Blocking IL-17A enhances tumor response to anti-PD-1 immunotherapy in microsatellite stable colorectal cancer

Abstract

Background: Immune checkpoint inhibitors (ICIs), including anti-PD-1 therapy, have limited efficacy in patients with microsatellite stable (MSS) colorectal cancer (CRC). Interleukin 17A (IL-17A) activity leads to a protumor microenvironment, dependent on its ability to induce the production of inflammatory mediators, mobilize myeloid cells and reshape the tumor environment. In the present study, we aimed to investigate the role of IL-17A in resistance to antitumor immunity and to explore the feasibility of anti-IL-17A combined with anti-PD-1 therapy in MSS CRC murine models.

Methods: The expression of programmed cell death-ligand 1 (PD-L1) and its regulation by miR-15b-5p were investigated in MSS CRC cell lines and tissues. The effects of miR-15b-5p on tumorigenesis and anti-PD-1 treatment sensitivity were verified both in vitro and in colitis-associated cancer (CAC) and APC^min/+ murine models. In vivo efficacy and mechanistic studies were conducted using antibodies targeting IL-17A and PD-1 in mice bearing subcutaneous CT26 and MC38 tumors.

Results: Evaluation of clinical pathological specimens confirmed that PD-L1 mRNA levels are associated with CD8+ T cell infiltration and better prognosis. miR-15b-5p was found to downregulate the expression of PD-L1 at the protein level, inhibit tumorigenesis and enhance anti-PD-1 sensitivity in CAC and APC^min/+ CRC models. IL-17A led to high PD-L1 expression in CRC cells through regulating the P65/NRF1/miR-15b-5p axis. Combined IL-17A and PD-1 blockade had efficacy in CT26 and MC38 tumors, with more cytotoxic T lymphocytes cells and fewer myeloid-derived suppressor cells in tumors.

Conclusions: IL-17A increases PD-L1 expression through the p65/NRF1/miR-15b-5p axis and promotes resistance to anti-PD-1 therapy. Blocking IL-17A improved the efficacy of anti-PD-1 therapy in MSS CRC murine models. IL-17A might serve as a therapeutic target to sensitize patients with MSS CRC to ICI therapy.

Keywords: combination; cytokines; drug therapy; gastrointestinal neoplasms; immunotherapy.

Figure 1

PD-L1 mRNA level is associated with CD8+ T cells infiltration and prognosis in MSS CRC. (A) Representative expression of PD-L1 protein and mRNA in MSS and MSI-H CRC tissues (scale bar, 100 µm; magnification scale bar, 200 µm). (B) Statistical analysis was conducted based on the level of PD-L1 protein and mRNA in patients with MSS and MSI-H. (C) Kaplan-Meier analysis of the overall survival rate of patients with MSS CRC, according to PD-L1 protein expression (left panel) and PD-L1 mRNA level (right panel). (D) Correlations of CD8+ cells densities with PD-L1 expression, according to PD-L1 protein expression (left panel) and PD-L1 mRNA level (right panel) in patients with MSS CRC (n=123). (E) Western Blotting detection showed that PD-L1 protein expression in MSS CRC tissues was higher than that in adjacent tissues (n=22). (F) RT-qPCR analysis showed that there was no significant difference in PD-L1 mRNA levels between MSS CRC tissues and adjacent tissues (n=54). P values and R values were calculated based on the analysis of Pearson’s correlation. The significance of survival difference was determined by the log rank test. Student’s t test. *P<0.05, **p<0.01, and ***p<0.001. CRC, colorectal cancer; MSI-H, microsatellite instability-high; MSS, microsatellite stable; PD-L1, programmed cell death ligand 1.

