Knock-out (KO) of FHL-associated genes in mouse CTLs and complementation of their function with human WT orthologues. (A) Validation of single guide RNA for each FHL gene: western immunoblots, performed under reducing conditions, show the loss of endogenous mouse protein expression following CRISPR/Cas9 gene KO, and the expression of human WT orthologues in these cells. β-actin is shown as loading control. The residual protein levels compared to NT control were (mean ± SEM): Munc13-4 - 8.8±1.3% (n = 10); Stxbp2 – 9.7% ± 1.8% (n = 6); Stx11 – 8.1% ± 3.6% (n = 5). (B) Gene KO–dependent reduction in CTL cytotoxicity and its restoration by human WT orthologues, measured by 51Cr-release from antigenic SIINFEKL peptide-pulsed EL4 target cells at indicated effector to target cell (E:T) ratios. Each value shown represents mean ± SEM of the following number of independent experiments: Prf1 KO and PRF1 WT, N = 10; Unc13d KO and UNC13D WT, N = 27; Stx11 KO and STX11 WT, N = 9; Stxbp2 KO and STXBP2 WT, N = 17. Shaded area represents the NT guide RNA (NT) control retrovirally transduced with empty MSCV GFP vector from N = 43 independent experiments (95% confidence interval is shaded gray), with the average value shown as a solid line. The levels of WT protein overexpression were (mean ± SEM): MUNC13-4 - 2.0 ± 1.0 (n = 6), STXBP2 – 3.3 ± 0.4 (n = 6), and STX11 – 4.2 ± 1.0 (n = 5). (C) Severely reduced degranulation in KO cells compared to NT control and restoration by human WT orthologues, using the same target cells as in panel B and measured as the externalization of LAMP-1/CD107a. (D) Summary of independent degranulation experiments: NT, Prf1 KO and PRF1 WT, N = 8; NT, Stx11 KO and STX11 WT, N = 10; NT, Unc13d KO and UNC13D WT, N = 12; NT, Stxbp2 KO and STXBP2 WT, N = 12. The results were analyzed using 1-way ANOVA; “ns” indicates no statistical difference, ∗∗ indicates P < .01, ∗∗∗ indicates P < .001, and ∗∗∗∗ indicates P < .0001. ANOVA, analysis of variance; GFP, green fluorescent protein; SEM, standard error of the mean.
