Extended Data Fig. 4: TLR9–MyD88-independent functions are B cell-intrinsic.

a-b, The competitive advantage of Tlr9^WT vs Tlr9^−/− or Tlr9^P915H/P915H in different subsets of immature (n = 11 mice from three cohorts), transitional and naïve B cells (n = 15 mice from three cohorts), at the late time point (19-20 weeks post chimerism). Values greater than one on the log₂ scale indicate TLR9^WT advantage and below one indicate Tlr9^−/− or Tlr9^P915H/P915H advantage ((a) T2, p = 0.066; FO, p < 0.0001; (b) Fr. A-C, p < 0.0434; Fr. D, p = 0.0063; Fr. E, p = 0.0092). c,d, The competitive advantage of Tlr9^WT vs Tlr9^−/− or Tlr9^P915H/P915H in different subsets of mature and differentiated B cells at the late time point (FO, MZ, GC, ABC, PB, CD80 + PDL2 + n = 15 mice from three cohorts; PC, n = 11 mice from three cohorts). Values greater than one on the log₂ scale indicate TLR9^WT advantage and below one indicate Tlr9^−/− or Tlr9^P915H advantage ((c) FO, p < 0.0001; CD80⁺PDL2⁺, p = 0.0162; PB, p = 0.0003; PC, p = 0.0356; FO versus MZ, p < 0.0001 (d) ABC, p = 0.0025; CD80⁺PDL2⁺, p = 0.0052; PB, p = 0.0014; PC, p = 0.0288). e, The competitive advantage of Tlr9^−/− vs Tlr9^P915H/P915H in different subsets of mature and differentiated B cells at the late time point (FO, MZ, GC, ABC, PB, CD80 + PDL2 + n = 15 mice from three cohorts; PC, n = 11 mice from three cohorts). Values greater than one indicate Tlr9^−/− advantage and below one indicate Tlr9^P915H advantage (FO, p < 0.0001; GC, p = 0.0002; ABC, p = 0.0005; CD80⁺PDL2⁺, p = 0.0002; PB, p < 0.0001; PC, p = 0.0056). (a-e) Data were pooled from three cohorts (two females and one male). Symbols represent individual mice and bars the mean ± SEM. Each group was compared to the hypothetical mean of no competitive advantage (one), by two-tailed one-sample t test (# p < 0.05; ## p < 0.01; ### p < 0.001; #### p < 0.0001), shown above each individual bar. Additionally, for (c), statistical significance between FO and the other B cell subsets was calculated by a mixed-effect analysis with Dunnett’s multiple comparisons test (* p < 0.05). f,g, Quantification of TLR7 (f) and TLR9 (g) in Tlr9^WT transitional (T1,T2) (TLR7 n = 5; TLR9, n = 5 mice), FO (TLR7, n = 18; TLR9, n = 12 mice), MZ (TLR7, n = 18; TLR9, n = 12 mice), ABC (TLR7, n = 18; TLR9, n = 12 mice) and memory-like CD80⁺PDL2⁺ (TLR7, n = 11; TLR9, n = 5 mice) B cells of the chimera mice, at the early time point. TLR7 expression is minimal in immature B cells and maximal in ABCs ((f) T1 versus FO, p = 0.0180; T1 versus MZ, p = 0.0122; T1 versus ABC, p = 0.0017; T1 versus CD80⁺PDL2⁺, p = 0.0059; T2 versus FO, p = 0.0127; T2 versus MZ, p = 0.0119; T2 versus ABC, p = 0.0015; T2 versus CD80⁺PDL2⁺, p = 0.0064; FO versus MZ, p < 0.0001; FO versus ABC, p < 0.0001; FO versus CD80⁺PDL2⁺, p < 0.0001; MZ versus ABC, p < 0.0001; ABC versus CD80⁺PDL2⁺, p < 0.0001; (g) T1 versus ABC, p = 0.0193; T2 versus ABC, p = 0.0240; FO versus ABC, p < 0.0001; MZ versus ABC, p < 0.0001; ABC versus CD80⁺PDL2⁺, p = 0.0144). ((f-g), Symbols represent individual mice and bars the mean ± SEM. ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001; ^**** p < 0.0001 Mixed-effects analysis between subsets, with Tukey’s multiple comparisons test). h,i, Quantification of TLR7 MFI (ratio of TLR7 MFI in Tlr9^−/− or Tlr9^P915H/P915H cells over Tlr9^WT subset per mouse), in FO (early, n = 18; late, n = 14), MZ (early, n = 18; late, n = 14), ABC (early, n = 18; late, n = 14) and CD80⁺PDL2⁺ (early, n = 11; late, n = 14) B cells, at the early (h) and late (i) time point. Each group was compared to the hypothetical mean of one (indicating similar TLR7 expression in mutated Tlr9 and Tlr9^WT mice), by two-tailed one-sample t test (# p < 0.05; ## p < 0.01; ### p < 0.001; #### p < 0.0001), shown above each individual bar; (Symbols represent individual mice and bars the mean ± SEM; (h) Tlr9^−/− FO, p < 0.0001; Tlr9^P915H/P915H FO, p = 0.0002; Tlr9^−/− MZ, p < 0.0001; Tlr9^P915H/P915H MZ, p < 0.0001; Tlr9^−/− ABC, p < 0.0001; Tlr9^P915H/P915H ABC, p = 0.0024; Tlr9^P915H/P915H CD80⁺PDL2⁺, p = 0.0174; (i) Tlr9^−/− FO, p = 0.0019; Tlr9^P915H/P915H FO, p < 0.0001; Tlr9^−/− MZ, p = 0.0008; Tlr9^P915H/P915H MZ, p < 0.0001; Tlr9^−/− ABC, p < 0.0001; Tlr9^P915H/P915H ABC, p = 0.0065). j,k, Quantification of TLR9 MFI in T1 (n = 5), T2 (n = 5), FO (n = 12), MZ (n = 12), ABC (n = 12) and CD80⁺PDL2⁺ (n = 5) B cells of the 3-way BM chimera mice at the early time point. Symbols represent individual mice and bars the mean ± SEM; For statistics, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, RM one-way ANOVA with Tukey’s multiple comparisons test; (j) T1, Tlr9^WT versus Tlr9^−/−, p = 0.0004; Tlr9^WT versus Tlr9^P915H/P915H, p = 0.0022; Tlr9^−/− versus Tlr9^P915H/P915H, p = 0.0004; T2, Tlr9^WT versus Tlr9^−/−, p = 0.0006; Tlr9^WT versus Tlr9^P915H/P915H, p = 0.0003; Tlr9^−/− versus Tlr9^P915H/P915H, p = 0.0009; FO, Tlr9^WT versus Tlr9^−/−, p < 0.0001; Tlr9^WT versus Tlr9^P915H/P915H, p < 0.0001; Tlr9^−/− versus Tlr9^P915H/P915H, p < 0.0001; (k) MZ, Tlr9^WT versus Tlr9^−/−, p < 0.0001; Tlr9^WT versus Tlr9^P915H/P915H, p < 0.0001; Tlr9^−/− versus Tlr9^P915H/P915H, p < 0.0001; ABC, Tlr9^WT versus Tlr9^−/−, p < 0.0001; Tlr9^WT versus Tlr9^P915H/P915H, p < 0.0001; Tlr9^−/− versus Tlr9^P915H/P915H, p < 0.0001; CD80⁺PDL2⁺, Tlr9^WT versus Tlr9^−/−, p = 0.0001; Tlr9^WT versus Tlr9^P915H/P915H, p = 0.0026; Tlr9^−/− versus Tlr9^P915H/P915H, p = 0.0006; l-p, Frequencies of T1 (n = 5), T2 (n = 3), FO (n = 4), ABC (n = 5) B cells and PB (n = 5) of the 3-way BM chimera mice in S or G2/M phase, determined by Ki67 and DAPI staining, and frequencies of activated caspase 3⁺ cells for each B cell subset (n = 6 mice). ((l-p), data points indicate individual mice and bars the mean ± SEM. For statistics * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, RM one-way ANOVA with Turkey’s multiple comparisons test. (l), G2/M T1, Tlr9^WT versus Tlr9^P915H/P915H, p = 0.0240; Caspase 3⁺ Tlr9^−/− versus Tlr9^P915H/P915H, p = 0.0020; Caspase 3⁺ Tlr9^WT versus Tlr9^P915H/P915H, p = 0.0076; (m), S T2, Tlr9^−/− versus Tlr9^P915H/P915H, p = 0.0485; G2/M T2, Tlr9^WT versus Tlr9^−/−, p = 0.0172; Caspase 3⁺ Tlr9^−/− versus Tlr9^P915H/P915H, p = 0.0333; Caspase 3⁺ Tlr9^WT versus Tlr9^P915H/P915H, p = 0.0361; (n) G2/M FO, Tlr9^WT versus Tlr9^−/−, p = 0.0109; Tlr9^WT versus Tlr9^P915H/P915H, p = 0.0113; (o) Caspase 3⁺ Tlr9^−/− versus Tlr9^P915H/P915H, p = 0.0473).

Source data