Study Of Genetic Profile Of AKIR And IKIR After Allo-HSCT And Their Clinical Significance At The MRNA Level

ObjectiveTo establish an experimental methodology for detecting expression of KIR at the mRNA transcription level.To choose patients who had received allogeneic hematopoietic stem cell transplantation(allo-HSCT)and their donors,and collect their blood samples and clinical data on the day of transplantation and at follow-up points.To dynamically detect expression of various activating KIR(aKIR)and inhibitory KIR(iKIR),and explore the impact of KIR expression on the prognosis of transplantation.MethodTo select 260 patients and their donors from August 2011 to May 2019,of which 252 pairs of recipient and donor were employed to study the dynamic detection of mRNA transcription level of KIR genes.Experimental and clinical data of 236 patients diagnosed as myeloid or lymphoblastic hematologic malignancies were employed to investigate the correlation between various KIR genotypes or expression and the prognosis of transplantation.The follow-up time was due on August 31,2019.PCR-SSO method was employed in the research of KIR genotyping and KIR/HLA interaction of donors and recipients.According to our original fluorescent quantitative PCR method,dynamic detection based on peripheral blood mononuclear cell at different time points was utilized in finding out the expression of aKIR(including 2DS1,2DS2,2DS3,2DS4,2DS5,3DS1 genes)and iKIR(including 2DL1,2DL2,3DL1 genes).Patients with grade Ⅱ-Ⅳ acute GvHD and moderate to severe chronic GvHD after transplantation were classified as the GvHD group,those without GvHD manifestations or with mild GvHD(including 0-Ⅰ grade acute GvHD and/or mild chronic GvHD)were classified as the tolerance groups.those whose peripheral blood or bone marrow was found to contain blast cells(proportion>5%)or extramedullary infiltration with leukemia cells was defined as the relapse group.Statistical analysis was exerted on the basis of the above groups.Result1.Among 236 patients with hematological malignancies,156 cases were diagnosed as myeloid malignancies,and 80 cases were lymphoid malignancies.One hundred and fourteen patients received haplo-identical HSCT and 122 patients were transplanted with HLA-identical unrelated or related donors.There were 42 female donor to male recipient transplants and 29,55,110 female to female,male to female and male to male combinations,respectively.One hundred and nineteen pairs of donor and recipient were ABO blood group compatibility and 117 pairs were incompatibility.To avoid the choice of female donor to male recipient transplant was a protective factor against relapse(HR:0.3,95%CI,0.2-0.8,P=0.01).Female donor to male recipient transplant had a significantly higher risk of cGvHD than other gender combinations(HR:3.4,95%CI,1.8-6.5,P<0.001).ABO blood group compatibility was a protective factor against relapse(HR:0.5,95%CI,0.2-0.9,P=0.04).CMV infection was dangerous factor for aGvHD(HR:2.5,95%CI,1.3-4.6,P=0.004).The incidence of aGvHD in patients receiving haploid transplantation(64/114,56.1%)was higher than that in patients receiving unrelated or related donor transplantation(63/122),51.6%),but the difference was not significant.Different transplantation types,diseases,gender compositions,ABO blood group compatibility,and CMV infection had no significant impacts on OS and LFS of patients.2.Regularity of KIR expression at the mRNA transcription level post-HSCT(1)The KIR genotyping method was used to confirm that the genetic profile of the recipient was transformed into the donor’s type.Specific donor-derived KIR was defined as KIR gene that was carried by the donor but not expressed in the recipient before transplantation.In the group of specific or non-specific donor-derived KIR,the mRNA expression of 2DS1,2DS3,2DS4,and 3DS1 all reached and exceeded the donor level in the 1M post-HSCT.2DS4 and 3DS1 reached the highest expression levels in the 3M,2DS3 and 2DS1 reached their peak values in the 1M and 2M,respectively.Then they gradually decreased and within 1 year after transplantation hovered around the level of the day when the donor was transplanted,attaining immune homeostasis.There was no statistical difference in the KIR expression at the same time point between the two groups.