Extended Data Fig. 3: Effects of TLR9 and gender on TLR7 expression and distribution.

a-d, Representative flow cytometry plots of live, singlet, TCRβ⁻CD19⁺ B cells of 13 week old BALB/c (a) or 5 week old MRL/lpr (d) mice. Numbers indicate the frequencies in the gate. b,c, Comparison of TLR7 and TLR9 MFI between Tlr9 ^WT male and Tlr9 ^WT female BALB/c mice is shown for each of the defined B cell subsets (male, n = 8; female, n = 8 from two experiments; (b) male versus female MZ B cells p = 0.0333; male FO versus male MZ B cells p < 0.0001; (c) male FO versus male MZ B cells p = 0.0009). e,f, Comparison of TLR7 and TLR9 MFI between Tlr9 ^WT male and Tlr9 ^WT female 5 week old MRL/lpr mice is shown for each of the defined B cell subsets (male, n = 9 mice; female, n = 9 mice from two experiments; (e) male versus female FO B cells p < 0.0001; male versus female MZ B cells p < 0.0001; male versus female ABC B cells p = 0.0282; male FO versus male MZ B cells p < 0.0001; male FO versus male ABC B cells p < 0.0001; male MZ versus male ABC B cells p = 0.0330 (f) male FO versus male MZ B cells p < 0.0001; male FO versus male ABC B cells p < 0.0001; male MZ versus male ABC B cells p < 0.0001). (For b,c,e,f, data points indicate individual mice and bars the mean ± SEM of two experiments pooled. * p < 0.05, **** p < 0.0001, two tailed unpaired t-test between male and female groups; b,c, ^# p < 0.05; ^## p < 0.01; ^### p < 0.001; ^#### p < 0.0001, two tailed paired-t-test between subsets in the male group; e,f ^# p < 0.05; ^## p < 0.01; ^### p < 0.001; ^#### p < 0.0001 RM one way ANOVA with Tukey’s multiple comparisons test between subsets in the male group.) g-i, Quantification of TLR7 MFI (ratio of TLR7 MFI over average TLR7 MFI of the corresponding male or female WT group), in FO, MZ and ABC B cells of 5 week old female and male MRL/lpr mice (Tlr9^K51E/K51E male, n = 4; female, n = 4; Tlr9 ^WT male, n = 4, female, n = 8; Tlr9^P915H/P915H male, n = 4, female, n = 7; Tlr9^+/− male, n = 3, female, n = 4; Tlr9^−/− male, n = 3, female, n = 7; (g, male) Tlr9^K51E/K51E versus Tlr9^P915H/P915H p = 0.0097; Tlr9^K51E/K51E versus Tlr9^−/− p = 0.0061, Tlr9^+/− versus Tlr9^−/− p = 0.0430; (g, female) Tlr9 ^WT versus Tlr9^+/− p = 0.0232; Tlr9 ^WT versus Tlr9^−/− p < 0.0001; Tlr9^K51E/K51E versus Tlr9^+/− p = 0.0386, Tlr9^K51E/K51E versus Tlr9^−/- p < 0.0001, Tlr9^P915H/P915H versus Tlr9 ^-/- p = 0.0002; (h, male) Tlr9 ^WT versus Tlr9^P915H/P915H p = 0.0018; Tlr9 ^WT versus Tlr9 ^-/- p = 0.0261; Tlr9^K51E/K51E versus Tlr9^P915H/P915H p < 0.0001; Tlr9^K51E/K51E versus Tlr9^−/− p = 0.0005; Tlr9^P915H/P915H versus Tlr9^+/− p = 0.0001; Tlr9^−/− versus Tlr9^+/− p = 0.0012; (h, female) Tlr9 ^WT versus Tlr9^−/− p = 0.0033; Tlr9^K51E/K51E versus Tlr9^−/− p < 0.0021; Tlr9^−/− versus Tlr9^+/− p = 0.0074; (i, male) Tlr9 ^WT versus Tlr9^K51E/K51E p = 0.0398, Tlr9^K51E/K51E versus Tlr9^P915H/P915H p = 0.0143, Tlr9^K51E/K51E versus Tlr9^−/− p = 0.0284; (i, female) Tlr9 ^WT versus Tlr9^+/− p = 0.0011, Tlr9 ^WT versus Tlr9^−/− P < 0.0001, Tlr9^K51E/K51E versus Tlr9^+/− p = 0.0004, Tlr9^K51E/K51E versus Tlr9^−/− p < 0.0001, Tlr9^P915H/P915H versus Tlr9^+/− p = 0.004, Tlr9^P915H/P915H versus Tlr9^−/− p = 