Figure 2

miR-15b-5p is a regulator of PD-L1 at post-transcriptional level in MSS CRC. (A) Heat map analysis of miRNA that gradually decreased from normal colon epithelial cells, to inflammation-associated colon epithelial cells, to cancer cells by GEO databases (GSE68306). (B) The Venn diagram indicates that miR-15b-5p, as a putative regulator of PD-L1 in CRC. (C) TCGA database analysis of miR-15b-5p expression decreased in MSS CRC samples compared with normal tissue. (D) RT-qPCR analysis that miR-15b-5p was downregulated in a plurality of MSS CRC cell lines compared with normal colon epithelial-derived FHC and NCM460 cell lines. (E) Schematic representation of predicted miR-15b-5p binding sites within the human and mouse PD-L1 3’-UTR. (F) Expression of PD-L1 in protein levels were analyzed by western blotting analysis in CT26 and MC38 cell lines treated with miR-15b-5p mimic or inhibitor. (G) PD-L1 expression on tumor cell surface was analyzed by flow cytometry in CT26 and MC38 cells after transfection of miR-15b-5p mimic or inhibitor. (H) Luciferase reporter activity was analyzed after cotransfection of miR-15b-5p mimic or negative control and WT PD-L1 3’-UTR luciferase reporter construct or mutant (Mut) construct into CT26 and MC38 cells. (I) Expression of PD-L1 in protein levels was analyzed by western blotting analysis after transfection of miR-15b-5p mimic or inhibitor into SW480 and SW1116 cell lines. (J) Luciferase reporter activity was analyzed after cotransfection of miR-15b-5p mimic or negative control and WT PD-L1 3’-UTR luciferase reporter construct or mutant (Mut) construct into SW1116 cells. The mean±SD from three independent experiments is represented. Data represent mean±SD. Results are representative of at least three separate experiments. Student’s t test. *P<0.05, **p<0.01, and ***p<0.001. CRC, colorectal cancer; MSS, microsatellite stable; PD-L1, programmed cell death ligand 1; UTR, untranslated region; WT, wild type.

Figure 3

miR-15b-5p inhibits CRC tumorigenesis and sensitizes anti-PD-1 therapy by targeting PD-L1 in murine model. (A) Schematic representation of a mouse model of CAC following AAV (miR-15b-5p sponge) infection. (B) Schematic representation of a mouse model of APC^min/+ colon cancer after infecting AAV (miR-15b-5p sponge). (C) Representative images were shown and observed colitis-associated tumor in the control and blocking miR-15b-5p group. (D) Representative images of H&E, miR-15b-5p, PD-L1, and CD8 +cells stained from CAC tumor tissues of control and blocking miR-15b-5p group mice (scale bar, 200 µm). (E) Representative images were shown and observed APC^min/+ tumor in the control and blocking miR-15b-5p group. (F) Representative images of H&E, miR-15b-5p, PD-L1, and CD8+ cells stained from APC^min/+ tumor tissues of control and blocking miR-15b-5p group mice (scale bar, 200 µm). (G) T cell-meditated tumor cell killing assay was performed in CT26 and MC38 cells infected with miR-15b-5p or control lentivirus. (H) Tumor growth of miR-sc, miR-15b-5p CT26 cells with or not PD-1 antibody treated in BALB/c mice (n=7 mice per group). (I) Survival of mice bearing syngeneic CT26 tumors following treatment with PD-1 antibody (n=9 mice per group). (J) Intracellular cytokine staining of CD8+ IFN-γ+ cells in the CD3+ T cell populations from isolated TILs. Data represent mean±SD. ANOVA followed by Tukey’s multiple comparison test was applied. Cumulative survival time was estimated by the Kaplan-Meier method, and the log-rank test was applied to compare the groups. *P<0.05, **p<0.01, and ***p<0.001. AAV, adeno-associated virus; ANOVA, analysis of variance; CAC, colitis-associated cancer; CRC, colorectal cancer; IFN, interferon; PD-L1, programmed cell death ligand 1; TIL, tumor-infiltrating lymphocyte.

Figure 4

IL-17A downregulates miR-15b-5p and enhances PD-L1 protein expression in MSS CRC cells. (A) RT-qPCR was used to analyze the effects of seven cytokines on the expression of miR-15b-5p in CT26 and MC38 cells. (B) RT-qPCR was performed to determine the expression of miR-15b-5p in CT26 and MC38 cells treated with IL-17A at indicated concentration for 12 hours. (C) RT-qPCR was performed to determine the expression of miR-15b-5p in CT26 and MC38 cells treated with IL-17A (100 ng/mL) for the indicated times. (D) SW1116 cells were treated with IL-17A in a dose-dependent manner for 12 hours and then the expression of miR-15b-5p was detected. (E) Four human MSS CRC cell lines were treated with human IL-17A (100 ng/mL), the expression of miR-15b-5p was detected by RT-qPCR. (F) CT26 and MC38 cell lines were treated with different concentrations of IL-17A, protein and mRNA levels of PD-L1 were detected by Western Blotting and RT-qPCR, respectively. (G) SW1116, HT29, SW480, and SW620 cell lines were treated with IL-17A (100 ng/mL), protein, and mRNA levels of PD-L1 were detected by Western Blotting and RT-qPCR, respectively. Data represent mean±SD. Student’s t test. *P<0.05, **p<0.01, and ***p<0.001. CRC, colorectal cancer; IL, interleukin; MSS, microsatellite stable; PD-L1, programmed cell death ligand 1.

