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Dynamic regulation of B cell complement signaling is integral to germinal center responses

Nature Immunology volume 22, pages 757–768 (2021)Cite this article

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Abstract

Maturation of B cells within germinal centers (GCs) generates diversified B cell pools and high-affinity B cell antigen receptors (BCRs) for pathogen clearance. Increased receptor affinity is achieved by iterative cycles of T cell–dependent, affinity-based B cell positive selection and clonal expansion by mechanisms hitherto incompletely understood. Here we found that, as part of a physiologic program, GC B cells repressed expression of decay-accelerating factor (DAF/CD55) and other complement C3 convertase regulators via BCL6, but increased the expression of C5b-9 inhibitor CD59. These changes permitted C3 cleavage on GC B cell surfaces without the formation of membrane attack complex and activated C3a- and C5a-receptor signals required for positive selection. Genetic disruption of this pathway in antigen-activated B cells by conditional transgenic DAF overexpression or deletion of C3a and C5a receptors limited the activation of mechanistic target of rapamycin (mTOR) in response to BCR–CD40 signaling, causing premature GC collapse and impaired affinity maturation. These results reveal that coordinated shifts in complement regulation within the GC provide crucial signals underlying GC B cell positive selection.

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Fig. 1: GC B cells downregulate expression of DAF (CD55) and other C3/C4 convertase inhibitors within the RCA.
Fig. 2: BCL6 transcriptionally represses DAF expression in GC B cells.
Fig. 3: Transgenic DAF overexpression or absence of C3ar1–C5ar1 on GC B cells aborts GC formation.
Fig. 4: Transgenic DAF overexpression or absence of C3ar1–C5ar1 in GC B cells limits affinity maturation.
Fig. 5: Defective expansion, delayed maturation and reduced competitive competencies of DAF-TMCγ1 and ΔC3ar1/C5ar1Cγ1 B cells during GC responses.
Fig. 6: DAF-TM and absence of C3aR–C5aR signaling in GC B cells reduce signals required for positive selection independently of CD40.

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Data availability

RNA-seq datasets were deposited in the GEO database under accession no. GSE148570 (reference series). Additional preprocessed data are provided in Supplementary Tables 1 and 2. Data from publicly available datasets were used for additional analyses, as specified in individual figure legends: GEO (https://www.ncbi.nlm.nih.gov/geo/) datasets and records GSE2350, GSE139833 (human tonsil B cell subsets); GSE68349 and GSE67494 (chromatin immunoprecipitation data for BCL6 and histone marks in human GC B cells); and the Immunological Genome Project (https://www.immgen.org) for mouse B cell subset gene expression data. Source data are provided with this paper.

References

  1. Victora, G. D. & Nussenzweig, M. C. Germinal centers. Annu. Rev. Immunol. 30, 429–457 (2012).

    Article  CAS  PubMed  Google Scholar 

  2. Shlomchik, M. J., Luo, W. & Weisel, F. Linking signaling and selection in the germinal center. Immunol. Rev. 288, 49–63 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ersching, J. et al. Germinal center selection and affinity maturation require dynamic regulation of mTORC1 kinase. Immunity 46, 1045–1058 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Luo, W., Weisel, F. & Shlomchik, M. J. B cell receptor and CD40 signaling are rewired for synergistic induction of the c-Myc transcription factor in germinal center B cells. Immunity 48, 313–326 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Schwickert, T. A. et al. A dynamic T cell–limited checkpoint regulates affinity-dependent B cell entry into the germinal center. J. Exp. Med. 208, 1243–1252 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Srinivasan, L. et al. PI3 kinase signals BCR-dependent mature B cell survival. Cell 139, 573–586 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Dominguez-Sola, D. et al. The proto-oncogene MYC is required for selection in the germinal center and cyclic reentry. Nat. Immunol. 13, 1083–1091 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Heeger, P. S. et al. Decay-accelerating factor modulates induction of T cell immunity. J. Exp. Med. 201, 1523–1530 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lalli, P. N. et al. Locally produced C5a binds to T cell–expressed C5aR to enhance effector T-cell expansion by limiting antigen-induced apoptosis. Blood 112, 1759–1766 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Strainic, M. G. et al. Locally produced complement fragments C5a and C3a provide both costimulatory and survival signals to naive CD4+ T cells. Immunity 28, 425–435 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Verghese, D. A. et al. C5aR1 regulates T follicular helper differentiation and chronic graft-versus-host disease bronchiolitis obliterans. JCI Insight, https://doi.org/10.1172/jci.insight.124646 (2018).

