Intrathymically injected HSPCs give rise to functional T cells. (A) Sublethally irradiated (700 cGy) C57BL/6 recipients received 6000 C57BL/6.CD45.1⁺ LSK cells via ITI 2 to 3 hours after irradiation. (i-iii) Percentage of naïve, central, and effector memory CD4⁺ and CD8⁺ T cells of host and donor origin. (i) A representative plot of 1 of 4 samples is presented. (ii-iii) Mean and SEM are presented (n = 4). (B) Sublethally irradiated C57BL/6 recipients received 5000 C57BL/6.Thy1.1⁺ LSK cells via ITI 2 hours after irradiation. Splenocytes were harvested on day 60 after radiation and were stimulated with 4-phorbol 12-myristate 13-acetate/ionomycin followed by intracellular staining for IL-2 and IFN-γ. CD4⁺ and CD8⁺ T cells of donor origin were gated on IL-2⁺ and IFN-γ⁺ cells. (i-ii) Representative plots of one of 4 samples are presented. (iii) Mean and SEM are presented (n = 4). (C) Animals were treated as described in panel B. Splenocytes were harvested on day 60 after radiation and were stained with V-β screening panel, and the TCR repertoire of CD4⁺ and CD8⁺ donor and host T cells was analyzed. Mean and SEM are presented (n = 4). (D) C57BL/6 recipients received 3000 C57BL/6.CD451.1⁺ LSK cells via ITI 2 hours after sublethal TBI or thymic irradiation or in the absence of radiation. In an additional group, C57BL/6 recipients received C57BL/6.CD45.1⁺ LSK cells via intravenous injection 2 hours after thymic irradiation. Thymuses and spleens were harvested on day 30 after LSK injection and analyzed for thymocytes and splenic T cells of donor origin. Mean and SEM are presented (n = 4). IT, intrathymic; IV, intravenous; XRT, radiation therapy.