Spleen MSCs suppress NK cell cytotoxicity in vivo. Tumor-bearing mice at 4 weeks after tumor injection were killed and spleen cells were isolated. (A) The percentages of DX5, F4/80, CD11b, and Gr-1⁺ cells were determined by FACS analysis as described in “Flow cytometry analysis.” The data represent the mean ± SEM from 5 mice from each group. (B) CD11b⁺Gr-1⁺ cells were isolated from the spleens of 9-week-old TS/A tumor-bearing BALB/c mice or naive mice. Increased numbers (1 × 10⁶, 3 × 10⁶, and 6 × 10⁶) of sorted CD11b⁺Gr-1⁺ MSCs were transferred intravenously into 2-month-old BALB/c female mice (n = 5 per group). Twenty-four hours after adoptive transfer, the efficiency of NK cell killing of injected YAC-1–Luc cells was determined by measuring the numbers of photons collected at 6 hours divided by the photons collected at 0 hours. *P < .05; **P < .001. (C) Female BALB/c mice (n = 4): TS/A tumor-bearing mice with tumor debulked surgically (surgery removal), tumor-intact mice (nonremoval), and non–tumor-bearing PBS control mice (naive mice). Two weeks later, an in vivo measurement of NK cell cytotoxicity was determined by injection of YAC-1–Luc using an identical protocol, as described in Figure 1. The efficiency of NK cell killing of injected YAC-1–Luc cells was determined by measuring the numbers of photons collected at 2 hours and 4 hours divided by the photons collected at 0 hours. *P < .05; **P < .001. The data represent the mean ± SEM of 2 independent experiments (n = 4) (C, right panel). (D) After imaged mice were killed, the percentages of leukocytes in the lung were determined in the gated R1 region of a FACS analysis (top left). The presence of CD11b⁺Gr-1⁺, DX5⁺, CD8⁺, and CD11c⁺ cells was determined (left, representative plots). Results obtained from 2 independent experiments with replica 4 mice in each experiment were pooled and are presented as the mean ± SEM. *P < .05; **P < .01.