Supplementary Figure 1 PTBPs expression in GC B cells.

(a) FPKMs of Ptbps in naïve and GC B cells49. Comparison of naive B cells to GC B cells (with DESeq2) revealed significant differences for Ptbp1 (padj <0.05), but not for Ptbp2 (0.19 padj) or Ptbp3 (0.78 padj). (b) Sashimi plots illustrating reads that map to exon-exon junctions (arches) and read coverage for the different Ptbps in mRNAseq data from naïve and GC B cells shown in a. Numbers above the arches show the number of reads mapping across two different exons. Numbers on the right in brackets are the maximum number of reads mapped to a particular genomic location. (c) FPKMs of the different Ptbps in GC B cells, which had received high levels of T cell help (Anti-DEC-OVA) or not (Anti-DEC-Ctrl)3. Comparison between GC B cells that received high levels of help to those that did not with DESeq2 resulted in 0.57, 0.99 and 0.99 padj values for Ptbp1, Ptbp2 and Ptbp3, respectively. (d) FPKMs of the different Ptbps in LZ and DZ GC B cell populations that are expressing or not c-MYC and AP47. Differential expression analysis of Ptbp1 and Ptbp3 is shown in Fig. 1a. For a, c and d, Smptb has two annotated gene copies in the Ensembl mouse genome (ENSMUSG00000083939 and ENSMUSG00000081486). No reads were found mapped to either of them. (e) Gating strategy used to identify GC B cells by flow cytometry from the spleen of mice immunised with NP-KLH (7 days post-immunisation) shown in Fig. 1b. (f) Flow cytometry histogram overlays show staining of the different PTBPs and respective isotype control stainings for GC and non-GC B cells shown in e and Fig. 1b.

Supplementary Figure 2 PTBPs expression in B cells from Cd79a+/+Ptbp1fl/fl and Cd79acre/+Ptbp1fl/fl mice.

(a) PTBP1, PTBP2 and PTBP3 protein expression in splenic B cells from Cd79a+/+Ptbp1fl/fl and Cd79acre/+Ptbp1fl/fl mice analysed by immunoblot. Numbers indicate proteins isolated from B cells of different mice. Names in brackets are the clone names for the different antibodies used. (b) Flow cytometry analysis of PTBP1, PTBP2 and PTBP3 in T (TCRβ+) and B (CD19+) cells using the antibodies shown in a. (c) Gating strategy used to identify developing B cells in bone marrow cells by flow cytometry shown in d. The first plots on the left were pre-gated on CD11b-, Gr1-, Ly6c-, F4/80-, CD8a-, CD3e- and Ter119- live cells. (d) PTBP1 and PTBP2 intracellular staining in the different populations of developing B cells shown in c. (e) Number of pre-pro B cells (B220low, surface IgM-, CD43high, CD25low, CD19-), pro/early pre B cells (B220low, surface IgM-, CD43high, CD25low, CD19+), pre B cells (B220low, surface IgM-, CD43low, CD25high, CD19+), immature B cells (B220low, surface IgM+) and mature B cells (B220high, surface IgM+) in bone marrows from control (Cd79a+/+Ptbp1fl/fl) and cKO (Cd79acre/+Ptbp1fl/fl) mice. Each symbol is data from an individual mouse. An unpaired two-tailed Student’s t-test was carried out and p-values are shown. (f) Number of T1 (B220+, CD19+, CD93high, IgMhigh and CD23-), T2 (B220+, CD19+, CD93high, IgMhigh and CD23+), T3 (B220+, CD19+, CD93high, IgMlow and CD23+), follicular (B220+, CD19+, CD93low, CD23high and CD21low) and marginal zone (B220+, CD19+, CD93low, CD23low and CD21high) B cells in the spleens of Cd79a+/+Ptbp1fl/fl and Cd79acre/+Ptbp1fl/fl mice. Each symbol is data from an individual mouse. P-values from unpaired two-tailed Student’s t-test are shown.

Supplementary Figure 3 Effects of Ptbp1 deletion in GC B cells.

