Effect of selected missense mutations in four FHL-associated genes expressed in mouse CTLs with endogenous orthologues knocked out. Cytotoxicity, degranulation, and protein expression data for PRF1: mutations from P3 and P8 from N = 2 independent experiments (see supplemental Figure 9 for additional data) (A), STX11: mutations from P2 and P19 from N = 2 independent experiments (B), STXBP2: mutations from P5 and P12 from N = 3 independent experiments (C), and UNC13D: compound heterozygous mutations from P10 from N = 3 independent experiments (D). 51Cr-release cytotoxicity assay was conducted on days 4 and 7 post activation using SIINFEKL peptide-pulsed EL4 target cells at indicated E:T ratios and represent mean ± SEM. Degranulation is measured by LAMP-1/CD107a externalization on effector cells on day 7 using SIINFEKL peptide-pulsed EL4 target cells. Western immunoblotting, performed on day 7 cells under reducing conditions, shows protein expression in KO, WT and mutant transduced cells. Equivalent cytotoxicity data for other mutations are shown in supplemental Figure 8. SEM, standard error of the mean.