Figure 5

NRF1 is the major transcription factor for IL-17A to accumulate PD-L1 protein. (A) Schematic representation of predicted NRF1 and YY1 binding sites in the promoter of mouse miR-15b-5p. (B) Ch-IP analyzes of NRF1 or YY1 binding to the miR-15b-5p promoter by antibodies against NRF1 or YY1 in CT26 and MC38 cells. (C, D) Effects of NRF1/YY1 overexpression or knockdown on miR-15b-5p expression through RT-qPCR analysis. (E, F) Modulation of the miR-15b-5p promoter activity by NRF1 overexpression or knockdown in CT26 cells. (G) The regulation of IL-17A/P65 signaling on NRF1 and PD-L1 expression in CT26 and MC38 cells. (H) The effect of the IL-17A/P65/NRF1 axis on miR-15b-5p expression in CT26 cells. Data represent mean±SD. Results are representative of at least three separate experiments. Student’s t test. *P<0.05, **p<0.01, and ***p<0.001. Ch-IP, chromatin immunoprecipitation; NRF1, nuclear respiratory factor 1; PD-L1, programmed cell death ligand 1.

Figure 6

IL-17A is elevated in MSS CRC and is associated with NRF1, miR-15b-5p, and PD-L1 expression in tumor tissues. (A) Two representative IHC or ISH staining results for IL-17A, miR-15b-5p, NRF1, and PD-L1 in tissues of patients with CRC (scale bar, 100 µm). (B) IL-17A positive cell count was positively correlated with NRF1 expression (p<0.001, R=0.355). (C) NRF1 expression was negatively correlated with miR-15b-5p expression (p<0.001, R=−0.4526). (D) miR-15b-5p expression was negatively correlated with PD-L1 expression (p<0.001, R=−0.4260). (E) IL-17A positive cells is elevated in tumors of MSS relative to MSI-H tumors (p<0.01). (F) miR-15b-5p expression is decreased in tumors of MSS relative to MSI-H tumors (p<0.01). (G) Correlations between IL-17A positive cells and CD3+ cells is in MSS CRC tissues (p=0.1079, R=0.235). (H) Correlations between IL-17A positive cells and CD33 positive cells is in MSS CRC tissues (p<0.001, R=0.2951). P values and R values were calculated based on the analysis of Pearson’s correlation. Student’s t test. *P<0.05, **p<0.01, and ***p<0.001. CRC, colorectal cancer; IHC, immunohistochemical staining; ISH, in situ hybridization; IL, interleukin; MSI-H, microsatellite instability-high; MSS, microsatellite stable; NRF1, nuclear respiratory factor 1; PD-L1, programmed cell death ligand 1.

Figure 7

Blocking IL-17A enhances the efficacy of anti-PD-1 therapy in murine model by promoting PD-L1 protein degradation. (A) Schematic diagram illustrating the treatment protocol of IL-17A antibody and PD-1 antibody in mice. (B) Tumor growth of CT26 cells with IL-17A antibody and/or PD-1 antibody treated in BALB/c mice (n=7 mice per group). (C) Survival of mice bearing CT26 tumors following treatment with IL-17A antibody and/or PD-1 antibody (n=9 mice per group). (D) IHC analysis of IL-17A and PD-L1 expression, ISH analysis of miR-15b-5p expression in CT26 tumors of each group of mice. (E) FACS analysis of staining of CD8+ IFN-γ+ cells in the CD3+ T cell populations from isolated tumor-infiltrating lymphocytes. (F) FACS analysis of staining of CD11b+ and Gr1+ cells in the tumor-infiltrating lymphocyte. (G) Immunofluorescence was used to analyze the staining of CD3+ cells and CD11b+ cells in CT26 tumor mice. (H) Waterfall plot showing change in 14 patients with MSS CRC received anti-PD-1 therapy tumor volume compared with baseline before treatment. Data represent mean±SD. ANOVA followed by Tukey’s multiple comparison test was applied. Cumulative survival time was estimated by the Kaplan-Meier method, and the log-rank test was applied to compare the groups. *P<0.05, **p<0.01, and ***p<0.001. ANOVA, analysis of variance; CRC, colorectal cancer; FACS, fluorescence-activated cell sorter; IFN, interferon; IHC, immunohistochemical staining; IL, interleukin; ISH, in situ hybridization; MSS, microsatellite stable; PD-1: programmed death1; PD-L1, programmed cell death ligand 1.