Panel A shows the percentage of interferon gamma-positive and tumor necrosis factor-positive mucosal-associated invariant T cells after stimulation with interleukin-18, interleukin-23, or interleukin-18 plus interleukin-23. The horizontal axis represents the different stimulation conditions, and the vertical axis represents the percentage of interferon gamma-positive or tumor necrosis factor-positive cells. Panel B shows similar data for V delta 2-positive gamma delta T cells. The horizontal axis represents the different stimulation conditions, and the vertical axis represents the percentage of interferon gamma-positive or tumor necrosis factor-positive cells. Panel C shows the percentage of interferon gamma-positive and tumor necrosis factor-positive natural killer cells under the same stimulation conditions. The horizontal axis represents the different stimulation conditions, and the vertical axis represents the percentage of positive cells. Panel D shows the levels of interferon gamma in the supernatant after 24 hours of stimulation with interleukin-23, interleukin-18, or interleukin-18 plus interleukin-23. The horizontal axis represents the different stimulation conditions, and the vertical axis represents interferon gamma concentration in picograms per milliliter. Panel E shows similar data after 48 hours of stimulation. The horizontal axis represents the different stimulation conditions, and the vertical axis represents interferon gamma concentration in picograms per milliliter. Each graph includes data points from different genotypes, and statistical significance is indicated by asterisks.
Impaired IL-23–dependent induction of IFN-γ in MAIT, Vδ2 + γδ T, and NK cells. (A–C) Percent intracellular IFN-γ+ induction was monitored by spectral flow cytometry in MAIT (A), Vδ2+ γδ T cells (B), and NK cells (C) after stimulation with IL-18 (200 ng/ml) and IL-23 (1 ng/ml), alone or in combination for 24 h, or with PMA/ionomycin stimulation for 6 h for frozen PBMCs. The percentages of IFN-γ+ and TNF+ cells were monitored as a control. 16 healthy controls, 2 IL23RG149R/G149R, 1 IL23RG300V/G300V, 7 IL23RR381Q/R381Q, 2 IL23R−/−, 1 IL12RB1−/−, and 4 TYK2P1104A/P1104A patients were included. The statistical significance of differences was assessed in unpaired Mann–Whitney U tests for comparisons of each variant with HD or WT as appropriate. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (D) IFN-γ levels in the supernatant were assessed after the stimulation of thawed PBMCs of the indicated genotypes for 24 h with IL-23 (1 ng/ml), IL-18 (200 ng/ml), or both. 14 healthy controls, 2 IL23RG149R/G149R, 1 IL23RG300V/G300V, 5 IL23RR381Q/R381Q, 1 IL23R−/−, 6 IL12RB1−/−, and 5 TYK2P1104A/P1104A patients were included. Nonparametric Mann–Whitney U tests were used for analysis, with *P < 0.05. (E) IFN-γ levels in the supernatant were assessed after the stimulation of thawed PBMCs of the indicated genotypes for 48 h with IL-23 (100 ng/ml), IL-18 (25 ng/ml), or both. 17 healthy controls, 2 IL23RG149R/G149R, 3 IL23RG300V/G300V, 4 IL23RR381Q/R381Q, 1 IL23R−/−, 2 IL12RB1−/−, and 8 TYK2P1104A/P1104A patients were included. Nonparametric Mann–Whitney U tests were used for analysis, with *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.