Anti–third-party Tcm administration induces LN accumulation of host anti-donor HTCs. Seven-hundred TCR transgenic 2c-Luc⁺ CD8⁺ cells were injected on day −1 into supralethally irradiated (4Gy on day − 5, and 10Gy on day − 2) C57BL/6 mice. One day after 2c administration, the mice received 3 × 10⁶ Balb/c-nude BM cells (“HTC only”) or BM+5 × 10⁶ (CB6)F1 Tcm (“HTC+Tcm”). (A-C) Representative IVIS images depicting the difference in 2c-Luc⁺ cell distribution within the body of untreated (A, “HTC only”) versus treated (C, “HTC and Tcm”) mice on day 10 after transplantation. Color intensities were adjusted according to signal for each individual image. (B-D) On day 9 after transplantation, organs (LN, spleen, BM, lung, and liver) were harvested from 5 animals from each group and numbers of live (7aad negative, viability staining solution) CD8⁺ 2c cells were assessed by FACS analysis. (B) Distribution of 2c cells in the recipients' organs in the absence of Tcm treatment. (D) Distribution of 2c cells in the recipients' organs in the presence of Tcm treatment. Data represent mean ± SD of total number of 2c cells of 5 animals in each group (*P < .05; **P < .01; ***P < .001).