Establishment of mixed hematopoietic chimerism and CML-like leukemia in mice. (A) Schematic diagram of the adoptive immunotherapy model system. TCD BM from 5-FU–treated Balb/c mice is transduced with retrovirus coexpressing BCR-ABL and GFP (top), then mixed with TCD BM from allogeneic C57Bl/6 donors (middle) and transplanted into lethally irradiated Balb/c recipients (right). Allogeneic splenocytes from the B6 donor (DLI) are infused into mice with mixed hematopoietic chimerism and established CML-like leukemia (bottom). In some experiments, allogeneic splenocytes are included in the initial BM graft (dotted arrow). (B) Flow cytometric analysis of peripheral blood leukocytes (PBLs) from a representative mouse that received TCD BCR-ABL–transduced syngeneic (Balb/c) and TCD allogeneic (B6) BM along with 1.5 × 10⁷ allogeneic B6 splenocytes as the initial graft, analyzed day 17 after transplantation (PBL count, 5.6 × 10⁹/L [5600/μL]). Hematopoietic chimerism (top panel) was analyzed with antibodies against H-2^b (x-axis) and H-2^d (y-axis), whereas CML-like leukemia (bottom panel) is indicated by cells coexpressing H-2^d (y-axis) and GFP (x-axis). The mouse was engrafted as a full allogeneic chimera with no signs of CML-like disease. (C) Flow cytometric analysis of PBLs, as in panel B, from a representative mouse that received TCD BCR-ABL–transduced syngeneic (Balb/c) and TCD allogeneic (B6) BM without splenocytes, analyzed day 19 after transplantation. The PBL count was 30 × 10⁹/L (30 000/μL), and the allogeneic chimerism was 35% H-2^b+; note the prominent population of H-2^d+/GFP⁺ cells (bottom panel), indicative of CML-like leukemia. (D) Wright-Giemsa–stained peripheral blood (PB) from a chimeric mouse with CML-like myeloproliferative disease, demonstrating increased circulating maturing myeloid cells (magnification × 400). (E-G) Coexpression of H-2^b and H-2^d in PBLs from mixed chimeras. Flow cytometric analysis of chimerism in a recipient of untransduced TCD BM from 5-FU–treated Balb/c donors and TCD normal B6 BM. Note the significant population of double-positive cells (E) that are not observed when PBLs from Balb/c and B6 mice are either stained for both antigens and mixed prior to analysis (F) or mixed followed by staining (G). In leukemic chimeras, this population included Bcr-Abl–expressing myeloid cells, as assessed by GFP expression and the forward-side scatter distribution.