In vitro restoration of Munc13-4 expression and cytotoxic function after gene transfer into Munc13-4–deficient T cells. (A) The level of Munc13-4 mRNA expression was 4.4 to 5.5 times higher in the patients’ H/F-LV-bulk–transduced cells than in healthy controls and nontransduced FHL3 cells, and was 1.5 times higher in VSVG-LV–transduced cells (MOI, 100) than in nontransduced cells. The experiments were performed on 3 different FHL3 samples (P5, P6, and P7). The data were normalized against GAPDH and are expressed as a fold-change relative to healthy control T cells. It is noteworthy that mRNA levels were similar in nontransduced patient cells and healthy control cells. (B) Munc13-4 protein expression 6 days after transduction (in the same 3 samples as described for panel A; only P6 and P7 are presented here). (C) Dot plots represent (for CD8⁺ gated cells) the number of Munc13-4/CFP⁺ cells as a percentage of the CD8⁺ population 6 days after transduction with H/F-LV at an MOI of 5 or with VSVG-LV at MOIs of 5 and 100; 1 representative experiment of 3 (same samples as in panel A) is shown. (D) The number of CD107a/b⁺ cells as a percentage of the CD8⁺ T-cell population after stimulation with 30 µg/mL anti-CD3. In transduced samples, the percentage of CD107a/b⁺ cells are presented with respect to CFP⁺ or Munc13-4/CFP⁺ gated cells. n = 7 different FHL3 samples for nontransduced cells (P1-P7); n = 5 for H/F-CFP as control (P3-P7); n = 3 for H/F (P5-P7); n = 6 for VSVG experiments (P1-P3, P5-P7). Data are presented as the mean ± standard deviation (SD). P values were calculated using an unpaired Student t test for mRNA expression and a 2-sided Mann-Whitney test for CD107a/b surface expression in the degranulation assay. *P < .05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, nonsignificant; NT, nontransduced.