Abstract
Plasma cell differentiation requires silencing of B cell transcription, while it establishes antibody-secretory function and long-term survival. The transcription factors Blimp-1 and IRF4 are essential for the generation of plasma cells; however, their function in mature plasma cells has remained elusive. We found that while IRF4 was essential for the survival of plasma cells, Blimp-1 was dispensable for this. Blimp-1-deficient plasma cells retained their transcriptional identity but lost the ability to secrete antibody. Blimp-1 regulated many components of the unfolded protein response (UPR), including XBP-1 and ATF6. The overlap in the functions of Blimp-1 and XBP-1 was restricted to that response, with Blimp-1 uniquely regulating activity of the kinase mTOR and the size of plasma cells. Thus, Blimp-1 was required for the unique physiological ability of plasma cells that enables the secretion of protective antibody.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Change history
17 March 2016
In the version of this article initially published, the label along the vertical axis of the left plot in Figure 3c was incorrect, as was the corresponding text in the legend, and there was an incorrect space between the horizontal axis and curves in the right histogram of Figure 5a. The correct label for Figure 3c is 'ER-Tracker'. The errors have been corrected in the HTML and PDF versions of the article.
References
Tarlinton, D., Radbruch, A., Hiepe, F. & Dorner, T. Plasma cell differentiation and survival. Curr. Opin. Immunol. 20, 162–169 (2008).
MacLennan, I.C. et al. Extrafollicular antibody responses. Immunol. Rev. 194, 8–18 (2003).
Hammarlund, E. et al. Duration of antiviral immunity after smallpox vaccination. Nat. Med. 9, 1131–1137 (2003).
Bettigole, S.E. & Glimcher, L.H. Endoplasmic reticulum stress in immunity. Annu. Rev. Immunol. 33, 107–138 (2015).
Nutt, S.L., Hodgkin, P.D., Tarlinton, D.M. & Corcoran, L.M. The generation of antibody-secreting plasma cells. Nat. Rev. Immunol. 15, 160–171 (2015).
Shi, W. et al. Transcriptional profiling of mouse B cell terminal differentiation defines a signature for antibody-secreting plasma cells. Nat. Immunol. 16, 663–673 (2015).
Kwon, H. et al. Analysis of IL-21-induced Prdm1 gene regulation reveals functional cooperation of STAT3 and IRF4 transcription factors. Immunity 31, 941–952 (2009).
Ochiai, K. et al. Transcriptional regulation of germinal center B and plasma cell fates by dynamical control of IRF4. Immunity 38, 918–929 (2013).
Sciammas, R. et al. Graded expression of interferon regulatory factor-4 coordinates isotype switching with plasma cell differentiation. Immunity 25, 225–236 (2006).
Kallies, A. et al. Initiation of plasma-cell differentiation is independent of the transcription factor Blimp-1. Immunity 26, 555–566 (2007).
Kallies, A. et al. Plasma cell ontogeny defined by quantitative changes in blimp-1 expression. J. Exp. Med. 200, 967–977 (2004).
Shapiro-Shelef, M. et al. Blimp-1 is required for the formation of immunoglobulin secreting plasma cells and pre-plasma memory B cells. Immunity 19, 607–620 (2003).
Lin, K.I., Angelin-Duclos, C., Kuo, T.C. & Calame, K. Blimp-1-dependent repression of Pax-5 is required for differentiation of B cells to immunoglobulin M-secreting plasma cells. Mol. Cell. Biol. 22, 4771–4780 (2002).
Lin, Y., Wong, K. & Calame, K. Repression of c-myc transcription by Blimp-1, an inducer of terminal B cell differentiation. Science 276, 596–599 (1997).
Piskurich, J.F. et al. BLIMP-I mediates extinction of major histocompatibility class II transactivator expression in plasma cells. Nat. Immunol. 1, 526–532 (2000).
Shaffer, A.L. et al. Blimp-1 orchestrates plasma cell differentiation by extinguishing the mature B cell gene expression program. Immunity 17, 51–62 (2002).
