Relationship Between The Genetic Background Of Donor KIR-recipient HLA And The Outcomes In HLA-identical Sibling HSCT

Allogenetic hematopoietic stem cell transplantation (alloHSCT) is a valuable therapy for malignant haematological diseases. One of the major challenges to successful outcome after alloHSCT is the need to overcome acute graft verse host disease (aGVHD). Effort to decrease the risk of aGVHD require optimal donor matching for human leukocyte antigen (HLA). Approximately 70 percent of the patients have no HLA-identical related donor, whom needs to be selected in unrelated volunteers. China is a multinational country and HLA distribution in it is complicated. Although studies of polymorphism and hyplotype character in HLA-ABDR have been carried thoroughly, research in HLA-Cw needs to be enhanced as it has been proved being the ligand of killer cell immunoglobulin -like receptor (KIR) and may regulate immune response. It possesses both scientific and practical importance to investigate HLA allele frequencies, polymorphism as well as their linkage disequilibrium in different Chinese population so as to provide information for donor selection.Another major challenge alloHSCT faced is the relapse of malignant diseases. Ithas been observed in clinical that some degree of aGVHD may be helpful in triggering graft verse leukemia reaction (GVLR). The ideal aim of alloHSCT is to lessen or avoid aGVHD while reserve and promote GVLR. Killer cell immunoglobulin -like receptor (KIR) makes it possible to approach the ideal aim. KIRs are members of the immunoglobulin superfamily expressed on the surface of natural killer (NK) cells and a subset of T cells. The function of NK-cell is regulated through the combination of KIR and HLA class I molecules distributed on target cells so that an inhibitory or active signal can be delivered. In normal inhibitory KIR binding with HLA class I is predominant and NK killing of autologous cells is prevented. When faced with mismatched allogeneic targets, NK cells sense the missing expression of self-HLA class I molecules and mediate alloreactions. In transplants between HLA non-identical donors and recipients, it has been observed that donor NK cells encountering recipient target cells lacking an corresponding HLA class I allele can mediate anti-leukemic effects in myeloid leukemias, if the class I mismatch predicts lack of ligand for donor inhibitory KIR. These anti-leukemic effects include lower rates for relapse, graft failure, and GVHD, ultimately translating into a higher overall survival (OS). Possibly, donor KIR and recipient HLA incompatibility may provide a new approach to improving alloHSCT outcome and immunomodulation therapy. It is known that the KIR genes and HLA genes are located on different human chromosome, and expressed separately. It is possible that KIR and HLA incompatibility appears between HLA-identical sibling donors and recipients. About 70 percent of alloHSCT were performed between HLA-identical sibling donors and recipients, however, it is still unclear about the relationship between the genetic background of donor KIR-recipient HLA and the outcome of it.This research includes three parts, HLA-A, B, Cw and DRB1 allele frequencies were detected, polymorphism and haplotypes of HLA class I and II were analyzed in Guangdong Han population in the first part so as to supply data for donor selection. In the second part HLA-Cw allele frequencies were surveyed and its allele polymorphism characteristics were explored in Guangdong Han population and the results were compared with those of some other populations. The recognitioncharacter between HLA-Cw and KIR in Guangdong Han population was analyzed. In the third part of this context a retrospective study was carried in order to analyze the outcomes of patients with various hematologic malignancies who received non T-cell depleted transplants from HLA-identical sibling donors, grouped according to lack or presence of recipient HLA ligand for donor inhibitory KIR. The results may offer molecular evidence in optimal donor selection directed by inheritance and may be advisory in clinical therapy.Methods1.Analysis of HLA class I and II polymorphism and haplotypes in Guangdong Han population160 cases of healthy donors from Guangdong Han population, all performed HLA type during 1996 to 2000 in Guangzhou tissue typing centre, Guangzhou blood centre, were studied. Venous blood was gathered and anticoagulated with ACD-B, DNA was extracted with QIAamp DNA extracting kit. Sequence specific oligonucleotide probes(SSOP, Dynal) and/or sequence specific primer polymerase chain reproduction (PCR-SSP, Biotest) was adopted to type HLA-A, B, Cw and DR. HLA-A, B, Cw and DR allele frequencies were calculated and linkage disequilibrium (LD) and relative linkage disequilibrium (RLD) were computed by serial analysis of gene expression (SAGE) software.2. The gene frequency of HLA-Cw in Guangdong Han population and its significance analysisVenous blood collection, DNA extracting and HLA-Cw genotyping were the same with part one. Hardy-Weinberg equilibrium test were performed using SAGE. Chi-square test was used in comparison of allele frequencies in different populations and t-test was used in comparison of allele frequencies between HLA-C1 and HLA-C2 group by SPSS 10.0.3. The relationship between genetic background of donor KIR-recipient HLA and the outcomes in HLA-identical sibling hematopoietic stem cell transplantationFifty-nine transplants performed between 1996 and 2000 were analyzed. Patientswith various hematologic malignancies received non T-cell depleted transplants from HLA- A, B, Cw, DR. identical sibling donors were observed and clinical data were