Prolonged survival of allogeneic chimeras engrafted with limiting numbers of LSCs. (A) Survival curve of Balb/c recipients of BCR-ABL1–transduced TCD BM from 5-fluorouracil–treated syngeneic donors injected with the indicated numbers of total BM cells. (B) Southern blot analysis of spleen genomic DNA from the cohorts shown in panel A. Lanes 1 and 2 were from recipients of 1 × 10³BCR-ABL1–transduced BM cells, lanes 3-5 received 3 × 10³ transduced cells, lanes 6-8 received 1 × 10⁴ transduced cells, lanes 9-11 received 3 × 10⁴ transduced cells, lanes 12-15 received 1 × 10⁵ transduced cells, whereas lanes 16 and 17 received 5 × 10⁵ transduced cells. Lanes 19-21 were from control cell lines each containing a single provirus. Note that reduction in BM cell dose to ≤ 1 × 10⁴ cells results in repopulation with 1-2 LSCs. (C) Survival curve of mixed allogeneic chimeras repopulated with limiting numbers of TCD LSCs, together with increasing numbers of TCD allogeneic BM cells. The numbers represent the ratio of LSCs in 1 × 10⁴BCR-ABL1–transduced TCD BM cells (approximately 2) to the number of allogeneic HSCs (assuming 1 HSC per 10⁵ BM cells). Control mice received 2 × 10⁵ syngeneic BCR-ABL1–transduced TCD BM cells only. (D) Percentage of circulating allogeneic cells and GFP⁺ cells versus time after BMT for the cohort that received the 1:500 mixture of BCR-ABL1–transduced and allogeneic BM from the experiment shown in panel C. Note the high level of allogeneic chimerism that persists until leukemic progression at 7 weeks. The GFP⁺ population between days 21 and 42 contained some cells that were allotype-positive because of phagocytosis of GFP⁺ leukemic cells by allogeneic macrophages.²¹