Fig 2: RIPK1 activation causes inflammation through T-Mono axis.a Schematic diagram of T cell-MDM co-culture system. b, c qRT-PCR analysis of TNF, IL1B and IL6 mRNA levels in GM-CSF-activated monocytes derived macrophages (MDMs) co-cultured with or without T cells for 6 h. GM-CSF-activated MDMs generated from (b, left) unaffected control 1 (C1) or (b, right) unaffected control 3 (C3) or P1 (c) were co-cultured with donor-matched T cells as well as T cells from other unaffected controls (C2 and C4) or patients as indicated. d Dot plot of IFNG, TNF in cell subtypes found in scRNA-seq of PBMCs from three unaffected controls and two patients. e Representative histograms and statistical analysis of TNF and IFNγ expression in CD8+ T cells in PBMCs isolated from unaffected controls (n = 6) and patients (n = 3 from P1 and P2) treated with 10 ng ml-1 TNF (T) with or without 250 nM SM-164 (S), 25 μM z-VAD-fmk (Z) for 24 h. f, T cells isolated from P2 were co-cultured with MDMs generated from unaffected control in the presence of 10 μg ml-1 adalimumab, 10 μg ml-1 emapalumab alone or in combination for 6 h. TNF, IL1B and IL6 mRNA levels of MDMs were analyzed by qRT-PCR. g Experimental timeline for generation of lineage negative bone marrow cells (Lin- BMs)-transplanted mice with conditional overexpression of human WT or mutated RIPK1 K377E or R390G in CD8+ T cell- or myeloid cell-specific lineage. h, i ELISA measurement of serum Tnf and Il6 levels in mice specifically overexpressing WT or mutated hRIPK1 in (h) CD8+ T cells (EV, n = 5; WT, K377E and R390G n = 7) or (i) myeloid cells (EV, n = 5; WT, K377E and R390G n = 6). All histogram graphs show mean ± SD. P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. Data in b, c, and f show the means ± SD of technical triplicates. The results shown in b were derived from two independent experiments using cells from different donors. The experiment in (c, h, i, f) is representative of two independent experiments. Data in e were pooled from two independent experiments.

Fig 3: T cells activate TNF/IFNγ pathway in RIPK1 activation-induced autoinflammatory diseases.a–d Pedigrees of two families with CRIA patients (CRIA-1 and CRIA-2). Patients were represented with filled symbols (a). Summary histograms of CD4/CD8 ratio in CD3+ T cells of unaffected controls (n = 12), CRIA-1 (n = 7) and CRIA-2 (n = 5) were shown (b). Representative contour plots of cl. Casp-3 (c) and histograms of pRIPK1(S166) (d) protein levels in CD8+ T cells in freshly isolated PBMCs from CRIA patients. e, f Representative contour plots depicting the percentages of CD4+ T, CD8+ T, double negative T (DNT) and double positive T (DPT) in CD3+ T cells gated from PBMCs of CRIA-1 (e) and SHARPIN deficiency (f) patient culturing in vitro for 24 h. g Work model: RIPK1 activation-dependent CD8+ T cell death-triggered inflammatory response of myeloid cells cause SAID. All histogram graphs show mean ± SD. P value was determined by two-tailed unpaired t-test. The experiments in c–f were not repeated due to the limited numbers of patient sample.