Extended Data Fig. 3: H1c^−/−H1e^−/− mature B cells show normal development in spleen and bone marrow.

a, mRNA of human H1B–H1E normalized to RPL13A in GC B cells relative to naive B cells (H1B, **P = 0.004; H1E, *P = 0.027), isolated from three independent specimens of human tonsils. Data are mean ± s.d., two-sided unpaired t-tests. b, Mouse H1b–H1e mRNA levels normalized to Gapdh in sorted GC B cells (n = 3) relative to naive B cells(n = 3) (H1b, ****P < 0.0001). Data are mean ± s.d., two-sided unpaired t-tests. c, Quantification of spleen/body weight ratios of two-month-old H1c^−/−H1e^−/− (n = 13) and wild-type littermate control (n = 14) mice. Data are pooled from two independent experiments. P < 0.05; NS, not significant; two-sided unpaired t-tests. Data are mean ± s.d. d, Quantification of GC area (Ki67 staining) in the spleens of H1c^−/−H1e^−/− (n = 10) and WT (n = 10) mice. ***P = 0.0005. Data are mean ± s.d., two-sided unpaired t-tests. e, f, Immunohistochemistry images of spleen sections of cleaved caspase-3 (e) and γ-H2AX (f) staining (left) and quantification (right) of positively stained follicular cells from H1c^−/−H1e^−/− (n = 3) and wild-type littermate control (n = 3) mice immunized with SRBCs and euthanized 10 days after immunization. Scale bars, 100 μm. P < 0.05 (not significant; NS), two-sided unpaired t-tests. Data are mean ± s.d. g, Flow cytometry analysis and quantification of (Fas⁺CD38⁻) GC B cells within total B cells from H1c^−/−H1e^−/− and wild-type mice (n = 10 per genotype). **P = 0.0018, two-sided unpaired t-tests. Data are mean ± s.d. h, Quantification of the percentage of B220⁺ splenocytes in H1c^−/−H1e^−/− (n = 10) and wild-type (n = 10) mice 9 days after SRBC immunization. P < 0.05, two-sided unpaired t-tests. Data are mean ± s.d. i, Flow cytometry analysis and quantification of GC B cells (Fas⁺GL7⁺) from H1c^−/−H1e^−/− (n = 10) and wild-type (n = 10) mice. Two-sided unpaired t-tests, *P = 0.041. Data are mean ± s.d. j, Flow cytometry analysis and quantification of mature B cells (B220⁺IgD⁺IgM⁺) and transitional B cells (B220⁺IgD^intIgM⁺) in spleens from H1c^−/−H1e^−/− (n = 10) and wild-type (n = 10) mice. P < 0.05, two-sided unpaired t-tests. Data are mean ± s.d. k, Flow cytometry quantification of follicular B cells (B220⁺D23⁺CD21⁺) and marginal zone B cells (B220⁺D23^loCD21⁺) in spleens from H1c^−/−H1e^−/− (n = 10) and wild-type (n = 10) mice. P < 0.05, two-sided unpaired t-tests. Data are mean ± s.d. l, Flow cytometry analysis gated on B220⁺CD24⁺ and quantification of ProBPreB (IgM⁻IgD⁻), immature (IgM⁻IgD^lo), transitional (IgD⁺IgM⁻) and early mature (IgD⁺IgM⁺) B cells in bone marrow of H1c^−/−H1e^−/− (n = 4) and wild-type (n = 5) mice. P < 0.05, two-sided unpaired t-tests. Data are mean ± s.d. m, Percentage of Ki67⁺early B cells (B220⁺CD24⁺) in bone-marrow of H1c^−/−H1e^−/− (n = 4) and wild-type (n = 5) mice, as well as naive B cells (***P = 0.0004)and marginal zone B cells (***P = 0.001) in the spleens of H1c^−/−H1e^−/− (n = 5) and wild-type (n = 5) mice. n, Schematic diagram of primary immunization with NP-KLH and secondary immunization 21 days after with NP-CGG. o, Ratio between high (NP₈) and low (NP₃₀) affinity NP-specific IgG1 antibody titres in sera of H1c^−/−H1e^−/− (n = 5) and wild-type (n = 5) mice by enzyme-linked immunosorbent assay (ELISA). P < 0.05, two-sided unpaired t-tests. Data are mean ± s.d. p, Enzyme-linked immunosorbent spot (ELISPOT) quantification of NP-specific (anti-NP₈ and anti-NP₃₀) IgG1-secreting cells from the bone marrow of H1c^−/−H1e^−/− (n = 5) and wild-type (n = 5) mice. P < 0.05, two-sided unpaired t-test. Data are mean ± s.d. Data are representative of two independent experiments. q, Representative images of anti-NP₈ and anti-NP₃₀ 96-well ELISPOT. r, Flow cytometry analysis and quantification of centroblasts within the dark zone (DZ) (CXCR4⁺CD86⁻) (***P = 0.0002) and centrocytes within the light zone (LZ) (CXCR4⁻CD86⁺) (***P = 0.0002) within GC B cells from H1c^−/−H1e^−/− (n = 10) and wild-type (n = 10) mice. Two-sided unpaired t-test. Data are representative of three independent experiments. s, Immunofluorescence confocal microscopy images of GCs at day 7 after immunization in mixed chimaeras. Scale bar, 50 μm. Images are representative of two independent experiments. t, Quantification from s of the fraction of PNA⁺CD45.1 or CD45.2 cells (17 GCs, n = 3 mice). Two-sided paired t-test, ***P = 0.0004. u, Relative EdU⁺ GC B cell/GC B cell fraction for wild-type CD45.1⁺and H1c^−/−H1e^−/− CD45.2⁺ cells at day 7 after immunization (n = 4 chimaeras). Two-sided paired t-test, **P = 0.0065. Data are representative of two independent experiments.