Figure 6.

Tumor-derived TGF-β1 plays a pivotal role in the induction of CD39⁺ γδTregs. (A, B) The relative mRNA levels of TGF-β1, TGF-β2, and TGF-β3 in SW480 cells were determined by RT-PCR and normalized to GAPDH (A). Concentrations of TGF-β1, TGF-β2, and TGF-β3 in SW480 cells-derived supernatant were detected by ELISA (B). Data are shown as mean ± SEM; n = 5; **p < 0.01. (C, D) The relative mRNA levels of TGF-β1 in SW480, vehicle pretreated SW480, control siRNA-transfected SW480, and TGF-β1-siRNA-transfected SW480 cells were determined by RT-PCR and normalized to GAPDH (C). Concentrations of TGF-β1 in supernatants derived from different treated SW480 cells were detected by ELISA respectively (D). Data are shown as mean ± SEM; n = 5; ns: no significance; RT-PCR: real-time PCR; **p < 0.01; ***p < 0.001. (E–G) Sorted CD39⁺ γδT cells from paired normal tissue were co-cultured with SW480 cells in the presence of TGF-β1 mAb or control mAb, vehicle pretreated SW480, and control siRNA or TGF-β1-siRNA-transfected SW480 cells in the presence of CD3 and CD28 mAbs. These CD39⁺ γδT cells were washed and harvested on day 4, and then co-cultured with CFSE-labeled allogeneic CD3⁺ T cells in the presence of CD3 and CD28 mAbs for additional 6 d; CD3⁺ T-cell proliferation was evaluated by FCM (E). Bar diagram summarizes the percentages of proliferated cells (CFSE^low) in CD3⁺ T cells (F). Meanwhile, the concentrations of adenosine in the supernatants of co-culture system were detected by HPLC (G). Data are shown as mean ± SEM; n = 4; ns: no significance; *p < 0.05; **p < 0.01.