| Objective: To explore the optimum dose of CD3?T lymphocyte to mix with purified CD34+ hematopoietic stem cells for preserving graft versus leukemia (GVL) while preventing graft versus host disease (GVHD) in haplotype hematopoietic stem cell transplantation. Methods: 1. Parental mice from C57BL/6(H-2b) X 61 5(H-2k) to produce F I (H-2kb) hybrids. F 1 (H-2kb) hybrids accepted the H-2 haploidentical graft from C57BL/6(H-2b). 2. Fl(H-2kb) hybrids were inoculated with L61 5 cells to develop a murine model of leukemia. 3. CD34+ hematopoietic stem cells from bone marrow and CD3+ T lymphocyte from spleen of C57BL/6 were purified using MinIMACS. FACS analysis was used to determine the subsets of CD34+ cells and CD3+ cells of the starting component and the final component. After separation, cell counting and viability staining with trypan blue were performed and the number of viable cells was determined . 4. Cohort:(DF1(H-2kb) hybrids bearing L615 cells without therapy.@)F1(H-2kb) hybrids bearing L6 15 cells with TBI.ighly purified CD34+ cells mixed with different doses of highly purified CD3+ cells were injected to F I (H-2kb) hybrids bearing L61 5 cells via tail vein and then the survival timex GVHD and GVL effect were evaluated. 5. Sixty days after transplantation, origin of the bone marrow cells responsible for the hematopoietic reconstitution were observed by detecting y-chromosome-positive cells. 6. The optimum dose is 5 X for every mouse in this model. Results: 1. A murine leukemia model of (C57BL/6X 61 5)F1(1-l-2kb) hybrids was developed successfully. The survival time of F1(H-2kb) hybrids correlate with the number of L615 cells. 2. Highly purified CD34+ cells from bone marrow and CD3?cells from spleen of C57BL/6 can be achieved by using MiniMACS. The mean purity of CD34+ cells and CD3+ cells of the final components was 81.5%(range 76.80?5.3%) and 95.35% (range 89.82-A 00%),the recovery of CD34+ cells and CD3+ cells was 47.54% (range 40.5054.31%) and 61.17% (range 55.41?9.70%) ,and the cells viability had not be affected (P>0.05). 3. The median survival time of Fl hybrids bearing 1 X 106 L6 15 cells without therapy and with TBI only was 7.70?1.Oldays and 14.33 ?1.92days respectively; The median survival time of Fl hybrids with the infusion of highly purified CD34?cells was 29.11 + 0.92days (compared with TBI ,P<0.0l) and all death from leukemia relapse; the median survival time of Fl hybrids with the infusion of CD34+ cells mixed with 1>( i07 CD3+ cells was 44.30?16.S5days (compared infusion CD34+ cells only, P<0.05) , 50% death from leukemia relapse, and 50% acquired long-term survival without GVHD or leukemia developed; the median survival time of Fl hybrids with the infusion of CD34+ cells mixed with 5 X l0~ CD3+ cells was long term survival and didn't develop GVHD or leukemia; the median survival time of Fl hybrids with the infusion of CD34+ cells mixed with 1 X 108 CD3+ cells was 48.88?.82days, 62.5% death from GVHD, 37.5% was long term survival(compared with CD34+ cells mixed with CD3+ cells 5X i07, P<0.0 1); the median survival time of Fl hybrids with the infusion of CD34+ cells mixed with 1.5 X 108 CD3+ cells was 23.75 ?.74days, (compared with CD34+ cells mixed with CD3+ cells I X 108, P(0.0l), 100% death from GVHD. 4. The bone marrow cells were 100% y-chromosome-positive after transplantation 60 days. Conclusion: 1. The murine leukemia model was developed successfully by injecting L615 ce...
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