Fig 2: Murine p40 with D36K/W37K/T38K substitutions interacts with murine p19 but not with murine IL-12Rβ1.A, structure of hIL-23 extracted from the structure of the IL-23:IL-23R complex structure (PDB 5mzv). A flexible linker sequence connecting p40 and p19 is indicated. B, Western blot analysis of secreted murine HIL-23, mp19 and mp40 variants from transfected CHO-K1 cells. The position of mp19 and mp40 variants is indicated by arrows. C, cellular proliferation of Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells. The cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or with the indicated cytokines (10% conditioned cell culture supernatant of transfected CHO-K1 cells). Parental Ba/F3-gp130 cells were used as controls. The results of one representative experiment of three are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3), ∗∗∗p ≤ 0.001. D, analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant of transfected CHO-K1 cells) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK1/2. Western blotting data show results of one representative experiment of two. E, co-IP of FLAG-tagged murine mp19 coexpressed with either mp40 or mp40D36K/W37K/T38K and full-length mIL-12Rβ1 or mIL-23R. The position of mIL-23R and mIL-12Rβ1 is indicated by arrows. One of two independent experiments is shown.

Fig 3: The N-terminal region of murine p40 from Y31 to T38 mediates binding to murine IL-12Rβ1.A, alignment of murine and human p40 N-terminal amino acids with analyzed mp40 deletion variants. B, crystal structure of the IL-23:IL-23R:IL-12Rβ1 complex (PDB 6WDQ). Insets show zoomed in views on the area surrounding residues D36-Y38. D36-Y38 are depicted in orange stick representation. p40 and IL-12Rβ1 residues in contact with D36-Y38 are displayed in stick representation. p40 mutations D36K, W37K and Y38K were visualized in the second inset. C, Western blot analysis of secreted murine HIL-23 variants from transfected CHO-K1 cells. D, cellular proliferation of Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells. The cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or with the indicated cytokines (10% conditioned cell culture supernatant of transfected CHO-K1 cells). Parental Ba/F3-gp130 cells were used as controls. The results of one representative experiment of four are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3), ∗∗∗p ≤ 0.001. E, analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant of transfected CHO-K1 cells) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK1/2. Western blotting data show results of one representative experiment of two. F, co-IP of FLAG-tagged murine HIL-23 variants (wild-type, ΔM23-D29, ΔM23-P39) and full-length mIL-12Rβ1. The position of mIL-12Rβ1 and HIL-23 variants is indicated by arrows. One of two independent experiments is shown. G, co-IP of FLAG-tagged murine HIL-23 variants (wild-type, ΔM23-D29, ΔM23-P39) and full-length mIL-23R. The position of mIL-23R and HIL-23 variants is indicated by arrows. One of two independent experiments is shown.

Fig 4: A, Relative expression of interleukin‐23 receptor (IL‐23R) in lung adenocarcinoma cell line A549, lung squamous carcinoma cell line H226, and lung immortalized cell line 16HBE. Original mean value of relative expression of 16HBE was 3.84 × 10−6. For simplification, we set the value of 16HBE as 1, and the other values of cell lines and tissues were corrected by the baseline. B, Each pair of data points connected by a line represent the relative expression of IL‐23R from tumor and adjacent noncancerous tissues of a single donor. Black line with gray block next to each group indicates median and 25%‐75% percentile. ***P < .001. C, Immunohistochemistry detected IL‐23R overexpression and sublocalization in lung cancer (LC) cell cytoplasm but not on membrane, stromal, or adjacent noncancer normal tissues. Tissue sections were derived from 2 patients with lung adenocarcinoma (T2aN1M1). The 10×, 20×, and 40 × microscopic field images were taken separately. D, Western blot analysis detected IL‐23R in lung adenocarcinoma tissues and paired noncancer tissues. Graph below shows relative expression of IL‐23R mRNA in the same patch of LC tissue pairs by quantitative PCR (qPCR). Values were corrected by the baseline of the relative expression level of 16HBE to 1. As internal standards, western blot analysis used β‐tubulin and qPCR used β‐actin. E, Interaction between IL‐23 and sIL‐23R in NSCLC patients’ serum. Optical density (OD) values of IL‐23 coated wells were significantly higher than control. ***P < .001

Fig 5: Effects of inhaled inactived Mycobacterium phlei (M. phlei) on the production of IL-17+γδT and IL-23R+γδT cells. Production of (A) IL-17+γδT cells and (B) IL-23R+γδT cells was assessed by fluorescence associated cell sorting analysis. (C) Quantitative analysis of the percentage of IL-17+γδT or IL-23R+γδT cells. Data are presented as the mean ± standard deviation and comparisons are indicated with lines: *P<0.05, **P<0.01, ***P<0.001. Group A is the normal control group, group B is the sensitized/M. phlei untreated group and group C is the sensitized/M. phlei treated group. IL-17, interleukin-17; IL-23R, interleukin-23 receptor.