Figure 4.

Pre-treatment with OSCC secretome affects Teff cell counts but CCR8, PD-1 and TIGIT expression is maintained after secretome removal in Treg and Teff. (A) Representative dot plots and cumulative data of Tregs and Teff cell counts after 48h of culture after removal of control or OSCC secretome. Briefly, sorted Tregs and Teff were pre-activated with anti-CD3/CD28 beads (1:5) in the presence of IL-2 (1000U). After activation, 1x10⁵ Tregs and Teff were co-cultured with secretomes from OSCC or control samples for 48h. Then, cells were washed and Tregs and Teff were cultured with anti-CD3/CD28 beads (1:5) in the presence of IL-2 (1000U) for 48h. (B) CCR6 and CCR8 expression was measured by flow cytometry in live Tregs and Teff. (C) PD-1 and TIGIT expression was measured by flow cytometry in live Tregs and Teff. (D) Supernatants of Tregs and Teff were collected and cytokines were measured with cytokine bead array. Data are presented as mean ± SEM using bars with scatter dot plots for phenotype and scatter dot plots for cytokine secretion (Unpaired t test). For all statistical tests, ^∗∗p < 0.01 and ^∗p < 0.05 were considered significant.