The Impact And Mechanism Of KIR Genotype On The Outcome Following Allogeneic Hematopoietic Stem Cell Transplantation

Killer cell immunoglobulin—like receptors(KIRs ) are a family of inhibitory and activating receptors expressed on natural killer(NK) cells and some T lymphocytes. They belong to the Ig superfamily,and are encoded by a compact cluster of genes located on chromosome 19q13.4.To date,at least 15 functional KIR genes and two pseudogenes have been identified.KIR gene family shows a high degree of genetic polymorphism.Significant differences in KIR genotypes exist among different ethnic populations.In general,two kinds of KIR haplotypes are recognized:group A and group B.KIRs interact with HLA I molecules,which transmit inhibitory or activating signal,thereby regulating the function of NK cells.KIRs play an important regulatory role in a variety of processes,including anti-infective immunity,tumor immunity,the clearance of aging cells,autoimmune diseases as well as hematopoietic stem cell transplantation.Allogeneic hematopoietic stem cell transplantation(allo-HSCT) is a successful curative therapy for a variety of hematological malignancies and non-hematologic malignancies.Especially for acute leukemia,more than 50%patients obtain a long-term disease-free survival after allogeneic hematopoietic stem cell transplantation.But allo-HSCT therapy is just a double-edged sword.On one hand,the transplant can effectively establish hematopoietic reconstitution and graft-versus-leukemia(GVL);on the other hand,it can also arouse a series of serious complications and result in patient's death.There are two major reasons for the patient's death:graft-versus-host disease (GVHD) and relapse after transplantation.Therefore,how to reduce GVHD and prevent relapse in allogeneic hematopoietic stem cell transplantation is in urgent need and addressing the key issues involved in it is of great importance.Some animal trials and clinical observation identify alloreactive NK cells caused by KIR-ligand mismatch can promote the implantation of bone marrow and hematopoietic reconstitution by releasing a variety of hematopoietic promoting factors and eliminate recipient antigen presenting cells(APC) which have been demonstrated to initiate T cell-mediated aGVHD.Meanwhile,alloreactive NK cells can kill residual leukemia cells,thereby can make patient avoid post-transplant relapse.These major discoveries indicate KIR-ligand mismatch will fundamentally solve the problem of contradiction between GVHD and GVL in bone marrow transplantation and bring new hope.The above-mentioned phenomenon was first discovered in haploidentical hematopoietic stem cell transplant.It was also observed in HLA-match unrelated donor transplantation and sibling transplantation in the following research.However,other studies have arrived at the opposite conclusion,particularly in unrelated donor HSCT (URD-HSCT),and have shown a deleterious effect of KIR-ligand mismatch.For example,KIR-ligand mismatch was associated with increased transplant- related mortality(TRM).In short,it is a hot area for research worldwide,but there are still many controversies over it.At present,KIR is not well studied in China.In order to investigate the effects of KIR genotype on outcome following hematopoietic stem cell transplantation as well as the mechanism.We conducted this study to address the following questions:a) Whether KIR genotype has the same effect on outcome in sibling or unrelated transplantation? b) What is the mechanism? c) How about the distribution of KIR genes in Eastern Chinese Han and Taiwanese Han populations?Section 1:The Effects of KIR and HLA Genotypes on Clinical Outcome Following Unrelated Donor or Sibling Allogeneic Hematopoietic Stem Cell TransplantationIn section 1,the effects of KIR and HLA genotypes on clinical outcome following unrelated-donor and sibling hematopoietic stem cell transplantation were investigated. Patient and donor DNA were prepared from peripheral