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

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.

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.

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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

Affiliations

Authors

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.

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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.

Source data

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 IgDFas+GL7+ GC B cells (middle) and IgDGL7+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 (CXCR4CD86+) 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+IgDGL7Fas 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+IgDGL7Fas 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 IgDGL7+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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