IDENTIFICATION OF HUMAN MINOR H ANTIGEN PEPTIDES & GENES

在动物模型中，次要组织相容性抗原(MIHA)一直被认为是导致同种异体器官移植排斥反应和移植物抗宿主病(GVHD)发生的关键因素。最近的证据表明，在人类中，MIHA还可以激发移植免疫，并在人类白细胞抗原相合的供受者对之间的骨髓移植中作为GVHD的靶点，导致15%-35%的受者需要终生接受药物免疫抑制。MIHA是一种来源于正常细胞蛋白的多肽，由I类和II类MHC分子呈递，并被MHC限制性T细胞识别，可从骨髓移植受者的外周血淋巴细胞中分离出来。然而，由于很难对这种抗原肽进行表征，因此MiHA在人类和小鼠体内的性质在很大程度上仍不清楚。目前关于人类MH基因座的总数、其等位基因形式、群体频率和免疫遗传潜力的信息很少。本申请回应的项目公告承认，这是理解这些抗原在人类移植物抗宿主病中所起作用的主要问题。本申请中所代表的3个实验室已经建立了合作，从而在人类中定义了4个MiHA，这代表了迄今为止为该物种阐明的此类抗原的整个数据库。这项申请建议对这项工作进行重大扩展，以显著增加已识别抗原的数量及其表达的遗传基础，以便能够系统地研究它们的免疫优势和参与GVHD。因此，本研究的具体目标是：(1)从表现为GVHD的人类白细胞抗原相合的骨髓移植患者中建立I类MHC限制性供体抗宿主T细胞系和克隆，并对其抗宿主特异性、MHC限制性、表型频率和组织特异性进行鉴定；(2)利用肽提取、T细胞表位重组和串联质谱仪对这些I类MHC限制性T细胞克隆识别的多肽抗原进行化学鉴定；(3)鉴定这些多肽抗原的编码基因，为人类群体内抗原表达的差异奠定基础，并开发MIHA等位基因的分子分型方法；(4)确定与单个I类MHC分子相关的一组多肽抗原中是否存在免疫优势抗原，以及它们是否与GVHD显著相关。

Minor histocompatibility antigens (MiHA) have long been known to be key factors in the rejection of solid organ allografts and in the development of graft-versus-host-disease (GVHD) following bone marrow transplantation (BMT) in animal models. Recent evidence suggests that MiHA in humans also provoke transplantation immunity and function as targets of GVHD in bone marrow transplants between HLA-identical donor/recipient pairs, necessitating life long pharmacologic immunosuppression of 15-35 percent of such recipients. MiHA have been shown to be peptides derived from normal cellular proteins that are presented by class I and class II MHC molecules and recognized by MHC-restricted T cells that can be isolated from peripheral blood lymphocytes of BMT recipients. However, because of the difficulty in characterizing such antigenic peptides, the nature of MiHA in both humans and mice has remained largely unknown. Little information is currently available on the total number of human mH loci, their allelic forms, population frequencies, and immunogenetic potential. The program announcement to which this application responds recognizes that this is a major problem in understanding the role that these antigens play in human GVHD. The 3 laboratories represented in this application have established a collaboration that has led to the definition of four MiHA in humans, which represents the entire database of such antigens so far elucidated for that species. This application proposes a major expansion of that work in order to dramatically increase the number of identified antigens and the genetic basis for their expression, in order to allow a systematic study of their immunodominance and involvement in GVHD. Accordingly, the specific aims of this proposal are: (1) To generate class I MHC restricted donor anti-host T cell lines and clones from HLA matched bone marrow transplant patients who exhibit GVHD, and to characterize these lines and clones for their anti-host specificity, MHC restriction, phenotype frequencies and tissue specificity; (2) To chemically identify the peptide antigens recognized by these class I MHC restricted T cell clones using peptide extraction, T cell epitope reconstitution, and tandem mass spectrometry; (3) To identify the genes encoding these peptide antigens, establish the basis for differences in antigen expression within the human population, and develop the means to molecularly type for MiHA alleles; (4) To establish whether there are immunodominant antigens among the group of peptide antigens associated with individual class I MHC molecule and whether they are significantly associated with GVHD.