Doody, G.M. et al. An extended set of PRDM1/BLIMP1 target genes links binding motif type to dynamic repression. Nucleic Acids Res. 38, 5336–5350 (2010).
Reimold, A.M. et al. Plasma cell differentiation requires the transcription factor XBP-1. Nature 412, 300–307 (2001).
Hu, C.C., Dougan, S.K., McGehee, A.M., Love, J.C. & Ploegh, H.L. XBP-1 regulates signal transduction, transcription factors and bone marrow colonization in B cells. EMBO J. 28, 1624–1636 (2009).
Shaffer, A.L. et al. XBP1, downstream of Blimp-1, expands the secretory apparatus and other organelles, and increases protein synthesis in plasma cell differentiation. Immunity 21, 81–93 (2004).
Taubenheim, N. et al. High rate of antibody secretion is not integral to plasma cell differentiation as revealed by XBP-1 deficiency. J. Immunol. 189, 3328–3338 (2012).
Todd, D.J. et al. XBP1 governs late events in plasma cell differentiation and is not required for antigen-specific memory B cell development. J. Exp. Med. 206, 2151–2159 (2009).
Shaffer, A.L. et al. IRF4 addiction in multiple myeloma. Nature 454, 226–231 (2008).
Shapiro-Shelef, M. & Calame, K. Regulation of plasma-cell development. Nat. Rev. Immunol. 5, 230–242 (2005).
Kallies, A., Xin, A., Belz, G.T. & Nutt, S.L. Blimp-1 transcription factor is required for the differentiation of effector CD8+ T cells and memory responses. Immunity 31, 283–295 (2009).
Klein, U. et al. Transcription factor IRF4 controls plasma cell differentiation and class-switch recombination. Nat. Immunol. 7, 773–782 (2006).
Seibler, J. et al. Rapid generation of inducible mouse mutants. Nucleic Acids Res. 31, e12 (2003).
Shapiro-Shelef, M., Lin, K.I., Savitsky, D., Liao, J. & Calame, K. Blimp-1 is required for maintenance of long-lived plasma cells in the bone marrow. J. Exp. Med. 202, 1471–1476 (2005).
Minnich, M. et al. Multifunctional role of the transcription factor Blimp1 in coordinating plasma cell differentiation. Nat. Immunol. doi:10.1038/ni.3349 (18 January 2016).
Peperzak, V. et al. Mcl-1 is essential for the survival of plasma cells. Nat. Immunol. 14, 290–297 (2013).
O'Connor, B.P. et al. BCMA is essential for the survival of long-lived bone marrow plasma cells. J. Exp. Med. 199, 91–98 (2004).
Bayles, I. & Milcarek, C. Plasma cell formation, secretion, and persistence: the short and the long of it. Crit. Rev. Immunol. 34, 481–499 (2014).
Martincic, K., Alkan, S.A., Cheatle, A., Borghesi, L. & Milcarek, C. Transcription elongation factor ELL2 directs immunoglobulin secretion in plasma cells by stimulating altered RNA processing. Nat. Immunol. 10, 1102–1109 (2009).
Park, K.S. et al. Transcription elongation factor ELL2 drives Ig secretory-specific mRNA production and the unfolded protein response. J. Immunol. 193, 4663–4674 (2014).
Brewer, J.W. Regulatory crosstalk within the mammalian unfolded protein response. Cell. Mol. Life Sci. 71, 1067–1079 (2014).
Yoshida, H., Matsui, T., Yamamoto, A., Okada, T. & Mori, K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107, 881–891 (2001).
Calfon, M. et al. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 415, 92–96 (2002).
Powell, J.D., Pollizzi, K.N., Heikamp, E.B. & Horton, M.R. Regulation of immune responses by mTOR. Annu. Rev. Immunol. 30, 39–68 (2012).
Edinger, A.L. & Thompson, C.B. Akt maintains cell size and survival by increasing mTOR-dependent nutrient uptake. Mol. Biol. Cell 13, 2276–2288 (2002).
