Fig 2: 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 Vα2+ Vβ5.1,5.2+ 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).

Fig 3: Tannic acid (TA) induced-TNFR1 shedding depends on the protein disulfide isomerase (PDI). A. Knockout effect of the PDI protein in L929 cells, as detected by western blotting. Knockout KO1 and KO2 are two different clones. B. L929 cells and PDI KO cells treated with TNF-α, as detected using a microscope. Cells treated with dimethyl sulfoxide (DMSO) (as the control) or TNF-α for 8 h. C. Activity of wild-type (WT) and PDI KO (KO-1 and KO-2) cells treated with TNF-α, as measured using MTS analysis. Cells treated with DMSO (as the control) or TNF-α for 8 h. D. The fluorescence intensity of TNFR1 on the membranes of L929 cells and PDI KO L929 cells, as detected by flow cytometry. E. Quantitative statistics of the TNFR1 fluorescence intensity on the cell membranes, as detected by flow cytometry. F. The fluorescence intensity of TNFR1 on the membranes of L929 cells treated with vinigrol (Vi) and TA for 8 h, as detected by flow cytometry. G. Quantitative statistics of the TNFR1 fluorescence intensity on cell membranes, as detected by flow cytometry. H. The change of soluble TNFR1 (sTNFR1) in the medium, as detected by ELISA. n = 3. Data are means ± SEM. A one-way ANOVA test was used, *p < 0.05, **p < 0.01, ***p < 0.001. n = the number of biological replicates. ns = no significant difference