Cotransplantation of allogeneic splenocytes with BCR-ABL1–transduced BM blocks the engraftment of LSCs. (A) Schematic diagram of secondary transplantation experiment. Primary Balb/c recipients (H-2^d) were lethally irradiated (700-900 cGy) and received BCR-ABL1–transduced TCD syngeneic BM and TCD allogeneic BM from MHC-matched (H-2^d), miHA-mismatched B10.D2 donors, with (dotted line) or without allogeneic splenocytes (DLI) administered at the time of transplantation (Early) or beginning 2 weeks after transplantation (Delayed). At different intervals after the primary transplantation, recipient BM was transplanted into sublethally (400-450 cGy) irradiated secondary Balb/c recipients, who were followed for development of CML-like leukemia. (B) Survival curve depicting the time to morbidity or death from CML-like disease of secondary recipients of BM from primary mice that received allogeneic splenocytes at the time of initial transplantation (+DLI; dashed lines) or did not receive allogeneic splenocytes (no DLI; solid lines). The secondary BMT was performed at 14 days (red curves), 7 days (green curves), or 4 days (blue curves) after the primary transplantation. Note that in this model, CML-like leukemia is not transplanted with 100% efficiency even from nonchimeric donors.²⁹  (C) Southern blot analysis of genomic DNA from spleens of secondary recipients in panel C (d14 cohort) harvested at day 56 after BMT. The blot was hybridized with a GFP probe that detects individual LSC clones (top panel), an ABL1 probe that detects murine c-Abl1 and BCR-ABL1, allowing quantification of proviral DNA content²³  (middle panels), and an SRY probe to detect male (donor) sex (bottom panel). Lanes 1-10 are from recipients of BM from DLI-treated primary mice, lanes 11-19 from recipients of primary mice who did not receive DLI, DNA from normal female and male Balb/c mice, and from a control cell line (C) containing a mixture of 2 proviral clones are on the right. Note that secondary recipients of BM from primary mice treated with early DLI did not engraft with any male BCR-ABL1⁺ stem cells. Similar results were obtained with analysis of the day 7 cohort (data not shown).