blood or bone marrow mononuclear cells prior to transplantation.HLA-C loci genes were analyzed using a PCR-SSP kit.HLA-C and HLA-B alleles were segregated into the epitope groups: HLA-C group 1,HLA-C group 2 and HLA-Bw4.KIR genotype analysis was performed by the PCR-SSP technique,using a KIR genotyping PCR-SSP kit.KIR genotyping was performed according to methods described previously.The inhibitory KIRs identified with HLA ligands are as follows: KIR2DL2/KIR2DL3,which bind group 1 HLA-C molecules(HLA-C1) characterized by serine at position 77 and asparagine at position 80,(Cw*01,03,07,08);KIR2DL1, which recognizes group 2 HLA-C molecules(HLA-C2) characterized by asparagine 77 and lysine 80(Cw*02,04,05,06);and KIR3DL1,which recognizes the HLA-Bw4 alleles.KIR-ligand mismatch was defined as the absence of one or more recipient HLA epitopes for the corresponding donor-inhibitory KIRs.Samples containing either one (A/B heterozygotes) or two(B/B homozygotes) group B haplotypes were assigned the genotype designation B/x.Samples that lacked all KIR B loci were assigned the genotype A/A.The results showed that in the setting of URD-HSCT,KIR-ligand mismatch was significantly associated with a decreased leukemic relapse risk(p=0.019;HR=0.329, 95%CI 0.131-0.830),mainly in myeloid disease(p=0.003;HR=0.193,95%CI 0.066-0.563).In myeloid disease,KIR-ligand mismatch also improved 5-year overall survival(OS)(p=0.031;HR=0.423,95%CI 0.194-0.924) and disease-free survival (DFS)(p=0.024;HR=0.445,95%CI 0.221-0.897).In lymphoid disease,KIR-ligand mismatch had not any beneficial effect on outcome.AA in the donor significantly decreased the cumulative incidence of relapse compared with Bx(12.9%vs.28.6%, P=0.024).We further found that more activating KIR genes in the donor resulted in an increased relapse risk(p=0.005;HR=1.463,95%CI 1.123-1.906).Meanwhile,the presence of donor activating KIR2DS3 was an independent factor associated with increased relapse risk(p=0.003;HR=5.045,95%CI 1.746-14.575) and decreased 5-year OS(p=0.006;HR=3.046,95%CI 1.378-6.732) and DFS(p=0.003;HR=2.919,95%CI 1.430-5.959) in myeloid disease.In the setting of sibling HSCT,KIR-ligand mismatch was not any beneficial effect on clinical outcome.In a multivariate analysis,heterozygous C1C2 in the patient resulted in increasedⅡ-ⅣaGVHD(p=0.032;HR=2.685,95%CI 1.087-6.635).This outcome also could be found in myeloid disease(p=0.024;HR=4.178,95%CI 1.206-14.475).The presence of donor activating KIR2DS1 was an independent factor associated with increasedⅡ-ⅣaGVHD(p=0.023;HR=11.86,95%CI 1.415-99.432) in the lymphoid disease.Section 2:Distribution of KIR Genes in Eastern Chinese Han and Taiwanese Han PopulationsIn section 2,we randomly selected 106 healthy unrelated individuals from Eastern Chinese Han and 97 from Taiwanese Han in order to detect the diversity of KIR gene content and the combination of haplotypes.KIR genotype analysis was performed by the PCR-SSP technique and the genotypes and haplotypes were assigned according to standard modal of Hsu et al.The results showed that all 17 KIR genes were observed in the two populations. Framework genes KIR2DL4,KIR3DL2,KIR3DL3 and KIR3DP1 were present in all individuals.The two populations had very similar frequencies for most loci,however, significant differences were noted in the frequencies of KIR3DS1 and KIR2DS4*003.A total of 35 genotypes were identified in the individuals from Eastern Chinese Han and 29 in the Taiwanese Han.Ten genotypes were also shared with other populations. These genotypes contained the most commonly observed genotypes,which were possessed by over 65%of Eastern Chinese Han and 71%of Taiwanese Han individuals. The most common KIR genotypes were(2,2)and(1,2),with frequencies of 23.6%and 19.8%,respectively,in the Eastern Chinese Han population;20.6%and 24.7%, respectively,in the Taiwanese Han population.Our data are consistent with the broad classification of KIR regions into A and B haplotypes,with a predominance of the