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Essential role for the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells

Nature Immunology volume 18, pages 442–455 (2017)Cite this article

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Abstract

Innate-like B-1a cells provide a first line of defense against pathogens, yet little is known about their transcriptional control. Here we identified an essential role for the transcription factor Bhlhe41, with a lesser contribution by Bhlhe40, in controlling B-1a cell differentiation. Bhlhe41−/−Bhlhe40−/− B-1a cells were present at much lower abundance than were their wild-type counterparts. Mutant B-1a cells exhibited an abnormal cell-surface phenotype and altered B cell receptor (BCR) repertoire exemplified by loss of the phosphatidylcholine-specific VH12Vκ4 BCR. Expression of a pre-rearranged VH12Vκ4 BCR failed to 'rescue' the mutant phenotype and revealed enhanced proliferation accompanied by increased cell death. Bhlhe41 directly repressed the expression of cell-cycle regulators and inhibitors of BCR signaling while enabling pro-survival cytokine signaling. Thus, Bhlhe41 controls the development, BCR repertoire and self-renewal of B-1a cells.

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Figure 1: B-1a cells depend on the transcription factors Bhlhe41 and Bhlhe40.
Figure 2: The residual DKO B-1a cells exhibit an altered BCR repertoire.
Figure 3: Regulation of B-1a cell development by Bhlhe41 and Bhlhe40.
Figure 4: Expression of pre-rearranged VH12 and Vκ4 transgenes fails to 'rescue' the DKO phenotype.
Figure 5: Identification of regulated Bhlhe41 target genes in B-1a cells.
Figure 6: Decreased BCR signaling in VH12Vκ4-transgenic DKO B-1a cells.
Figure 7: DKO B-1a cells exhibit increased proliferation.
Figure 8: Bhlhe41 and Bhlhe40 regulate the self-renewal of B-1a cells.

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References

  1. Hayakawa, K., Hardy, R.R., Parks, D.R. & Herzenberg, L.A. The “Ly-1 B” cell subpopulation in normal immunodefective, and autoimmune mice. J. Exp. Med. 157, 202–218 (1983).

    Article  CAS  Google Scholar 

  2. Baumgarth, N. The double life of a B-1 cell: self-reactivity selects for protective effector functions. Nat. Rev. Immunol. 11, 34–46 (2011).

    Article  CAS  Google Scholar 

  3. Montecino-Rodriguez, E. & Dorshkind, K. B-1 B cell development in the fetus and adult. Immunity 36, 13–21 (2012).

    Article  CAS  Google Scholar 

  4. Casola, S. et al. B cell receptor signal strength determines B cell fate. Nat. Immunol. 5, 317–327 (2004).

    Article  CAS  Google Scholar 

  5. Hardy, R.R. & Hayakawa, K. B cell development pathways. Annu. Rev. Immunol. 19, 595–621 (2001).

    Article  CAS  Google Scholar 

  6. Hoffmann, A. et al. Siglec-G is a B1 cell-inhibitory receptor that controls expansion and calcium signaling of the B1 cell population. Nat. Immunol. 8, 695–704 (2007).

    Article  CAS  Google Scholar 

  7. Pan, C., Baumgarth, N. & Parnes, J.R. CD72-deficient mice reveal nonredundant roles of CD72 in B cell development and activation. Immunity 11, 495–506 (1999).

    Article  CAS  Google Scholar 

  8. Binder, C.J. Natural IgM antibodies against oxidation-specific epitopes. J. Clin. Immunol. 30, 56–60 (2010).

    Article  Google Scholar 

  9. Wang, H. & Clarke, S.H. Positive selection focuses the VH12 B-cell repertoire towards a single B1 specificity with survival function. Immunol. Rev. 197, 51–59 (2004).

    Article  CAS  Google Scholar 

  10. Sohlenkamp, C., López-Lara, I.M. & Geiger, O. Biosynthesis of phosphatidylcholine in bacteria. Prog. Lipid Res. 42, 115–162 (2003).

    Article  CAS  Google Scholar 

  11. Lalor, P.A., Herzenberg, L.A., Adams, S. & Stall, A.M. Feedback regulation of murine Ly-1 B cell development. Eur. J. Immunol. 19, 507–513 (1989).

    Article  CAS  Google Scholar 

  12. Kristiansen, T.A. et al. Cellular barcoding links B-1a B cell potential to a fetal hematopoietic stem cell state at the single-cell level. Immunity 45, 346–357 (2016).

