Fig 1: Rapid production of TNF-a mediates and amplifies the ablation of BTLAhi cDC1s(A) Wild-type mice were treated with PBS or LPS (10 µg/mouse) at the indicated time points before analysis of splenocytes by flow cytometry. Graph shows percentages of BTLAhi cDC1s among all cDCs as a percentage of the baseline (average of PBS-treated mice for each time point) in the indicated groups (n = 4–6 mice per group pooled from two independent experiments per time point). Significance is shown for the comparison of PBS and LPS groups at each time point.(B and C) Wild-type mice were treated with PBS or LPS (10 µg/mouse) 1 h before analysis of splenocytes by flow cytometry.(B) Plots (representative of multiple independent experiments) show anti-XCR1 and anti-TNF-a intracellular staining intensity in all cDCs.(C) Graph shows percentages of TNF-a+ cells within the cDC1 (XCR1+ CD172aneg) and cDC2 (CD172a+XCR1neg) subsets, as indicated, from mice treated with LPS (n = 6 mice pooled from three independent experiments).(D and E) Wild-type mice were treated with PBS (0 h) or LPS (10 µg/mouse) at the indicated time points before analysis of splenocytes by flow cytometry. Graphs show percentages of TNF-a+ cells within cDC1 (D) or cDC2 (E) subsets for each time point (n = 2–6 mice per group pooled from three independent experiments). Significance is shown for each time point compared with the baseline (0 h).(F and G) Tlr4+/+ and Tlr4?CD11c were treated with LPS (10 µg) 1 h before analysis of splenocytes by flow cytometry.(F) Plots (representative of multiple independent experiments) show anti-XCR1 and anti-TNF-a intracellular staining intensity in all cDCs.(G) Graph shows percentages of TNF-a+ cells within the cDC1 subset in the indicated groups (n = 5–6 mice per group pooled from three independent experiments).(H) Wild-type mice were treated with PBS or LPS (10 µg/mouse) 1 h before analysis of splenocytes by flow cytometry. Graph shows percentages of TNF-a+ cells within the indicated populations of cDC1s (n = 6 mice pooled from three independent experiments).(I) Wild-type mice were treated with anti-TNF-a or isotype control antibody as indicated and LPS (10 µg/mouse) 24 h before analysis of splenocytes by flow cytometry. Graph shows percentages of BTLAhi cDC1s among all cDCs in the indicated groups (n = 5–6 mice per group pooled from two independent experiments).(J) Lethally irradiated Tlr4+/+ mice were reconstituted with a 1:1 mix of congenically labeled Tlr4-/- and Tlr4+/+ BM. The reconstituted mixed BM chimera mice were treated with anti-TNF-a or isotype control antibody and PBS or LPS (10 µg/mouse) as indicated 24 h before analysis of splenocytes by flow cytometry. (Left) General experimental outline is shown. (Right) Graph shows percentages of BTLAhi cDC1s among Tlr4-/- cDCs in the indicated groups (n = 3–7 mice per group pooled from two independent experiments).(K and L) Wild-type mice were treated with PBS or recombinant mouse TNF-a 24 h before analysis of splenocytes by flow cytometry.(K) (Top) General experimental outline is shown. (Bottom) Representative plots show anti-XCR1 and anti-BTLA staining intensity in all cDCs.(L) Graph shows percentages of BTLAhi cDC1s among all cDCs in the indicated groups. Results represent one of two similar experiments (n = 3–5 mice per group).(M) Lethally irradiated Tlr4+/+ mice were reconstituted with the indicated ratios of congenically labeled Tlr4-/- and Tlr4+/+ BM. The reconstituted mixed BM chimera mice were treated with LPS (10 µg/mouse) 24 h before analysis of splenocytes by flow cytometry. (Left) General experimental outline is shown. (Right) Graph shows percentages of BTLAhi cDC1s among Tlr4-/- cDCs in the indicated groups (n = 4–7 mice per group pooled from two independent experiments).(B, F, and K) Numbers next to outlined regions indicate corresponding percentages. (A, D, and E) Graphs show mean ± standard error of mean (SEM).(C, G–J, L, and M) Graphs show mean ± SD. *p < 0.05, **p < 0.01, and ****p < 0.0001 determined by two-way ANOVA with Šídák’s multiple comparisons (A), unpaired two-tailed t test (C, G–I, and L), or one-way ANOVA with Tukey’s multiple comparisons (D, E, J, and M).