INITIATOR CELLS IN DELAYED-TYPE HYPERSENSITIVITY

迟发型超敏反应的起始细胞

• 批准号: 2063491
• 负责人: PHILIP William ASKENASE
• 金额: $ 24.49万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2063491
• 关键词: T cell receptor; T lymphocyte; cellular immunity; chlorobenzenes; clone cells; delayed hypersensitivity; flow cytometry; gene expression; gene rearrangement; genetic library; hamsters; immunomodulators; interleukin 3; laboratory mouse; molecular cloning; nucleic acid sequence; tissue /cell culture

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.

迟发型超敏反应（DTH）是一种CD 4 + T细胞免疫反应模型。 招募到组织中的免疫抵抗和过敏， 自身免疫性疾病 致敏的CD 4+、DTH效应T细胞的存在 不足以募集到组织部位 用抗原（Ag）攻击。 另一种Ag特异性细胞称为DTH- 需要启动细胞。 这些细胞产生银特异性因子 其类似于IgE抗体并通过导致 局部释放血管活性胺血清素。 使用一组 单克隆抗体消耗DTH起始活性，一种不寻常的 发现Ag特异性细胞的表型：Thy-1+，CD 5+，CD 4-，CD 8-， CD3 β-、sIg-、B220+、I1-2R-和IL-3R+。 此外，DTH启动 细胞在无胸腺裸鼠中诱导，但在SCID小鼠中未诱导，导致 结论DTH起始细胞是原始的，相对胸腺 独立的，Ag特异性细胞，采用重排基因编码 介导DTH起始的Ag特异性因子。 使用裸鼠 在没有污染的情况下免疫和加强DTH起始细胞， CD 4 + DTH效应T细胞，或CD 8+抑制性T细胞， DTH起始细胞，以及DTH起始细胞的体外系和克隆 产生了。 通过FACS测定DTH克隆的表型， 分子分析证实DTH起始细胞是原始的Ag- 独特混合表型的特定细胞：对于T样标记：Thy-1+， CD 5+、CD 4-、CD 8-、表面和mRNA CD 3-、表面和mRNA α TCR- 和δ TCR mRNA+;对于B样标志物：表面和mRNA IG-，B220+， CD 23-;以及对于Mphi标志物Fc γ R+，Mac 1+，II类+。 目前的具体目标是：1）开发DTH启动克隆， 另一种Ag特异性，以确认在 克隆水平 2)为了克隆、测序和表达TCR-1的产物， 在DTH起始克隆中转录的δ杂交基因。 3)目的：克隆编码迟发型超敏反应起始因子的基因。 一个 将从DTH起始克隆构建未扩增的cDNA文库 将通过几种方法进行筛选，以尝试分离 编码抗原特异性DTH起始因子的cDNA。 探测将是 尝试用DTH起始因子的多克隆抗体， 配体探针由相关半抗原的缀合物组成 与白蛋白偶联，白蛋白还与碱性磷酸酶连接。 尝试 将继续生化纯化DTH引发因子， 氨基酸序列信息以构建寡核苷酸探针。 最后，仓鼠单克隆抗体将被开发用于DTH启动 因子以提供单特异性试剂来筛选文库。 因此 希望知识将先进的重要体内现象， DTH-生物学分子克隆水平的启动 相关分子