  12. Medof, M. E., Kinoshita, T. & Nussenzweig, V. Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes. J. Exp. Med. 160, 1558–1578 (1984).

    Article  CAS  PubMed  Google Scholar 

  13. Carroll, M. C. et al. Organization of the genes encoding complement receptors type 1 and 2, decay-accelerating factor, and C4-binding protein in the RCA locus on human chromosome 1. J. Exp. Med. 167, 1271–1280 (1988).

    Article  CAS  PubMed  Google Scholar 

  14. Jacobson, A. C. & Weis, J. H. Comparative functional evolution of human and mouse CR1 and CR2. J. Immunol. 181, 2953–2959 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. Kennedy, D. E. et al. Novel specialized cell state and spatial compartments within the germinal center. Nat. Immunol. 21, 660–670 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Basso, K. & Dalla-Favera, R. Roles of BCL6 in normal and transformed germinal center B cells. Immunol. Rev. 247, 172–183 (2012).

    Article  PubMed  CAS  Google Scholar 

  17. Kitano, M. et al. Bcl6 protein expression shapes pre-germinal center B cell dynamics and follicular helper T cell heterogeneity. Immunity 34, 961–972 (2011).

    Article  CAS  PubMed  Google Scholar 

  18. Dominguez-Sola, D. et al. The FOXO1 transcription factor instructs the germinal center dark zone program. Immunity 43, 1064–1074 (2015).

    Article  CAS  PubMed  Google Scholar 

  19. Basso, K. et al. Integrated biochemical and computational approach identifies BCL6 direct target genes controlling multiple pathways in normal germinal center B cells. Blood 115, 975–984 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Cardenas, M. G. et al. Rationally designed BCL6 inhibitors target activated B cell diffuse large B cell lymphoma. J. Clin. Invest. 126, 3351–3362 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Rickert, R. C., Roes, J. & Rajewsky, K. B lymphocyte-specific, Cre-mediated mutagenesis in mice. Nucleic Acids Res. 25, 1317–1318 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Casola, S. et al. Tracking germinal center B cells expressing germ-line immunoglobulin γ1 transcripts by conditional gene targeting. Proc. Natl Acad. Sci. USA 103, 7396–7401 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Lalli, P. N., Strainic, M. G., Lin, F., Medof, M. E. & Heeger, P. S. Decay accelerating factor can control T cell differentiation into IFN-γ-producing effector cells via regulating local C5a-induced IL-12 production. J. Immunol. 179, 5793–5802 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Verghese, D. A. et al. T cell expression of C5a receptor 2 augments murine regulatory T cell (TREG) generation and TREG-dependent cardiac allograft survival. J. Immunol. 200, 2186–2198 (2018).

    Article  CAS  Google Scholar 

  25. Mayer, C. T. et al. The microanatomic segregation of selection by apoptosis in the germinal center. Science 358, eaao2602 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Hebell, T., Ahearn, J. M. & Fearon, D. T. Suppression of the immune response by a soluble complement receptor of B lymphocytes. Science 254, 102–105 (1991).

    Article  CAS  PubMed  Google Scholar 

  27. Wentink, M. W. et al. CD21 and CD19 deficiency: two defects in the same complex leading to different disease modalities. Clin. Immunol. 161, 120–127 (2015).

    Article  CAS  PubMed  Google Scholar 

  28. Jolly, C. J., Klix, N. & Neuberger, M. S. Rapid methods for the analysis of immunoglobulin gene hypermutation: application to transgenic and gene targeted mice. Nucleic Acids Res. 25, 1913–1919 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Inoue, T. et al. The transcription factor Foxo1 controls germinal center B cell proliferation in response to T cell help. J. Exp. Med. 214, 1181–1198 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Pereira, J. P., Kelly, L. M., Xu, Y. & Cyster, J. G. EBI2 mediates B cell segregation between the outer and centre follicle. Nature 460, 1122–1126 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Klein, U. et al. Transcriptional analysis of the B cell germinal center reaction. Proc. Natl Acad. Sci. USA 100, 2639–2644 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Gatto, D., Wood, K. & Brink, R. EBI2 operates independently of but in cooperation with CXCR5 and CCR7 to direct B cell migration and organization in follicles and the germinal center. J. Immunol. 187, 4621–4628 (2011).

