High Doses Of Mhc-mismatched Donor Cell Infusion Induce Donor Cell Engraftment And Avoid Gvhd Animal Studies

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy for a wide variety of diseases, including hematological malignancies, metabolic diseases, immune deficiencies, autoimmune diseases and solid tumors. It has long been considered that high-strength pretreatment and the depletion of bone marrow is the prerequisite for donor cells to engraft and induce graft versus leukemia. But pretreatment related toxicity graft-versus-host disease (GVHD), graft rejection and infections after transplantation seriously restrain the curative effect of allo-HSCT. Toxicity-reduced conditioning regimens such as nonmyeloablative conditioning cause much less transplantation related complications than traditional myeloablated regimens, but radiation and chemotheraphy included in pretreatment, application of immunity inhibitor after transplantation, serious infections of bacteria, fungus and virus caused by immunosuppression are still big treats to life and health of the patients. So reductions of transplantation related complications and fatality rate are clinic problems urgently needed to be solved since they are the key factors of curative effect.This study was aimed to achieve stable implantation of donor cells and prevent GVHD by means of minimum pretreatment and adjusting the quantity of donor cells, which was based on the research of myeloablated, nonmyeloablative and MHC haploid matched transplantation. This study was trying to research the engraftment of allogenic donor cells and immune tolerance from another perspective, problems and mechanism related to GVHD were also included. Feasibility and safety of allo-HSCT without pretreatment were discussed to provide experiment evidence for clinic application of allo-HSCT.Flow cytometry(FCM)was used to detect macrochimerism and Real-time quantitative polymerase chain reaction(Real-time PCR)for microchimerism of donor cells, their efficiency were evaluated. Then the principle of how different levels of TBI (8GY~1GY) and different donor cells of transfusion(1×10~7～9×10~7)affect murine engraftment and GVHD was studied in the H-2 haploid matched transplantation. Donor's engraftment situation and GVHD of mice inoculated with different quantity of H-2 haploid matched donor cells had been analyzed to verify feasibility and safety of H-2 haploid matched transplantation without pretreatment. And distribution of donor cells, T-lymphocyte subpopulation of peripheral blood cells and cytokine were studied to figure out possible mechanism of stable engraftment induced by infusion of H-2 haploid matched donor cells without pretreatment.The phenotype of recipients H-2 was detected by FCM and expression of SRY gene of recipients was detected by Real-time PCR to determine the rate of donor chimerism. Primer and probe were designed on base ofβ-actin gene and sry gene and amplified by PCR. Then Electrophoresis product was recycled to build plasmid and standard curve was drawn after correct sequencing. Real time PCR was used for amplification and the method was optimized. At last DNA samples of male mice were used as positive reference and female for negative reference to verify sensitivity, specificity and repeatability of Real-time PCR. H-2 haploid t matched transplantation with TBI 6GY and 2GYwere selected as models and Efficiency of FCM together with Real-time PCR was verified by DC detection of 2 groups of mice after transplantation.H-2 haploid matched transplantation of HSCT was investigated when parental generation and filial generation mice were used as donors and recipients respectively; total body irradiation(TBI) was used as a conditioning regimen. Mice were divided into 5 groups according to different intensity of TBI(8GY,6GY,4GY,2GY,1GY) and infused with 1×10~7 mobilized donor spleno mononuclear cells. Mice of control group were infused with equal quantity of normal sodium. The difference of engraftment, incidence of GVHD and survival in different TBI group were analyzed. Experimental mice were divided into 4 groups according to different quantity of donor cells (1×10~7,3×10~7,6×10~7,9×10~7) and same TBI intensity, and then difference of engraftment, incidence of GVHD and survival were analyzed. Another experiment was to divide mice into