(2)The regularity of KIR gene expression showed that the transcription levels of iKIRs,e.g.,2DL1,2DL2,and 3DL1 gradually increased after transplantation,and in the 3M reached the highest value.The levels of 3DL1 in the 2M and 2DL1 in the 3M were obviously higher than that of the donor(P<0.001).In aKIR,2DS3 and 2DS5 were in the 1M reached the peak value,2DS1 was in the 2M,while 2DS2,2DS4,and 3DS1 were in the 3M,respectively.The expression levels of 2DS2 and 2DS3 in the 2M were significantly higher than that of donors(P<0.05).The mRNA transcription level of either iKIR or aKIR gradually decreased after reaching its highest value,and reached the donor’s level to keep a stable state in the first year post-HSCT.3.Influences of KIR genotype and KIR/HLA interaction on hematopoietic reconstruction after HSCT(1)When the donor’s genotype was KIR-Bx,the expression levels of 2DL1 and 3DL1 in patients failing in engraftment were obvious lower than those succeeding in engraftment(P<0.05),but the differences had no statistical significance in the donor with KIR-A A genotype.(2)The mean time for engraftment of platelets for patients with HLA-Bw4/Bw4 homozygosity was 16.0 days,for Bw4/Bw6 heterozygosity was 19.5 days,and for Bw6/Bw6 homozygosity was 20.3 days.The disparity in three groups was significant(P=0.02).4.Comparison of KIR transcription levels in the GvHD group,the tolerance group,and the relapse group(1)The median expression levels of 2DS4,2DL1,and 3DL1 in the GvHD group were lower than those in the tolerance group in either KIR genotype Bx or AA.When the donor was KIR-Bx,the expression of 2DS4 between 1M and 2M was statistically different(P<0.05),the levels of 2DL1 and 3DL1 were not statistically different between the two genotypes.(2)The expression levels of 2DL1/2DS1,2DL2/2DS2 in Bx genotype at each time point in the GvHD group was lower than those in the tolerance group,especially that of 2DS1 in the 2M(P=0.04).3DL1/3DS1 showed different expressional trends from the 1M to 3M in GvHD group.The median transcription level of 3DL1 was lower than that of the tolerance group,whereas the expression of 3DS1 was higher than that of the tolerance group.There was no significant difference between the two groups.In the relapse group,the levels of 2DL1/2DS1 and 3DL1/3DS1 were higher than those in the tolerance group.The expression levels of 2DL1,2DS1,and 3DL1 in the relapse group were higher than those in the GvHD group,especially for 2DS1 in the 2M(P=0.03),whereas there was no statistical difference in the expression of other KIR genes among the three groups.(3)The median transcription levels of aKIR,e.g.,2DS3,2DS4,and 2DS5,in the GvHD group were reduced compared to the tolerance group.The levels of 2DS4 in the 1M and 2DS5 in the 2M in the GvHD group were significantly lower than those of the tolerance group(P=0.04),and the expression of 2DS3 showed no significant difference between the two groups.(4)When the donors were KIR-AA and KIR-Bx genotypes,the 2-year LFS of the patients was 56.6%and 71.0%,and the 2-year OS was 60.5%and 71.7%,respectively.There was no statistical difference in KM survival analysis between the two groups(P=0.08,P=0.06).The 2-year OS of 3DS1 in high expression group and low expression group were 92.5%and 38.6%,respectively,and the difference was statistically significant(P<0.001).ConclusionOur original RT-qPCR method can be used to detect the mRNA transcription levels of various aKIR and iKIR after our rigorous evaluation.The KIR genotype of patients was transformed into the one of donors after transplantation.The transcription levels of iKIR and aKIR all reached and exceeded the donor level in the 1M,and aKIRs showed a higher expressional tendency than iKIRs in the early stage after transplantation.Compared with the tolerance group,the KIR expression was lower in the GvHD group and higher in the relapse group.The changes of mRNA transcription levels of 2DS1,2DS4,and 3DS1 going from low to high and remaining high expression had significant impacts on clinical application for the prediction of the occurrence of GvHD,relapse,OS,and LFS post-HSCT.Therefore,the study results provided a new indicators for dynamiccally monitoring and evaluating GvHD and relapse.Meanwhile,experimental evidence was produced for studying the effects of different aKIR and iKIR genes on the prognosis of transplantation.