Clinical And Experimental Study Of HLA Haploidentical Family Donor Allogeneic Peripheral Blood Stem Cell Transplantation For The Treatment Of Hematologic Malignancies

Allogeneic hematologic stem cell transplantation(allo-HSCT)is the most effective treatment for hamatologic malignancies,several studies suggest that allogeneic peripheral blood stem cell transplantation(allo-PBSCT)has the advantages of faster engraftment, fewer infection and rapid immune reconstitution;but it can not be applied to every patient because of the difficulty to fred a HLA identical donor and the complications including acute graft versus host disease(aGVHD),chronic GVHD(cGVHD),rejection and relapse after transplantation.HLA haploidentical family donor transplantation is a good way to enlarge the source of donor,but we should establish the immune tolerance for increasing the long-term leukemia-free survival rate and life quality of the patients.With the development in clinical and basic immunology in recent years,the research in induction of immune tolerance is a key point to reduce the complications,although many inducing methods are still practiced in animal and clinical experiment.This study aimed to find suitable and effective ways to induce the immune tolerance to patients with hamatologic malignancies,especially those who had HLA hapioidentical family donors and explore the feasibility through the clinical effect,chimarism,cytokine and immune reconstitution.PART 1 Clinic analyse of HLA haploidentical family donor allogeneic peripheral blood stem cell transplantation for the treatment of hematologic malignanciesObjective:To explore the effect of HLA haploidentical family donor allo-PBSCT without T cell depletion(TCD)for the treatment of hematological malignancies.Methods: 20 patients were treated by haploidentical family donor allo-PBSCT(haploidentical group) without TCD,the clinical outcomes were compared with those of 25 consecutive HLA identical sibling transplantation(identical group).The donors were given granulocyte-colony stimulating factors(G-CSF)mobilization.Mononuclear cell (MNC)and CD34~+ cell were detected in peripheral blood and peripheral blood stem cell(PBSC)graft before and after the mobilization.A combination of cyclosporine(CsA) and methotrexate(MTX)was the standard regimen to prevent GVHD in identicai group. Myclophenolate mofetil(MMF),Anti-thymocyte globulin(ATG)and anti-CD25 monoclonal antibody were added to the host in haploidentical group according to the HLA disparity.Results:45 patients were engrafted,the median number of MNC in the graft were 14.62(8.59-31.78)×10~8/kg in haploidentical group and 14.45(7.62-19.9)×10~6/kg in identical group,the median number of CD34~+ cell in the graft were 9.13(4.57-41.78)×10~6/kg in haploidentical group and 6.58(2.31-18.89)×10~6/kg in identical group respectively.The median time of hematopoietic reconstitution was 13(10～20)d and 14(8～17)d in haploidentical group and identical group respectively,the median follow-up duration was 21(2～64)months in haploidentical group and 23(6～14)months in identical group.17 cases(85%)in haploidentical group are disease-free now,20 cases (80%)in identical group are disease-free now.The two year probability,of disease-free survival is 76.4%in haploidentical group and 77.2%in identical group respectively(P＞0.05).The incidence of aGVHD and cGVHD(35%,45%)in haploidentical group did not differ with that(8%,40%)in identical group(P＞0.05).There was no correlation between the number of MNC and CD34~+ cell and the incidence of aGVHD and cGVHD(P＞0.05). Conclusions:1)The incidence and severity of GVHD can be reduced by the different combination of intensive immunosuppression for the HLA haploidentical family donor transplantation.2)The major histoincompatibility barrier in the haploidentical family donor transplantation might be crossed by donor stimulated with G-CSF and combined GVHD prophylaxis program.PART 2 Application of sequential monitoring of chimerism in HLA haploidentical family donor allogeneic peripheral blood stem cell transplantationObjective:To establish multiple short tandem repeat(STR)amplification by fluorescence labeling polymerase chain reaction(PCR)combined with capillary electrophoresis for quantitative determination of chimerism,and to evaluate the relation between the type of chimerism and the outcome of allo-PBSCT.Methods:Thirty-five patients were analyzed,including twenty with HLA identical sibling donors and fifteen with HLA haploidentical family donors,ABI PRISM 3130XL was used to genotype 15 microsatellite loci for D8S1179,D21S11,D7S820,D3S1358,CSFIPO,THO1,D13S317,D16S539,D2S1338,D19S433,VWA,TPOX,D18S51,D5S818,FGA and sexual locus Amelogenin.The results of the donor and recipient in pre-transplantation and post-transplantation were analyzed to determine the type of chimerism.The feasibility and accuracy of this assay were tested in-vitro by using serial DNA mixtures from unrelated individuals.Resultes:Discrimination between donor and recipient was possible in all patients except for 3 patients whose pre-transplantation samples were not available, complete donor chimerism(CC)were detected in all patients at 1 month post-transplantation,STR results were the same to the transformation of red blood group,sexual