Delayed DLI and imatinib therapy leads to prolonged leukemia-free survival in MHC-matched/miHA-mismatched chimeras with BCR-ABL1–induced CML-like disease. (A) Survival curve for cohorts of Balb/c recipients of BCR-ABL1–transduced Lin⁻ Balb/c BM (1 × 10⁴ cells) mixed with 500-fold excess of MHC-matched allogeneic stem cells (B10.D2→Balb/c donors). All recipients developed mixed hematopoietic chimerism with CML-like leukemia (GFP⁺ myeloid cells) at day 14 after transplantation. Beginning at day 14, mice were treated with repeated weekly infusions of allogeneic (B10.D2) splenocytes (3 × 10⁷ cells per treatment, total of 5 infusions per recipient), treated with high-dose imatinib (100 mg/kg once daily by oral gavage), or a combination of the 2 (DLI + imatinib). Imatinib treatment was discontinued at day 49 (indicated by the vertical dotted line and asterisk). The addition of delayed DLI to imatinib resulted in superior survival compared with imatinib alone (P < .0001, Mantel-Cox test), whereas the survival difference between cohorts receiving DLI + imatinib and DLI only was of borderline significance (P = .072). (B) Flow cytometric analysis of peripheral blood leukocytes from 5 representative mice each from the 4 cohorts in panel A, analyzed at 5 weeks after transplantation (after 3 DLI doses). Allogeneic chimerism (Y-axis) was detected by a polymorphism in β2-microglobulin as described in “Methods,” whereas GFP⁺ cells (X-axis) represent BCR-ABL1–expressing leukemic cells. The percentage of allotype-positive and GFP⁺ cells in each plot is indicated. Note the eradication of leukemia in the majority of recipients treated with DLI + imatinib compared with the persistent leukemia in control or imatinib-only mice. (C) Southern blot analysis of genomic DNA from the cohorts in panel A, harvested at day 120 after BMT. Paired samples from the spleen (S) and BM (B) of the same individual mouse are grouped by the bars; all other samples are from the spleen. Lanes with DNA from normal female mice are indicated, whereas C indicates DNA from cell lines containing 1 or 2 proviral copies. The blot was hybridized with probes for GFP and ABL1 as in Figure 1C. The band indicated by the asterisk is a background band.