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Figure S1. The scheme illustrates the two-step transplantation strategy. CD45.1⁺ donor thymi were transplanted for 5 h and 1, 2, 3, and 4 d under the kidney capsule of one CD45.1/CD45.2 F1 recipient mouse before retransplant under the kidney capsule of a second CD45.1⁺ recipient mouse. Transplanted thymi were analyzed after 17 d, and the detected presence or absence of CD45.2⁺ double-positive thymocytes derived from the first recipient is indicated by a plus or a minus, respectively. Results are based on at least two independent experiments with at least three lobes per experiment.

To further characterize the transplantation model for thymus homing, we determined the time point after transplantation when recipient T cell lineage precursors entered the graft. This was done by serial transplantation of thymic lobes to understand if the simple exposure of the graft to recipient blood gave rise to an outgrowth of recipient-type thymocytes. Transplanted CD45.1⁺ donor thymi were transplanted back into a CD45.1⁺ recipient after they had been under the kidney capsule of the CCR9^EGFP/+//CD45.1/CD45.2 F1 recipient for 5 h or 1, 2, 3, or 4 d. The analysis of serially transplanted thymi for CCR9^EGFP+ CD45.2⁺ recipient double-positive thymocytes after a total of 17 d revealed that the immigration of CD45.1/CD45.2 T cell lineage precursors into transplanted newborn thymi required 2 d and did not occur in transplants that remained in the CCR9^EGFP/+//CD45.1/CD45.2 F1 recipient for only 5 h or 1 d. This finding indicated that homing of T cell lineage precursors into the transplanted thymi is specific and does not occur simply by exposing newborn thymi to recipient blood during transplantation; rather, it appears that this process requires resident time of the transplant in the recipient, presumably for microvascular reconnect.