FHL3 model CTL lines for functional analysis of UNC13D variants. Allo13 (A) or HVS-T1 (B) cells transfected with FLAG-tagged cDNA carrying wild-type UNC13D sequences linked to the T2A peptide and EGFP were stimulated with P815 cells and an anti-CD3 mAb (OKT3). CD3⁺CD8⁺EGFP⁺ cells were gated, and degranulation was assessed by flow cytometry. Results are representative of 3 independent experiments. (C) HVS-T1 cells were transfected with FLAG-tagged cDNA carrying wild-type UNC13D sequences linked to the T2A peptide and EGFP. EGFP⁺ cells were sorted and incubated with P815 (target cells). Data represent the mean ± SD of 3 independent experiments. *P < .05; **P < .01; ***P < .001. E:T ratio, effector-to-target ratio.