IDENTIFICATION OF HUMAN MINOR H ANTIGEN PEPTIDES & GENES

长期以来，在动物骨髓移植（BMT）后，微小组织相容性抗原（MiHA）一直被认为是实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，MiHA在人类中也引起移植免疫，并在HLA相同的供体/受体对之间的骨髓移植中作为GVHD的靶点发挥作用，需要对15- 35%的此类受体进行终身药理学免疫抑制。 MiHA已被证明是衍生自正常细胞蛋白的肽，其由I类和II类MHC分子呈递并由可从BMT接受者的外周血淋巴细胞分离的MHC限制性T细胞识别。 然而，由于难以表征这些抗原肽，MiHA在人类和小鼠中的性质在很大程度上仍然未知。 目前关于人类mH基因座总数、等位基因形式、人群频率和免疫遗传潜力的信息很少。 本申请响应的程序公告认识到这是理解这些抗原在人类GVHD中所起作用的主要问题。本申请中代表的3个实验室已建立合作关系，从而定义了人类中的4种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。 本申请提出了该工作的重大扩展，以显著增加鉴定的抗原的数量及其表达的遗传基础，从而允许系统地研究其免疫显性和参与GVHD。 （1）从HLA匹配的GVHD骨髓移植患者中产生I类MHC限制性供体抗宿主T细胞系和克隆，并表征这些系和克隆的抗宿主特异性、MHC限制性、表型频率和组织特异性;（2）采用肽段提取、T细胞表位重构和串联质谱法对这些I类MHC限制性T细胞克隆识别的肽段抗原进行化学鉴定;（3）鉴定编码这些肽抗原的基因，建立人群中抗原表达差异的基础，并开发MiHA等位基因分子分型的方法;（四）为了确定在与单个I类MHC分子相关的肽抗原组中是否存在免疫显性抗原，以及它们是否与免疫缺陷相关。GVHD。

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.