    Article  CAS  Google Scholar 

  13. Yang, Y. et al. Distinct mechanisms define murine B cell lineage immunoglobulin heavy chain (IgH) repertoires. eLife 4, e09083 (2015).

    Article  Google Scholar 

  14. Gu, H., Förster, I. & Rajewsky, K. Sequence homologies, N sequence insertion and JH gene utilization in VHDJH joining: implications for the joining mechanism and the ontogenetic timing of Ly1 B cell and B-CLL progenitor generation. EMBO J. 9, 2133–2140 (1990).

    Article  CAS  Google Scholar 

  15. Düber, S. et al. Induction of B-cell development in adult mice reveals the ability of bone marrow to produce B-1a cells. Blood 114, 4960–4967 (2009).

    Article  Google Scholar 

  16. Herzenberg, L.A. & Herzenberg, L.A. Toward a layered immune system. Cell 59, 953–954 (1989).

    Article  CAS  Google Scholar 

  17. Montecino-Rodriguez, E., Leathers, H. & Dorshkind, K. Identification of a B-1 B cell-specified progenitor. Nat. Immunol. 7, 293–301 (2006).

    Article  CAS  Google Scholar 

  18. Montecino-Rodriguez, E. et al. Distinct genetic networks orchestrate the emergence of specific waves of fetal and adult B-1 and B-2 development. Immunity 45, 527–539 (2016).

    Article  CAS  Google Scholar 

  19. Haughton, G., Arnold, L.W., Whitmore, A.C. & Clarke, S.H. B-1 cells are made, not born. Immunol. Today 14, 84–87 (1993).

    Article  CAS  Google Scholar 

  20. Hayakawa, K. et al. Positive selection of natural autoreactive B cells. Science 285, 113–116 (1999).

    Article  CAS  Google Scholar 

  21. Berland, R. & Wortis, H.H. Origins and functions of B-1 cells with notes on the role of CD5. Annu. Rev. Immunol. 20, 253–300 (2002).

    Article  CAS  Google Scholar 

  22. Zhou, Y. et al. Lin28b promotes fetal B lymphopoiesis through the transcription factor Arid3a. J. Exp. Med. 212, 569–580 (2015).

    Article  CAS  Google Scholar 

  23. Yuan, J., Nguyen, C.K., Liu, X., Kanellopoulou, C. & Muljo, S.A. Lin28b reprograms adult bone marrow hematopoietic progenitors to mediate fetal-like lymphopoiesis. Science 335, 1195–1200 (2012).

    Article  CAS  Google Scholar 

  24. Humbert, P.O. & Corcoran, L.M. oct-2 gene disruption eliminates the peritoneal B-1 lymphocyte lineage and attenuates B-2 cell maturation and function. J. Immunol. 159, 5273–5284 (1997).

    CAS  PubMed  Google Scholar 

  25. Pedersen, G.K. et al. B-1a transitional cells are phenotypically distinct and are lacking in mice deficient in IκBNS. Proc. Natl. Acad. Sci. USA 111, E4119–E4126 (2014).

    Article  CAS  Google Scholar 

  26. Kato, Y., Kawamoto, T., Fujimoto, K. & Noshiro, M. DEC1/STRA13/SHARP2 and DEC2/SHARP1 coordinate physiological processes, including circadian rhythms in response to environmental stimuli. Curr. Top. Dev. Biol. 110, 339–372 (2014).

    Article  CAS  Google Scholar 

  27. Ow, J.R., Tan, Y.H., Jin, Y., Bahirvani, A.G. & Taneja, R. Stra13 and Sharp-1, the non-grouchy regulators of development and disease. Curr. Top. Dev. Biol. 110, 317–338 (2014).

    Article  CAS  Google Scholar 

  28. Sun, H., Lu, B., Li, R.Q., Flavell, R.A. & Taneja, R. Defective T cell activation and autoimmune disorder in Stra13-deficient mice. Nat. Immunol. 2, 1040–1047 (2001).

    Article  CAS  Google Scholar 

  29. Yang, X.O. et al. Requirement for the basic helix-loop-helix transcription factor Dec2 in initial TH2 lineage commitment. Nat. Immunol. 10, 1260–1266 (2009).