    Article  CAS  PubMed  Google Scholar 

  33. Cyster, J. G. et al. Follicular stromal cells and lymphocyte homing to follicles. Immunol. Rev. 176, 181–193 (2000).

    Article  CAS  PubMed  Google Scholar 

  34. Green, J. A. et al. The sphingosine 1-phosphate receptor S1P2 maintains the homeostasis of germinal center B cells and promotes niche confinement. Nat. Immunol. 12, 672–680 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Gatto, D., Paus, D., Basten, A., Mackay, C. R. & Brink, R. Guidance of B cells by the orphan G protein-coupled receptor EBI2 shapes humoral immune responses. Immunity 31, 259–269 (2009).

    Article  CAS  PubMed  Google Scholar 

  36. Zhao, R. et al. A GPR174–CCL21 module imparts sexual dimorphism to humoral immunity. Nature 577, 416–420 (2020).

    Article  CAS  PubMed  Google Scholar 

  37. Lu, P., Shih, C. & Qi, H. Ephrin B1–mediated repulsion and signaling control germinal center T cell territoriality and function. Science 356, eaai9264 (2017).

    Article  PubMed  CAS  Google Scholar 

  38. Pae, J. et al. Cyclin D3 drives inertial cell cycling in dark zone germinal center B cells. J. Exp. Med. 218, 20201699 (2021).

    Article  CAS  Google Scholar 

  39. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Kitamura, D., Roes, J., Kuhn, R. & Rajewsky, K. A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene. Nature 350, 423–426 (1991).

    Article  CAS  PubMed  Google Scholar 

  41. Ahearn, J. M. et al. Disruption of the Cr2 locus results in a reduction in B-1a cells and in an impaired B cell response to T-dependent antigen. Immunity 4, 251–262 (1996).

    Article  CAS  PubMed  Google Scholar 

  42. Sivasankar, B. et al. CD59a deficient mice display reduced B cell activity and antibody production in response to T-dependent antigens. Mol. Immunol. 44, 2978–2987 (2007).

    Article  CAS  PubMed  Google Scholar 

  43. Wiede, F. et al. CCR6 is transiently upregulated on B cells after activation and modulates the germinal center reaction in the mouse. Immunol. Cell Biol. 91, 335–339 (2013).

    Article  CAS  PubMed  Google Scholar 

  44. Suan, D. et al. CCR6 defines memory B cell precursors in mouse and human germinal centers, revealing light-zone location and predominant low antigen affinity. Immunity 47, 1142–1153 (2017).

    Article  CAS  PubMed  Google Scholar 

  45. Carroll, M. C. & Isenman, D. E. Regulation of humoral immunity by complement. Immunity 37, 199–207 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Paiano, J. et al. Follicular B2 cell activation and class switch recombination depend on autocrine C3ar1/C5ar1 signaling in B2 cells. J. Immunol. 203, 379–388 (2019).

    Article  CAS  PubMed  Google Scholar 

  47. Dernstedt, A. et al. Regulation of decay accelerating factor primes human germinal center B cells for phagocytosis. Front. Immunol. 11, 599647 (2020).

    Article  CAS  PubMed  Google Scholar 

  48. Biram, A., Davidzohn, N. & Shulman, Z. T cell interactions with B cells during germinal center formation, a three-step model. Immunol. Rev. 288, 37–48 (2019).

    Article  CAS  PubMed  Google Scholar 

  49. Wu, Y. L. & Rada, C. Molecular fine-tuning of affinity maturation in germinal centers. J. Clin. Invest. 126, 32–34 (2016).

    Article  PubMed  Google Scholar 

  50. Arbore, G. et al. T helper 1 immunity requires complement-driven NLRP3 inflammasome activity in CD4+ T cells. Science 352, aad1210 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Liszewski, M. K. et al. Intracellular complement activation sustains T cell homeostasis and mediates effector differentiation. Immunity 39, 1143–1157 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. West, E. E., Afzali, B. & Kemper, C. Unexpected roles for intracellular complement in the regulation of Th1 responses. Adv. Immunol. 138, 35–70 (2018).

