ITI of multipotent HSPCs results in long-lasting generation of donor T cells. (A) Lethally irradiated C57BL/6 recipients were transplanted with C57BL/6.CD45.1⁺ Lin⁻ BM cells and received 3000 C57BL/6.Thy1.1⁺ luciferase-expressing LSK cells via ITI 2 hours after irradiation. The whole-body distribution of LSK-derived cells at the indicated time points after BMT was monitored using in vivo BLI. Pseudocolor images superimposed on conventional photographs are shown. A total of 4 representative animals of 10 are presented. (B) Animals were treated as described in panel A. Thymuses were harvested 6 months after BMT and analyzed for early T-cell progenitor populations (CD25⁻ LSK cells were resolved into subpopulations based on c-kit and IL-7Rα expression) gated on cells of BM donor and LSK donor origin. (i-ii) Representative plots of 1 of 4 LSK group samples are presented. (iii) Mean and SEM are presented (n = 4). (C) Animals were treated as described in panel A. Thymuses were harvested 6 months after BMT and analyzed for long-term HSC populations of BM donor and LSK donor origin (LSK cells were resolved into subpopulations based on CD48 and CD150 expression). (i-ii) Representative plots of 1 of 4 LSK group samples are presented. (iii) Mean and SEM are presented (n = 4). (D) Syngeneic BMT: C57BL/6 recipients were transplanted as described in panel A. Allogeneic BMT: lethally irradiated BALB/c recipients were transplanted with C57BL/6 TCD BM cells and received 5000 C57BL/6.CD45.1⁺ LSK cells via ITI 2 hours after irradiation. Thymuses were harvested 2 months and 6 months after BMT and analyzed for CD4⁻CD8⁻CD11b⁺ populations of LSK donor origin. Representative plots of 2 to 5 LSK group samples are presented.