Abstract
When B cells encounter an antigen, they alter their physiological state and anatomical localization and initiate a differentiation process that ultimately produces antibody-secreting cells (ASCs). We have defined the transcriptomes of many mature B cell populations and stages of plasma cell differentiation in mice. We provide a molecular signature of ASCs that highlights the stark transcriptional divide between B cells and plasma cells and enables the demarcation of ASCs on the basis of location and maturity. Changes in gene expression correlated with cell-division history and the acquisition of permissive histone modifications, and they included many regulators that had not been previously implicated in B cell differentiation. These findings both highlight and expand the core program that guides B cell terminal differentiation and the production of antibodies.
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Acknowledgements
We thank D. Emslie, T. Kratina, M. Everest and S. Heinzel for technical assistance and J. Vasiliadis, J. Leahy and T. Camilleri for animal care. Socs2−/− mice were a gift from W. Alexander (The Walter and Eliza Hall Institute, Parkville, Victoria, Australia). The institutional flow cytometry facility provided essential services. Supported by the National Health and Medical Research Council (NHMRC) of Australia (NHMRC IRIISS grant 361646; Program Grants 575500 and 1054925 to D.M.T., P.D.H., S.L.N. and L.M.C.; Program Grant 1054618 to G.K.S.; Project Grant 1023454 to G.K.S. and W.S.; and fellowships to G.K.S., D.M.T., P.D.H. and L.M.C.), the Multiple Myeloma Research Foundation (Senior Award to S.L.N.), the Australian Research Council (ARC Future Fellowship to S.L.N.) and the Victorian State Government through an Operational Infrastructure Support grant.
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W.S., Y.L., G.K.S. and M.I. did the bioinformatic and statistical analyses; S.N.W. and N.T. did experiments relating to in vitro differentiation and cell-division studies; S.L.N., D.M.T., P.D.H. and L.M.C. contributed to the experimental design and analysis, and provided mouse models; L.M.C. carried out many of the experiments; W.S., S.L.N. and L.M.C. wrote the manuscript; all authors had editorial input.
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Integrated supplementary information
Supplementary Figure 1 Fraction of absolutely expressed genes in each cell population.
A detection P value cutoff of ≤0.05 was applied for calling absolutely expressed genes. 26k NBCI RefSeq genes were included in the analysis. See the Online Methods for a description of the algorithm used for calling absolutely expressed genes.
Supplementary Figure 2 Expression profiles of selected genes encoding chemokines, cytokines and their receptors in ex vivo B cell and ASC populations.
Three groups of genes are highlighted here: (a) Sphingosine-1-phosphate receptor, (b) chemokines and chemokine receptors and (c) cytokine receptors. Normalized FPKM values of genes are shown.
Supplementary Figure 3 Patterns of gene expression change during the cell-division process.
This figure is the same as Fig. 6f in the main text, except that gene expression values are shown instead of expression fold changes. The k-means clustering algorithm was used to classify the genes, which had ≥2.5-fold changes at the division stage (2,095 genes), into nine clusters based on stepwise expression changes from Div 0 to Div 7. These nine clusters were then further grouped into six distinct patterns. Numbers shown in upper corners of the figures indicate the number of genes included in each cluster.
Supplementary Figure 4 Chromatin changes during B cell differentiation.
(a) Correlation of gene expression changes and the prevalence of H3K4me3 and H3K4me1 in the A20 (GC-like B lymphoma) and MPC-11 (plasmacytoma) cell lines. The top 500 genes most differentially expressed between A20 and MPC11 were included in the analysis. Values of Pearson correlation coefficients calculated with these genes are shown at the top left corner of each graph. The red diagonal lines indicate the slope of linear regression of histone mark changes on gene expression changes. (b) Correlation of the H3K4me3 and H3K4me1 data from the A20 and MPC-11 cell lines with gene expression changes between their primary cell counterparts, GC B cells and BMPCs, respectively. Here the top 500 most differentially expressed genes between GC B cells and BMPCs were included in the graphs. (c) Barcode plot showing that super-enhancer–associated genes (756 genes) tend to be upregulated in FoBs relative to BMPCs. The horizontal axis shows TREAT t-statistics for all genes comparing FoBs to BMPCs, and the vertical bars show the ranks of the super-enhancer genes. The worm shows enrichment of super-enhancer genes relative to random ordering. Roast P values are shown here and for d. (d) Barcode plot showing that super-enhancer–associated genes tend to be upregulated in GC B cells relative to BMPCs.
Supplementary Figure 5 Expression profiles of genes encoding transcription factors and chromatin-associated proteins that are specifically expressed in B cells.
(a–d) Normalized FPKM values of genes in ex vivo B cell and ASC populations: (a) B cells including FoBs, MZBs, B1 cells and GC B cells; (b) GC B cells; (c) MZBs; and (d) B1 cells.
Supplementary Figure 6 Expression profiles of genes encoding transcription factors and chromatin-associated proteins that are specifically expressed in ASCs.
Shown are normalized FPKM values of genes in ex vivo B cell and ASC populations.
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Supplementary Text and Figures
Supplementary Figures 1–6 (PDF 794 kb)
Supplementary Table 1
The list of ASC signature genes discovered in the study (XLSX 83 kb)
Supplementary Table 2
Lists of genes that are shared between or unique to different ASC populations on the basis of comparison with FoBs. These gene lists correspond to different sets of genes included in Fig. 4e,f. (XLSX 488 kb)
Supplementary Table 3
Lists of genes that are shared between or unique to TI and TD plasmablasts on the basis of comparison with B cell blasts. These gene lists correspond to different sets of genes included in Fig. 5a,b. (XLSX 327 kb)
Supplementary Table 4
Summary of library sizes, read mapping percentages and read quantification results for RNA-seq data. Column "% mapped" gives the percentage of mapped reads. Column "% assigned" gives the percentage of fragments assigned to NCBI RefSeq genes, out of all the fragments included in each library. In each paired-end library, a fragment represents a pair of reads. In each single-end library, a fragment represents a read. (XLSX 51 kb)
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Shi, W., Liao, Y., Willis, S. et al. Transcriptional profiling of mouse B cell terminal differentiation defines a signature for antibody-secreting plasma cells. Nat Immunol 16, 663–673 (2015). https://doi.org/10.1038/ni.3154
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DOI: https://doi.org/10.1038/ni.3154
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