Lee, J.H., Budanov, A.V. & Karin, M. Sestrins orchestrate cellular metabolism to attenuate aging. Cell Metab. 18, 792–801 (2013).
Chen, C.C. et al. FoxOs inhibit mTORC1 and activate Akt by inducing the expression of Sestrin3 and Rictor. Dev. Cell 18, 592–604 (2010).
Leung-Hagesteijn, C. et al. Xbp1s-negative tumor B cells and pre-plasmablasts mediate therapeutic proteasome inhibitor resistance in multiple myeloma. Cancer Cell 24, 289–304 (2013).
Lohr, J.G. et al. Widespread genetic heterogeneity in multiple myeloma: implications for targeted therapy. Cancer Cell 25, 91–101 (2014).
Brinkmann, V. & Heusser, C.H. T cell-dependent differentiation of human B cells into IgM, IgG, IgA, or IgE plasma cells: high rate of antibody production by IgE plasma cells, but limited clonal expansion of IgE precursors. Cell. Immunol. 152, 323–332 (1993).
Gass, J.N., Gifford, N.M. & Brewer, J.W. Activation of an unfolded protein response during differentiation of antibody-secreting B cells. J. Biol. Chem. 277, 49047–49054 (2002).
Benhamron, S., Pattanayak, S.P., Berger, M. & Tirosh, B. mTOR activation promotes plasma cell differentiation and bypasses XBP-1 for immunoglobulin secretion. Mol. Cell. Biol. 35, 153–166 (2015).
Goldfinger, M., Shmuel, M., Benhamron, S. & Tirosh, B. Protein synthesis in plasma cells is regulated by crosstalk between endoplasmic reticulum stress and mTOR signaling. Eur. J. Immunol. 41, 491–502 (2011).
Hasbold, J., Corcoran, L.M., Tarlinton, D.M., Tangye, S.G. & Hodgkin, P.D. Evidence from the generation of immunoglobulin G-secreting cells that stochastic mechanisms regulate lymphocyte differentiation. Nat. Immunol. 5, 55–63 (2004).
Liao, Y., Smyth, G.K. & Shi, W. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 41, e108 (2013).
Liao, Y., Smyth, G.K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
Law, C.W., Chen, Y., Shi, W. & Smyth, G.K. voom: Precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 15, R29 (2014).
Ritchie, M.E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).
Smyth, G.K. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, Article3 (2004).
Wu, D. et al. ROAST: rotation gene set tests for complex microarray experiments. Bioinformatics 26, 2176–2182 (2010).
Ebert, A. et al. The distal VH gene cluster of the Igh locus contains distinct regulatory elements with Pax5 transcription factor-dependent activity in pro-B cells. Immunity 34, 175–187 (2011).
Acknowledgements
We thank U. Klein (Columbia University) for Irf4fl/fl mice; L. Glimcher (Weill Cornell Medical College) for Xbp1fl/fl mice; S. Wilcox, M. Chopin and C. Seillet for technical assistance; and J. Leahy for animal care. Supported by the National Health and Medical Research Council of Australia (G.K.S. and S.L.N.; 361646, 575500 and 1054925 to S.L.N.; 1054618 to G.K.S.; 1023454 to G.K.S. and W.S.; and 1049416 to A.K.), the Sylvia and Charles Viertel Foundation (A.K.), the Multiple Myeloma Research Foundation (S.L.N.), Boehringer Ingelheim (Busslinger laboratory) and the European Research Council (291740-LymphoControl for the Busslinger laboratory), and made possible through Victorian State Government Operational Infrastructure Support.
Author information
Authors and Affiliations
Contributions
J.T. performed most experiments; W.S., Y.L. and G.K.S. performed the bioinformatics and statistical analyses; M.M. and M.B. provided data for chromatin immunoprecipitation followed by deep sequencing; S.C. performed electron microscopy; A.K. provided mouse models; S.L.N. supervised the study; and J.T. and S.L.N. wrote the manuscript, for which all authors provided editorial input.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Kinetics of PC survival in the absence of IRF4 or Blimp-1.