chromosome and HLA phenotype,PAGE and silver staining.The result showed a clear correlation between the percentage of donor or recipient DNA and the proportion of mixed sample,with the limit of detection for a minor DNA percemage being 5%. Conclusions:1)this approach allows a rapid,sensitive,automated and non-isotopic STR-PCR testing,and provide a reliable alternative for assessment of the status of chimerism after allo-PBSCT;2)The measurement taken for the induction of immune tolerance in our study could induce CC successfully.PART 3 Th1/Th2 type cytokine expression in peripheral blood lymphocyte of patients underwent HLA haploidentical family donor allogeneic peripheral blood stem cell transplantation and their clinical significances.Objective:To explore the expression of Th1 type cytokine(IFN-γ)and Th2 type cytokine(IL-4)and their clinical significances in peripheral blood lymphocyte of patients underwent haplidentical family donor allo-PBSCT.Method:Twenty-eight patients were included in this study,including 13 with HLA haploidentical family donors (haploidentical group)and 15 with HLA identical sibling donors(identical group).Flow cytometry was used to kinetically analyze the expression of IFN-γand IL-4.Result:The kinetic study in haploidentical group showed that the expression of IFN-γdecreased gradually post transplantation,and had been statistically lower than that before transplantation since 6 month post transplantation(P＜0.05),the expression of IL-4 was statistically lower at 1,3,6 month(P＜0.05)but higher at 12,18 month post transplantation(P＜0.05)than that before transplantation;the kinetic study in identical group showed that the expression of IFN-γdecreased in the same way with that in haploidentical group(P＜0.05),but the expression of IL-4 increased gradually post transplantation(P＜0.05).Both of the expressions of IFN-γand IL-4 showed no difference between the patients in haploidentical group and identical group(P＞0.05).All patients achieved engraftment,5 patients developed aGVHD,8 patients developed cGVHD,the expression of IFN-γin patients with aGVHD was significantly higher than that in patients without aGVHD(P＜0.05).IL-4 expression showed no difference between the two groups(P＞0.05).Both of the expressions of IFN-γand IL-4 showed no difference between the patients with and without cGVHD(P＞0.05).Conclusion:1) IFN-γplay an important role in the development of human aGVHD,It can interfere the development of immune tolerance;the kinetic measurement of IFN-γafter allo-PBSCT could provide predictive marker for aGVHD.2)The combined GVHD prophylaxis program did not affect the expression of IFN-γand IL-4 in haploidentical family donor allo-PBSCT.PART 4 The kinetic study on the immune reconstitution after HLA haploidentical family donor allogeneic peripheral blood stem cell transplantation.Objective:to study the recovery of the peripheral lymphocyte subsets in 15 patients underwent haploidentical family donor allo-PBSCT(haploidentical group)and guide the prevention and treatment of infection.Methods:Indirect immunofluorescence assay was used to detect the lymphocyte subsets,such as T cell subsets(CD3,CD4,CD8),B cell (CD19)and natural killer(NK)cell(CD56)at 1,3,6,12,18 month post transplantation. The outcomes were compared with those of 25 consecutive HLA identical sibling transplantation(identical group),lymphocyte subsets of 32 samples from healthy blood donors were tested as normal control values.Results:1)CD3~+,CD4~+ and CD4~+/CD8~+ cells were significantly decreased than normol control at 1 month post transplantation,the recovery of CD3~+ cells was within 3～12 months post transplantation.The CD4~+ T cells was still statistically lower than normol control at 3 month post transplantation and recovered to normol during 6～12 months post transplantation;CD8~+ T cells recovered quickly,it had been significantly higher than normal control since 3 months post transplantation and recovered to normal at 12 month post transplantation;CD4~+/CD8~+ ratio decreased gradually post transplantation and was significantly lower than that of normal control at 1,3,6 months post transplantation,it reversed at 3 and 6 month and recovered to normal at 12 months post transplantation;CD 19~+ T cells did not decrease and was significantly higher than normal control at 1,18 months post transplantation;CD56~+ T cells did not decrease and was significantly higher than normal control at 3 months post transplantation;2)The CD3~+,CD8~+ T cells were statistically higher but CD4~+/CD8~+ ratio was statistically lower in haploidentical patients than that in identical patients at 3 months post transplantation,there were no difference between the two groups at 1,6,12,18 months post transplantation;3)There was no difference in all of the lymphocyte subsets between the aGVHD~+ group and aGVHD group;The patients with cGVHD had significantly higher CD4~+ cells than those without cGVHD at 1 month post transplantation,there was no significant difference in all of the lymphocyte subsets between them at 3,6,12,18 month after transplantation.Conclusion:HLA haploidentical family donor allo-PBSCT without TCD has a hastened immune reconstitution,which is not delayed by the combined immunesupressing treatment and the development of aGVHD and cGVHD.