Figure 1. The suppression of responder T cells mediated by human tumor-derived γδ Treg cells is due to the induction of cell G0/G1 cycle arrest.

(A) Suppression of naïve T cell proliferation by γδ Treg cells. CD4-C1 effector T cells served as a negative control displaying no suppressive activity. Naïve CD4⁺ or CD8⁺ T cells were co-cultured with γδ Treg or control T cells at a ratio of 10:1. The proliferation of naïve T cells in the presence of anti-CD3 antibody was determined by [³H]-thymidine incorporation assays. (B) The suppression of naïve CD4⁺ T cell proliferation mediated by γδ Treg cells is not due to the induction of apoptosis. (C) γδ Treg cells promoted the accumulation of naïve CD4⁺ T cells in G0/G1 cell cycle arrest. Naïve CD4⁺ T cells were co-cultured with CFSE-labeled γδ Treg or CD4-C1 cells in the presence of plate-bound anti-CD3 antibody. Apoptosis in naïve CD4⁺ T cells was analyzed after staining with PE-labeled Annexin V and 7-AAD gating on CFSE negative cell populations (in B). Cell cycle distribution in naïve CD4⁺ T cells was analyzed after incubation with 10 µg/ml propidium iodide and 100 µg/ml RNase A (in C). Naïve CD4⁺ T cells co-cultured with or without CD4-C1 T cells served as controls. (D) Cell cycle regulatory molecules p21, p16, and p53 were involved in human γδ Treg-induced T cell senescence. Cell treatment and procedure were the same as in (B) and (C). Co-cultured naïve CD4⁺ T cells were purified by FACS and then lysates prepared for Western blot analyses. Data shown in (A) to (D) are representative of three independent experiments with similar results.