    Article  CAS  PubMed  Google Scholar 

  53. Rossbacher, J. & Shlomchik, M. J. The B cell receptor itself can activate complement to provide the complement receptor 1/2 ligand required to enhance B cell immune responses in vivo. J. Exp. Med. 198, 591–602 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Bird, L. Switch to antitumour B cells. Nat. Rev. Immunol. 20, 274–275 (2020).

    Article  CAS  PubMed  Google Scholar 

  55. Lu, Y. et al. Complement signals determine opposite effects of B cells in chemotherapy-induced immunity. Cell 180, 1081–1097 (2020).

    Article  CAS  PubMed  Google Scholar 

  56. Heng, T. S. et al. The Immunological Genome Project: networks of gene expression in immune cells. Nat. Immunol. 9, 1091–1094 (2008).

    Article  CAS  PubMed  Google Scholar 

  57. Zhang, J. et al. Disruption of KMT2D perturbs germinal center B cell development and promotes lymphomagenesis. Nat. Med. 21, 1190–1198 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Zhang, M. et al. The role of natural IgM in myocardial ischemia–reperfusion injury. J. Mol. Cell. Cardiol. 41, 62–67 (2006).

    Article  CAS  PubMed  Google Scholar 

  59. Llaudo, I. et al. C5aR1 regulates migration of suppressive myeloid cells required for costimulatory blockade-induced murine allograft survival. Am. J. Transpl. 19, 633–645 (2019).

    Article  CAS  Google Scholar 

  60. Hovingh, E. S., van den Broek, B. & Jongerius, I. Hijacking complement regulatory proteins for bacterial immune evasion. Front. Microbiol. 7, 2004 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  61. Mashiko, D. et al. Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Sci. Rep. 3, 3355 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Kwan, W. H. et al. Antigen-presenting cell-derived complement modulates graft-versus-host disease. J. Clin. Invest. 122, 2234–2238 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Le, T. V., Kim, T. H. & Chaplin, D. D. Intraclonal competition inhibits the formation of high-affinity antibody-secreting cells. J. Immunol. 181, 6027–6037 (2008).

    Article  CAS  PubMed  Google Scholar 

  64. Phan, R. T. & Dalla-Favera, R. The BCL6 proto-oncogene suppresses p53 expression in germinal-centre B cells. Nature 432, 635–639 (2004).

    Article  CAS  PubMed  Google Scholar 

  65. Gitlin, A. D., Shulman, Z. & Nussenzweig, M. C. Clonal selection in the germinal centre by regulated proliferation and hypermutation. Nature 509, 637–640 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Jacob, J. & Kelsoe, G. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. II. A common clonal origin for periarteriolar lymphoid sheath-associated foci and germinal centers. J. Exp. Med. 176, 679–687 (1992).

    Article  CAS  PubMed  Google Scholar 

  67. Lefranc, M. P. et al. IMGT, the international ImMunoGeneTics information system. Nucleic Acids Res. 37, D1006–D1012 (2009).

    Article  CAS  PubMed  Google Scholar 

  68. Alamyar, E., Duroux, P., Lefranc, M. P. & Giudicelli, V. IMGT((R)) tools for the nucleotide analysis of immunoglobulin (IG) and T cell receptor (TR) V-(D)-J repertoires, polymorphisms, and IG mutations: IMGT/V-QUEST and IMGT/HighV-QUEST for NGS. Methods Mol. Biol. 882, 569–604 (2012).

    Article  CAS  PubMed  Google Scholar 

  69. MacCarthy, T. et al. V-region mutation in vitro, in vivo, and in silico reveal the importance of the enzymatic properties of AID and the sequence environment. Proc. Natl Acad. Sci. USA 106, 8629–8634 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. 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).

    Article  CAS  PubMed  Google Scholar 

  72. 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  PubMed  PubMed Central  CAS  Google Scholar 

  73. Reich, M. et al. GenePattern 2.0. Nat. Genet. 38, 500–501 (2006).

    Article  CAS  PubMed  Google Scholar 

  74. Faul, F., Erdfelder, E., Lang, A. G. & Buchner, A. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav. Res. Methods 39, 175–191 (2007).

