Article Figures & Data
Figures
- FIGURE 1.
Expression of IL-35 in human cancer tissues. H&E staining and IHC were performed on paraffin-embedded serial tissue sections of human large B cell lymphoma (A, B), nasopharyngeal carcinoma (C, D), skin melanoma (E, F), and lymph node metastatic melanoma (G, H). The anti-human IL-35 mAb 15K8D10 was used to stain human cancer tissues. (A), (C), (E), and (G) show H&E staining; (B), (D), (F), and (H) show IL-35–specific staining. Scale bars, 100 μm.
- FIGURE 2.
Generation of IL-35–producing J558 and B16.F10 cells. Mouse plasmacytoma J558 cells or B16F10 melanoma cells were cotransfected with an expression vector pORF9-IL-35 and a selection vector pCDNA-neo or the control expression vector pORF9 and pCDNA-neo. Stable cell lines that were resistant to G418 were generated. RT-PCR was used to detect the expression of transcripts for recombinant IL-35, IL-12A, EBI3, and tumor Ag P1A in J558 cells (A) and IL-35, IL-12A, and EBI3 transcripts in B16F10 cells (E). Immunofluorescence staining and ELISA revealed that IL-35 protein was produced by the generated J558 (B) and B16.F10 (F) cells. Flow cytometry was used for the analysis of MHC class I expression on the generated J558 cells (C) and B16 cells (G). MTT proliferation assay was used to measure growth and proliferation of J558 cells (D) and B16 cells (H). Bars indicate SD of triplicates. Original magnifications ×400 for (B) and (F).
- FIGURE 3.
Expression of IL-35 in the tumor microenvironment enhances tumorigenesis. A total of 5 × 106 J558-IL-35 or J558-Ctrl cells was injected s.c. into each BALB/c mouse (A) or Rag2−/−BALB/c mouse (C). The tumor growth was observed over time; at the end of the experiments, tumors were removed from sacrificed mice and photographed. (B) J558–IL-35 and J558-Ctrl tumors removed from BALB/c mice (left panel). ELISA was used to quantify IL-35 concentration in lysates of representative tumors (right panel). Each Rag2−/−BALB/c mouse was inoculated s.c. with 5 × 106 J558–IL-35 cells in the presence of anti–IL-35 (V1.4C4.22; Shenandoah Biotechnology) or an isotype-matched control mAb (IgG2b; Bio X Cell) at a concentration of 50 μg/ml. Mice were observed for tumor growth over time. Bars indicate SD of three mice in each group, and data shown represent two experiments with similar results. A total of 1 × 105 B16–IL-35 or B16-Ctrl cells was injected s.c. into each C57BL/6 mouse (E) or Rag1−/−C57BL/6 mouse (F). The tumor growth was observed over time. Bars in (A) and (C–F) indicate SD of 5 mice/group. Data shown represent three to five experiments with similar results. (G) A total of 1 × 105 B16–IL-35 or B16-Ctrl cells was injected i.v. into each C57BL/6 mouse. Eighteen days after tumor cell injection, lungs from the recipient mice were removed, photographed (left panel), and weighed, and lung/body weight ratios were calculated and plotted (right panel). *p < 0.05, **p < 0.01, Student t test.
- FIGURE 4.
IL-35 production in the tumor microenvironment enhances angiogenesis. J558 tumors (A, B) from Rag2−/− mice and B16 tumors from C57BL6 mice (C, D) were analyzed for the expression of CD31 and VEGF by immunofluorescent staining and microscopy. Scale bars, 200 μm. Mean vessel wall area and numbers of VEGF+ cells for each tumor were analyzed and quantified using ImageJ software. Three random fields from each slide/tumor were analyzed, and each symbol represents data from one microscope field. **p = 0.001, ***p < 0.0001, Student t test.
- FIGURE 5.
