Functional properties of MUNC13-4 mutants identified in case #1 (P6) and #2 (P11) and reconstituted in Unc13d KO mouse CTLs. (A) ⁵¹Cr-release assay using murine CTLs (as described in Figure 2) was conducted on day 4 of activation and shows ablated activity of G863D-MUNC13-4 and WT levels of cytotoxicity of R411Q-MUNC13-4 identified in case #1. Shown is mean ± SEM of N = 4 independent experiments. (B) Degranulation assay of case #1 MUNC13-4 mutations, as measured by LAMP-1/CD107a externalization. The degranulation levels mirror the cytotoxicity levels shown in panel A. SIINFEKL peptide-pulsed EL4 cells were used as targets. (C) Western immunoblot, performed under reducing conditions, showing endogenous mouse Munc13-4 protein expression, its level after CRISPR/Cas9 KO, and the overexpression of human WT-MUNC13-4 and R411Q-MUNC13-4, and G863D-MUNC13-4. β-actin is shown as loading control. (D) The effect of case #2 mutation E379K-MUNC13-4 on mouse Unc13d KO CTLs was assessed using ⁵¹Cr-release assay on days 4, 5, 6 and 7 of cell activation, using SIINFEKL peptide-pulsed EL4 target cells, at indicated E:T ratios. Each value shown represents mean ± SEM of N = 4 (for day 4), N = 2 (for day 5), N = 3 (for day 6) and N = 3 (for day 7) independent experiments. (E) Degranulation assay, as measured by LAMP-1/CD107a externalization of Unc13d deficient mouse CTLs transduced with E379K-MUNC13-4, using SIINFEKL peptide-pulsed EL4 target cells. (F) Western immunoblotting, performed on day 7 cells under reducing conditions, shows endogenous mouse Munc13-4 and the recombinant human WT-MUNC13-4 and E379K-MUNC13-4 protein expression; shown is 1 out of 5 independent experiments. E379K-MUNC13-4 expression level was 69% ± 11% compared to WT-MUNC13-4 (SEM of N = 5 independent experiments; P = .06, paired t test). β-actin is shown as loading control. SEM, standard error of the mean.