Figure 4.

Long-term in vitro mTORi treatment improves proliferation and viability of Vδ2^neg γδ T cells and CMV-specific αβ T cells. PBMCs from KTRs who were R+ were incubated with IL-2, with or without IL-15, for Vδ2^neg γδ T cells, and IL-2 alone for CMV-specific αβ T cells, and with the indicated doses of EVR. Proliferation and viability of Vδ2^neg γδ T cells at day 14, 21, and 28 of culture and of CMV-specific αβ T cells at day 9 and 16 of culture were performed. (A) Representative donor for proliferation of Vδ2^neg γδ T cells. (B) Representative flow cytometry staining of S6 and phospho-S6 (p-S6) among Vδ2^neg γδ T cells at day 14 of culture. (C) Proliferation of Vδ2^neg γδ T cells, represented as fold increases normalized to culture with medium alone, at day 14 (0.5 nM EVR, n=13; 10 nM EVR, n=6), 21 (0.5 nM EVR, n=15; 10 nM EVR, n=8), and 28 (0.5 nM EVR, n=12; 10 nM EVR, n=6). (D) Vδ2^neg γδ T-cell viability tested by flow cytometry live-dead staining (n=5). (E) Proliferation of CMV-specific αβ T cells, represented as fold increases normalized to culture with medium alone (0.5 nM EVR, n=5). (F) Viability of CMV-specific αβ T cells tested by flow cytometry live-dead staining (n=5). For (C), (D), (E), and (F), each symbol represents an individual donor. *P<0.05, **P<0.01, ***P<0.001, as determined by Wilcoxon test.