IL-17 inhibits CXCL9/10-mediated recruitment of CD8+ cytotoxic T cells and regulatory T cells to colorectal tumors

Abstract

Background: The IL-17 family cytokines are potent drivers of colorectal cancer (CRC) development. We and others have shown that IL-17 mainly signals to tumor cells to promote CRC, but the underlying mechanism remains unclear. IL-17 also dampens Th1-armed anti-tumor immunity, in part by attracting myeloid cells to tumor. Whether IL-17 controls the activity of adaptive immune cells in a more direct manner, however, is unknown.

Methods: Using mouse models of sporadic or inducible colorectal cancers, we ablated IL-17RA in the whole body or specifically in colorectal tumor cells. We also performed adoptive bone marrow reconstitution to knockout CXCR3 in hematopoietic cells. Histological and immunological experimental methods were used to reveal the link among IL-17, chemokine production, and CRC development.

Results: Loss of IL-17 signaling in mouse CRC resulted in marked increase in the recruitment of CD8⁺ cytotoxic T lymphocytes (CTLs) and regulatory T cells (Tregs), starting from early stage CRC lesions. This is accompanied by the increased expression of anti-inflammatory cytokines IL-10 and TGF-β. IL-17 signaling also inhibits the production of T cell attracting chemokines CXCL9 and CXCL10 by tumor cells. Conversely, the inability of hematopoietic cells to respond to CXCL9/10 resulted in decreased tumor infiltration by CTLs and Tregs, decreased levels of IL-10 and TGF-β, and increased numbers of tumor lesions. Blockade of IL-17 signaling resulted in increased expression of immune checkpoint markers. On the other hand, treatment of mouse CRC with anti-CTLA-4 antibody led to increased expression of pro-tumor IL-17.

Conclusion: IL-17 signals to colorectal tumor cells and inhibits their production of CXCL9/10 chemokines. By doing so, IL-17 inhibits the infiltration of CD8⁺ CTLs and Tregs to CRC, thus promoting CRC development. Cancer immunotherapy may be benefited by the use of anti-IL-17 agents as adjuvant therapies, which serve to block both IL-17-mediated tumor promotion and T cell exclusion.

Keywords: And colorectal cancer; CXCL10; CXCL9; Interleukin-17; Regulatory T cell.

Fig. 1

IL-17 inhibits the infiltration of CD4⁺ T cells, and the production of TGF-β and IL-10 in CRC. a and b q-RT-PCR analyses of the indicated mRNAs in designated tissues of control and IL-17RA-deficient Cdx2-Cre⁺/Apc^F/+ mice (a, n = 11), and Cdx2-Cre-ERT2⁺/Apc^F/F mice (b, n = 5 for MLN, 11 for tumor). Mice in b received tamoxifen injection and were kept for 5 weeks for the development of early CRC tumors. Tumor-adjacent colonic tissues were used as “normal” control. c CD4⁺ T cells (CD45⁺ CD3⁺ CD4⁺), CD8⁺ T cells (CD45⁺ CD3⁺ CD8⁺), B cells (CD45⁺ CD19⁺), monocytes (CD45⁺ CD11b⁺ Ly-6C^High), neutrophils (CD45⁺ CD11b⁺ Ly-6C^Low, Ly-6G⁺), macrophages (CD45⁺ CD11b⁺, F4/80⁺), fibroblasts (CD45⁻ EpCam⁻), and tumor cells (CD45⁻ EpCam⁺) were FACS-sorted from pooled colonic tumors of 10 Il17ra^+/+/Cdx2-Cre⁺/Apc^F/+ mice. Purified cells were subjected to q-RT-PCR analysis, and the levels of each individual mRNA were shown as “1” in the cell type of highest expression. d Cdx2-Cre-ERT2⁺/Apc^F/F mice that were Il17ra^−/− or Il17ra^+/− were given i.p. injection of tamoxifen (75 mg/kg body weight) daily for 3 consecutive days. Mice were sacrificed 5 weeks after tamoxifen-induced Apc ablation, and their mesenteric lymph nodes (MLN) and tumors were subjected to flow cytometry analysis. n = 5. Cells were isolated following collagenase digestion of the indicated tissues, followed by 4-h in vitro stimulation with PMA and ionomycin in the presence of Brefeldin A and monensin. e Representative flow cytometry plots for tumors samples from d. Data represent means ± S.E.M. *p < 0.05 in Students’ t test

Fig. 2

IL-17 blocks the accumulation of CD8⁺ T lymphocytes starting from early stage CRC. a Immunostaining of colon tumors from 5-month-old Cdx2-Cre⁺/Apc^F/+ mice that were heterozygous (+/−) or null (−/−) for Il17ra. Scale bar = 100 μm. b Statistics for the percentages of CD8⁺ cells shown in a. n = 8. c-e: Cdx2-Cre-ERT2⁺/Apc^F/F mice that were Il17ra^+/− or Il17ra^−/− were sacrificed 5 weeks after tamoxifen-induced Apc ablation, and their MLN (c and d) and tumors (c, d, and e) were subjected to flow cytometry (c and e, n = 4 for Il17ra^+/−, 10 for Il17ra^−/−) and q-RT-PCR (d, n = 5 for MLN, 11 for tumor) analyses. Data represent means ± S.E.M. *p < 0.05 in Students’ t test