(a-e) In vivo gene ablation. (a-b) Schematic representation of the experimental plan. Cell transfer (a, c) and intact mice (b, d) as in Fig. 1a, b. (c) Left panel, frequency of Irf4fl/+CreERT2 or Irf4fl/-CreERT2 GFP+ spleen PCs (out of total spleen cells) was determined at the indicated day after mice were treated with tamoxifen treatment to induce Irf4 inactivation (reported by GFP expression). PCs were identified as CD138+B220lo. Right panel, frequency of Prdm1+/gfpCreERT2 (+/gfp) or Prdm1fl/gfpCreERT2 (fl/gfp) spleen PCs at the indicated day after tamoxifen treatment. PCs were identified as CD138+Blimp1-GFP+. (d) Frequency of spleen PCs from Prdm1+/gfpCreERT2 or Prdm1fl/gfpCreERT2 mice at the indicated day after tamoxifen treatment. Symbols represent data from a single mouse. Mean value is shown by a horizontal line. (e) Prdm1+/gfpCreERT2 or Prdm1fl/gfpCreERT2 mice were immunized with NP-KLH precipitated in alum and then treated with tamoxifen to induce Prdm1 inactivation 28 days later. PCs were identified as CD138+Blimp1-GFP+. Graphs show the proportion of PCs in spleen (left) and BM (right) at different time points after tamoxifen treatment. Each symbol represents an individual recipient mouse. Horizontal line is the mean. (f) In vitro gene inactivation. B cells were isolated from Prdm1+/gfpCreERT2 or Prdm1fl/gfpCreERT2 spleens and cultured with CD40L, IL-4 and IL-5 for 5 days. Cells were treated with 4-hydroxytamoxifen (100nM) at the indicated time points to induce Prdm1 inactivation. Cytometry profiles at day 5 of a representative experiment of 4 experiments. Boxes indicate the proportion of CD138+B220low PBs. P values compare the indicated groups using a paired t-test. * P<0.05, ** P<0.005.
Supplementary Figure 2 Efficient inactivation of Prdm1 and Xbp1 after tamoxifen treatment.
(a) Rag1−/− mice injected with isolated Prdm1+/gfpCreERT2 (+/gfp) or Prdm1fl/gfpCreERT2 (fl/gfp) B cells were treated with tamoxifen 2 days after transfer to induce Prdm1 inactivation. Splenic B cells (CD19+CD22+) and PCs (CD138+Blimp1-GFP+) 7 days after tamoxifen treatment are shown for a representative of 2 experiments. Each symbol represents a single recipient mouse. Horizontal line shows the mean. (b) PCR analysis showing the loss of the Prdm1 floxed allele (fl, 765 bp) in BM B cells (CD19+CD22+) and PCs (CD138+Blimp1-GFP+) from intact mice of the indicated genotypes, 21 days after tamoxifen treatment (as in Supplementary Fig. 1b). The location of the wild type (+, 611bp) and deleted alleles (-, 645bp) are indicated. Tail DNA from an untreated mouse was used as a control. (c) Abundance of junction read spanning exons 5-6 or exons 4-6, in Prdm1+/gfpCreERT2 or Prdm1fl/gfpCreERT2 PC RNAseq data. Shown is the number of junction reads per million mapped reads (RPM), excluding Ig sequences. Junction reads were identified using the Subjunc aligner. Data are the mean of two samples for each genotype. Comparison of exon 5-6 reads from Prdm1+/gfpCreERT2 or Prdm1fl/gfpCreERT2 indicates that the efficiency of deletion of the Prdm1 exon 5 was 87%. Reads spanning exons 4-6 only occur after exon 5 excision and are shown as a specificity control. (d) RNA sequencing tracks for Xbp1 in Xbp1+/+Prdm1+/gfpCreERT2 and Xbp1fl/flPrdm1+/gfpCreERT2 PCs 21 days after mice were treated with tamoxifen. The tamoxifen treatment has induced the complete deletion of Xbp1 exon 2. Data is representative of 2 experiments.