collected. Venous blood collection, DNA extracting and HLA genotyping were the same with part one. KIR genotyping was realized by SSP-PCR. Various grouped definition were listed as bellow:HLA-KIR model: the inhibitory KIR with identified HLA ligands are KIR2DL2 and KIR2DL3, which recognize the HLA-C group 1-related alleles such as HLA-Cw 01,03,07,08 characterized by an asparagine residue at position 80 of the a-1 helix (HLA-C^"80); The inhibitory KIR with identified HLA ligand is KIR2DL1, which recognizes the HLA-C group 2-related alleles such as HLA-Cw*02,04,05,06 characterized by a lysine residue at position 80 (HLA-CLys8°); and KIR3DL1, which recognizes the HLA-BBw4 alleles.HLA-KIR group: KIR ligand absence ("missing KIR ligand") is defined as the absence of one or more recipient HLA epitopes for the identified donor inhibitory KIR. KIR ligand presence is defined as the presence of all the recipient HLA epitopes for the identified donor inhibitory KIR. KIR ligand absence is further classified as C2, Bw4,C2-Bw4 absence according to the absence of specific recipient HLA-ligand C2, Bw4,C2-Bw4 respectively.KIR-HLA match refers to the presence of at least one recipient HLA epitopes for the identified donor inhibitory KIR. KIR-HLA mismatch refers to the absence of all the recipient HLA epitopes for the identified donor inhibitory KIR. KIR-HLA matched group is further classified as Cl, Bw4, Cl-Bw4 and Cl-C2-Bw4 subgroup according to the presence of specific recipient HLA-ligand Cl, Bw4, Cl-Bw4 and Cl-C2-Bw4 C2 respectively.The log-rank statistic of Kaplan-Meier method was used to evaluate the univariate effects on overall survival and disease-free survival. Chi-square test was used in comparison of occurrence of aGVHD, 5 -year relapse, treatment related mortality and the incidence of infections.Results1. Analysis of HLA class I, II polymorphism and haplotypes in Guangdong Han population14 HLA-A, 23 B, 11 Cw and 13 DRB1 alleles were obtained, Among which the following allele had higher frequency distribution: HLA-A* 11 (0.3292), A*02 (0.2929), A*24 (0.1340), A*33 (0.1268), HLA-B*60 (0.1268), B*75 (0.1197), B*58 (0.1161), B*46 (0.1197), HLA-Cw*03 (0.2580), Cw*07 (0.2016), Cw *01 (0.1708) Cw*08 (0.1056), HLA-DRB1*12 (0.1708), DRB1*14 (0.1197), DRB1*O9 (0.1197) .A total of 9 HLA-A-B, 20 Cw-B, 7 A-Cw, and 8 A-DRB1, 9 B-DRB1, 10 Cw-DRBl haplotypes were found. List as bellow were the haplotypes with very strong linkage disequilibrium (LD): A*02-B*46, A*33-B*58, B*13-Cw*03, B*46-Cw*01, B*51-Cw*14, B*58-Cw*03, B*75-Cw*08, A*02-Cw*01, A*33-Cw*03,A*33-DRBl*17, B*58-DRB1*17, Cw*03-DRBl*17, CwO8-DRBl*122. The gene frequency of HLA-Cw in Guangdong Han population and its significance analysis2.1 The allele frequencies of HLA-Cw*03, 07, 01, 08, 14, 04, 15, 12, 06, 05, 16 were 0.2580, 0.1887,0.1732, 0.1071, 0.0654, 0.0587, 0.0453, 0.0387, 0.0322,0.0127, 0.0032 respectively. It was tested that HLA-Cw in Guangdong Han population was shown to be in Hardy-Weinberg equilibrium (x2=60.35, u=54, P>0.1).2.2 The Cw*06 allele frequency in Guangdong Han population was only 0.0322, much lower than that in Shanghai Han population (P<0.001). The Japanese Cw* 12, 14 gene frequencies were higher than that in Guangdong Han population (P<0.00l). The Cw*01, 03, 08, 14 allele distribution were higher ,while the Cw* *16 frequency was lower in Guangdong Han population than that in European and African. The allele frequencies of Cw*05, 06, 07 in European and Cw*02, 04, 17 in African are higher than that in Guangdong Hanpopulation respectively(P<0.001).2.3 There was a significant difference in allele frequencies between HLA-C1 group (0.8310) and HLA-Cw2 group (0.1399) in Guangdong Han population3. The relationship between genetic background of donor KIR-recipient HLA and the outcomes in HLA-identical sibling hematopoietic stem cell transplantationIncidence of grade II -IV aGVHD was lower in patients with KIR-HLA match than that in patients with KIR-HLA mismatch. Incidence of grade II -IV aGVHD and fungus infection were lower in Bw4 matched group than that in Bw4 mismatched group. Incidence of grade II -IV aGVHD was lower in patients of Cl-Bw4 subgroup than that in CK Bw4> Cl-C2-Bw4 subgroups. Over all survival in patients of Cl-C2-Bw4 subgroup was higher than that in Cl, Bw4 or Cl-Bw4 subgroups. Fungus infection ratio was higher in patients of Cl subgroup than that in other subgroups. In myeloid disease, patients with Bw4 match had lower incidence of grade II - IV aGVHD and fungus infection when compared with Bw4 mismatched patients, and patients with C2 match had higher overall survival and disease free survival simultaneously when comparing with C2 mismatched patients.Conclusionsl.HLA class I and II alleles in Guangdong Han population have richer polymorphisms. The haplotype distribution possesses territory characteristic.2.It is easier to select a suitable donor in Guangdong Han population for some patients carrying HLA hyplorypes with strong linkage disequilibrium such as A*02-B*46> B*46-Cw*0K A*02-Cw*01> A*33-DRB1*17, B*58- DRBl*^ Cw*03-DRBl*17 and so on .3. HLA-Cw allele in Guangdong Han population is observed through Hardy-Weinberg equilibrium test being in genetic equilibrium. It has richer polymorphisms and character.4.HLA-C1 gene is predominant compared with HLA-C2 gene in Guangdong Han population.5.Donor KIR-recipient HLA genetic background is related with the outcome in HLA-identical sibling HSCT. Donor selection referring to Bw4 or C2 match maydecrease the incidence of aGVHD or improving the OS and DFS accordingly in patients. Selecting Bw4 matched donor may decrease the incidence of fungus infection, but selecting only Cl matched donor may increase the incidence of fungus infection.