    Article  CAS  Google Scholar 

  30. Lin, C.-C. et al. Bhlhe40 controls cytokine production by T cells and is essential for pathogenicity in autoimmune neuroinflammation. Nat. Commun. 5, 3551 (2014).

    Article  Google Scholar 

  31. Vilagos, B. et al. Essential role of EBF1 in the generation and function of distinct mature B cell types. J. Exp. Med. 209, 775–792 (2012).

    Article  CAS  Google Scholar 

  32. Heng, T.S. & Painter, M.W. The Immunological Genome Project: networks of gene expression in immune cells. Nat. Immunol. 9, 1091–1094 (2008).

    Article  CAS  Google Scholar 

  33. Baier, P.C. et al. Mice lacking the circadian modulators SHARP1 and SHARP2 display altered sleep and mixed state endophenotypes of psychiatric disorders. PLoS One 9, e110310 (2014).

    Article  Google Scholar 

  34. Honma, S. et al. Dec1 and Dec2 are regulators of the mammalian molecular clock. Nature 419, 841–844 (2002).

    Article  CAS  Google Scholar 

  35. Wöhner, M. et al. Molecular functions of the transcription factors E2A and E2–2 in controlling germinal center B cell and plasma cell development. J. Exp. Med. 213, 1201–1221 (2016).

    Article  Google Scholar 

  36. Seimiya, M. et al. Clast5/Stra13 is a negative regulator of B lymphocyte activation. Biochem. Biophys. Res. Commun. 292, 121–127 (2002).

    Article  CAS  Google Scholar 

  37. Arnold, L.W., Pennell, C.A., McCray, S.K. & Clarke, S.H. Development of B-1 cells: segregation of phosphatidylcholine-specific B cells to the B-1 population occurs after immunoglobulin gene expression. J. Exp. Med. 179, 1585–1595 (1994).

    Article  CAS  Google Scholar 

  38. Jolma, A. et al. DNA-binding specificities of human transcription factors. Cell 152, 327–339 (2013).

    Article  CAS  Google Scholar 

  39. Buenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y. & Greenleaf, W.J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).

    Article  CAS  Google Scholar 

  40. Holodick, N.E., Tumang, J.R. & Rothstein, T.L. Continual signaling is responsible for constitutive ERK phosphorylation in B-1a cells. Mol. Immunol. 46, 3029–3036 (2009).

    Article  CAS  Google Scholar 

  41. Wong, S.-C. et al. Peritoneal CD5+ B-1 cells have signaling properties similar to tolerant B cells. J. Biol. Chem. 277, 30707–30715 (2002).

    Article  CAS  Google Scholar 

  42. Chew, V. & Lam, K.-P. Leupaxin negatively regulates B cell receptor signaling. J. Biol. Chem. 282, 27181–27191 (2007).

    Article  CAS  Google Scholar 

  43. Rickert, R.C., Rajewsky, K. & Roes, J. Impairment of T-cell-dependent B-cell responses and B-1 cell development in CD19-deficient mice. Nature 376, 352–355 (1995).

    Article  CAS  Google Scholar 

  44. Yoshida, T. et al. Defective B-1 cell development and impaired immunity against Angiostrongylus cantonensis in IL-5Rα-deficient mice. Immunity 4, 483–494 (1996).

    Article  CAS  Google Scholar 

  45. Moon, B.-g., Takaki, S., Miyake, K. & Takatsu, K. The role of IL-5 for mature B-1 cells in homeostatic proliferation, cell survival, and Ig production. J. Immunol. 172, 6020–6029 (2004).

    Article  CAS  Google Scholar 

  46. Scott, C.L. et al. Reassessment of interactions between hematopoietic receptors using common β-chain and interleukin-3-specific receptor beta-chain-null cells: no evidence of functional interactions with receptors for erythropoietin, granulocyte colony-stimulating factor, or stem cell factor. Blood 96, 1588–1590 (2000).

    CAS  PubMed  Google Scholar 

  47. Arnold, L.W., McCray, S.K., Tatu, C. & Clarke, S.H. Identification of a precursor to phosphatidyl choline-specific B-1 cells suggesting that B-1 cells differentiate from splenic conventional B cells in vivo: cyclosporin A blocks differentiation to B-1. J. Immunol. 164, 2924–2930 (2000).

    Article  CAS  Google Scholar 

  48. Guo, B. & Rothstein, T.L. RasGRP1 Is an essential signaling molecule for development of B1a cells with autoantigen receptors. J. Immunol. 196, 2583–2590 (2016).

