The generation of an immunosuppressive microenvironment by soluble factors in head and neck squamous cell carcinoma

Tumour infiltrating lymphocytes (TIL) in head neck squamous cell carcinoma (HNSCC) are enriched in Treg, a finding confirmed in the current study, which is thought to be a contributing factor to immunosuppression and tumour evasion. Soluble factors released by malignant HNSCC cells may contribute to the enrichment of Treg by inducing differentiation from naïve T cells and their migration to the tumour. Using multi-colour flow cytometry, increases in CD4+CD25hi, CD4+CD25+CD39+, CD4+CD25+CD26− and CD4+CD25+FoxP3+ Treg phenotypes were observed in TIL from HNSCC compared to PBMC. Following culture of CD4+CD25− T cells with conditioned medium (CM) collected from dispersed tumour samples, there was an increase in CD39 expression but not FoxP3 compared to control cultures that were cultured in complete growth medium. Furthermore these cells were unable to suppress the proliferation of CD4+CD25− T cells in CFSE assays. Soluble factors in CM from UMSCC cell lines and tumour-derived fibroblasts were unable to induce the expression of any Treg markers following culture with CD4+CD25− T cells. Culture with CM also had no effect on T cell apoptosis, with no significant increase in PI-AnexinV+ of CaspACE-binding in T cells cultured with CM compared to controls. The expression of the chemokine receptors, CXCR3, CCR4, CCR5 and CCR6 was analysed on T cell populations from HNSCC PBMC and TIL and healthy control PBMC using five-colour flow cytometry. Increased proportions of CXCR3+ and CCR5+ T cells were observed in HNSCC TIL compared to HNSCC PBMC but were no different between the patient and healthy control PBMC. CCR4 and CCR6 were expressed on a higher proportion of Treg from HNSCC PBMC compared to healthy controls but no difference was observed on CTL. In TIL the percentage expression of CCR4 and CCR6 were no different in that of HNSCC patient PBMC. Despite these observed differences in receptor expression, soluble factors in tumour dispersed CM was unable to induce T cell chemotaxis. Overall, although limited effects were observed from soluble factors in tumour CM, the different expression of chemokine receptors suggests there may be a role for soluble factors in Treg recruitment. However whether this is responsible for Treg accumulation or general to all T cells is unclear.