Tcms, in contrast to CTLs, populate the recipient LNs and proliferate extensively shortly after the BMT. Lethally irradiated (8 Gy) BALB/c (H-2^d) mice received 4 × 10⁶ C57BL/6-NUDE (H-2^b) BM cells and 1 × 10⁴ syngeneic HTCs. Mice then received a transplant of 10⁷ DIR-labeled, CB6 (H-2^bd)–derived, purified anti–third-party Tcms or CTLs. After 2 or 7 days, selected recipients were killed; the peripheral LNs (pLNs), mesenteric LNs (mLNs), spleen, liver, and BM were extracted; cells were purified from the different organs; and the purified cells were analyzed by FACS. To obtain absolute values of cells, samples were suspended in constant volume and flow cytometric counts for each sample were obtained during a constant, predetermined period of time and were compared with flow cytometric counts obtained from control samples that were set up with fixed volume and fixed numbers of input cells. (A) Representative FACS analysis demonstrating the presence of CD8⁺ and alive (7AAD⁻) CB6-derived cells (Tcms or CTLs) in the peripheral LNs of the recipients 2 days after transplantation. (B-C) Quantification of the FACS analysis as described in panel A, demonstrating the distribution of CB6-derived cells in various organs at 2 (B) and 7 (C) days after transplantation. (D) The sum of the total number of Tcms or CTLs, harvested from all organs tested, at 2 or 7 days after transplantation. In panels B-D, results shown represent average ± SD of pooled data from 6 animals from each group in 2 independent experiments. *P < .05.