    Article  CAS  Google Scholar 

  49. Seimiya, M. et al. Impaired lymphocyte development and function in Clast5/Stra13/DEC1-transgenic mice. Eur. J. Immunol. 34, 1322–1332 (2004).

    Article  CAS  Google Scholar 

  50. Hipfner, D.R. & Cohen, S.M. Connecting proliferation and apoptosis in development and disease. Nat. Rev. Mol. Cell Biol. 5, 805–815 (2004).

    Article  CAS  Google Scholar 

  51. Rossner, M.J. et al. Disturbed clockwork resetting in Sharp-1 and Sharp-2 single and double mutant mice. PLoS One 3, e2762 (2008).

    Article  Google Scholar 

  52. Kwon, K. et al. Instructive role of the transcription factor E2A in early B lymphopoiesis and germinal center B cell development. Immunity 28, 751–762 (2008).

    Article  CAS  Google Scholar 

  53. Tallquist, M.D. & Soriano, P. Epiblast-restricted Cre expression in MORE mice: a tool to distinguish embryonic vs. extra-embryonic gene function. Genesis 26, 113–115 (2000).

    Article  CAS  Google Scholar 

  54. Kretschmer, K., Stopkowicz, J., Scheffer, S., Greten, T.F. & Weiss, S. Maintenance of peritoneal B-1a lymphocytes in the absence of the spleen. J. Immunol. 173, 197–204 (2004).

    Article  CAS  Google Scholar 

  55. Parkhomchuk, D. et al. Transcriptome analysis by strand-specific sequencing of complementary DNA. Nucleic Acids Res. 37, e123 (2009).

    Article  Google Scholar 

  56. Schebesta, A. et al. Transcription factor Pax5 activates the chromatin of key genes involved in B cell signaling, adhesion, migration, and immune function. Immunity 27, 49–63 (2007).

    Article  CAS  Google Scholar 

  57. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).

    Article  CAS  Google Scholar 

  58. Feng, J., Liu, T., Qin, B., Zhang, Y. & Liu, X.S. Identifying ChIP-seq enrichment using MACS. Nat. Protoc. 7, 1728–1740 (2012).

    Article  CAS  Google Scholar 

  59. Revilla-i-Domingo, R. et al. The B-cell identity factor Pax5 regulates distinct transcriptional programmes in early and late B lymphopoiesis. EMBO J. 31, 3130–3146 (2012).

    Article  CAS  Google Scholar 

  60. Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  61. Aszodi, A. MULTOVL: fast multiple overlaps of genomic regions. Bioinformatics 28, 3318–3319 (2012).

    Article  CAS  Google Scholar 

  62. Machanick, P. & Bailey, T.L. MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics 27, 1696–1697 (2011).

    Article  CAS  Google Scholar 

  63. Trapnell, C., Pachter, L. & Salzberg, S.L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).

    Article  CAS  Google Scholar 

  64. Cunningham, F. et al. Ensembl 2015. Nucleic Acids Res. 43, D662–D669 (2015).

    Article  CAS  Google Scholar 

  65. Anders, S., Pyl, P.T. & Huber, W. HTSeq–a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).

    Article  CAS  Google Scholar 

  66. Love, M.I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    Article  Google Scholar 

  67. Wagner, G.P., Kin, K. & Lynch, V.J. Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples. Theory Biosci. 131, 281–285 (2012).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S.H. Clarke and S.-R. Lee (University of North Carolina at Chapel Hill, USA) and H. Wang (National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA) for VH12- and Vκ4-transgenic mice; K. Rajewsky (Max Delbrück Center for Molecular Medicine) for anti-VH12; K. Schindler for help with signaling experiments; M. Fischer for bioinformatics analysis; C. Theussl for the generation of transgenic mice; K. Aumayr and colleagues for sorting by flow cytometry; and A. Sommer and colleagues for Illumina sequencing. Supported by Boehringer Ingelheim, the European Community's Seventh Framework Program (European Research Council Advanced Grant 291740-LymphoControl to M.B.), the Austrian Industrial Research Promotion Agency (Headquarter Grant FFG-852936 to M.B.), the Austrian Science Fund (P28841 to T.K.) and the Marie Sklodowaska-Curie action of the European Community's Framework Program (PIIF-GA-2013-628065 to T.K.).