Supplementary Figure 3 Points at which Blimp-1 regulates the processing of Igh mRNA, the UPR and the mTOR pathway.
(a) Blimp-1 promotes the expression of the secreted form of Igh. In PCs two isoforms exist for each Ig isotype. Using Ighm as an example the minority membrane-bound isoform includes, in addition to the first four exons, two membrane specific exons encoding the transmembrane domain. The truncated secreted form uses an earlier polyadenylation signal sequence and is comprised of the first four exons and a specific secreted sequence upstream of the new polyadenylation site. Ell2, a direct activated target of Blimp-1, favors the processing of the secreted form of the Igh mRNA in PCs. (b) Blimp-1 regulation at the apex of the unfolded protein response (UPR). The UPR is composed of three arms; each one triggered through a transmembrane sensor (Ire1, Perk, Atf6) leads to the activation of a specific transcription factor (Xbp1s, Atf4, cleaved Atf6). Blimp-1 directly induces Atf6 and Ern1/Ire1 expression. Blimp-1 loss also significantly impacts Atf4 transcripts, through an indirect mechanism. As a result, Blimp-1 promotes the activity of all three arms of the UPR. (c) Blimp-1 promotes mTOR activity through the control of the upstream regulators of the pathway. Blimp-1 activates several crucial amino acid carriers (CD98, Asct2, PAT1) and the transferrin receptor (CD71) that activate mTORC1 kinase activity. In parallel, Blimp-1 represses transcription of Sestrin-1 and -3 (Sesn), preventing the inhibitory action of AMPK. Solid orange lines indicate direct regulation (as evidenced by Blimp-1 binding in the respective loci) and dashed lines denote indirect regulation.
Supplementary Figure 4 Blimp-1 and Xbp1 regulate distinct sets of target genes.
Whole genome RNA-sequencing analysis on BM PCs, analyzed as described in Fig. 2 and 6. (a) Graph shows the log2-Fold change (FC) of expression of the unfolded protein response (UPR) genes (as listed in Supplementary Table 3) for BM PCs from Prdm1fl/gfpCreERT2 versus Prdm1+/gfpCreERT2 and Xbp1fl/flPrdm1+/gfpCreERT2 versus Xbp1+/+Prdm1+/gfpCreERT2 mice. Genes are categorized depending the process their encoded protein is involved in. The mean FC for each genotype and category is shown by a horizontal line. (b) Venn diagram showing overlap and differences between Blimp-1 and Xbp1 target genes (FDR <0.05, normalized average expression ≥4 RPKM in at least one sample). (c) Heat map shows the expression of the signature genes of wild type follicular B cells (FoB)5 and BM PCs5 from Xbp1+/+Prdm1+/gfpCreERT2 (+/+) and Xbp1fl/flPrdm1+/gfpCreERT2 (fl/fl) mice. Data derive from 2 experiments.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–4 and Supplementary Tables 1–3 (PDF 1719 kb)
Rights and permissions
About this article
Cite this article
Tellier, J., Shi, W., Minnich, M. et al. Blimp-1 controls plasma cell function through the regulation of immunoglobulin secretion and the unfolded protein response. Nat Immunol 17, 323–330 (2016). https://doi.org/10.1038/ni.3348
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ni.3348
This article is cited by
-
An epithelial cell-derived metabolite tunes immunoglobulin A secretion by gut-resident plasma cells
Nature Immunology (2023)
-
Clinical Relevance of Interferon Regulatory Family-4 (IRF4) Expression in Newly Diagnosed Patients with Multiple Myeloma
Indian Journal of Hematology and Blood Transfusion (2023)
-
UTX inactivation in germinal center B cells promotes the development of multiple myeloma with extramedullary disease
Leukemia (2023)
-
RNA sequencing identifies novel regulated IRE1-dependent decay targets that affect multiple myeloma survival and proliferation
Experimental Hematology & Oncology (2022)
-
Transcriptome signatures preceding the induction of anti-stalk antibodies elicited after universal influenza vaccination
npj Vaccines (2022)