    Article  PubMed  Google Scholar 

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Acknowledgements

The authors thank the Mount Sinai Biorepository and Pathology Core, The Mount Sinai Mouse Genetics Core (K. Kelley, director), The Mount Sinai Flow cytometry core, The Mount Sinai Microscopy Core and the Genomics Core for their technical assistance. The authors thank Y. Garcia-Carmona, L. Anderson, D. Peace and N. Samuel-Stokes (Icahn School of Medicine at Mount Sinai) for technical assistance, and C. Cunningham-Rundles (Icahn School of Medicine at Mount Sinai), R. Fairchild (Cleveland Clinic, Cleveland, OH) and F. Lin (Cleveland Clinic, Cleveland, OH) for critical comments/advice. This research was funded through the NIH (no. R01-AI141434, awarded to P.S.H. and D.D.-S., and no. R21 AI 126009, awarded to P.S.H., D.H. and S.A.L.) and NIH/NCI Cancer Center Support (grant no. P30-CA196521 to the Tisch Cancer Institute at Mount Sinai). A.C. was supported by a fellowship grant from the American Society of Transplantation, G.V. by a postdoctoral fellowship of the Lymphoma Research Foundation and M.P.R. by a Ruth L. Kirchstein National Service Award Institutional Research Training Grant (no. T32-CA078207). F.O. was supported by an Institutional Research Training Grant (no. T32-CA078207).

Author information

Author notes

  1. David Heja

    Present address: eGenesis Inc., Cambridge, MA, USA

  2. Gabriele Varano

    Present address: Department of Translational Medicine, Laboratory for Advanced Therapy Technologies, University of Ferrara, Ferrara, Italy

  3. These authors jointly supervised this work: D. Dominguez-Sola and P. S. Heeger.

Authors and Affiliations

  1. Renal Division, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Arun Cumpelik, David Heja, Yuan Hu, Farideh Ordikhani & Peter S. Heeger

  2. Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Arun Cumpelik, David Heja, Yuan Hu, Farideh Ordikhani & Peter S. Heeger

  3. Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Gabriele Varano, Mark P. Roberto & David Dominguez-Sola

  4. Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Farideh Ordikhani & David Dominguez-Sola

  5. Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Mark P. Roberto

  6. Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Zhengxiang He, Dirk Homann, Sergio A. Lira, David Dominguez-Sola & Peter S. Heeger

  7. Department of Pathology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    David Dominguez-Sola

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Contributions

A.C. contributed to the study design, performed the majority of in vivo and in vitro studies, prepared figures and wrote and edited the manuscript. D.H. and Z.H designed and prepared the DAF-TM targeting construct and performed in vitro characterization of the DAF-TM gene product in founder mice. Y.H and G.V. performed studies on DAF gene regulation by BCL6 experiments, BCR-seq and, together with M.P.R., performed RNA-seq analyses and reviewed and edited the manuscript. F.O. performed experiments, including all studies with B1-8hi mice, and reviewed and edited the manuscript. D.H. and S.A.L. outlined the strategy for DAF-TM generation, served as critical reviewers of data and edited the manuscript. D.D.-S. and P.S.H. conceptualized, designed and supervised the project, reviewed all data, wrote and edited the manuscript and provided funding.

Corresponding authors

Correspondence to David Dominguez-Sola or Peter S. Heeger.

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The authors declare no competing interests.

Additional information

Peer review information Nature Immunology thanks Anne Astier, Michael Carroll and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Distribution of complement regulator expression in mature B cell subsets.

a,b, Gating strategy for flow cytometry analysis of murine (a) and human (b) naïve, GC and memory B cells, with representative histograms of DAF expression across B cell subsets. c, DAF expression on light (LZ), dark (DZ) and grey zone (GZ) GC B cells defined by CXCR4 and CD86 expression (left) with representative histograms (middle) and quantitation (right). d, Representative flow plots of mouse B cell subsets for CR1/2, Crry, and CD59 expression. e, Representative histograms of human tonsillar B cell subsets for CR1(CD35), CD46, CD59 and CR2(CD21) expression, with quantitation of CR2/CD21 expression (right panel). f, Heatmap, source: ImmGen database, with row-normalized mRNA expression of complement regulators and Bcl6 in different murine B cell subsets. g, Representative histograms showing C3b staining on murine (left) and human (right) B cell subsets. h, Representative (3 individual experiments) image of human tonsil staining with anti-C9 showing positive staining of vascular endothelium (positive control for Fig. 1g), scale bar 50 μm. Data are presented as MFI +/− SEM, *p < 0.05, **p < 0.01, ***p < 0.001 by ANOVA with Bonferroni post-test (c,e). Each dot represents a biological replicate. n.s., not significant.

