Abnormally differentiated ALPS DNT cells show high proliferative activity in vivo. (A) PBMC from patients with ALPS and healthy controls were gated for CD4⁺, CD8⁺, and DNT cells and analyzed for Ki67 expression. Histograms show a representative experiment, y-axis is normalized to mode. Cumulative data of all untreated patients with ALPS and healthy controls are graphed; each symbol represents an individual subject. ***P < .001 (Mann-Whitney U test). (B) Expression of proliferating cell nuclear antigen and cyclin A was determined in ALPS CD4⁺ (gray, filled), CD8⁺ (black, dotted), and DNT (black, solid) cells. Overlays show representative data from 1 of 5 independent experiments with similar results. (C) PBMC of patients with ALPS (n = 12) were gated for Ki67⁺ lymphocytes. Frequencies of B cells (CD3⁻/CD19⁺), NK cells (CD3⁻/CD56⁺), NKT cells (CD3⁺/CD56⁺ or TCRVα24⁻Jα18⁺), γδ T cells (CD3⁺/TCRγδ⁺), CD4⁺ T cells (CD3⁺/TCRαβ⁺/CD4⁺), CD8⁺ T cells (CD3⁺/TCRαβ⁺/CD8⁺), and DNT cells (CD3⁺/TCRαβ⁺/CD4⁻/CD8⁻) among all Ki67⁺ lymphocytes were determined by flow cytometry. Box plots depict the 75th percentile, median, and 25th percentile values; whiskers represent maximum and minimum values. (D) Correlation of percentage of Ki67⁺ DNT cells and frequency of CCR7⁻/CD45RA⁺ DN T cells among all ALPS DNT cells is shown; r = .7471; ***P < .001 (Spearman’s test). (E) Expression of CD27, CD28, CD57, and Ki67 were determined in ALPS DNT cells. Plots show representative data from 1 of 3 independent experiments with similar results.