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  1. Bojan Vilagos & Hiromi Tagoh

    Present address: Present addresses: Research Center for Molecular Medicine, Austrian Academy of Sciences, Vienna, Austria (B.V.), and Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK (H.T.).,

Authors and Affiliations

  1. Research Institute of Molecular Pathology, Vienna Biocenter, Campus-Vienna-Biocenter 1, Vienna, Austria

    Taras Kreslavsky, Bojan Vilagos, Hiromi Tagoh, Daniela Kostanova Poliakova, Tanja A Schwickert, Miriam Wöhner, Markus Jaritz & Meinrad Busslinger

  2. Department of Molecular Immunology, Helmholtz Center for Infection Research, Braunschweig, and Institute of Immunology, Medical School, Hannover, Germany

    Siegfried Weiss

  3. Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

    Reshma Taneja

  4. Department of Psychiatry, Ludwig Maximillian University of Munich, Munich, Germany

    Moritz J Rossner

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Contributions

T.K. designed and did most experiments; B.V. identified Bhlhe41 as a B-1-cell-specific transcription factor; H.T. performed the ATAC-seq experiments; D.K.P. generated the targeted Bhlhe41Tag embryonic stem cells; T.A.S. and M.W. provided advice and help with immunization experiments; M.J. performed the bioinformatics analysis of the ChIP-seq data; S.W. provided PtC-containing liposomes; R.T. and M.J.R. provided the Bhlhe40−/− mice and Bhlhe41−/− mice, respectively; and T.K. and M.B. planned the project and wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Expression of Bhlhe41 and Bhlhe40 in B cell development and mature B cell subsets.

(a) Scatter plot showing differential expression of genes between splenic B-1a cells and follicular (FO) B cells. RNA-seq data (Vilagos et al., J. Exp. Med. 209, 775-792) are shown for known and predicted genes with regulatory functions. RPKM, reads per kilobase of exon per million mapped sequence reads. (b) Expression of Bhlhe41 (red) and Bhlhe40 (gray) across the Immgen Database. Several replicates are shown for each cell type. Fraction D (FrD), pre-B cells; fraction E (FrE), immature B cells. (c) Expression (RNA-seq) of Bhlhe41 and Bhlhe40 by the indicated precursor and B cell populations. TPM, transcripts per million. LMPP, lymphoid-primed multipotent progenitors; ALP, all-lymphoid progenitor; BLP, B-cell-biased lymphoid progenitor; pre-B(L), large pre-B cells; pre-B(S), small pre-B cells; PC, plasma cells; MZ B, marginal zone B cells; GC B, germinal center B cells; FL, fetal liver; PP, Peyer’s patches; PerC, peritoneal cavity. (d,e) Cells from adult spleen (d), bone marrow (e) and peritoneal cavity (e) as well as neonatal liver (e, bottom row; day 1 after birth) of Bhlhe41-Cre-hCD2 transgenic and wild-type mice were stained with antibodies against human CD2 and markers defining the indicated cell populations. The flow cytometric definition of all cell types is described in the Online Methods. Histograms comparing hCD2 expression of the indicated cell types are shown. ΔMFI, difference in the median fluorescence intensity between Bhlhe41-Cre-hCD2 transgenic and wild-type cells of the indicated cell populations for the pair of wild-type and reporter mice shown in the plot. A representative result of two independent experiments is shown. (f) Expression (RNA-seq) of Bhlhe41 and Bhlhe40 in FO B cells stimulated with LPS for the indicated time (Wöhner et al., J. Exp. Med. 213, 1201-1221). (g) hCD2− follicular B cells were sorted by flow cytometry from spleens of Bhlhe41-Cre-hCD2 transgenic and wild-type mice and then cultured in the presence of LPS, anti-IgM plus IL-4 or anti-CD40 plus IL-4 for 2 or 4 days. One representative result of two independent experiments is shown.

Supplementary Figure 2 Bhlhe41 and Bhlhe40 are dispensable for B cell development.