Source data

Extended Data Fig. 2 BCL6 is inversely correlated with DAF expression and binds to regulatory regions of RCA genes.

a, Schematic diagram illustrating effects of absent DAF with persistent CD59 expression on complement activation products on naïve (top) vs GC (bottom) B cells. b, Gating strategy for DAF/CD55 expression on immunized BCL6-YFP+ reporter mice, d3 post-immunization with SRBCs including d10 naïve and GC B cell controls (complementary to Fig. 2a). c, Heat map depicting relative mRNA expression levels (row-normalized) for BCL6 and complement regulators on human B cell lymphoma cell lines (Diffuse Large B cell lymphoma and selected Burkitt lymphoma cell lines: Raji, BL70, P3HR1; and Multiple Myeloma cell lines: KMS26, KMS27 and MOLP2). Data extracted from the Cancer Cell Line Encyclopedia (CCLE) repository1,2. d-e, Schematic depiction of ChIP-seq tracks for BCL6 and selected histone marks at the human RCA (d) and CD59 (e) gene loci. Data extracted from GEO records GSE68349, GSE674943,4.

Source data

Extended Data Fig. 3 Design and validation of conditional DAF-TM transgenic mice.

a, Schematic of Crispr/Cas9n strategy for producing DAF-TM transgenic mouse. The sequence inset shows a segment of the WT Rosa26 locus and our targeting gRNA design. The PAM (Protospacer Adjacent Motif) is highlighted in red. The 5’–NGG–3’ sequence is the PAM consensus for binding of S. pyogenes Cas9 and Cas9D10A (Cas9n) nickase variant. The sequences in blue adjacent to the PAM sequences (Target L and Target R) indicate the target sites for Cas9n mediated cleavage. These sequences are identical with the spacer sequences in gRNA-A and gRNA-B, respectively. Cas9n nicks the target DNA at sites indicated with red triangles. Offset nicking induces recombination between the genomic Rosa26 locus and the homology arms in the repair plasmid that results in insertion of the DAF-TM transgenic construct between the WT Rosa26 segments indicated with red and black boxes. b-c, Representative flow cytometry plots (b) and quantified results (c) of DAF staining on naïve and GC B cells from DAF-TMCD19 mice (DAF-TM x CD19-Cre+/–) and CD19+/– control mice in the absence or presence of phospholipase C (PLC), n = 5 independent biological replicates. Note that PLC totally removes native surface DAF from Control (CD19-Cre+/–) B cells. In DAF-TMCD19 B cells, PLC removed native (GPI-anchored) DAF leaving lower but detectable levels of transgenic DAF resistant to PLC cleavage. d, Representative histograms (top) and quantification (bottom) depicting lower C3b deposition on B cell subsets from DAF-TMCD19 compared to control CD19-Cre+/–- mice. e-f, Representative flow cytometry plots (e) and quantified results (f) of CD59 staining on naïve and GC B cells from DAF-TMCD19 (DAF-TM x CD19-Cre+/–) and CD19-Cre+/– control mice in the absence or presence of phospholipase C (PLC), n = 5 independent biological replicates. PLC removed the GPI-anchored CD59 from the surfaces of GC B cells in both CD19-Cre+/– and DAF-TMCD19 B cells.; All data are presented as MFI +/− SEM, *p < 0.05,**p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Bonferroni post-test (c,d,f). n.s., not significant. Each dot represents a biological replicate.

