Inhibition of IL-17A in Tumor Microenvironment Augments Cytotoxicity of Tumor-Infiltrating Lymphocytes in Tumor-Bearing Mice

<div><p>It remains controversial whether IL-17A promotes or inhibits cancer progression. We hypothesized that IL-17A that is locally produced in the tumor microenvironment has an important role in angiogenesis and tumor immunity. We investigated the effect of inhibiting IL-17A at tumor sites on tumor growth and on local and systemic anti-tumor immunity. MC38 or B16 cells were inoculated subcutaneously into mice, and intratumoral injection of an adenovirus vector expressing siRNA against the mouse IL-17A gene (Ad-si-IL-17) significantly inhibited tumor growth in both tumor models compared with control mice. Inhibition of IL-17A at tumor sites significantly suppressed CD31, MMP9, and VEGF expression in tumor tissue. The cytotoxic activity of CD8⁺ T cells from tumor-infiltrating lymphocytes in mice treated with Ad-si-IL-17 was significantly higher than in control mice; however, CD8⁺ T cells from splenocytes had similar activity levels. Suppression of IL-17A at tumor sites led to a Th1-dominant environment, and moreover, eliminated myeloid-derived suppressor cells and regulatory T cells at tumor sites but not in splenocytes. In conclusion, blockade of IL-17A at tumor sites helped suppress tumor growth by inhibiting angiogenesis as well as cytotoxic T lymphocytes activation at tumor sites.</p> </div

The levels of each immune subset in splenocytes and TILs (MC38).

<p>Spleens were filtered through mesh, and live splenocytes were collected by 100% Ficoll gradient centrifugation. Tumor tissues were dissected and digested, and filtered through mesh, and TILs were collected by 80%/100% Ficoll gradient centrifugation. Splenocytes or TILs were stained with anti-CD4, anti-IFN-γ, anti-IL-4, anti-Foxp3, anti-Gr-1, anti-CD11b and anti-CD45 mAb. Data are representative of two independent experiments. Values represent the mean percentage ± SE in CD4⁺ or CD45⁺ population. (<i>n</i> = 5 per group, *<i>P</i><0.05 (Student's <i>t</i> test), compared with Ad-SNC).</p

Inhibition of IL-17A in tumor sites increases Th1 cells in tumor microenvironment.

<p>Splenocytes and TILs were collected from spleens or tumor tissues of MC38 subcutaneous tumor model. Th1/Th2 cells were detected by intracellular staining assay of anti-IFN-γ mAb and anti-IL-4 mAb. Representative flow cytometry analysis profiles gated on anti-CD4 mAb. Ad-si-IL-17 treatment increased Th1 phenotype of TILs compared to control, but that of splenocytes was similar level in both treatments. Th2 phenotype was similar ratio of TILs and splenocytes in both treatment. Data are representative of two independent experiments (<i>n</i> = 5).</p

Inhibition of IL-17A at tumor sites suppresses tumor growth.

<p>(A, left panel) 1×10⁶ B16 tumor cells were injected subcutaneously into C57BL/6 mice, and PBS, Ad-SNC, or Ad-si-IL-17 with 1×10⁹ PFU was injected intratumorally at 7, 10, and 13 d. Data represent means ± SE (<i>n</i> = 7 mice per group of two independent experiments). *<i>P</i><0.05, one-way ANOVA. (A, right panel) 5×10⁵ MC38 tumor cells were injected subcutaneously into mice, in the same way adenovirus vectors were injected at 5, 8, and 11 d. Data represent means ± SE (<i>n</i> = 7 mice of two independent experiments). *<i>P</i><0.05, one-way ANOVA. (B).Levels of IL-17A protein were measured by ELISA in tumor lysates from mice after having been treated with PBS, Ad-SNC, or Ad-si-IL-17. In B16 and MC38 tumor tissues, Ad-si-IL-17 treatment showed lower levels of IL-17A compared with PBS or Ad-SNC treatment. Data are representative of two independent experiments. Data are presented as means ± SE (<i>n</i> = 4). *<i>P</i><0.05, Student's <i>t</i> test.</p

Inhibition of IL-17A at tumor sites decreases the intratumoral microvessel density.

