摘要:本計畫的目的是要研究 HLA-C功能性對偶基因多形性對 Killer immunoglobulin-like receptors (KIRs)的反應性及自然殺手(Natural Killer-NK)細胞功能的影響。NK細胞是淋巴球細胞的一支,和 T淋巴球不同的是,NK cells並非以辨認外來的抗原 (foreign antigens),而是辨認細胞表面的 MHC分子,來區別是”正常健康的”還是”不正常、不健康”的細胞,之後再加以毒殺,這中間需要 NK細胞表面的抑制性接受器跟它的配位體(ligand)MHC class I分子的作用。 KIRs是一個包含有抑制性跟活化性接受器的家族。KIRs有表現在 NK細胞的表面,可以辨認人類 MHC(HLA) class I分子為它的配位體,並調節 NK細胞的功能,包括對人類病毒感染、自體免疫疾病、懷孕、癌症及骨髓移植都有影響。KIRs跟 HLA class I的基因都具有多形性(polymorphism),也因此他們之間的交互作用,讓人類的免疫系統更加多樣性。許多研究也顯示,在人體中 HLA class I和 KIRs的不同組合,跟人類對病毒的抵抗力、自體免疫疾病的產生、懷孕的併發症、骨髓移植的預後,都有關聯性。此外,在 NK細胞發育過程中,KIRs跟 HLA class I分子結合的強弱,不僅影響產生的下游細胞訊息強度,也會影響 NK細胞對不正常細胞的反應能力,稱為 NK細胞的”education”。 在 HLA class I分子中,HLA-C是最主要跟 KIRs結合的配位體,也對 NK細胞功能調節影響最大。藉由 HLA-C跟 KIRs結合的特異性,HLA-C分成 C1跟 C2兩種抗原性,其差異主要在 HLA-C上面第 80個胺基酸的位置。HLA-C跟 KIRs之間結合的強弱,很容易受到雙方胺基酸序列上單個胺基酸分子替換的影響。而且 KIRs跟 HLA-C交互作用來調節 NK細胞反應功能時,其結果並不是二元性的有或無,而是藉由精細的微調產生不同的下游細胞訊息強度造成 NK細胞反應功能連續性的差異。不同的 HLA-C跟 KIRs,產生不同程度的交互作用進而影響 NK細胞反應功能的現象,也用來解釋在人體中 HLA class I和 KIRs的不同組合,跟人類對病毒感染、骨髓移植的預後及懷孕的成功與否的臨床相關性。但在探討不同的 HLA-C跟 KIRs的交互作用對 NK細胞功能產生時,卻只是以 C1、C2抗原性來區分,尚未把同是 C1或是 C2的 HLA-C多形性對 KIR交互作用甚至 NK細胞功能的影響做進一步探討。 我們初步的實驗結果顯示,不同的 HLA-C1對偶基因對其接受器,KIRDL2/3,確實產生不同的反應性,並進一步影響 NK細胞的功能。造成這個現象的原因很可能是因為同是 C1的 HLA-C對偶基因彼此之間的氨基酸序列有些許的差異。因此我們提出假說,在 HLA-C除了不同的 C1、C2抗原性對 NK細胞功能有不同的影響外,C1、C2中不同的對偶基因多型性對其接受器 KIRs會有不同的反應性,進而對 NK細胞功能產生不同的影響。本研究的目的就是想證實此一假說,同時探討其影響是否由某些 HLA-C上面的氨基酸序列所決定。 因此我們主要的目標為:1.確認除了 HLA-C1外,不同的 HLA-C2對偶基因對其接受器,KIRDL1,也會產生不同的反應性,並進一步影響 NK細胞的功能。2.找出決定上述影響的 HLA-C氨基酸序列。3.確認 HLA-C對偶基因多形性和 KIRs,跟血液幹細胞移植預後及人工受孕成功的臨床關聯性。 一旦找出決定影響 NK細胞功能的 HLA-C胺基酸序列,我們可以發展快速有便宜的方法,來檢驗正常人跟有疾病的人。這個研究的成果可以運用到臨床方面,像是選擇血液幹細胞移植或NK細胞治療的捐贈者、或是評估不同免疫相關疾病的易感性及預後。
Abstract: The goal of this proposal is to study the effects of functional allelic polymorphism of HLA-C on the reactivity with killer immunoglobulin-like receptors (KIRs) and Natural killer (NK) cell functions. NK cells kill a target cell in a spontaneous fashion that did not require any priming and distinguish between normal healthy cells and abnormal cells by involving inhibitory NK cell receptor that engage MHC class I molecules. KIRs, which include inhibitory and activating receptors, are a family of receptors expressed on the surface of NK cells that can recognize human MHC (HLA) class I molecules. KIRs regulate NK cell functions which play important roles in human health and diseases, including viral infections, autoimmunity, pregnancy, cancer and bone marrow transplantation. Both KIRs and HLA class I molecules are polymorphic and their interactions diversify human immune system. Some studies have showed that combinations of HLA class I and KIR variants are associated with resistance to infections, susceptibility to autoimmune diseases, complications of pregnancy, and outcome of hematopoietic stem cell transplantation (HSCT). In addition, the signal strength during NK cell education, which is a process during their development through engagement of KIRs with their cognate HLA class I ligands and can be altered by receptor-ligand binding affinities, is relevant to the effector capacity of NK cells. Among HLA class I molecules, HLA-C plays a leading role in providing KIR ligands and regulating NK cell functions. In the context of KIR recognition of HLA-C, two mutually exclusive groups of HLA-C allotypes, HLA-C1 and C2, are defined by the dimorphism at position 80 in the amino-acid sequence. HLA-C and KIRs interaction is exquisitely sensitive to single amino-acid substitutions. Besides, KIR-mediated NK cell control involves continuum along which the strengths of the inhibitory KIR/HLA-C interaction vary. These differences in the observed inhibitory capacities of HLA-C reactive KIRs are hypothesized to explain clinical associations with the response of viral infection, outcome of hematopoietic stem cell transplantation and reproductive success. However, beyond the allotypic delineation between C1 and C2, understanding how HLA-C polymorphism affects NK cell functions is limited. Our preliminary results showed different HLA-C1 alleles have differential reactivity with KIR2DL2/3 which have effects on NK cell functions and some of the amino acid positions of HLA-C1 may be responsible for above effects. We therefore hypothesized that beyond the allotypic delineation between C1 and C2, allelic polymorphism of HLA-C can lead to differential reactivity with KIRs and thus differential effects on NK cell functions. In this proposal, we will study the effects of the allelic polymorphism of HLA-C in the reactivity with KIRs and NK cell functions as well as the molecular determinants on HLA-C which are responsible for their functional diversity. Our specific study aims are 1. Verify the functional diversity of HLA-C2 alleles in reactivity with KIR2DL1 and effects on NK cell functions. 2. Establish the molecular determinants of functional allelic polymorphism of HLA-C1/C2 alleles in the reactivity with KIRs and effects on NK cell functions. 3. Verify the association of functional allelic polymorphism of HLA-C with KIRs in the clinical outcome of hematopoietic stem cell transplantation and success of pregnancy during in vitro fertilization (IVF). Once the principal amino-acid residues of HLA-C are determined, we can develop a novel and high-throughput laboratory assay to allow a rapid and affordable testing for the presence of these key residues that regulate NK functions in health and disease. These results of this study could also be applied in the clinical aspects, such as donor selection in HSCT and NK cell therapy, and susceptibility and outcome prognostications in various immune-related health conditions.