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Extended Data Fig. 4 Extended characterization of GC and antibody responses in GC- (Cγ1-Cre) and B cell-specific (CD19-cre) DAF-TM and ΔC3aR1, ΔC5aR1 mice.

a, representative histograms (left) and kinetics of total surface DAF and DAF-TM (PLC-resistant) expression on IgD–Fas+GL7+ GC B cells (middle) and IgD–GL7+CCR6+CD38+ B cells (right) in DAF-TMCγ1 (DAF TM) and control Cγ1-Cre+/– (Cγ1 Ctrl) mice. Note the progressive accumulation of DAF-TM+ B cells over time, following the kinetics of Cγ1-Cre-driven recombination5. b–d, Quantified surface expression of Crry (b) CR1/2 (c), and CD59 (d) proteins on B cell subsets from Cγ1-Cre+/– control, DAF-TMCγ1 and, ΔC3ar1/C5ar1Cγ1 mice. e-f, Relative % (e) and absolute frequencies (f) of splenic GC B cells in d12 SRBC-immunized DAF-TMCγ1 (DAF TM), ΔC3ar1/C5ar1Cγ1 (ΔC3aRΔC5aR) C3ar1Cγ1 (ΔC3aR), ΔC5ar1Cγ1 (ΔC5aR), and control Cγ1-Cre+/– (Cγ1 Ctrl) mice. g, Kinetics of relative GC B cells frequencies in SRBC immunized DAF-TMCγ1, ΔC3ar1/C5ar1Cγ1 and Cγ1-Cre+/– mice. h, Ratios of DZ (CXCR4+CD86+) vs LZ (CXCR4−CD86+) GC B cells d10 post-immunization with NP-KLH (left) or SRBC (right). i, Representative flow cytometry plots for CD38+NP+ memory B cells (Bmem), within B220+IgD–GL7–Fas– spleen cell populations of NP-KLH-immunized Cγ1-Cre+/– control, DAF-TMCγ1 and ΔC3ar1/C5ar1Cγ1 mice (d12 post-immunization) (see also Fig. 3f). j-k, Representative flow cytometry plots (j) and quantified results (k) for CD38+CD73+ Bmem gated on B220+IgD–GL7–Fas– spleen cells in NP-KLH-immunized Cγ1-Cre+/– control, DAF-TMCγ1 and ΔC3ar1/C5ar1Cγ1 mice on d12. Data are presented as MFI (b-d) or mean (a, e-h, k) +/− SEM *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by ANOVA with Bonferroni post-test (a-h, k), For kinetics in (g), 3 genotypes were compared at each time point. Each dot represents a biological replicate. n.s., not significant.

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Extended Data Fig. 5 Analysis of C3aR1 and C5aR1 Expression in mouse and human B cell subsets.

a-b, Representative histograms (left panels) and quantified results (right panels) for C3aR1 (a) or C5aR1 (b) expression on splenic B cell subsets of immunized control Cγ1-Cre+/– mice. c-d, Representative histograms (left panels) and quantified results (right panels) for C3aR1 (c) and C5aR1 (d) expression on human tonsil B cell subsets. e, Representative histograms (left panels, Cγ1-Cre+/– mice) and quantified results (right panel) for C5aR2 (C5L2) expression on B cell subsets from DAF-TMCγ1 (DAF TM), ΔC3ar1/C5ar1Cγ1 (ΔC3aRΔC5aR), and control Cγ1-Cre+/– (Cγ1 Ctrl) mice on d10 after NP-KLH immunization. (f) Quantified GC sizes on d10 post-immunization and (g) representative IF images of GCs from d6 and d10 ΔC3ar1/C5ar1Cγ1 and Cγ1-Cre+/– mice post-SRBC immunization (spleen). Dotted lines in (g) outline GCs. Scale bar 50 μm. Data derived from 3 different tissue sections from each of 3 individual animals. Data are presented as MFI (a-e) or mean (f) ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 by ANOVA with Bonferroni post-test (a-e) or Students t-test (f). n.s., not significant. Each dot represents a biological replicate.