<p>(A) The endothelial maker CD31-stained sections of tumor tissue are shown in Ad-SNC and Ad-si-IL-17 treatment models, Ad-si-IL-17 treatment decreased the intratumoral microvessel density compared with Ad-SNC. Vessels in five high-power fields (×200 magnification) were counted. Positive cells were quantified by an image-processing application. Data are representative of two independent experiments. Data are presented as means ± SE (<i>n</i> = 5). *<i>P</i><0.05, Student's <i>t</i> test. (B) Intratumoral MMP-9 expression was decreased by the inhibition of IL-17. MMP-9 stained sections of tumor tissue were shown in Ad-SNC and Ad-si-IL-17 treatment models. Ad-si-IL-17 treatment decreased the intratumoral MMP-9 density compared with Ad-SNC. MMP-9 in five high-power fields (×200 magnification) was counted. Positive cells were quantified by an image-processing application. Data are representative of two independent experiments. Data are presented as means ± SE (<i>n</i> = 5). *<i>P</i><0.05, Student's <i>t</i> test. (C) VEGF and IL-17A levels produced by MC38 cells were measured by ELISA. MC38 cells did not secretion IL-17A protein and produce VEGF protein. Levels of VEGF in vivo were measured by ELISA in tumor lysates from mice at 14 d after having been treated with Ad-SNC, or Ad-si-IL-17. In tumor tissues, Ad-si-IL-17 treatment showed lower levels of VEGF compared with Ad-SNC treatment. Data are representative of two independent experiments. Data are presented as means ± SE (<i>n</i> = 4). *<i>P</i><0.05, Student's <i>t</i> test. ND: not detected.</p

Inhibition of IL-17A in tumor sites promotes CTLs activation, especially in the tumor microenvironment.

<p>The cytotoxicity assay used CD8⁺ T cells from splenocytes or TILs in MC38 subcutaneous tumor treated Ad-si-IL-17 and Ad-SNC. (A) The cytotoxic activities against MC38 cells of CD8⁺ T cells from splenocytes in mice treated with intratumoral injection of Ad-si-IL-17 were almost the same as those in control mice (<i>n</i> = 5). The cytotoxic activities against B16 cells were less than 10% in both groups (<i>n</i> = 5). (B) The cytotoxic activities against MC38 cells in CD8⁺ T cells from TILs in mice treated with intratumoral injection of Ad-si-IL-17 were significantly higher than those with Ad-SNC (<i>n</i> = 3). Data are representative of two independent experiments. Data are presented as means ± SE. *<i>P</i><0.05, Mann-Whitney test.</p

The recognition of HIG2 and HLA-A*02:01-expressing cells by a HIG2-9-4 peptide-specific CTL clone.

<p>(a) A HIG2-9-4 peptide-specific CTL clone was stimulated with COS7 cells expressing both HIG2 and HLA-A*02:01 (close diamond), or either HIG2 alone (open circle) or HLA-A*02:01 alone (open triangle), then the IFN-γ production was examined by ELISA. R/S ratio; responder/stimulator ratio. (b) The cytotoxic activity of the HIG2-9-4 peptide-specific CTL clone was examined against HLA-A*02:01-positive HIG2-expressing A498 cells (closed diamond) or HLA-A*02:01-negative HIG2-expressing Caki-1 cells (open circle). E/T ratio; effector/target ratio. All experiments were performed in triplicate. Representative results from three independent experiments are shown. *; <i>P</i><0.001.</p

Candidate peptides derived from HIG2 restricted with HLA-A*02:01.

<p>The binding score was obtained from the BIMAS website (<a href="http://www-bimas.cit.nih.gov/molbio/hla_bind" target="_blank">http://www-bimas.cit.nih.gov/molbio/hla_bind</a>).</p

The expression of a HIG2-9-4 peptide-specific T cell receptor on CD8+ T cells.

<p>The expression of the HIG2-9-4 peptide-specific T cell receptor was examined on CD3⁺CD4⁻ cells following CTL expansion culture of HIG2-9-4 peptide-induced CTLs. (a) A quadrant gate was set based on the staining results with the HIV peptide/HLA-A*02: 01 tetramer. (b) CD8⁺ T cells expressing the HIG2-9-4 peptide/HLA-A*02: 01-specific T cell receptor were detected. Similar results were obtained from three independent experiments.</p

The HLA-A*02:06-restricted response of a HIG2-9-4 peptide-specific CTL clone.

<p>(a) A HIG2-9-4 peptide-specific CTL clone was induced from HLA-A*02:06-positive PBMCs, and stimulated with HLA-A*02:06-positive PSCCA0922 cells pulsed with the HIG2-9-4 peptide (close diamond) or HIV peptide (open square). (b) The HIG2-9-4 peptide-specific CTL clone was stimulated with COS7 cells expressing both HIG2 and HLA-A*02:06 (close diamond), or either HIG2 alone (open circle) or HLA-A*02:06 alone (open triangle). The IFN-γ production in the culture supernatant was examined by ELISA. R/S ratio; responder/stimulator ratio. The representative results from three independent experiments are shown.</p