The fundamental basis for immunotherapy of leukemia is that leukemia cells express specific antigens that are not expressed by normal hematopoietic cells. However, the host immune system appears to be tolerant of leukemia cells. To overcome this immune tolerance, we transduced WEHI-3B mouse monocyte leukemia cells (1) with a transmembrane form of GM-CSF (tmGM-CSF). The tmGM-CSF was constructed using the pDisplay vector for cell-surface targeting (Invitrogen) into the pLOX lentivirus gene transfer vector (2). After infection of WEHI-3B cells with a recombinant lentivirus encoding tmGM-CSF, nearly all the transduced cells expressed tmGM-CSF on the cell-surface, as determined by flow cytometry analysis using anti-GM-CSF. To determine whether vaccination with tmGM-CSF expressing WEHI-3B cells would prevent leukemia formation, immunocompetent BALB/c mice were immunized with lethally-irradiated WEHI-3B cells (106, 3 times 7 day intervals), which express tmGM-CSF, prior to challenging vaccinated mice with WEHI-3B cells (5x104) that express GFP as a marker. 100% of vaccinated mice were protected from leukemia. Non-vaccinated mice succumbed to leukemia within 50–55 days. Vaccination of mice with lethally-irradiated WEHI-3B cells expressing CD40L protected 80% of the mice from leukemia. In contrast, mice immunized with lethally-irradiated WEHI-3B/GFP cells lacking tmGM-CSF were not protected. Mice vaccinated three times at 5,12, 19 days after challenge with WEHI-3B/GFP cells had a significant increase in survival in that 60% of mice were alive and healthy at 16 days (to this date) after all control non-vaccinated mice had died. Similar vaccine studies were performed with BCR-ABL (b3a2)+ 32D cells (106) in immunocompetent C3H/HeJ mice (3). These mice die of leukemia within 35 days. After infection of BCR-ABL+ 32D cells with the lentivirus encoding tmGM-CSF/GFP, tmGM-CSF was expressed on the cell-surface. The C3H/HeJ mice challenged with BCR-ABL+32D/GFP cells (106) showed a significant level of protection by vaccination with lethally-irradiated tmGM-CSF+ 32D BCR-ABL cells (106, 2 times at 7 day intervals); 40% of the vaccinated mice remained healthy; all non-vaccinated mice died of leukemia. There was a significant difference in survival (P=0.03) between the vaccinated and non-vaccinated groups. Interestingly, the spleens of vaccinated C3H/HeJ mice that died of leukemia at the same time as non-vaccinated mice approached normal size whereas non-vaccinated mice had enlarged spleens. Our findings suggest that over-expression of cell-surface tmGM-CSF in leukemia cells can overcome immune tolerance, allowing the immune system to efficiently recognize and destroy the leukemia cells, providing extended survival of vaccinated mice. Because significant protection from death was achieved by vaccination after challenge with leukemia cells, tmGM-CSF expression in leukemia cells has potential as a therapeutic strategy for treatment of leukemia.