Transgenic mice expressing the t(15;17) translocation fusion genes PML-RARA (PR) and RARA-PML (RP) under control of human cathepsin G (hCG) regulatory sequences have been generated in our laboratory and intercrossed to study the role of both fusion proteins in development of acute promyelocytic leukemia (PNAS 96, 15103, 1999). We have recently developed a tumor bank by cryopreserving cells in a DMSO-based medium, and utilized banked samples to define the stage of myeloid differentiation, and to establish APL in cohorts of secondary recipients. Transfer of 200,000 freshly-thawed APL cells into (C3H x B1/6)F1 or C3H SCID animals intraperitoneally results in lethal APL in 100% of recipients after 40-100 days; however, at doses of 103 - 104 APL cells, SCID mice are more susceptible to APL than (C3H x B1/6)F1 mice. This could be explained by functional cellular immunity in immunocompetent mice, leading to clearance of small numbers of APL cells. DNA immunization with a plasmid expression vector containing PR cDNA leads to 2030% survival to lethal tumor challenges, compared to vector immunized cohorts. To begin to define the nature of immune responses to APL cells, (C3H x B1/6)F1 animals that survived an initial tumor challenge of 10M04 cells were re-challenged 6 months later with 100,000 cells from the same APL sample. All animals were evaluated sixty days after the second challenge. Six of ten animals which had previously survived APL challenge remained healthy, with normal white cell counts and differentials and an absence of transgene-positive cells in the blood (sensitivity of the PCR-based assay is 1 tumor cell in 100,000 total cells). We are now evaluating 3H-Thymidine (3H-T) incorporation to assess cellular proliferation from spleen cells obtained from DNA immunized mice in response to a dendritic cell monolayer pulsed with tumor cell lysates. We have used both bone marrow-derived dendritic cells (DCs) from syngeneic C3HxB6 Fl animals, or DCs derived from cryopreserved APL tumor populations (tumor DCs), as stimulators. DCs were generated by 6-12 days of culture in recombinant murine (rm)GM-CSF and rmlL-4. Tumor DCs were morphologically indistinguishable from bone marrow-derived DCs, and expressed similar levels of the cell surface antigens MHC Class II, B7.2, and CDllc, but still retained some Gr-1 surface expression. Studies are currently being conducted to determine whether tumor DCs more effectively present tumor antigens to spleen cell populations from immunized animals.
|Issue number||11 PART II|
|State||Published - Jan 1 2000|