Fig 1: The mesothelial cells in peritoneum produce IL-20 and IL-24 when challenged by disseminated ovarian cancer (OC) cells in the peritoneal cavity.(A, B) qRT-PCR analysis of IL20RA ligands (Il19, Il20, and Il24) in peritoneal organs (intestinal, abdominal wall, ovary) (A) or peritoneal macrophages (CD11b+ F4/80+) (B) taken from C57BL/6 mice with intraperitoneal injection of ID8 cells (OC) or phosphate-buffered saline (PBS) control (Ctrl) 9 days before. Data are shown as means ± SEM, n = 18 for Ctrl group and n = 6 for OC group, ***p<0.001, ns, not significant, by unpaired two-sided Student’s t-test. (C) qRT-PCR analysis of IL20RA ligands in IL20RA-reconstituted or control (Vec) ID8 cells (means ± SEM, ns, not significant). (D) ELISA measurement of IL-20 and IL-24 in peritoneal flushing fluid from mice with intraperitoneal injection of ID8 cells (OC) or PBS control (Ctrl) 9 days before. n = 6 for each group. Data are shown as means ± SEM, ***p<0.001, by unpaired two-sided Student’s t-test. (E) qRT-PCR analysis of the abdominal walls dissected from C57BL/6 mice and co-cultured with medium (Ctrl) or ID8 cells for 48 hr (means ± SEM from three independent experiments, ***p<0.001, ns, not significant, by unpaired two-sided Student’s t-test). (F) Hematoxylin and eosin staining of the abdominal wall of C57BL/6 mice. Scale bar: 50 µm (upper panel); 20 µm (lower panel). (G) Immunohistochemical staining of IL-20 and IL-24 in abdominal walls dissected from mice with intraperitoneal injection of ID8 cells (OC) or PBS control (Ctrl) 9 days before. Scale bar: 20 µm. Figure 6—source data 1.An Excel sheet with numerical quantification data.
Fig 2: Inhibition of Foxo1 decreases the antitumor efficacy of Th9 cells in vivoNaive CD4+ T cells from OT-II mice or OT-II-Il24−/− mice were cultured under Th9-polarizing conditions for 2 days with or without the addition of Foxo1i.(A and B) After 5 days of i.v. injection of 1 × 106 B16-OVA cells into C57BL/6 mice, 5 × 106 Th9 cells were injected i.v. into tumor-bearing mice. Mice given PBS served as controls. The experiments were performed twice with a total of 10 mice per group (n = 10). (A) Images showing lung tumor development in mice of different treatment groups. (B) Statistics of lung weight in different treatment groups in A.(C) After 5 days of s.c. injection of 2 × 105 B16-OVA cells into C57BL/6 mice, 5 × 106 Th9 cells were injected i.v. into tumor-bearing mice. Mice given PBS served as controls. The experiments were performed twice with a total of 10 mice per group (n = 10). Shown are the tumor growth curves. Data are presented as mean ± SD of the combined experiments. ∗∗p < 0.01. See also Figure S8.
Fig 3: IL-24 is highly expressed in Th9 cells(A–E) Naive CD4+ T cells isolated from spleens of mice were cultured under Th0- or Th9-polarizing conditions for 2 days.(A) Cell samples (S1-3) were collected and analyzed by RNA-seq. S1-3: Samples from three independent experiments. The heatmap shows the log2-fold change of the significantly differentially expressed genes of cytokines and chemokines.(B) The expression of Il24 in cultured T cells was assessed by qPCR.(C) IL-24 secretion in the cultures was assessed by ELISA.(D) Intracellular IL-24 expression was analyzed by Flow cytometry. Numbers in the dot plots represent the percentages of IL-24+CD4+ T cells. Right, summarized results of three independent experiments obtained as at left.(E) Western-blots examined IL-24 and β-actin in T cells.(F and G) Naive CD4+ T cells isolated from spleens of mice were cultured under Th0- or Th9-polarizing conditions. Cells samples were collected at the specified time points.(F) The expression of Il24 in cultured T cells was assessed by qPCR.(G) The secretion of IL-24 and IL-9 in the cultures was analyzed by ELISA. Data are representative of three (D, E) independent experiments or presented as mean ± SD of three (B-D, F-G) independent experiments. ∗p < 0.05; ∗∗p < 0.01. See also Figures S1–S3.
Fig 4: Foxo1 contributes to IL-24 expression in Th9 cells(A) Another analysis was performed using the RNA-seq data in Figure 1A. The heatmap shows the log2-fold change of the differentially expressed genes of transcription factors.(B–D) Mouse naive CD4+ T cells from WT or Il24−/− mice and cultured under Th0- or Th9-polarizing conditions for 2 days. The mRNA levels of the indicated transcription factors were analyzed by qPCR (C and D) Mouse naive CD4+ T cells were cultured under Th0 or Th9 polarizing conditions with or without the addition of FOXO1 inhibitor (Foxo1i). qPCR analysis of Il9 (C) and Il24 (D) in CD4+ T cells.(E) Naive CD4+ T cells labeled with CFSE were cultured under Th0- or Th9-polarizing conditions with or without (DMSO) the addition of Foxo1i for 3 days. CFSE-stained T cells were analyzed by Flow cytometry. Numbers in the histograms represent the fluorescence intensity of CFSE-stained T cells. Right, summarized results of three independent experiments obtained as at left.(F) Mouse naive CD4+ T cells were cultured under Th0- or Th9-polarizing conditions with or without the addition of Foxo1i for 3 days. Annexin V+ PI+ T cells were analyzed by Flow cytometry. Numbers in the dot plots represent the percentages of Annexin V+ PI+ T cells. Right, summarized results of three independent experiments obtained as at left. Data are representative of three (E, F) independent experiments or presented as mean ± SD of three (B-F) independent experiments. NS, non-significant; ∗p < 0.05; ∗∗p < 0.01.
Fig 5: Knockout of IL-24 decreases Th9 cell survival in vivo(A–D) Naive CD4+ T cells from OT-II mice or OT-II-Il24−/− mice were cultured under Th9 polarizing conditions for 2 days 5 × 106 Th9 cells were labeled with CFSE and injected i.v. into C57BL/6 mice. CFSE-stained Th9 cells from spleen, lymph nodes and lung were analyzed by Flow cytometry at day 5 (A) and day 10 (C). Numbers in the dot plots represent the percentages of CFSE+CD4+ T cells (B and D). Data are representative of three (A and C) independent experiments or presented as mean ± SD of three (B and D) independent experiments. NS, non-significant; ∗p < 0.05; ∗∗p < 0.01.
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