six groups according to different quantity of donor cell (1×10~7,3×10~7,6×10~7,9×10~7,12×10~7,15×10~7) after infusion of H-2 haploid matched donor cells without conditioning, then difference of engraftment , incidence of GVHD and survival in different group were analyzed. T lymphocyte subpopulation, cytokine, distribution of tissue and DC of cell line were detected. The method of detection for donor chimersm was successfully created. Flow cytometry (FCM) performed well in the detection of macrochimerism in 6GY group (DC>90%, 2 weeks after transplantation), while Real-time PCR had poor accuracy. Real-time PCR is good at detecting microchimerisms in 2GY group (DC<1%, I month after transplantation), while FCM was not sensitive. Combination of the two methods can afford sensitive and specific tool of quantitative chimerism analysis for different chimerism after transplantation.In the same transplantation condition(donor cells:1×10~7), complete chimerism (CC) and GVHD sign were detected in the mice of group of 8GY at 2 weeks after transplantation . all mice in 8GY group died at 18d～25d after transplantation, while 6GY group also got CC 6 weeks later and survived for 3 months. Only 3/8 of the mice had slight GVHD. 4GY group only got MC and 1/8 of the mice had slight GVHD, and the whole group survived for a long time. No GVHD was detected in the 2GY and 1GY group.In the condition of TBI-6GY it took 3 weeks for mice group infused with 3×10~7 donor cells to transfer from MC to CC. 6×10~7 group needed 2.5 weeks and 9×10~7 group needed 2 weeks. All group got GVHD and range of fatality rate was 75%～100%. In the condition of TBI-4GY, 3 groups got CC and the fatality rate were 25%, 50%, 100% respectively. In the condition of TBI-2GY/1GY, 3×10~7 group got MC and transcient donor chimerism, no GVHD had been detected. 6×10~7 group and 9×10~7 group got CC and GVHD sign in the condition of 2GY. The incidence rates of GVHD were 62.5% and 100% respectively and fatality rate were 25% and 50%.In the condition of 1GY 6×10~7 group and 9×10~7 group got CC and slight GVHD, and the incidence rate of GVHD were 25% and 62.5% respectively, all mice survived.The transient donor microchimerism were detected in the mice after infusion of 1×10~7~6×10~7H-2 haploid matched donor cells without pretreatment, median time of existent of donor cells in the recipients were 5 weeks, 7 weeks, and 9 weeks respectively. No GVHD had been detected in these groups. Stable complete chimerism were detected in 5/8 mice of 9×10~7group and all mice in the group 12×10~7and 15×10~7. No GVHD were detected in the mice of group 1×10~7~12×10~7 except 25% mice of group 15×10~7. But the mice of mild GVHD recovered gradually and survived for 6 months without therapy.Haematogenesis and T lymphocyte were suppressed transiently and recovered to normal in the mice after infusion of H-2 haploid matched donor cells without conditioning. Serum cytokine of TH1 and inflammation TNF-αdecreased while serum cytokine of TH2 increased comparing to control group. Mixed lymphocyte reaction(MLR) essay indicated the responsibility of recipient to donor cells was lower in the 6×10~7and 12×10~7 group than that of normal mice, while the responsibility of recipient to the third independent cells was same. DC detected in the subgroup and tissue indicated that 12×10~7 group got multi-chimerism. The engraftment of donor cells and distribution of immune organ was similar to the mice of conditioning group.In a word, this research successfully established a quantitative method for detection of murine donor chimerism rate with FCM and Real-time PCR. And it demonstrated that TBI intensity and quantity of donor cells were both key factor for engraftment and GVHD. the mice model of H-2 haploid t matched transplantation without conditioning was also successfully established, the proper quantity of donor cells for successful engraftment and none GVHD without pretreatment had been confirmed. The regimen free of conditioning can possible induce engraftment and achieve immune tolerance by forming mixed chimerism(MC) in the tissue, adjusting of balance of TH1/TH2, increasing regulated T lymphocyte. This model and related mechanism research could provide foundation of animal experiment and theory evidence, revealing new strategy for the immune tolerance of transplantation.