group A haplotypes in our two populations. Group A outnumbered group B haplotypes with a frequency of 68.1%in the population of Eastern Chinese Han and 74.1%in the Taiwanese Han.Four new KIR genotypes in every population could not be assigned to the haplotypes,according to standard method of Hsu.Section 3:Effects on Activity of Human NK Cells by KIR Functional ModificationIn section 3,the effect of human NK cells on leukemic cells and allogeneic dendritic cells(DC) by blocking the inhibitory receptors KIR2DL1 and KIR2DL2/2DL3 with monoclonal antibody was observed.And we aim to further clarify the possible mechanism about NK cells achieving the separation of GVL and GVHD through KIR functional modification.Human peripheral blood NK cells are isolated by Rosettesep human NK sorting kit. Using lactate dehydrogenase(LDH) release assay is to detect NK cells killing activity against different leukemic cells(human tumor cell lines NB4,K-562 and Raji cells) and against allogeneic mature or immature DC.And to observe the effect on killing activity of NK cells by KIR2DL1 and KIR2DL2/2DL3 blockade,we established NK cell and T lymphocyte reaction system in vitro.The proliferation of T lymphocytes was detected using CCK-8 reagent.The level of TGF-β1 were detected by ELISA method.The results showed that the cytotoxicity of NK cells to NB4 cells was augmented, and the clone-forming capacity of NB4 cells was inhibited remarkably with the increase of concentration of the antibody.Combination with both antibodies can enhance NK cell killing activity.NK cells have a strong ability of killing K-562 cells.However,the cytotoxicity to K-562 cells was not a little of amplification with the inhibitory receptors blockade.The cytotoxicity to Raji cells was not evidently augmented,and statistical difference was observed only between groups of 0μg/ml and 20μg/ml,p＜0.05.The cytotoxicity to immature DC was also not evidently augmented,and statistical difference was not observed.But the cytotoxicity of NK cells to mature DC was augmented remarkably with the increase of concentration of the antibodies (2.20%±1.10%vs.37.59%±5.06%,p＜0.05).In mixed lymphocyte reaction,NK cells by the two antibodies blockade can evidently inhibit T cell proliferation(77.85%±8.31% vs.43.05%±5.95%,p＜0.05).The content of TGF-β1 in the supernatant was increased with the increase of concentration of the antibodies.In summary:1) In the setting of unrelated HSCT for myeloid leukemia,KIR-ligand mismatch was associated with a long-term survival advantage and decreased relapse.2) In the setting of unrelated HSCT,low number of activating KIRs genes in the donor could reduce the risk of relapse;The presence of KIR2DS3 in the donor was an important risk factor for myeloid leukemia.3) In the setting of sibling HSCT,heterozygous C1C2 in the patient was associated increasedⅡ-ⅣaGVHD;The presence of donor activating KIR2DS1 was also an independent factor associated with increasedⅡ-ⅣaGVHD for lymphoid leukemia.4) The KIR gene polymorphisms in Eastern Chinese Han and Taiwanese Han populations were similar to those of other Chinese Han populations.5) Some differences of the distribution of KIR genes in Eastern Chinese Han and Taiwanese Han populations may be related to "genetic drift".6) The study of the distribution of KIR genes will help choose a suitable donor and predict risks of transplantation in the two communities.7) The cytotoxic effects of human NK cells by the inhibitory receptors KIR2DL1 and KIR2DL2/2DL3 blockade against leukemic cells,especially acute myeloid leukemia, were significantly enhanced.This is demonstrated in vitro,alloreactive NK cells by KIR functional modification have a stronger GVL effect;8) Human NK cells by KIR2DL1 and KIR2DL2/2DL3 blockade significantly enhanced the cytotoxic effects against allograft mature DC and also secreted the protein of TGF-β1,which further inhibited T cells proliferation.This might be one of the mechanisms in preventing the T cell-mediated aGVHD.