Extended Data Fig. 1: TLR9 has a scaffold protective role in lupus.

a-b, Proportion of activated CD21^int (a) or CD21^hi (b) B cells with nuclear NF-κB (black gate, Fig. 1c) before (black bar) and after (red bar) stimulation. Five week old MRL/lpr mice, n = 3 per group, (* p < 0.05; *** p < 0.001, two-tailed paired-t test between the unstimulated and stimulated conditions for each Tlr9 genotype; (a) Tlr9 ^WT unstimulated versus stimulated p = 0.0007, Tlr9^K51E/K51E unstimulated versus stimulated p = 0.0452, (b) Tlr9 ^WT unstimulated versus stimulated p = 0.0013, Tlr9^K51E/K51E unstimulated versus stimulated p = 0.0480; ^# p < 0.05, ^### p < 0.001, one-way ANOVA between the stimulated conditions). c-g, Evaluation of the clinical parameters, separated by gender (Tlr9^WT male, n = 54; Tlr9^−/− male, n = 21; Tlr9^K51E/K51E male, n = 15; Tlr9^WT female, n = 46; Tlr9^−/− female, n = 22; Tlr9^K51E/K51E female, n = 16; (c female) Tlr9^WT versus Tlr9^−/− p < 0.0001, Tlr9^−/− versus Tlr9^K51E/K51E p = 0.0001; (d) Tlr9^WT versus Tlr9^−/− p = 0.0056, Tlr9^−/− versus Tlr9^K51E/K51E p < 0.0001,Tlr9^WT versus Tlr9^K51E/K51E p = 0.038; (e) Tlr9^WT versus Tlr9^−/− p < 0.0001, Tlr9^−/− versus Tlr9^K51E/K51E p < 0.0001; (f) Tlr9^WT versus Tlr9^−/− p = 0.0173, Tlr9^−/− versus Tlr9^K51E/K51E p = 0.0003; (g) Tlr9^WT versus Tlr9^−/− p = 0.0632, Tlr9^−/− versus Tlr9^K51E/K51E p = 0.0115). h, Representative gating strategy of B cell populations (upper panel) and T cell (lower panel) subsets i, Percent TCR⁻CD19 + B cells of live splenocytes in male and female mice of the indicated genotypes (Tlr9^WT, n = 86; Tlr9^−/−, n = 36; Tlr9^K51E/K51E, n = 29; (i) Tlr9^WT versus Tlr9^−/− p = 0.0420, Tlr9^−/− versus Tlr9^K51E/K51E p = 0.0078). j-k, Percent CD21^hi CD23^lo MZ, and CD11b⁺ CD11c⁺ ABC of live B cells in mice of the indicated genotypes. ((j) Tlr9^WT, n = 86; Tlr9^−/−, n = 36; Tlr9^K51E/K51E, n = 29; Tlr9^WT versus Tlr9^−/− p = 0.0081, Tlr9^−/− versus Tlr9^K51E/K51E p = 0.0196; (k) Tlr9^WT, n = 86; Tlr9^−/−, n = 36; Tlr9^K51E/K51E, n = 24; (k) Tlr9^WT versus Tlr9^−/− p = 0.0871, Tlr9^−/− versus Tlr9^K51E/K51E p = 0.0215) l, Percent TCR⁻CD44^hiCD138^hi plasmablasts of live splenocytes in mice of the indicated genotypes (Tlr9^WT, n = 86; Tlr9^−/−, n = 36; Tlr9^K51E/K51E, n = 29; Tlr9^WT versus Tlr9^−/− p = 0.0372, Tlr9^−/− versus Tlr9^K51E/K51E p = 0.0015). m, Percent TCR⁺CD4⁺ and TCR⁺CD8⁺ cells of live splenocytes in male and female mice of the indicated genotypes (Tlr9^WT, n = 79; Tlr9^−/−, n = 36; Tlr9^K51E/K51E, n = 28; for CD4 + , Tlr9^−/− versus Tlr9^K51E/K51E p = 0.0002, Tlr9^WT versus Tlr9^K51E/K51E p = 0.0023; for CD8 + , Tlr9^WT versus Tlr9^−/− p = 0.001, Tlr9^−/− versus Tlr9^K51E/K51E p = 0.0304). n-o, Percent naïve (CD62L^hi CD44^lo), activated (CD62^hi CD44^hi) and memory (CD62L^lo CD44^hi) of CD4 and CD8 splenocytes (Tlr9^WT, n = 79; Tlr9^−/−, n = 36; Tlr9^K51E/K51E, n = 28; for naïve CD4 + , Tlr9^WT versus Tlr9^−/− p = 0.0017, Tlr9^−/− versus Tlr9^K51E/K51E p = 0.0071; for activated CD4 + , Tlr9^WT versus Tlr9^−/− p < 0.0001, Tlr9^−/− versus Tlr9^K51E/K51E p = 0.0089; for naïve CD8 + , Tlr9^WT versus Tlr9^−/− p = 0.0012; for memory CD8 + , Tlr9^WT versus Tlr9^−/− p = 0.0095). For all panels, data points indicate individual mice and bars the mean ± SEM. For statistics in (c-o) * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test).

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