Fig. 3

IL-17 inhibits the production of CXCL9 family chemokines. a q-RT-PCR analyses of the indicated mRNAs in normal colon and colorectal tumor tissues of 5-month-old control (Il17ra^+/−) and IL-17RA whole body knockout (Il17ra^−/−) mice that also harbor Cdx2-Cre⁺/Apc^F/+ genotypes (n = 12). b Colonic tumors from control (Il17ra^+/−) and IL-17RA-deficient (Il17ra^−/−) Cdx2-Cre⁺/Apc^F/+ mice were cultured in Opti-MEM medium for 24 h. Concentrations of chemokines were tested using a bead-based immunoassay (Biolegend, for CXCL9), or plate based ELISA (R&D systems, for CXCL10). Data are shown as pg/ml chemokine per mg tumor in culture (n = 13). c 5-month-old Cdx2-Cre⁺/Apc^F/+ mice were treated with i.p. injection of isotype or anti-IL-17A antibodies (100 μg per injection, every 3 days) for two weeks. Colonic tumors were harvested at the end of the study, and analyzed by q-RT-PCR for indicated mRNAs. n = 13. d Cdx2-Cre-ERT2⁺/Apc^F/F mice (that were Il17ra^−/− or Il17ra^+/−) were sacrificed 5 weeks after tamoxifen-induced Apc ablation. Mouse MLN and tumors were subjected to q-RT-PCR analysis (n = 5 for MLN, 11 for tumor). Data represent means ± S.E.M. *p < 0.05 in Students’ t test

Fig. 4

IL-17 signals to transformed epithelial cells to suppress CXCL9 family chemokine production. a Cdx2-Cre⁺/Apc^F/+ mice were crossed to Il17ra-flox mice to generate a conditional ablation of Il17ra gene in colorectal epithelial cells and tumor cells. These mice carry Cdx2-Cre⁺/Apc^F/+/Il17ra^F/− genotypes and are labeled as “Il17ra^F/−”. Cdx2-Cre⁺/Apc^F/+/Il17ra^F/+ mice (labeled as Il17ra^F/+) were used as controls. Both groups of mice were sacrificed at 5 months of age. Colorectal tumors and adjacent normal colon tissues were harvested for q-RT-PCR analysis. n = 6. b Tumor cells were isolated from colorectal tumors of Cdx2-Cre-ERT2⁺/Apc^F/F mice 4 weeks after tamoxifen injection. Cells were then cultured a 3-D system to allow their development into primary tumor spheres. Tumor spheres were subsequently treated with vehicle control (PBS with 0.1% BSA) or 100 ng/ml recombinant human IL-17A, C, or F for 24 h, followed by q-RT-PCR analysis (n = 3, and data represent one of three consistent tests). Data represent means ± S.E.M. *p < 0.05 in Students’ t test

Fig. 5

CXCR3 mediates the attraction of CD8⁺ CTLs and Treg cells, and inhibits the CRC development. a FACS-purified cells (as depicted Fig. 1c) from colonic tumors of Cdx2-Cre⁺/Apc^F/WT mice were subjected to q-RT-PCR analysis. b-e Bone marrow cells were harvested from WT and Cxcr3^−/− mice, and transferred into lethally irradiated 6–8-week-old Cdx2-Cre⁺/Apc^F/WT mice. Recipient mice were sacrificed at 5 months of age, and their colorectal tumors were used for flow cytometry (b and c, n = 7), q-RT-PCR (d, n = 16), and tumor statistics (e, n = 9). Cells shown in c were gated as live/CD45⁺. Data represent means ± S.E.M. *p < 0.05 in Students’ t test

Fig. 6

Blockade of IL-17 signaling in CRC results in enhanced immune checkpoint signaling. a q-RT-PCR analysis of normal colon tissue and colorectal tumors from Cdx2-Cre⁺/Apc^F/+ mice that harbor heterozygous or null alleles of Il17ra gene (n = 12). b 4-month-old Cdx2-Cre⁺/Apc^F/+ mice received i.p. injection of 100 μg isotype or anti-IL-17A antibodies every 3 days for 1 month. Mice were sacrificed for q-RT-PCR analysis (n = 9). c Cdx2-Cre-ERT2⁺/Apc^F/F mice that were Il17ra^+/− or Il17ra^−/− were sacrificed 5 weeks after tamoxifen-induced Apc ablation, and their MLN and tumors were subjected to q-RT-PCR analysis (n = 5 for MLN, 11 for tumor). d Cdx2-Cre-ERT2⁺/Apc^F/F mice that were Il17ra^−/− or Il17ra^+/− were sacrificed 5 weeks after tamoxifen-induced Apc ablation, and their MLN and tumors were subjected to flow cytometry analysis. n = 5. Data represent means ± S.E.M. *p < 0.05 in Students’ t test

Fig. 7

Blockade of CTLA-4 induced the expression of IL-17 in CRC. a, b 5-month-old Cdx2-Cre⁺/Apc^F/+ mice received i.p. injection of 100 μg isotype or blocking antibodies against CTLA-4 (a, n = 24) or PD-1 (b, n = 8) every 3 days for 2 weeks, and were sacrificed for q-RT-PCR analysis. Data represent means ± S.E.M. *p < 0.05 in Students’ t test. c: IL-17 signals directly to tumor cells in CRC to inhibit the production of CXCL9 and CXCL10. These two chemokines are required for the recruitment of CD8⁺ CTLs and Tregs, which inhibits CRC development by targeting cancer cells and dampening tumor-promoting inflammation, respectively