(a) Percentages of PTBP1 or PTBP2 positive GC (CD95+ CD38low B220+ CD19+) or non GC B (CD95- CD38high B220+ CD19+) cells analysed by flow cytometry from splenocytes of mice 7 days following immunisation with NP-KLH. The same number of events is shown in each dot plot. Numbers are percentages of gated cells. (b) Flow cytometry analysis of splenocytes 7 days after immunisation with NP-KLH. Events shown in upper pseudocolour plots are gated on B220+CD4-CD8a-CD11b- cells. (c) Percentages and numbers of GC B cells, DZ and LZ GC B cells identified as shown in b. Each symbol shows data from an individual mouse. (d) Flow cytometry of splenocytes. NP-KLH of B6.SJL mice reconstituted with bone marrow cells from B6.SJL and Cd79a+/+Ptbp1fl/fl or Cd79acre/+Ptbp1fl/fl mice at a 1:1 ratio were immunised with NP-KLH (day 7 post immunisation). Upper plots show pre-gated CD45.2+ or CD45.2- B cells (B220+, CD4-, CD8α- and CD11b-). Numbers in cytometry plots are percentages. (e) Percentages of cells shown in d. Dots shown in bar graphs represent data from individual mice. (f) Anti-NP20 (high + low affinity) or anti-NP2 (high affinity only) IgG1 end-point titres measured by ELISA in the sera of mice 42 days following immunization with NP-KLH. (g) High affinity (NP2) vs. high and low affinity (NP20) anti-NP IgG1 ratio calculated from the data shown in f. (h) Flow cytometry analysis of IgG1+ GC B cells (CD95+CD38lowB220+) from spleens of Cd79acre/+Ptbp1fl/fl or Cd79a+/+Ptbp1fl/fl mice immunised with NP-KLH 7 days previously. Histograms show GC B cells from two representative animals. Dots in bar graphs represent individual animals from the same experiment. (i) Proportion of cDNA clones containing mutated JH4 intronic regions. Genomic DNA was isolated from FACS-sorted GC B cells from Cd79acre/+Ptbp1fl/fl or Cd79a+/+Ptbp1fl/fl mice immunised with NP-KLH (day 7). Numbers in the centre of the graphs show the number of individual clones analysed pooled from two independent experiments. (j) Mutation frequencies in the JH4 intronic region calculated from the data shown in i.The two independent experiments are shown with individual points. (k) Numbers of GC B cells (CD19+ CD38low CD95+), LZ (CD86high CXCR4low), and DZ (CD86low CXCR4high) GC B cells from spleens of mice immunised with SRBCs (day 8). Dots show data from individual mice. In c,e-h,j and k bar graphs show the mean and P-values from unpaired two-tailed Student’s t-test are shown for the comparisons indicated.

Supplementary Figure 4 Ptbp1 deletion results in changes in mRNA abundance and alternative splicing.

(a) MA plots showing genes with different (padj <0.1) mRNA abundance when LZ GC B cells are compared to DZ GC B cells in control Cd79a+/+Ptbp1fl/fl or Cd79acre/+Ptbp1fl/fl cKO mice. Grey dots are all the genes with different mRNA abundance (padj <0.1). Blue dots are those genes that have different mRNA abundance in our mRNAseq libraries and were increased in LZ GC B cells compared to DZ GC B cells in the study published by Victora et al.33. Red dots are the genes that have different mRNA abundance in our mRNAseq libraries and were reduced in LZ GC B cells compared to DZ GC B cells in the study published by Victora et al. (b) Correlation of Log2 fold changes of differentially abundant genes (shown in Fig. 4a) when LZ are compared to DZ GC B cells from Cd79a+/+Ptbp1fl/fl control and Cd79acre/+Ptbp1fl/fl cKO mice. R = Pearson’s correlation coefficient. (c) Correlation of Log2 fold changes of genes that have different (padj <0.1) mRNA abundance due to the lack of PTBP1 in either LZ or DZ shown in Fig. 4b. R = Pearson’s correlation coefficient. (d) Proportions of genes with different (padj <0.1) mRNA abundance (DA) which are bound by PTBP1 to the 3’UTR. (e) The different types of alternatively spliced events analysed with rMATS60. (f) Example of inclusion level (proportion of transcripts including a particular event) and inclusion level difference calculation showing exon 10 of Cers5, which is frequently skipped in control GC B cells. Read coverage is shown for one replicate per condition. Arches with numbers depict the number of reads that map across an exon-exon junction. (g) Overlap of the number of differentially AS events (shown in Fig. 4c,d) observed when LZ GC B cells were compared to DZ GC B cells from Cd79a+/+Ptbp1fl/fl control mice (DS controls DZ - LZ), Cd79a+/+Ptbp1fl/fl control LZ GC B cells were compared to Cd79acre/+Ptbp1fl/fl cKO LZ GC B cells (DS LZ Ctrl – P1 cKO) and Cd79a+/+Ptbp1fl/fl control DZ GC B cells were compared to Cd79acre/+Ptbp1fl/fl cKO DZ GC B cells (DS DZ Ctrl – P1 cKO). (h) Proportions of differentially AS events that are bound by PTBP1. One event was considered to be bound by PTBP1 as detailed in the methods section: Analysis of mRNAseq libraries generated in this study.

Supplementary Figure 5 PTBP1 controls nucleotide biosynthesis pathways.

(a) Biological process gene ontology terms found to be significantly enriched in genes that have different mRNA abundance (DA) due to PTBP1 absence in LZ or DZ GC B cells as shown in Fig. 4b. Numbers in brackets represent the p-value and the FDR q-value, respectively, from the gene ontology enrichment analysis. (b) Log2 fold changes of genes with different (padj<0.1) mRNA abundance, which are part of GO pathways enriched due to Ptbp1 deletion in LZ GC B cells shown in a. (c) Log2 fold changes of genes with different (padj<0.1) mRNA abundance, which are part of GO pathways enriched due to PTBP1 absence in DZ GC B cells shown in a. In b and c genes highlighted in fuchsia were bound by PTBP1 at the 3’UTR, genes highlighted in blue were alternatively spliced with an inclusion level difference >10% and genes highlighted in purple were bound by PTBP1 at the 3’UTR and also alternatively spliced with an inclusion level difference > than 10%.

