| [Purpose] Hematopoietic stem cell transplantation (HSCT) is one of the most effective treatments for leukemia. Although the match of human leukocyte antigen (HLA) is the most critical criteria for the selection of donors, the clinical outcome varies in HLA matched HSCT. Killer cell immunoglobulinlike receptors (KIRs) are a diverse family of receptors on natural killer cells. The KIR genes display a high degree of variability at the level of gene presence or absence. The distribution of KIR genes in Chinese Han population is unclear. There are two KIR haplotypes: haplotype A and haplotype B.2DS4 is the only activating KIR gene in haplotype A. Only 2DS4*001(includes 2DS4*0010101, *0010102, *0010103, *00102, and *00103) encodes for cell surface receptors while the remaining alleles, 2DS4*003, *004, *006, *007, *008, and *009, carry a 22-base pair deletion in exon 5, which causes a frame shift, yielding a truncated KIR2DS4 protein with loss of the transmembrane and cytoplasmic domains of the full-length KIR2DS4 protein. The deleted variants of KIR2DS4 with deletions are not anchored to the cell membrane but encode a soluble form of the protein that is potentially secreted. Recent understanding of KIR recognition and lysis of tumor cells suggests that allo-reactivity of NK cells induced by HLA-KIR mismatch could facilitate engraftment and enhance graft-versus-leukemia (GVL) effect. The regulation of activation of NK cells by activating KIR receptors (aKIR) may benefit in separation of graft-versus-host disease (GVHD) and GVL.[Methods] The method was applied to 150 Chinese Han individuals: 75 patients who received allo-HSCT and their unrelated donors, establishing frequencies of KIR gene and 2DS4 alleles within the local population. All the patients were undergoing transplantation for CML (n=24), AML (n=19), ALL (n=29) and other malignancies (n=3).In this study, first, high resolution typing of HLA-A, -B, -C, -DRB1, and -DQB1 for the 75 donor-recipient pairs were performed by sequence-based typing (SBT), respectively; the KIR gene typing for the 150 individuals was performed by PCR-sequence-specific primers (PCR-SSP). Of the 75 cases, 60 donor-recipient pairs were HLA-A, B, C, DRB1 and DQB1 high-resolution typing identical; 15 had an allelic mismatch at the HLA-C locus. The KIR gene frequencies and the distribution of KIR haplotypes in Chinese-Han population was evaluated and compared with other populations. Second, to characterize the allelic diversity of KIR2DS4, a sequence-based testing and TOPO TA cloning system identifying and distinguishing alleles of the KIR2DS4 gene was established. The full-length 2DS4 allele and the deleted variants were detected and then the distribution of 2DS4 alleles in Chinese Han population was analyzed. Third, we studied the KIR-ligand model in the 75 pairs and considered the association of"missing ligand"model with the clinical outcomes. For 30 transplants characterized by"KIR-HLA mismatch", RT-PCR was used for quantification of KIR2DL1, 2DS1, 3DL1, 3DS1 at different time points after HSCT. Furthermore, we studied the role of KIR haplotypes in HSCT and analyzed the association of full-length 2DS4 and the expression of KIR2DL1/3DL1 with the occurrence of aGVHD.[Results]⑴All 17 KIR genes were observed and the framework genes 3DL3, 3DP1, 2DL4, and 3DL2 were positive in all samples. The 17 KIR loci were found in 21.3%–100% of individuals. The frequencies of 2DL1 and 2DP1 genes were high in the Chinese Han population, whereas the frequencies of 2DL2 and 2DS3 genes were low. We classified the different KIR haplotypes of 150 individuals into two groups: A/A and B/x. The percentages of individuals who were A/A and B/x were 44.0% and 56.0%, respectively. In comparison with other populations, it was remarkable that the KIR phenotype and haplotype frequencies of the Chinese Han population were similar to that of Japanese, but different from Caucasoid and African American populations.