Increased numbers of CD11b+Gr1+ myeloid cells in the tumor microenvironment of IL-35+ tumors. (A) J558–IL-35 and J558-Ctrl tumors from Rag2−/− mice were analyzed for the infiltration of myeloid cells by immunofluorescence staining and microscopy. Scale bars, 200 μm. Frozen tissue sections were colabeled for CD11b (Alexa Fluor 488) and Gr1 (Texas Red), and images were analyzed and quantified using ImageJ software. Three random fields from each slide/tumor were analyzed, and each symbol represents data from one microscope field. Arrows indicate CD11b and Gr1 double positive MDSCs. **p < 0.001, Student t test. (B–D) Flow cytometry was also used for the analysis of myeloid cells in IL-35+ and IL-35− tumors. Single-cell suspensions were prepared from tumors grown in Rag2−/− (B), BALB/c (C), and C57BL6 (D) mice and stained for CD11b and Gr-1, followed by flow cytometry analysis. Each symbol represents data from a single tumor. *p < 0.05, **p < 0.01, ***p < 0.001, Student t test. (E) A total of 5 × 106 J558–IL-35 or J558-Ctrl cells was injected s.c. into each BALB/c mouse, followed by treatment with 250 μg/mouse of anti-Gr1 mAb (RB6-8C5; Bio X Cell) i.p. on days 0, 5, and 10. Mice were observed for tumor growth over time. Five mice/group were used for this experiment, and the data shown represent two experiments with similar results.
- FIGURE 6.
IL-35 does not increase migratory activity of myeloid cells. Migration assay using a Transwell system, with or without IL-35, was performed to test the migration capacity of Raw264.7 cells (A), P338D1 cells (B), spleen MDSCs (C), and bone marrow Gr1+ cells (D). Cells that migrated to the bottom side of the Transwell membrane were stained with DAPI, and random fields from each well were photographed under a fluorescence microscope. The numbers of cells in each field were quantified using ImageJ software. Each symbol represents data from one microscopic field, and data shown represent three experiments with similar results. Original magnification for (A): ×200.
- FIGURE 7.
Expression of IL-35 contributes to an immune-suppressive microenvironment. J558 cells or B16 cells, with or without IL-35 expression, were injected s.c. into each BALB/c or C57BL6 mouse. When tumors were fully established (∼1 cm in length), mice were sacrificed, and T cell responses in tumors were evaluated by flow cytometry. (A–E) T cell responses in IL-35+ and IL-35− J558 tumors from BALB/c mice. (F–H) T cell responses in IL-35+ and IL-35− B16 tumors from C57BL6 mice. Each symbol represents data from a single mouse/tumor. *p < 0.05, **p < 0.01, Student t test.
- FIGURE 8.
IL-35 does not directly affect differentiation of tumor Ag–specific CTL. Splenocytes from P1CTL-transgenic mice were activated with P1A peptide (0.2 μg/ml) in the presence or absence of IL-35. [3H]tritium-incorporation assay (A) and MTT assay (B) were used to determine cell proliferation and survival. (C) Intracellular staining and flow cytometry were used to determine IFN-γ and granzyme B expression in activated P1CTL cells. (D) [51Cr] release assay was used to determine cytotoxicity of activated P1CTL cells to P815 target cells. Data shown represent at least three experiments with similar results.
- FIGURE 9.
IL-35 induces tumor cell resistance to CTL destruction. P1CTL cells were activated with P1A peptide (0.2 μg/ml) for 5 d. [51Cr] release assay was used to determine cytotoxicity of activated P1CTL cells to J558–IL-35/J558-Ctrl cells (A) and P815 cells treated or not with IL-35 (B). Data shown represent three experiments with similar results. (C) Frozen tissue sections from J558–IL-35 or J558-Ctrl tumors grown in BALB/c mice were labeled for TUNEL; images were photographed under a fluorescent microscope and quantified using ImageJ software (left panels). Scale bars, 200 μm. Three random fields from each slide/tumor were analyzed, and each symbol represents data from one microscope field (right panel). RT-PCR was used to detect IL-35R subunits in B16–IL-35/B16-Ctrl and J558–IL-35/J558-Ctrl cells (D) or in J558 and P815 cells treated or not with IL-35 (E). Data shown in (D) and (E) represent three experiments with similar results. *p < 0.05, Student t test.
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