Expansion and characteristics of human T regulatory type 1 cells in co-cultures simulating tumor microenvironment

Objective Chronic inflammation and cancer development are associated with dysregulated immune responses and the presence of regulatory T cells (T_reg). To study the role of T_reg in tumor cell escape from immune surveillance, an in vitro model simulating the tumor microenvironment and promoting the induction and expansion of IL-10⁺ T_reg type 1 (Tr1) was established. Methods An in vitro co-culture system (IVA) included an irradiated head and neck squamous cell carcinoma cell line, immature dendritic cells (iDC), CD4⁺CD25⁻ T cells and cytokines, IL-2 (10 IU/ml), IL-10 (20 IU/ml), IL-15 (20 IU/ml) ± 1 nM rapamycin. Autologous iDC and CD4⁺CD25⁻ T cells were obtained from the peripheral blood of 15 normal donors. Co-cultures were expanded for 10 days. Proliferating lymphocytes were phenotyped by multi-color flow cytometry. Their suppressor function was measured in CFSE inhibition assays ± neutralizing anti-IL-10 mAb and using transwell cultures. Culture supernatants were tested for IL-4, IL-10, TGF-β and IFN-γ in ELISA. Results In the IVA, low doses of IL-2, IL-10 and IL-15 promoted induction and expansion of CD3⁺CD4⁺CD25⁻IL2Rβ⁺IL2Rγ⁺FoxP3⁺CTLA-4⁺IL-10⁺ cells with suppressor activity (mean suppression ± SD = 58 ± 12%). These suppressor cells produced IL-10 (mean ± SD = 535 ± 12 pg/ml) and TGF-β (mean ± SD = 512 ± 38 pg/ml), but no IL-4 or IFN-γ. Suppressor function of co-cultures correlated with the percent of expanding IL-10⁺ Tr1 cells (r ² = 0.9; P < 0.001). The addition of rapamycin enriched Tr1 cells in all co-cultures. Neutralizing anti-IL-10 mAb abolished suppressive activity. Suppression was cell-contact independent. Conclusion The tumor microenvironment promotes generation of Tr1 cells which have the phenotype distinct from that of CD4⁺CD25^highFoxP3⁺ nTreg and mediate IL-10 dependent immune suppression in a cell-contact independent manner. Tr1 cells may play a critical role in cancer progression.