Fig 1: TNFR1 orchestrates the ablation of tolerogenic BTLAhi cDC1s(A and B) Spleens from untreated wild-type mice were analyzed by flow cytometry.(A) Plot (left) shows anti-XCR1 and anti-BTLA staining intensity in all cDCs. Shaded regions specify gating of populations denoted in the overlaid histograms (right) that show anti-TNFR1 staining intensity of the indicated populations. Results are representative of multiple independent experiments.(B) Graph shows median fluorescence intensity (MFI) of anti-TNFR1 staining in the indicated populations (gated as in A; n = 4 mice pooled from three independent experiments).(C–E) Lethally irradiated Tlr4+/+Tnfrsf1a+/+ mice were reconstituted with a 1:1 mix of congenically labeled Tlr4-/-Tnfrsf1a+/+ and Tlr4+/+Tnfrsf1a+/+ or Tlr4-/-Tnfrsf1a-/- and Tlr4+/+Tnfrsf1a+/+ BM.(C) General experimental outline for mixed BM chimeras.(D) Reconstituted mixed BM chimera mice were treated with PBS or LPS (10 µg/mouse) 24 h before analysis of splenocytes by flow cytometry. Graph shows percentages of BTLAhi cDC1s among cDCs within the indicated genotype for each treatment (n = 3–8 mice per group pooled from two independent experiments).(E) Reconstituted mixed BM chimera mice were treated with PBS or LPS (10 µg/mouse) 48 h before analysis of peripheral lymph nodes by flow cytometry. Graph shows percentages of BTLAhiCCR7lo cDC1s among cDCs within the indicated genotype for each treatment (n = 4–7 mice per group pooled from three independent experiments).(F–H) Lethally irradiated Tlr4+/+Tnfrsf1a+/+ mice were reconstituted with a 2:1 mix of congenically labeled Tlr4-/-Tnfrsf1a+/+ and Tlr4+/+Tnfrsf1a+/+ or Tlr4-/-Tnfrsf1a-/- and Tlr4+/+Tnfrsf1a+/+ BM. Sorted Foxp3negCD25neg T cells from OT-II TCR tg Foxp3RFP mice were adoptively transferred into the reconstituted mixed BM chimera mice that were treated with anti-DEC-OVA and LPS (10 µg/mouse) 14 to 15 days before analysis of spleens and peripheral lymph nodes by flow cytometry.(F) General experimental outline for adoptive transfers into reconstituted mixed BM chimeras.(G and H) Graphs show percentages of Foxp3(RFP)+ cells among Va2+ Vß5.1,5.2+ CD4+ T cells in the indicated groups of recipients among splenocytes (G) or lymph node cells (H) (n = 10–12 mice per group pooled from three independent experiments).(B, D, E, G, and H) Graphs show mean ± SD. *p < 0.05, **p < 0.01, and ****p < 0.0001 determined by one-way ANOVA with Tukey’s multiple comparisons (B, D, and E) or unpaired two-tailed t test (G and H).