INITIATOR CELLS IN DELAYED-TYPE HYPERSENSITIVITY

迟发型超敏反应(DTH)是一种CD4+T细胞模型 在免疫抵抗和过敏反应中向组织中募集 自身免疫性疾病。致敏的CD4+、DTH效应T细胞的存在 在循环中不足以重新进入组织部位 用抗原(Ag)攻击。另一种称为DTH的银特异性细胞- 需要启动电池。这些细胞阐述了Ag特异的因子 类似于IgE抗体，并通过导致 局部释放血管活性胺5-羟色胺。使用一个面板 耗尽迟发型超敏反应启动活性的单抗是一种不同寻常的 发现一种抗原特异性细胞的表型：Thy-1+，CD5+，CD4-，CD8-， CD3epsilon-、Sig-、B220+、I1-2R-和IL-3R+。此外，潜伏期启动 在裸鼠体内可以诱导出细胞，但在SCID鼠中不能诱导出细胞，导致 结论：迟发型超敏反应启动细胞是原始的，相对胸腺细胞 独立的、抗原特异的细胞，利用重排基因来编码 介导迟发型超敏反应启动的抗原特异性因子。使用裸鼠 在无污染的情况下免疫和增强迟发型超敏反应启动细胞 CD4+DTH效应T细胞，或下调CD8+抑制T细胞 迟发型超敏反应启动细胞及其体外品系和克隆 都被生成了。用流式细胞仪和扫描电子显微镜测定了DTH克隆的表型 分子分析证实，迟发型超敏反应启动细胞是原始的Ag- 一种独特混合表型的特定细胞：对于T样标记物：THY-1+， CD5+，CD4-，CD8-，表面和mRNA CD3-，表面和mRNAαβTCR- 和Delta TCR mRNA+；对于B样标记物：表面和mRNA Ig-，B220+， CD23-；Mphi标记FcGammaR+、MAC1+、Class II+。 目前的具体目标是：1)开发引发潜伏期的克隆 另一种确认迟发型超敏反应启动的抗原特异性的抗原特异性 克隆水平。2)克隆、测序和表达TCR- 在DTH启动克隆中转录的Delta杂交基因。 3)克隆潜伏期诱发因子的编码基因(S)。一个 未扩增的cDNA文库将从DTH启动克隆中构建 在lambdalZAP中，并将通过多种手段进行筛选，以尝试分离 编码抗原特异性潜伏期启动因子的基因(S)。探测将会是 尝试使用针对DTH启动因子的多克隆抗体，并且还 使用由相关半抗原的共轭组成的配基探针 与白蛋白偶联，白蛋白也与碱性磷酸酶相连。尝试 将继续生化提纯DTH启动因子以获得 氨基酸序列信息构建寡核苷酸探针。 最后，将发展仓鼠单抗以启动DTH。 提供单特异性试剂以筛选文库的因子。它是这样的 希望对重要的体内现象有更深入的认识 潜伏期引发到生物分子克隆的水平 相关分子。

Delayed-type hypersensitivity (DTH) responses are a model of CD4+ T cell recruitment into the tissues in immune resistance and in allergic and autoimmune diseases. The presence of sensitized CD4+, DTH effector T cells in the circulation is not sufficient for recruitment into a tissue site challenged with antigen (Ag). Another Ag-specific cell called DTH- initiating cells is required. These cells elaborate Ag-specific factors that are analogous to IgE antibody and mediate DTH-initiation by leading to local release of the vasoactive amine serotonin. Using a panel of monoclonal antibodies to deplete DTH-initiating activity, an unusual phenotype for an Ag-specific cell was found: Thy-1+, CD5+, CD4-, CD8-, CD3epsilon-, sIg-, B220+, I1-2R-, and IL-3R+. Furthermore, DTH-initiating cells were induced in athymic nude mice but not in SCID mice, leading to the conclusion that DTH-initiating cells were primitive, relatively thymic independent, Ag-specific cells that employed rearranging genes to encode the Ag-specific factors that mediate DTH-initiation. Nude mice were used to immunize and boost DTH-initiating cells in the absence of contaminating CD4+ DTH-effector T cells, or CD8+ suppressor T cells that down-regulate DTH-initiating cells, and in vitro lines and clones of DTH-initiating cells were generated. the phenotype of the DTH- clones determined by FACS and molecular analysis confirmed that DTH-initiating cells are primitive Ag- specific cells of a unique mixed phenotype: for T-like markers: Thy-1+, CD5+, CD4-, CD8-, surface and mRNA CD3-, surface and mRNA alphabeta TCR- and delta TCR mRNA+; for B-like markers: surface and mRNA Ig-, B220+, CD23-; and for Mphi markers FcgammaR+, Mac1+, Class II+. The current specific aims are to: 1) Develop DTH-initiating clones of another Ag specificity to confirm Ag-specificity of DTH-initiation at the clonal level. 2) To clone, sequence, and express the product of the TCR- delta hybridizing gene that is transcribed in the DTH-initiating clones. 3) To clone the gene(s) that encode the DTH-initiating factors. An unamplified cDNA library will be constructed from the DTH-initiating clone in lambdalZAP and will be screened by several means to attempt to isolate cDNA(s) encoding the Ag specific DTH-initiating factor. Probing will be attempted with a polyclonal antibody for DTH-initiating factors, and also with a ligand probe consisting of a conjugate of the relevant hapten coupled to albumin to which is also linked alkaline phosphatase. Attempts will continue to biochemically purify the DTH-initiating factor to obtain amino acid sequence information to construct an oligonucleotide probe. Finally, hamster monoclonal antibodies will be developed to DTH-initiating factor to provide monospecific reagents to screen the library. It is thus hoped that knowledge will be advanced of the important in vivo phenomena of DTH-initiation to the level of molecular cloning of the biologically relevant molecule.