Figure 4.
Figure 4. Expression level of the transcribed munc13-4 protein determines the pathogenicity of an UNC13D variant. HVS-T1 cells were transfected with FLAG-tagged cDNA carrying wild-type or missense UNC13D sequences linked to the T2A peptide and EGFP. CD3+CD8+EGFP+ cells were gated, and expression of munc13-4 (A) and EGFP (B) in cells transfected with missense UNC13D variants relative to that in cells transfected with the wild-type UNC13D sequence was analyzed by flow cytometry. Relative expression of munc13-4 protein (A) was calculated as follows: MFI of anti-FLAG–stained cells transfected with UNC13D variants − MFI of anti-FLAG–stained mock-transfected cells/MFI of anti-FLAG−stained cells transfected with wild-type UNC13D − MFI of anti-FLAG–stained mock-transfected cells. Relative expression of EGFP (B) was calculated as follows: MFI of EGFP in cells transfected with UNC13D variants/MFI of EGFP in cells transfected with wild-type UNC13D. Values represent the mean ± SD of 3 independent experiments. (C) Allo13 or (D) HVS-T1 cells were transfected with FLAG-tagged cDNA carrying wild-type or missense UNC13D sequences linked to the T2A peptide and EGFP, followed by incubation with P815 and an anti-CD3 mAb (OKT3). CD3+CD8+EGFP+ cells were gated, and degranulation was assessed by flow cytometry. Relative degranulation was calculated as follows: MFI of CD107a expression by cells transfected with UNC13D variants − MFI of CD107a expression by mock-transfected cells/MFI of CD107a expression by cells transfected with wild-type UNC13D − MFI of CD107a expression by mock-transfected cells. Data represent the mean ± SD of 3 independents experiments. (E) HVS-T1 cells were transfected with FLAG-tagged cDNA carrying wild-type or missense UNC13D sequences linked to the T2A peptide and EGFP. EGFP+ cells were sorted and incubated with P815 cells and an anti-CD3 mAb (OKT3) at the indicated effector-to-target (E:T) ratios. Specific lysis was then calculated. Data represent the mean ± SD of 3 independent experiments. *P < .05, †P < .01, ‡P < .001, compared with the wild-type UNC13D construct.

Expression level of the transcribed munc13-4 protein determines the pathogenicity of an UNC13D variant. HVS-T1 cells were transfected with FLAG-tagged cDNA carrying wild-type or missense UNC13D sequences linked to the T2A peptide and EGFP. CD3+CD8+EGFP+ cells were gated, and expression of munc13-4 (A) and EGFP (B) in cells transfected with missense UNC13D variants relative to that in cells transfected with the wild-type UNC13D sequence was analyzed by flow cytometry. Relative expression of munc13-4 protein (A) was calculated as follows: MFI of anti-FLAG–stained cells transfected with UNC13D variants − MFI of anti-FLAG–stained mock-transfected cells/MFI of anti-FLAG−stained cells transfected with wild-type UNC13D − MFI of anti-FLAG–stained mock-transfected cells. Relative expression of EGFP (B) was calculated as follows: MFI of EGFP in cells transfected with UNC13D variants/MFI of EGFP in cells transfected with wild-type UNC13D. Values represent the mean ± SD of 3 independent experiments. (C) Allo13 or (D) HVS-T1 cells were transfected with FLAG-tagged cDNA carrying wild-type or missense UNC13D sequences linked to the T2A peptide and EGFP, followed by incubation with P815 and an anti-CD3 mAb (OKT3). CD3+CD8+EGFP+ cells were gated, and degranulation was assessed by flow cytometry. Relative degranulation was calculated as follows: MFI of CD107a expression by cells transfected with UNC13D variants − MFI of CD107a expression by mock-transfected cells/MFI of CD107a expression by cells transfected with wild-type UNC13D − MFI of CD107a expression by mock-transfected cells. Data represent the mean ± SD of 3 independents experiments. (E) HVS-T1 cells were transfected with FLAG-tagged cDNA carrying wild-type or missense UNC13D sequences linked to the T2A peptide and EGFP. EGFP+ cells were sorted and incubated with P815 cells and an anti-CD3 mAb (OKT3) at the indicated effector-to-target (E:T) ratios. Specific lysis was then calculated. Data represent the mean ± SD of 3 independent experiments. *P < .05, †P < .01, ‡P < .001, compared with the wild-type UNC13D construct.

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