FIGURE 4 from Exercise-induced β2-adrenergic Receptor Activation Enhances the Antileukemic Activity of Expanded γδ T-Cells via DNAM-1 Upregulation and PVR/Nectin-2 Recognition

ISO infusion enhances the antileukemic activity of ex vivo expanded Vγ9Vδ2+ T-cells in vitro and in vivo. A, Schematic of the experimental design for in vitro and in vivo experiments. B, The total number (1 × 10⁶) of Vγ9Vδ2+ cells isolated before (REST) and during the final 5 minutes of ISO infusion at day 0, and the total number of Vγ9Vδ2+ T-cells generated in the expanded cell products after stimulation with ZOL+IL2 for 14 days. The number of Vγ9Vδ2+ cells generated at day 14 relative to the number of γδ cells in the PBMC fractions at day 0. The cellular composition of the expanded Vγ9Vδ2+ T-cell products at day 14. The proportions of naïve (N), CM, EM, and CD45RA+ EMRA cells among Vγ9Vδ2+ T-cells after 14 days of expansion (n = 7). C, The in vitro anti-tumor activity of ex vivo expanded Vγ9Vδ2+ T-cells against K562, Daudi, and U266 cells via flow cytometry–based assays. Significance indicated by ** (P < 0.01) and *** (P < 0.001). D, To determine the in vivo GvL effect of ISO expanded Vγ9Vδ2+, NSG-IL15 mice were injected with REST or ISO expanded Vγ9Vδ2+ (10 × 10⁶) and challenged with 1 × 10⁶ luciferase tagged human chronic myeloid leukemia cells (K562-luc). A subset of mice in each group were also provided weekly injections of ZOL (REST+ZOL; ISO+ZOL; VEHICLE +ZOL). The BLI (photons/second) scores, TFS, change in BW, and probability of survival after injection of expanded Vγ9Vδ2+ T-cells with or without ZOL sensitization (n = 7–8/group). BLI significant difference from EX+ZOL and EX conditions indicated by * and ^#, respectively (all conditions were significantly different from vehicle controls). TFS, BW, and OS significant difference from vehicle controls were indicated by * (TFS; EX, EX+ZOL, and REST+ZOL significance indicated by a single marker). E, Representative bioluminescence images of leukemia-bearing mice that received (from left to right) vehicle, vehicle + ZOL, REST expanded Vγ9Vδ2+ T-cells, REST expanded Vγ9Vδ2+ T-cells + ZOL, ISO expanded Vγ9Vδ2+ T-cells, and ISO expanded Vγ9Vδ2+ T-cells + ZOL. BLI intensity on a scale from low (purple) to high (red). Data are represented as mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Students two-tailed paired t test (B); Repeated measures two-way ANOVA or LMM with Bonferroni post hoc test (C–D); log-rank (Mantel–Cox) test (D).