Fig 2: Reduced Th17, Th1*, and MAIT cells are characteristic for IL23R deficiency. a CD3+ T cells, b CD4+ T cells, c CD8+ T cells, f mucosal-associated invariant T (MAIT) cells (CD8+CD161+Va7.2+), e proportion of T effector memory (TEM), T central memory (TCM), Naïve T cells and TEM re-expressing CD45RA (TEMRA) cells from CD3+CD4+ T cells. f proportion of Th1 (CCR4-CXCR3+ROR?T-), Th1* (CCR4-CXCR3+ROR?T+), Th2 (CCR4+CXCR3-ROR?T-), and Th17 (CCR4+CXCR3-ROR?T+) cells from live CD14-CD3+CD4+CD45RA- cells. Pooled data from 3 independent experiments. Unpaired Student T test was used for comparison between groups. g IL-17 secretion after 7 days in vitro differentiation of naïve CD4.+ T cells (Th0) to Th17 cells. Pooled data from 3 independent experiments. Paired Student T-test was used for comparison within groups

Fig 3: Cell-type specific Ca2+/NFAT activation by 2D216. (A) The response ratios of Ramos or Jurkat NFAT reporter cells treated with 3.16 µM 2D216 were normalized to the positive controls (100%). The response ratio of anti-IgM and anti-CD3/anti-CD28 antibody-treated cells were 8.00 ± 0.41 and 3.88 ± 0.27, respectively. Data represent mean ± SD of two independent experiments showing similar results. **p < 0.01 by one-way ANOVA with Tukey’s post hoc test. (B) Ramos and Jurkat cells were treated with 2D216 (5 µM) or ION (1 µM) for 15 and 30 min, and NFATc2 activation was assessed by immunoblot. Slower and faster migrating bands represent phosphorylated (inactive) and dephosphorylated (active) NFATc2, respectively. (C) Cell-type specific Ca2+ influx by 2D216. Ramos and Jurkat cells were loaded with Fura-2 and treated with ION (1 µM), TG (5 µM), or 2D216 (5 µM) for 60 min. The arrow indicates the addition of compounds. Data shown are representative of three independent experiments.

Fig 4: Impaired IFN-? and IL-17 production in response to IL-23 stimulation. a FACS plots of IFN-? secretion by CD4+ T cells in 1 HC and patient, pre-activated PBMCs with anti-CD3/CD28 with or without addition of IL-23 (100 ng/mL), IL-6 (10 ng/mL), or IL-12 (40 ng/mL) for 7 days. FMO IFN-? was used for proper gating. Representative of 3 independent experiments b–d % of IFN-? secreting CD8+, CD4.+, and MAIT cells, pooled from 3 independent experiments. To assess differences between conditions within HCs or patient, a Friedman test (p < 0.0001) with post hoc Dunn’s multiple comparison test was used. To compare HC and patient groups, a Mann–Whitney U test was used for each condition. e IFN-? measured from supernatant of PBMCs in co-culture with Mycobacterium abscessus ATCC19977 for 3 days with or without IL-23 or IL-12. PMA ionomycin was used as a positive control condition. Data from 1 experiment. f IL-17 secreted by stimulated PBMCs with CD3/CD28 with or without addition of IL-23 (100 ng/mL), data from 2 experiments. To assess response in HCs, a paired Student T-test was used

Fig 5: Impaired STAT3 but preserved STAT 4 phosphorylation responses in patient EBV-B cells and PBMCs. a FACS plot of 1 HC and patient for pSTAT3 on CD8 + T cells after IL-23 (100 ng/mL), IL-6 (10 ng/mL) or IFN-a (10 ng/mL) stimulation for 60 min. FMO pSTAT3 was used for proper gating. Representative of 2 independent experiments b pSTAT3 histograms from 3 HCs, patient and an IL12RB1 -/- patient, representative of 2 independent experiments c Western blot on EBV-B cells of 2 HC and patient for pSTAT3, stimulation as indicated in Fig. 3a. Representative of 3 independent experiments. d Western blot on PBMCs of 1 HC and patient for pSTAT3, stimulation as indicated in Fig. 3a. Representative of 3 independent experiments. e Western blot showing pSTAT4 after IL-23 (100 ng/mL) and IL-12 (40 ng/mL) stimulation of anti-CD3/CD28 pre-activated PBMCs from 2 HCs, patient and an IL12RB1 -/- patient. Data from 1 experiment. f pSTAT3 after lentiviral vector transduction of patient cells using stimulation as indicated in Fig. 3a, representative of n = 2 independent experiments. *N-glycosylated IL23R, UT = untransduced. EV = empty vector