⑵We examined the KIR-ligand pairs in these 75 transplants. KIR-HLA mismatching status was determined using high-resolution HLA-B and HLA-C typing. Allo-reactive KIRs were defined as donor 2DL1 lacking HLA group C2 alleles on the recipient's cells as well as 2DL2/L3 lacking HLA group C1 alleles and 3DL1 lacking HLA-Bw4 alleles. Of the 66 pairs with potential allo-reactive KIR, 53 pairs had no C2 group allele in the recipient for donor KIR2DL1; 2 pairs, no C1 group allele for donor KIR2DL2 and/or 2DL3; and 24 pairs, no HLA-Bw4 in the recipient for donor KIR3DL1.⑶The distribution of KIR2DS4 allells is distinct in the Chinese Han population. A majority (139) of the 150 samples (92.7%) were positive for KIR2DS4. Sequencing of the coding regions of this gene detected four of the nine known KIR2DS4 alleles, KIR2DS4*00101, *003, *004, and *007. The KIR2DS4 allele frequencies showed some differences in populations. We examined the frequency of the non-deleted and deleted versions of KIR2DS4. The KIR2DS4-deleted variant was found in 44.0% of the 150 individuals. The ratio of deleted to non-deleted versions of KIR2DS4 is approximately 1:2. Of the A/A population (n =66), 12.1 % (8 of 66) individuals had the KIR2DS4-deleted version only and 42.4% (28 of 66) individuals were full-length/deleted 2DS4 alleles heterozygous. The percentages were 11.0% (8 of 73) and 30.1% (22 of 73) in B/x population. A decreased frequency of carrying two 2DS4-deleted variants was observed in populations having KIR2DS4 as the only activating KIR gene. Three novel 2DS4 alleles were identified.⑷To analyze the role of killer Ig-like receptors (KIR) in SCT, it was noticed that, among the 75 donor-recipient pairs, 60 were HLA 10/10 matched and 15 had one allelic mismatch at HLA-C. In A/A group, 88.6% transplants carried KIR-HLA mismatch, compared to 87.5% in B/x group. Analysis of the association of KIR-HLA mismatch with clinical outcomes after HSCT showed no statistical difference. To analyze the role of killer Ig-like receptor haplotype in donor , we found that transplants from KIR haplotype B/x group donors showed significantly higher overall survival (OS) rates compared with those from KIR haplotype A/A donors (relative risk (RR) 3.1 (95% confidence interval (CI) 1.1–8.6), P=0.007). Of the 75 recipient/donor pairs, 35 cases who received transplants from KIR haplotype A/A donors were then further analyzed. In transplants, when donors carried two KIR2DS4-full-length alleles (n=14), the aGVHD rate was much higher, compared with those in which the donors carried one or two 2DS4-deleted variants (n=21) (RR 9.0 (95% CI 1.2–66.9), P=0.01). The risk of grade III–IV aGVHD was also increased significantly in the KIR2DS4-full subgroup (5 of 14), compared with the 2DS4-deletion subgroup (0 of 21) (P=0.006). For the 30 transplants characterized by HLA-KIR mismatch, the mRNA expression of 2DL1 increased significantly in patients with aGVHD 30–90 days after transplantation compared with patients without aGVHD (P=0.021). The same difference was observed for 3DL1 (P=0.005). The results suggested the association of a high expression of KIR2DL1 and 3DL1 in early stages (<day 90) post transplantation with the occurrence of aGVHD. This conclusion was more obvious when the recipient/donor pairs were all KIR haplotype A/A. A significant statistical difference of 2DL1 and 3DL1 copy numbers could be found by 90 days post transplantation between the groups with and without aGVHD (z=2.558, P=0.011).[Conclusion] Our findings suggest that the Chinese Han population is distinct in KIR gene frequencies and 2DS4 allele frequencies in comparison with some other populations. Transplants from donors who carried at least one KIR haplotype B had improved OS rates. A significant association of full-length KIR2DS4 or KIR2DL1/3DL1 expression with the occurrence of aGVHD was indicated in this study. KIR2DS4 allele typing and dynamic detection of KIR2DL1/3DL1 expression would be beneficial for prediction of aGVHD after transplantation. In aggregate these results suggested that combining KIR and HLA genotyping could help in the selection of transplant donors and improve the outcome of transplantation. |
PDF Full Text Request