Trafficking of donor T cells to recipient SLTs 24 hours after transplantation, and their subsequent in vivo expansion. (A) B6D2 recipients were lethally irradiated and then administered 4 × 10⁶ non–eGFP-expressing B6.PL-Thy1^a/CyJ Thy1.1⁺ T_n plus either 4 × 10⁶ eGFP⁺ WT B6 T_n or 4 × 10⁶ eGFP⁺ CCR7^−/− B6 T_n cells. Recipient animals were killed after 24 hours, and lymphocytes were extracted from the spleen and MLNs. These cells were then stained for flow cytometry using PerCp-Cy5.5–conjugated anti-Thy1.1 and APC-conjugated anti-CD3 antibodies. The ratios of CD3⁺eGFP⁺/CD3⁺Thy1.1⁺ events were then determined for all of the animals in each treatment group. For spleens, n = 6 per group. For MLNs, n = 3 LN pairs per group. *P < .001. **P = .005. Error bars indicate SEM. (B-C) B6D2 recipients were lethally irradiated and then administered 3 × 10⁶ unlabeled TCD BM cells from WT B6 donors plus 3 × 10⁶ CFSE-labeled T_n cells from either WT or CCR7^−/− B6 donors. Recipient animals were killed on transplantation day +3, and lymphocytes were extracted from the spleen for flow cytometry. (B) Histograms of cells isolated from the spleen from 1 of 3 representative WT T_n cell recipients and from 1 of 3 representative CCR7^−/− T_n cell recipients are depicted, using a CD3⁺ K^d-negative (donor cell) live lymphocyte gate. (C) The mean percentage of proliferating donor T cells within the spleens of WT (left) and CCR7−/− T_n cell (right) recipients are also shown. Error bars indicate SEM. *P = .014. (D) Recipient animals were killed on transplantation day +3, and the MLNs from each of 3 animals per treatment group were pooled for flow cytometry. MLN plots are depicted using a CD3⁺ K^d-negative (donor cell) live lymphocyte gate.