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Extended Data Fig. 6 Characterization of mouse GC and antibody responses in absence of CD21 or complement component expression.

a, Relative (left), absolute frequency (middle) of GC B cells and serum anti-TNP antibodies (right panel) in groups of WT and germline, congenic, cohoused C3–/– and Cr1–/– (Cr2–/–, CD21 null) mice on d14 after immunization with TNP-KLH (CR1 and CR2/CD21 derive from alternatively spliced transcripts from a single gene in mice). b, Quantified GC B cells (left 2 panels) and serum anti-NP antibodies (total, 3rd panel, high affinity 4th panel) in C3 BM chimeras and controls (see Methods). c, Relative (left), absolute frequency (middle) of GC B cells and serum anti-TNP antibodies (right panel) in groups of WT and germline congenic cohoused C3–/–, fB–/–, C1q–/– and Mbl1–/–Mbl2–/– (Mbl–/–) mice on d14 after immunization with TNP-KLH. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by ANOVA with Bonferroni post-test. n.s. not significant. Each dot represents a biological replicate.

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Extended Data Fig. 7 Extended data for RNA-seq and surface marker signatures of GC B cells in DAF-TMCγ1, ΔC3ar1/C5ar1Cγ1 and Cγ1-Cre+/– mice.

a, General summary of curated pathways up- or downregulated in RNA-seq gene expression datasets (relative enrichment) from DAF-TMCγ1 (DAF TM) and control Cγ1-Cre+/– (Cγ1 Ctrl) mice (see also Extended Data Table 2). p-values hypergeometric distribution based on gene overlaps, with FDR q-value <0.05 (p-value after Benjamini and Hochberg correction for multiple hypothesis testing). b, Representative flow cytometry plots depicting the percentage of CD62L+ GC B cells from DAF-TMCγ1 (DAF TM), ΔC3ar1/C5ar1Cγ1 (ΔC3aRΔC5aR), and control Cγ1-Cre+/– (Cγ1 Ctrl) mice on d10 after immunization with SRBC. c, Representative flow cytometry histograms for TLR7, CCR7 and S1P1 gated on IgD−GL7+Fas+ GC B cells from DAF-TMCγ1, ΔC3ar1/C5ar1Cγ1 and Cγ1-Cre+/– mice on d10 after immunization with SRBC (numbers correspond to MFI values).

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Extended Data Fig. 8 Extended data and experimental controls for RNA-seq analysis and mTOR signaling responses to CD40 and C3aR1/C5aR1 ligation.

a, GSEA enrichment plot for E2F gene signature in DAF-TMCg1 vs. Cγ1-Cre+/– mice. NES, normalized enrichment score; FDR, false discovery rate (see also Extended Data Table 2). b, Representative histograms (left) and MFI (right) of pS6 levels in GC B cells (filled histograms) from DAF-TMCγ1 (DAF TM), ΔC3ar1/C5ar1Cγ1 (ΔC3aRΔC5aR), and control Cγ1-Cre+/– (Cγ1 Ctrl) at d10 post-SRBC immunization (without anti-CD40 or anti-IgM F(ab’)2 stimulation). c-d, Representative histograms for pS6 levels in naïve (left) or GC B cells (right) from DAF-TMCγ1, ΔC3ar1/C5ar1Cγ1 and Cγ1-Cre+/– mice on d10 after SRBC immunization and 4 h after i.v. anti-CD40 antibody at the indicated dose (c), or anti-CD40+ anti-IgM F(ab’)2 as indicated. e-g, 2 × 107 WT or C3aR1–/–C5aR–/– B cells were transferred into μMT recipients, which were subsequently immunized with SRBC. Levels of pS6 were quantified in GC (e) or naïve B cells (f) d10 post-immunization and 4 h after i.v anti-CD40/anti-IgM F(ab’)2 stimulation. g, ELISA for serum IgM in adoptive hosts (d10), including μMT negative and a WT B6 positive controls. h, Representative pS6 staining histograms of in vitro cultured naïve (top) and GC (bottom) B cells, stimulated for 20 min ± recombinant C3a, C5a (alone), without anti-CD40/ anti-IgM F(ab’)2 stimulation. n = 5/group, 2 independent experiments. Data are presented as MFI (b, e-f) or mean (g) +/- SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by ANOVA with Bonferroni post-test (b, e-f). n.s, not significant. Each dot represents a biological replicate.

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Cumpelik, A., Heja, D., Hu, Y. et al. Dynamic regulation of B cell complement signaling is integral to germinal center responses. Nat Immunol 22, 757–768 (2021). https://doi.org/10.1038/s41590-021-00926-0

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