Study On Relationship Between KIR2DS4 And Outcome Of Patients After HLA-identical Sibling Hematopoietic Stem Cell Transplantation

PART I The Relationship between KIR2DS4/KIR1 D genotype and Outcome of Patients after HLA-identical Sibling Hematopoietic Stem Cell Transplantation【Objective】To investigate the relationship between KIR2DS4 genotype and outcome of patients after HLA-identical sibling hematopoietic stem cell transplantation(sib-HSCT).【Methods】Using the sequence specific primers(PCR-SSP), KIR genotyping of 267 Chinese Han donor-patient pairs were analyzed, and their KIR2DS4 allele’s frequencies and genotype were calculated.【Results】There were no statistical differences in KIR2DS4 alleles frequencies and phenotype between patients and donors(P>0.05).Of all the 165 patients, one patient died before hematopoietic reconstruction. There were no differences in neutrophil engraftment and platelet recovery time among the three groups.KIR2DS4 and its variant KIR1 D were associated with grade III-IV a GVHD(P=0.0159), while not associated with II-IV a GVHD(P>0.05). 1D+1D+ allele was an independent high risk factor for III-IV a GVHD(HR=4.221, 95%CI 1.470-12.124, P=0.0070). There were no statistical differences in the cumulative incidences of c GVHD among the three groups(P>0.05).The incidence of CMV infection was lower in 2DS4+1D+ group(20.16%) than in 2DS4+2DS4+ group(46.96%), and 1D+1D+ group(63.54%)(P<0.05), indicating that 2DS4+1D+ allele was an independent protective factor for CMV reactivation(HR=0.268, 95%CI 0.125-0.574, P=0.0010).The cumulative relapsed rate at three years were 21.41%, 21.41% and 35.06% in the 2DS4+2DS4+ group, 2DS4+1D+ group and 1D+1D+ group, respectively(P>0.05). Simultaneously, there were no statistical differences in the cumulative TRM(P>0.05).The OS rates of three groups at three years showed no statistical significance, while the OS rate at one year was higher in 2DS4+2DS4+ group than in other two groups(P<0.05). The DFS at three years were 58.45%, 48.27% and 31.25% in 2DS4+2DS4+, 2DS4+1D+ and 1D+1D+ groups, respectively, and there were no statistical differences, while in advanced cases, the DFS at three years were lower in 1D+1D+ group than in the other two groups( P=0.0314).【Conclusion】The distribution of KIR2DS4 allele’s frequencies in the Chinese Han population is different with other populations. KIR2DS4 and its variant KIR1 D are associated with outcomes for patients whose donors belong to the KIR gene haplotype AA. The donors from the sibling with 1D+/1D+ KIR are not suitable for patients with an advanced stage of diseases.PART Ⅱ The Relationship between KIR2DS4 m RNA Expression and Outcome of Patients after Allogeneic Hematopoietic Stem Cell Transplantation【Objectives】To explore the relationship between KIR2DS4 m RNA expression at different time points and outcomes of patients after allo-HSCT.【Methods】Using Q-PCR, KIR2DS4 m RNA expression at different time points of 60 patients was detected, and its association with outcomes of patients after allo-HSCT was analyzed.【Results】In immune tolerance group after sib-HSCT, patients after HSCT on reconstitution and day 30 were significantly lower than that of healthy control(P<0.05), then slowly raised to the level of healthy people after 3M of sib-HSCT.Among the three groups, KIR2DS4 m RNA expression of KIR2DS4 group was the lowest, 2DS4+2DS4+ group showed the highest level, which was statistical significant in each group.In the a GVHD patients, the KIR2DS4 m RNA expression was higher than that of non-a GVHD cases(P=0.011). While there were no significant differences between CMV infection and CMV negative patients. In patients relapsed after sib-HSCT, the KIR2DS4 m RNA expression level was similar to the immune tolerant patients after sib-HSCT.【Conclusions】The m RNA expression levels of KIR2DS4 are different in three KIR2DS4 genotypes, and are associated with outcomes for patients whose donors belong to the KIR gene haplotype AA in transplantations involving HLA-identical siblings.PART Ⅲ The membrane KIR2DS4 expression of Patients after Allogeneic Hematopoietic Stem Cell Transplantation【Objectives】To explore whether the membrane expression level of 2DS4 influence the function of NK cells and outcomes of HLA-identical sibling allo-HSCT.【Methods】A total of 12 patients were performed the function of NK cells and membrane expression level of KIR2DS4 at different time points after HSCT using flow cytometry, and then we studied the correlation between membrane expression level of KIR2DS4 and patients’ outcomes post Hematopoietic Stem Cell Transplantation.【Results】The membrane expression level of KIR2DS4 in 2DS4+2DS4+ group was significantly higher than that in 2DS4+1D+ group(P=0.021).The level of IFN-r on NK cells in 2DS4+2DS4+ group was significantly higher than that in 2DS4+1D+ group(P=0.0060). There was no statistical difference in the levels of IFN-r on NK cells between 2DS4+2DS4+ group and 1D+1D+ group(P<0.05).There was no statistical difference in the levels of Perforin and CD107 a on NK cells among the three groups(P>0.05).In patients of a GVHD, the membrane level of KIR2DS4 on day-3~0 prior to GVHD was significantly higher than that in immune tolerance group(P<0.0210). After treatment, the membrane level of KIR2DS4 was significantly lower than that in immune tolerance group(P=0.0200).When a GVHD was under control, the membrane level of KIR2DS4 began to rise, with no statistical difference than healthy controls(P>0.05)In patients of a GVHD, the level of IFN-r on NK cells on day-10~-7 prior to GVHD was significantly higher than that in immune tolerance group(P<0.0430).After treatment, the level of IFN-r on NK cells showed no statistical difference than that in immune tolerance group(P>0.05). When a GVHD was under control, the level of IFN-r on NK cells began to rise, with no statistical difference than that in immune tolerance group(P>0.05).In patients of a GVHD, the level of Perforin on NK cells on day-10~-7 prior to GVHD showed no statistical difference than that in immune tolerance group(P>0.05).After treatment, the level of Perforin on NK cells began to decline, significantly lower than that in immune tolerance group(P=0.0210).When a GVHD was under control, the level of Perforin on NK cells began to rise, with no statistical difference than that in immune tolerance group(P>0.05).In patients suffering a GVHD, the level of CD107 a on NK cells on day-10~-7 prior to GVHD was significantly higher than that in immune tolerance group(P=0.0210). After treatment, the level of CD107 a on NK cells began to decline, significantly lower than that in immune tolerance group(P=0.0210).When a GVHD was under control, the level of CD107 a on NK cells began to rise, with no statistical difference than that in immune tolerance group(P>0.05).In patients suffering relapse, the membrane levels of KIR2DS4 on day-10 prior to relapse, on the day diagnosed with molecular relapse and on the day diagnosed with extramedullary relapse, respectively, were higher than those in immune tolerance group(P>0.05).In patients suffering a GVHD, the levels of IFN-r, Perforin and CD107 a on NK cells on day-10 prior to relapse, on the day diagnosed with molecular relapse and on the day diagnosed with extramedullary relapse, respectively, were higher than those in immune tolerance group(P>0.05).【Conclusions】The membrane expression levels of KIR2DS4 may affect function of NK cells, and are associated with outcomes for patients whose donors belong to the KIR gene haplotype AA in transplantations involving HLA-identical siblings.