Extended Data Fig. 2 |. iRHOM2 deficiency impairs ADAM17-dependent TNF shedding.

(a) Quantitative RT-PCR (Q-PCR) of ADAM17 mRNA isolated from healthy control (HC) and patient (P) T cells. β-actin, GAPDH, or 18 S served as the endogenous control. Data of three independent experiments. (b) Western blot of TNF and Na +/K + ATPase Alpha 1 expression in membrane fractions of patient and healthy T cells stimulated with anti-CD3 for four hours. (c) Membrane TNF on live-gated CD14⁺ monocytes from HCs and P1 either untreated or stimulated with lipopolysaccharide (LPS) for 4 hours measured as in Fig. 1 (n = 4 for HC group). (d) Flow cytometry as in b, for HC cells either with or without treating with control or iRhom2 (RHBDF2)-targeting small interfering RNAs (siRNAs) as indicated. (e) Knockdown and knockout efficiency are shown by Q-PCR of RHBDF2 using RNA isolated from T cells. β−actin was the endogenous control. RQ, relative quantitation (n = 3 per group). (f) Knockout efficiency shown by western blot of ADAM17 and β-actin loading control using T cells from HC. Asterisk, pro-ADAM17; arrowhead, mature ADAM17. (g) mTNF (mean fluorescence intensity) MFI for T cells from HC and Ps stimulated with anti-CD3 antibody. Data of three independent experiments. (h) mTNF MFI for HC T cells treated with control sgRNA and iRHOM2 sgRNA and the stimulated with 10 μg/ml anti-CD3 antibody for 4 hours (n = 3 per group). (i) Q-PCR of RHBDF2 mRNA isolated from T cells treated with negative (control) or wild-type RHBDF2 coding sequence. RNA was isolated 5 days after lentiviral transduction. β−actin was the endogenous control (n = 3 per group). (j) Flow cytometry dot plots of cell side scatter (SSC) mTNF expression (percentage given in the expression gate) by live CD2⁺ HC T cells transduced with empty or iRHOM2 overexpressing lentiviral vector and then stimulated with anti-CD3 (10 μg/ml) for 4 hours. (k) Flow cytometric mTNF measurements from isolated cell populations from either WT or Rhbdf2^−/− mice (Gating strategy was shown in Extended Data Fig. 6); Error bars represent standard error of the mean (n = 6 per group). (l) CD4 T cells (upper panel) and naïve CD8 T cells (lower panel) from HC and STAT3-deficient HIES patients stimulated with PMA (20 ng/ml) and ionomycin (750 ng/ml) or dimethyl sulfoxide (DMSO) vehicle for 4 hours and analyzed. Scatter plots were generated by gating on live CD3⁺CD4⁺, CD3⁺CD8⁺, CD4⁺, or CD8⁺ T cells (n = 2 per group). All data are mean ± s.d. and were analyzed by two-tailed, unpaired Student’s t-test (g, h, k).