Supplementary Figure 6 Relevant genes for GC B cell responses in the absence of PTBP1.

(a) Overlaps of genes induced upon GC B cell selection and differentially expressed or alternatively spliced due to Ptbp1 deletion in LZ GC B cells. Supplementary Table 5 contains the different lists of genes used in the overlaps and information on how these lists were generated. (b) Phospho-S6 (Ser240/244) analysed by flow cytometry in non GC B cells (CD19+CD38+CD95-), LZ GC B cells (CD19+CD38lowCD95+CD86highCXCR4low) and DZ GC B cells (CD19+CD38lowCD95+CD86lowCXCR4high) of mice immunised with SRBC (Day 8). Each symbol in bar graphs shows data from an individual mouse. P-values from two-tailed unpaired Student’s t-test are shown for the indicated comparisons. (c) DEseq2 normalised read counts of LZ and DZ GC B cells from Cd79a+/+Ptbp1fl/fl and Cd79acre/+Ptbp1fl/fl mice. Padj values are shown above the comparisons made between cells from Cd79a+/+Ptbp1fl/fl and Cd79acre/+Ptbp1fl/fl mice. Dots depict individual biological replicates. Bars show mean values. (d) gMFI of BCL6 intracellular antibody staining of non GC (CD95-CD38highB220+) and GC (CD95+CD38lowB220+) B cells from Cd79a+/+Ptbp1fl/fl and Cd79acre/+Ptbp1fl/fl mice immunised with NP-KLH (day 7). (e) gMFI of FOXO1 intracellular antibody staining in non CG (CD95-CD38highCD19+), LZ (CD86hiCXCR4low) and DZ (CD86lowCXCR4hi) GC (CD95+CD38lowCD19+) B cells from Cd79a+/+Ptbp1fl/fl and Cd79acre/+Ptbp1fl/fl mice immunised with SRBC (day 8). For c and d, dots shown in bar graphs represent data from individual mice. Bars depict the geometric mean fluorescence mean. P-values from unpaired two-tailed Student’s t-test are shown for the comparisons indicated.

Supplementary Figure 7 PTBP1 regulates cell proliferation in GC B cells.

(a) Gating strategy for cells at different stages of the cell cycle within LZ (CD86hi CXCR4low) or DZ (CD86low CXCR4high) GC B cells (CD95+ CD38low CD19+ B220+) based on BrdU and DNA (7AAD) staining by flow cytometry in cells from spleen 7 days after immunisation with NP-KLH. Numbers shown in cytometry plots are percentages. Graphs show percentages of cells in each phase of the cell cycle. Individual data points represent individual mice. Bars depict the mean. P-values from a two-tailed Student’s t-test are shown above the comparisons made. (b) BrdU and DNA (7AAD) staining of GC B cells (CD95+ CD38low B220+ CD19+) from spleens of mice immunised with SRBCs (day 8). (c) BrdU and DNA (7AAD) staining of CD45.2- or CD45.2+ GC (CD95+ CD38low B220+) B cells from the bone marrow chimera experiment shown in Supplementary Fig. 3d. (d) Analysis of cell cycle progression in developing B cells. Left plots show the gating strategy of Pro, Early and Late Pre B cells. Contour plots show pre-gated B220+ IgD- surface IgM- events from the bone marrow. BrdU and DNA staining shown on the right depict gating strategy of cells in G1/G0, S and G2/M phases. Bar graphs show the mean. Each dot represents data from an individual mouse. P-values from unpaired two-tailed Student’s t-test are shown for the comparisons indicated. In a-c BrdU was administered 1.5 hours before analysis. In a-c Numbers in cytometry plots show percentages. Dots shown in bar graphs represent data from individual mice. Bars depict the mean. P-values from unpaired two-tailed Student’s t-test are shown for the comparisons indicated. (e) DESeq2 normalised read counts of LZ and DZ GC B cells from Cd79a+/+Ptbp1fl/fl control and Cd79acre/+Ptbp1fl/fl cKO mice. Padj values are shown above the comparisons made.

Supplementary Figure 8 Ptbp1 deletion results in PKM1 expression.

Full images from the cropped immunoblot images presented in Fig. 8d.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1-8

Life Sciences Reporting Summary

Supplementary Table 1

PTBP1 iCLIP

Supplementary Table 2

Differential mRNA abundance analysis

Supplementary Table 3

Differential AS analysis

Supplementary Table 4

GOrilla GO enrichment analysis

Supplementary Table 5

Overlap between the c-MYC-dependent gene expression program induced during positive selection and PTBP1-dependent genes

Supplementary Table 6

Reagents including antibodies used in this study

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Monzón-Casanova, E., Screen, M., Díaz-Muñoz, M.D. et al. The RNA-binding protein PTBP1 is necessary for B cell selection in germinal centers. Nat Immunol 19, 267–278 (2018). https://doi.org/10.1038/s41590-017-0035-5

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