Fig 2: NK cells from tumor-infiltrated lymph nodes have strong cytotoxic potential(a, b) Cytotoxicity of NK cells against autologous melanoma cell lines. The graph reports the mean percentage specific lysis ±SD of at least 3 individual representative experiments performed at the indicated Effector:Target (E:T) ratios. The figure compares killing by autologous NK cells isolated from patients’ TILN (circle symbol) and TFLN (black square) or allogenic NK cells isolated from healthy donors’ PBL (H-PBL, black triangle) (a). Comparison between cytotoxicity of autologous NK cells isolated from TILN (circle symbol, n=6), patients’ PBL (P-PBL, black triangle, n=4) and allogenic NK cells isolated from healthy donors’ PBL (H-PBL, white triangle, n=4) (b). Statistical analysis is indicated (ANOVA). (c) Basal CD107a surface expression on TILN- and TFLN-NK cells from 2 representative (AMM18 and AMM21) patients, as assessed by FACS analysis. Numbers in dot plots indicate the percentage of CD107a positive cells within CD56dim and CD56bright NK subsets (upper panels) or alternatively within CD57+CD56+ NK cells (lower panels). The dot plot quadrants are set according to the isotype IgG control for TILN and TFLN as shown in the figure. In the CD56 dot plots two additional regions identify bright and dim NK cell subsets. The bar diagrams (right) refer to the mean of values ± SD of experiments performed in 5 metastatic melanoma patients (ANOVA). (d) Two representative metastatic melanoma patients (AMM17 on the left and AMM20 on the right) FACS analysis of the CD107a mobilization assay gating on CD56+CD3− TILN and TFLN NK cells after K562 pulsing at different E:T ratios. Quadrants are drawn according to the IgG isotype control for TILN and TFLN, as shown. The bar diagram (right) shows the mean of values ± SD (n=4) expressed as fold increase of CD107a expression in target pulsed NK cells over NK cells alone. (e) Ki67 intracellular staining TILN CD57+CD56+ NK. The bar diagram (right) shows the mean of values ± SD (n=5).

Fig 3: Frequency and phenotype of human NK cells in melanoma patients(a) Representative example (left) for cytofluorimetric analysis of CD56+CD3- NK cell subsets distribution from TILN, TFLN and PBL (P-PBL) in a metastastic melanoma patient in comparison with healthy donor PBL (H-PBL), in the same experimental conditions. Two regions were selected to represent respectively CD56bright and CD56dim populations. Bar graph (right) report mean±SD of the frequency of CD56dim and CD56bright NK subsets within TILN (black column, n=31) and TFLN (white column, n=12) gated on lymphocytes of all metastatic melanoma patients tested. In parallel light grey columns show the frequency of CD56dim and CD56bright NK subsets within peripheral blood lymphocytes of patients (P-PBL, n=10) and dark grey columns that of healthy controls (H-PBL, n=3). (b) Expression of CD57, CD16 and CD158b on CD56dim and CD56bright NK cell subsets from TILN, TFLN and P-PBL. Dot plots from one representative patient are presented. The quadrant marker has been set according to the isotype controls; to corroborate the validity of the markers T cells where used as internal negative control in vertical and the bright/dim boundary in horizontal. The identical quadrant marker has been applied on all the plots. (c) Expression of CD57, CD16, CD158b, CCR7, CD69 on CD56dim and CD56bright NK cell subsets. Bar graphs report mean±SD of the percentage of positive cells for each indicated marker in gated NK cells from TILN and TFLN of metastatic melanoma patients (n=12). (d) CD69+ CD56+CD3- NK cell subsets frequencies from TILN (n=31), TFLN (n=12), P-PBL (n=10) and H-PBL (n=3). Values shown are the mean±SD of percentages of double positive cells indicated. In all experiments shown P value is calculated by ANOVA followed by post-hoc Bonferroni test.

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: 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