Preferential recruitment of Th17 cells to cervical cancer via CCR6-CCL20 pathway

Abstract

Our previous studies suggest that Th17 cells accumulate within tumor tissues and correlate with recurrence of cervical cancer patients. However, the source of the increased tumor-infiltrating Th17 cells remains poorly understood. We investigated the prevalence, phenotype and trafficking property of Th17 cells in patients with cervical cancer. Our results showed that Th17 cells highly aggregated within tumor tissues in an activated phenotype with markedly increased expression of CCR6. Correspondingly, level of CCL20 in the tumor tissues was significantly higher than that in non-tumor and normal control tissues, and strongly positively associated with Th17 cells. Further, in vitro migration assay showed CCL20 had effective chemotaxis to circulating Th17 cells. In conclusion, Th17 cells are recruited into tumor tissues preferentially through CCR6-CCL20 pathway, which can serve as a novel therapeutic target for cervical cancer.

Fig 1. Th17 cells are highly enriched in tumors of patients with cervical cancer.

Th17 cells were gated from CD3⁺ T cells by flow cytometry. (A) Representative IL-17 expression profiles in CD4⁺ T cells from the four studied groups. The percentages represent the frequency of Th17 cells among CD4⁺ T cells. (B) Statistical analysis show that the frequency of Th17 cells was higher in patients with cervical cancer, especially among tumor-infiltrating lymphocytes (n = 35). **P < 0.01, ***P < 0.001. (C) Representative images for Th17 cells (IL-17⁺) and Tregs (FoxP3⁺) infiltration in cervical cancer tissue from the same patient. Immunostained cells (brown, indicated by black arrow) and tumor cells (blue). Magnification,×100.

Fig 2. Association of intratumoral Th17-cell prevalence with clinical parameters.

Fig 3. Phenotypic analysis of Th17 cells in patients with cervical cancer.

(A) Representative expression profiles of CD45RO, HLA-DR, Granzyme B and PD-1 in tumor-infiltrating Th17 cells. The percentages represent the frequencies of various markers in Th17 cells. (B) Representative expression profiles of CCR4, CCR6 and CD49d on Th17 cells from peripheral blood (long dotted line), non-tumor (dotted line) and tumor tissues (solid line). The percentages represent the frequencies of various markers on tumor-infiltrating Th17 cells. (C) Statistical analysis of surface expression of CCR4, CCR6, CD49d on Th17 cells from peripheral blood, non-tumor and tumor tissues (n = 25). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4. Associations of chemokine levels with intratumoral Th17 cells.

(A) Real-time PCR of chemokine CCL17, CCL20 and CCL22 in tumor (T) and non-tumor (nT) regions of patients with cervical cancer and in normal control (NC). The value was normalized to GAPDH, multiplied by 10⁵, and log transformed. *P < 0.05, **P < 0.01. (B, C, D) Correlations of chemokine CCL17 (B), CCL20 (C) and CCL22 (D) with frequency of tumor-infiltrating Th17 cells. Statistical analysis showed a strong correlation of CCL20 with Th17 cells in tumor.

Fig 5. Chemokine effects on Th17 cell recruitment.

Migration assays were performed in a Transwell system. (A) Th17 cells migrate in response to recombinant human CCL17, CCL20 and CCL22 in dose-dependent manner (n = 5). Specific antibodies to chemokines significantly inhibit Th17 cell migration.***P < 0.001. (B and C) Expression of CCL20 by HeLa, Siha, and C-33A cells detected using Real-time PRC (B) and ELISA (C). All the three cervical cells could highly secrete CCL20 with highest level of CCL20 by HeLa cells. (D) Th17 cells also migrate toward culture supernatants of HeLa, Siha, and C-33A cells, which can be efficiently blocked by antibody against CCL20 alone or in combination with CCL17 and CCL22, but significantly less effectively by antibody against CCL17 or CCL22 (n = 3). *P < 0.05, **P < 0.01,***P < 0.001.