Flow cytometry plots for father's CD3 negative CD56 positive CD107a positive cells on Day 0 and Day 2. Axes represent CD56 and CD107a. Day 0 shows 7.5 percent stimulated cells. Day 2 shows 71 percent unstimulated and 19 percent stimulated cells. Flow cytometry plots for father's CD3 positive CD8 positive CD57 positive CD107a positive cells on Day 2. Axes represent CD57 and CD107a. Day 2 shows 19 percent stimulated cells. Flow cytometry plots for mother's CD3 negative CD56 positive CD107a positive cells on Day 0 and Day 2. Axes represent CD56 and CD107a. Day 0 shows 29 percent stimulated cells. Day 2 shows 85 percent unstimulated and 9.1 percent stimulated cells. Flow cytometry plots for mother's CD3 positive CD8 positive CD57 positive CD107a positive cells on Day 2. Axes represent CD57 and CD107a. Day 2 shows 9.1 percent stimulated cells. Flow cytometry plots for Patient 1's CD3 negative CD56 positive CD107a positive cells on Day 0 and Day 2. Axes represent CD56 and CD107a. Day 0 shows 4 percent stimulated cells. Day 2 shows 52 percent unstimulated and 0.69 percent stimulated cells. Flow cytometry plots for Patient 1's CD3 positive CD8 positive CD57 positive CD107a positive cells on Day 2. Axes represent CD57 and CD107a. Day 2 shows 0.69 percent stimulated cells. Flow cytometry plots for control's CD3 negative CD56 positive CD107a positive cells on Day 0 and Day 2. Axes represent CD56 and CD107a. Day 0 shows 38 percent stimulated cells. Day 2 shows 89 percent unstimulated and 21 percent stimulated cells. for control's CD3 positive CD8 positive CD57 positive CD107a positive cells on Day 2. Axes represent CD57 and CD107a. Day 2 shows 21 percent stimulated cells.
NK and CD8 + T cell degranulation assays in patient 1 and family members. Representative flow cytometric plots from patient 1, a healthy control, and the patient’s parents. PBMCs were analyzed either at day 0 without prior cytokine stimulation or after 48 h of culture with IL-2 (day 2). Degranulation was assessed after 2 h under unstimulated conditions or following target-cell stimulation. Left and middle panels show NK cell degranulation, quantified as CD3−CD56+CD107a+ cells. NK cell degranulation was assessed at day 0 (without IL-2) and at day 2 (after 48 h of IL-2 culture), under unstimulated conditions or after co-culture with K562 target cells. Right panels show CD8+ cytotoxic T cell degranulation, quantified as CD3+CD8+CD57+CD107a+ cells, assessed at day 2 after IL-2 culture, under unstimulated conditions or after co-culture with P815 cells alone or with P815 cells in the presence of anti-CD3 (OKT3). Percentages of CD107a-positive cells are indicated within the gated regions. Markedly reduced degranulation responses were observed only in the patient, whereas normal degranulation was preserved in the healthy control and both parents, including the father carrying p.Gly94Ser and p.Trp73Arg in cis. Experiments were performed once for each individual due to clinical constraints. The patient 1 and healthy control plots on day 0 shown here are reproduced from Fig. 1 for direct comparison with the parental samples.