Fig 2: FasL and TRAIL compete for DR5 binding and exert similar effects on cell death.(A, B) FasL–Fc binding to hFLSCs or EL4 cells transfected with human FAS, or TNFRSF10B after preincubation with human sTRAIL or sFasL. (C) Model of FasL and DR5 derived from the crystal structure of the FasL/DcR3 complex (Protein Data Bank: 4 MSV) and TRAIL/DR5 complex (Protein Data Bank: 1D4V). (D, E) Flow cytometric analysis of FasL–Fc or DR5–Fc binding to EL4 cells transfected with human WT or mutated TNFRSF10B or FASLG. (F, G) Comparison of the effects of sFasL and sTRAIL on (F) apoptosis and (G) necroptosis in hFLSCs. (H) Joint swelling and clinical scores in Faslgld/gld mice injected with Z–VAD–FMK and/or sFasL (n = 6 per group). Data were pooled from four (A, B, and D–G) or three (H) independent experiments and are presented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way ANOVA.Figure 2—source data 1.Numerical data obtained during experiments represented in Figure 2, Figure 2—figure supplement 3.

Fig 3: Death receptor (DR5) is a Fas-independent receptor for soluble Fas ligand (sFasL) that promotes arthritis.(A) Joint swelling and clinical scores in wild-type (WT), Faslpr/lpr, Faslgld/gld, and Fas–/– mice (n = 6 per group). (B) Joint swelling and clinical scores in WT, Fasl?m/?m, Fasl?s/?s, and Fasl?s/?s mice injected with sFasL (n = 6 per group). (C, D) Gross and microscopic examination of arthritis (magnified 10× in the upper panel and 200× in the lower panel). Scale bars: 1 cm (C), 200 µm (D, upper panel), and 100 µm (D, lower panel). (E) Tandem mass spectra of unique DR5 peptides. (F) Transcript levels of Tnfrsf10b in synovial CD45+ immune cells and CD45– non-immune cells from WT mice with or without AIA. (G) Immunohistochemistry of DR5 expression in joint tissue from a healthy control subject and a patient with rheumatoid arthritis (n = 3; magnified 400×, scale bar: 50 µm). (H) Flow cytometric analysis of biotinylated protein binding to EL4 cells transfected with human WT TNFRSF10B preincubated with recombinant hTRAIL, or simultaneously incubated with anti-FasL, or anti-DR5 antibodies. (I) Flow cytometric analysis of biotinylated FasL binding on hFLSCs with FAS and/or TNFRSF10B knockout, and TNFRSF10B and/or FAS overexpression in FAS and TNFRSF10B double knockout (DKO) cells. (J) hLFSCs were preincubated with TNF-a (as a negative control), FasL, or TRAIL and cross–linked with BS3. Lysates from these cells were immunoprecipitated with anti–DR5 or control IgG antibody and immunoblotted with anti-DR5, TNF-a, FasL, or TRAIL antibodies. (K) Flow cytometric analysis of DR5–Fc binding on EL4 cells transfected with human WT FASLG in the presence of recombinant hTRAIL, anti-DR5, or FasL antibodies. Data were pooled from three (A, B, and D–G) or four (H, K) independent experiments and are presented as mean ± standard error of the mean (SEM). *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way analysis of variance (ANOVA).Figure 1—source data 1.Numerical data obtained during experiments represented in Figure 1, Figure 1—figure supplements 1, 3, and 4.

Fig 4: FAS Y232C reduces FAS signaling activity and promotes resistance to FASL-mediated apoptosis(A) Diagram of Fas ligand-based killing assay. (B) Percent live cells remaining after Fas ligand-based killing assay. (C) ICS of downstream protein target. T cells were either exposed to Fas ligand and a conjugating His-Tag antibody for 24 h or just the His-Tag antibody and then ICS was performed for cleaved caspase 3. n = 2 donors. ns = not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; (B) two-way ANOVA with Dunnett’s multiple comparisons test against no gRNA control; and (C) two-way ANOVA with uncorrected Fisher’s LSD.