0.0002). j-l, Quantification of TLR9 MFI (ratio of TLR9 MFI over mean TLR9 MFI of the corresponding male or female WT group), in FO, MZ and ABC B cells of 5 week old female and male MRL/lpr mice (Tlr9^K51E/K51E male, n = 4; female, n = 4; Tlr9 ^WT male, n = 4, female, n = 8; Tlr9^P915H/P915H male, n = 4, female, n = 7; Tlr9^+/− male, n = 3, female, n = 4; Tlr9^−/− male, n = 3, female, n = 7; (j, male) Tlr9 ^WT versus Tlr9^P915H/P915H p = 0.0001, Tlr9 ^WT versus Tlr9^+/− p < 0.0001, Tlr9 ^WT versus Tlr9^−/− p < 0.0001, Tlr9^K51E/K51E versus Tlr9^P915H/P915H p = 0.0004, Tlr9^K51E/K51E versus Tlr9^+/− p < 0.0001, Tlr9^K51E/K51E versus Tlr9^−/− p < 0.0001, Tlr9^P915H/P915H versus Tlr9^−/− p = 0.0004, Tlr9^+/− versus Tlr9^−/− p = 0.0104; (j, female) Tlr9 ^WT versus Tlr9^P915H/P915H p < 0.0001, Tlr9 ^WT versus Tlr9^+/− p < 0.0001, Tlr9 ^WT versus Tlr9^−/− p < 0.0001, Tlr9^K51E/K51E versus Tlr9^P915H/P915H p < 0.0001, Tlr9^K51E/K51E versus Tlr9^+/− p < 0.0001, Tlr9^K51E/K51E versus Tlr9^−/− p < 0.0001, Tlr9^P915H/P915H versus Tlr9^−/− p < 0.0001, Tlr9^+/− versus Tlr9^−/− p < 0.0001; (k, male) Tlr9 ^WT versus Tlr9^P915H/P915H p < 0.0001, Tlr9 ^WT versus Tlr9^+/− p < 0.0001, Tlr9 ^WT versus Tlr9^−/− p < 0.0001, Tlr9^K51E/K51E versus Tlr9^P915H/P915H p < 0.0001, Tlr9^K51E/K51E versus Tlr9^+/− p < 0.0001, Tlr9^K51E/K51E versus Tlr9^−/− p < 0.0001, Tlr9^P915H/P915H versus Tlr9^−/− p = 0.0008, Tlr9^+/− versus Tlr9^−/− p = 0.0015; (k, female) Tlr9 ^WT versus Tlr9^P915H/P915H p < 0.0001, Tlr9 ^WT versus Tlr9^+/− p < 0.0001, Tlr9 ^WT versus Tlr9^−/− p < 0.0001, Tlr9^K51E/K51E versus Tlr9^P915H/P915H p < 0.0001, Tlr9^K51E/K51E versus Tlr9^+/− p < 0.0001, Tlr9^K51E/K51E versus Tlr9^−/− p < 0.0001, Tlr9^P915H/P915H versus Tlr9^+/− p = 0.0467, Tlr9^P915H/P915H versus Tlr9^−/− p < 0.0001, Tlr9^+/− versus Tlr9^−/− p < 0.0001; (l, male) Tlr9 ^WT versus Tlr9^P915H/P915H p < 0.0001, Tlr9 ^WT versus Tlr9^+/− p < 0.0001, Tlr9 ^WT versus Tlr9^−/− p < 0.0001, Tlr9^K51E/K51E versus Tlr9^P915H/P915H p < 0.0001, Tlr9^K51E/K51E versus Tlr9^+/− p < 0.0001, Tlr9^K51E/K51E versus Tlr9^−/− p < 0.0001, Tlr9^P915H/P915H versus Tlr9^−/− p < 0.0001, Tlr9^+/− versus Tlr9^−/− p < 0.0001; (l, female) Tlr9 ^WT versus Tlr9^P915H/P915H p < 0.0001, Tlr9 ^WT versus Tlr9^+/− p < 0.0001, Tlr9 ^WT versus Tlr9^−/− p < 0.0001, Tlr9^K51E/K51E versus Tlr9^P915H/P915H p = 0.0018, Tlr9^K51E/K51E versus Tlr9^+/− p = 0.0108, Tlr9^K51E/K51E versus Tlr9^−/− p < 0.0001, Tlr9^P915H/P915H versus Tlr9^−/− p = 0.0008, Tlr9^+/− versus Tlr9^−/− p = 0.0024). (For g-l, data points indicate individual mice and bars the mean ± SEM of one or two experiments pooled; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test). m, The percent of TLR7 positivity in EEA1⁺ or LAMP-1⁺ endosomes of TLR9^WT B cells is unchanged after a 2 hour stimulation with TLR7 agonist (CL097). (TLR7⁺EEA1⁺ unstimulated B cells, n = 17 from one mouse; TLR7⁺EEA1⁺ stimulated B cells, n = 25 from one mouse; TLR7⁺LAMP1⁺ unstimulated B cells, n = 27 from two mice; TLR7⁺LAMP1⁺ stimulated B cells, n = 40 from two mice). Data points indicate individual fields (1-3 cells per field) analyzed, and bars the mean ± SEM of two mice.

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