Fig. 2 |. Defective ADAM17 maturation and activity in patient T cells.

a, Immunoblot of ADAM17 and β-actin loading control using T cells from HCs, patients, WT sibling and heterozygous parents. Asterisk, pro-ADAM17; arrowhead, mature ADAM17. Representative result of three independent experiments. b, Flow cytometry dot plots quantitating surface mTNF expression on anti-CD3-stimulated live-gated CD2+ T cells from human subjects (patients) versus side scatter (SSC). Red, percentage of cells in positive gate. c, Quantification of mTNF measured in b (n = 3 per group). d, Level of soluble TNF in culture supernatants (n = 3 per group). e, Flow cytometry as in b, for HC cells with or without treatment with CRISPR–Cas9 sgRNA complexes (n = 3 per group). f, Flow cytometry as in e. g, Quantification of samples as in e and f and Extended Data Fig. 2d that were without stimulation or re-stimulated with anti-CD3 for 4 h (n = 3 per group). h, Flow cytometry as in b, for P1 T cells transduced with iRHOM2-expressing or empty lentiviral vectors and re-stimulated with anti-CD3 antibody for 4 h (n = 3 per group). i, Quantification of mTNF measured in g (n = 3 per group). j, Quantitative PCR of RHBDF2 isolated from WT CD8 T cells and then stimulated with 10 μg ml⁻¹ anti-CD3 antibody and anti-CD28 antibody for 12 h. 18S as endogenous control. RQ, relative quantitation (n = 3 per group). k, Measurements of TNF in patient or HC serum by bead-based multiplex assay (n = 4 for each HC and patient group). l, Fraction of T cells expressing CD62L in blood following anti-CD3 stimulation for 4 h (n = 3 per group). All experiments are representative of at least two (P2 and P3) or three (others) independent experiments with similar findings. All data are mean ± s.d. and were analyzed by two-tailed, unpaired Student’s t-test (c–d,g,i–l). APC, Allophycocyanin; stim, stimulation; UT, untreated.