(a) Flow cytometric analysis showing CD19 and IgM expression (top) and Kit and CD2 expression (bottom) on CD19+IgM− cells from E18.5 fetal livers of wild-type (WT) and DKO embryos. One representative result of two independent experiments is shown. (b) Flow cytometric analysis showing IgM and IgD expression on CD19+ cells (top) and Kit and CD2 expression on CD19+IgM−IgD− cells (bottom) from the bone marrow of mice with the indicated genotypes. One representative result of two independent experiments is shown. (c) Absolute numbers of the pro-B, pre-B, and immature B cells analyzed in (b). (d) Absolute numbers of peritoneal B-1b and B-2 cells (left) and splenic FO and MZ B cells (right) from adult mice of the indicated genotypes. The gating was preformed as shown in Fig. 1b,c. Horizontal bars indicate mean value, and error bars represent s.d. NS P > 0.05, * P < 0.05, ** P < 0.01 as determined by the Student’s t-test. Five age-matched mice were analyzed per genotype. (e) Expression of CD5 and CD23 on peritoneal CD19+ B cells from wild-type and Bhlhe40−/− mice. One representative result of two independent experiments is shown. (f) T, NK and IgM+ B cell-depleted bone marrow cells from wild-type (WT) and DKO mice were mixed at a 1:1 ratio and injected into lethally irradiated Rag2−/− recipients. Chimeras were analyzed 14-16 weeks later, as described in the legend of Fig. 1f,g and Online Methods. Five chimeric mice were analyzed. Horizontal bars indicate mean value, and error bars represent s.d. NS P > 0.05, ** P < 0.01, *** P < 0.001 as determined by the Student’s t-test. One representative result of two independent experiments is shown.

Supplementary Figure 3 Analysis of B-1a cells and their progenitors in early ontogeny and adult mice.

(a) RNA-seq datasets of the indicated wild-type (FO B, MZ B, and B-1a cells) and DKO B-1a cell populations from adult mice were examined by principal component analysis based on the 500 most differentially expressed genes among the wild-type B cell subsets. (b) Identification of B220lo/– B-1-specified progenitors as described by Montecino-Rodriguez et al. (Nat. Immunol. 7, 293-301). The gating strategy for identifying the Lin−IgM−CD93+CD19+B220lo/– progenitors is shown for adult bone marrow and fetal liver (FL) cells (E15.5). Expression of the Bhlhe41-Cre-hCD2 reporter gene was analyzed in Lin−IgM−CD93+CD19+B220lo/– progenitors of the E15.5 fetal liver (bottom row). (c) Cells with the B-1a phenotype and PtC-binding specificity emerge in the spleen at postnatal day 9. Cells from the indicated organs of wild-type neonatal and adult mice were stained with antibodies against CD19, B220, CD5 and VH12 as well as with PtC liposomes prior to flow cytometry.

Supplementary Figure 4 Analysis of B-1a cells from VH12- and VH12Vκ4-transgenic mice.

(a) Peritoneal cells from mice of the indicated genotypes were stained with antibodies against VH12, IgM, CD5, CD23, CD19, and B220 and were analyzed by flow cytometry. All plots are gated on CD19+ cells. One representative result of four (VH12 transgenic) or at least seven (non-transgenic) independent experiments is shown. (b) Principal component analysis of RNA-seq data of the indicated wild-type populations (FO B, MZ B and polyclonal B-1a cells) and VH12/Vκ4 transgenic (Tg) B-1a cells based on the 500 most differentially expressed genes for the wild-type B cell subsets. (c) Bone marrow cells from mice of the indicated genotypes were stained with anti-IgM and anti-VH12 antibodies and analyzed by flow cytometry. One representative result of two independent experiments is shown.

Supplementary Figure 5 Generation and characterization of Bhlhe41Tag/Tag mice.

(a) Schematic domain structure (not drawn to scale) of the tagged Bhlhe41 protein that is expressed from the Bhlhe41Tag allele. The tag sequences added at the last codon of Bhlhe41 contained epitopes for Flag and V5 antibodies, two cleavage sites for the TEV protease and a biotin acceptor sequence for biotinylation by the Escherichia coli biotin ligase BirA. (b) Frequency of peritoneal and splenic B-1a cells in wild-type and Bhlhe41Tag/Tag mice. Three mice of each genotype were analyzed. Horizontal bars indicate mean value, and error bars represent s.d. (c) Gene set enrichment analysis (GSEA). The Bhlhe41/Bhlhe40-repressed genes identified in VH12/Vκ4 transgenic B-1a cells were compared to the ranked log2-fold gene expression changes between polyclonal DKO and wild-type B-1a cells (left). Likewise, the Bhlhe41/Bhlhe40-repressed genes identified in polyclonal B-1a cells were compared to the ranked log2-fold gene expression changes between VH12/Vκ4 transgenic DKO and wild-type B-1a cells (right). (d) The average density of open chromatin (determined by ATAC-seq) at all genomic Bhlhe41 peaks (left) or at the 117 Bhlhe41 peaks present at repressed target genes was assessed for a region extending from -1 kb to +1 kb relative to the Bhlhe41 peak summit.

