Effect Of HLA Haplotype And Anti-HLA Antibody In Allogeneic Hematopoietic Stem Cell Transplantation

Part I:The impact of HLA haplotype and alleles mismatches of donor-recipient pairs on outcome of haplo-identical hematopoietic stem cell transplantation with a third part cord blood unitObjectives:Analyzed allele mismatches of HLA-A,-B,-C,-DRB1,-DQB1 and haplotype mismatches of donor-recipient pairs on the outcome of haploidentical transplantation combined with a third part cord blood unit.Methods: 230 pairs of donor-recipient were performed HLA-A,B,C,DRB1,DQB1 typing using SBT and SSOP methods from January 2012 to December 2014.Results: HLA-5/10、6/10、7/10 and≥8/10 matched group divided according to HLA-A,B,C,DRB1,DQB1 high-resolution typing, the 3-year probability of overall survival(OS) for each group were 48.7%、59.3%、71.1%、38.3%(P=0.068). HLA>6/10 matched group associated with significant favourable effect on OS compared with HLA-5/10 matched(P=0.041).When the HLA class I antigen matched on the recipient and donor, it is improved the OS and event free survival(EFS) in HLA-6/10 matched than HLA-5/10 matched group(P=0.017, P=0.088), especially in single HLA-A loci allele matched group(P=0.013, P=0.013). As to the third part cord blood unit, share the same haplotype with the recipient-donor pairs turned out better platelet recovery than the misfit one(95.3% vs 86.2%,P=0.007), similar result was found in neutrophil recovery(98.8% vs 96.1%,P=0.022).Conclusions: HLA Allele mismatch and haplotype mismatch of the donor and recipient should be useful for selection of the most optimum donor. Co-infused of an unrelated cord blood unit shared the same haplotype with the recipient-donor pairs could improve hematopoietic recovery. Part II:The mechanism of endothelial cell injury caused by the donor-specific anti-HLA antibodies under different MFI valuesObjectives: To further discuss the mechanism of donor specific anti-HLA antibodies activated the PI3K/AKT signal pathway under different MFI values on HUVEC, to clarify the DSA under different MFI is the main cause of cell proliferation, which induce the cell damage under certain transplantation.Furthermore, to provide experimental evidence that DSA plays an important influence on the prognosis of allogeneic hematopoietic stem cell transplantation.Methods:Two different HLA typed HUVECs were apply to the the experiment.Different concentrations of DSA were detected by Luminex method in order to confirm corresponding MFI values. An isotype Ig G was used as a control. Negative, positive, intermediately positive, strongly positive and super-strongly positive were in the treatment group.The protein prepared from HUVEC treated with different MFI values,then subjected to Western Blotting to measure the phosphorylation of S6 RP in PI3K/AKT signal pathway.Meanwhile, we utilize a flow cytometric assayexploiting the dilution of fluorescent stained by carboxy-fluorescein succinimidy ester(CFSE) during cell division to measure cell proliferation index.Results:When the concentration of anti-class A2(human) antibodies were 0.00375 ug/m,0.015 ug/ml,0.03 ug/ml,0.06 ug/ml,0.25 ug/ml, respectively, relevant MFI value were negative,positive, intermediately positive, strongly positive and super-strongly positive.As for anti-class Cw1 antibodies,same groups were separately divided when the concentration was 0.09 ug/ml,0.18 ug/ml,0.375 ug/ml,0.75 ug/ml,1.5 ug/ml. Treatment of HUVEC with above group of anti-class A2(human)antibody respectively for 15 min stimulated phosphorylation of S6 RP at Ser235/236,followed the phosphorylation percentage of each group were 78.2%, 83.6%, 90.6%, 93.0%, 100%, 94.3%.Moreover,such rising trend could also be observed in anti-class Cw1 antibodies,of which,the phosphorylation percentage of each set were 67.4%, 75.9%, 89.3%, 89.7%, 93.6%, 100%. After CFSE stained endothelial cell co-cultured with different values of MFI of anti-class A2 antibodies or anti-class Cw1 antibodies.Flow cytometry instrument was applied to detect fluorescence intensity decline.The proliferation ratio of negative, positive, intermediately positive, strongly positive and super-strongly positive treated with anti-class A2 antibodies and anti-class Cw1 antibodies respectively were 38.9%,58.4%,29.6%,60.1%,66.9% and 20%, 10%, 15%, 35%, 90%.Conclusions:On the basis of prophase studies in this laboratory, we in-depth researched the difference between anti-class A2 and Cw1 antibodies under different intensity of MFI to activated the PI3K/AKT signal pathway. There was an obvious increase of S6 RP Ser235/236 and cell proliferation when the MFI strength increased.