CTL- vs T_reg lymphocyte-attracting chemokines, CCL4 and CCL20, are strong reciprocal predictive markers for survival of patients with oesophageal squamous cell carcinoma

Background: Tumoural infiltration of T lymphocytes is determined by local patterns of specific chemokine expression. In this report, we examined the roles of CCL4 and CCL20 in the accumulation of CD8⁺ cytotoxic T lymphocytes (CTLs) and regulatory T (T_reg) lymphocytes in oesophageal squamous cell carcinoma (ESCC), and determined the correlations between chemokine expressions and ESCC patients' survival. Methods: Reverse transcriptase-PCR and immunohistochemistry (IHC) staining were performed to examine the expressions of interested genes. Flow cytometry was adopted to check the expressions of CCL4- and CCL6-specific receptors, CCR5 and CCR6, on CTLs and T_reg cells. In addition, transwell assay was carried on. Results: The CCL4 expression was significantly correlated with the expression of CTL markers (CD8 and Granzyme B), whereas CCL20 was positively correlated with T_reg markers (FoxP3 and IL-10). Consistently, CCR5 was found to be mainly expressed on CD8⁺ T lymphocytes, while CCR6 showed prevalence on T_reg lymphocytes and the frequencies of CCR5⁺ CD8⁺ CTLs and CCR6⁺ T_reg cells were higher in TIL compared with PBMC. Respectively, CCL4 and CCL20 recruited CD8⁺ and regulatory T cells in vitro. Importantly, high levels of CCL4 in the lesions of ESCC patients predicted prolonged survival. Furthermore, CCL4^high/CCL20^low group demonstrated better overall survival, whereas CCL4^low/CCL20^low and CCL4^low/CCL20^high groups showed the worst overall survival. Conclusions: Our data showed that CCL4 and CCL20 recruit functionally different T lymphocyte subsets into oesophageal carcinoma, indicating CCL4 and CCL20 are potential predictors of ESCC patients' survival.