Supplementary Figure 6 BCR signaling in VH12Vκ4-transgenic B-1a cells.

(a) Intracellular Ca2+ flux in VH12/Vκ4 transgenic wild-type and DKO splenic B-1a cells, as measured by the change in fluorescence emission of a Ca2+ sensor dye after addition (arrow) of a goat anti-mouse IgM F(ab’)2 fragment. (b) Scatter plot of gene expression differences between ex vivo sorted VH12/Vκ4 transgenic wild-type and DKO peritoneal B-1a cells. Genes encoding components of the BCR signaling pathway (GO:0050853 “B cell receptor signaling pathway” plus manually added genes) are highlighted in the left panel, while genes implicated in B-1a cell development and homeostasis as well as genes encoding the α- and β-chains of the IL-5, IL-3 and GM-CSF receptor family are indicated in the right panel. Genes that are discussed in the text are marked in red. RPM, reads per million mapped sequence reads. (c) Expression of the immunoglobulin μ-heavy chain (Ighm) mRNA (RNA-seq; left) and the cell-surface IgM protein (right) is shown for peritoneal B-1a cells from VH12/Vκ4 transgenic wild-type and DKO mice. The Ighm mRNA data were determined by two independent RNA-seq experiments per cell type, and error bars indicate s.e.m. * P < 0.05 as determined by the Student’s t-test. TPM, transcripts per million. (d) Peritoneal cells from VH12/Vκ4 transgenic DKO and wild-type mice as well as peritoneal B-2 cells from wild-type mice were stained on ice for CD19, B220 and CD5, fixed, permeabilized and stained intracellularly (ic) for IgM and phosphorylated epitopes of the indicated signal transducers. Overlay contour plots displaying IgM expression and the phosphorylation status of the indicated BCR signaling components are shown for polyclonal B-2 cells (CD19+CD5−B220hi) as well as for VH12/Vκ4 transgenic wild-type and DKO CD19+ B cells. One representative result of three independent experiments is shown. (e) Frequency of VH12− and VH12+ PtC liposome-binding cells (gated as shown in Fig. 6e) of peritoneal wild-type and Cd19−/− IgM+CD5+ B-1a cells. Horizontal bars indicate mean value, and error bars represent s.d. NS P > 0.05, * P < 0.05 as determined by the Student’s t-test. The data of eight wild-type and seven Cd19−/− mice analyzed in three independent experiments are shown.

Supplementary Figure 7 Bhlhe41 represses genes encoding cell-cycle regulators.

(a) Expression of genes encoding the indicated cell cycle regulators in VH12/Vκ4 transgenic and polyclonal wild-type and DKO B-1a cells, as determined by RNA-seq. The data are from two independent RNA-seq experiments per cell type, and error bars indicate s.e.m. TPM, transcripts per million. (b) Bhlhe41 binding (ChIP-seq) at the genes indicated in (a). RPM, reads per million mapped sequence reads.

Supplementary Figure 8 Bhlhe41 and Bhlhe40 regulate expression of the receptors for IL-5 and IL-3.

(a) Expression of the Tnfrsf13b (TACI) and Tnfrsf13c (BAFF-R) genes in VH12/Vκ4 transgenic (top) and polyclonal (bottom) wild-type and DKO B-1a cells, as determined by two independent RNA-seq experiments per cell type. Error bars indicate s.e.m. TPM, transcripts per million. (b) Surface IL-5Rα and IL-3Rα expression on polyclonal B-1a cells of the indicated genotypes (top) as well as surface IL-5Rα expression on VH12/Vκ4 transgenic B-1a cells (bottom left) and B-1a cells from mixed fetal liver (FL) chimeras (bottom right). One representative result of at least two independent experiments is shown.

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Kreslavsky, T., Vilagos, B., Tagoh, H. et al. Essential role for the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells. Nat Immunol 18, 442–455 (2017). https://doi.org/10.1038/ni.3694

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