ANTI-PEPTIDE ANTIBODIES TO HUMAN T CELL RECEPTORS

人类T细胞受体目前已被定义为诱导物、抑制物和 I类和II类特异性细胞毒T淋巴细胞与90KD T_3相关 克隆型分子。它们由一个49-51KD阿尔法和43KD组成 二硫键连接的β亚基。多肽图谱分析研究 建议每个亚基由恒定结构域和可变结构域组成。 此外，Beta中这种差异的确切分子基础 亚基已经被DNA克隆研究定义，表明类似于 免疫球蛋白，特定的V，D，J和C片段融合形成活性钛 β基因。在胸腺中，这种二硫键连接的异二聚体是 仅限于少量表面细胞的表达 T3+。然而，关于Alpha合成的精确分子细节 和β亚基以及细胞质和表面形式的存在 由于缺乏适当的能够反应的试剂而没有定义 与单独的分离亚基结合。为了更详细地了解 胸腺内个体发育过程中人类T细胞受体表达的性质 将涉及三个领域：1)制作和表征 针对钛α和β亚基恒定区的抗肽抗血清 同时使用异种免疫和单抗 技术；2)胸腺内个体发育的分析 含特异性探针的亚基；3)抗肽抗血清的利用 以进一步描绘T3-Ti分子络合物的结构。 首先，与个别亚基区域相对应的多肽可能 基于Kyte-Doolitel和Hopps和Wood预测的免疫原性将 合成并用于异源抗血清和单抗的生产 抗体。然后将针对免疫肽对试剂进行筛选 以及变性的分离亚基，随后评估 胸腺细胞和T细胞表面天然分子的反应性 淋巴细胞。其次，一旦单抗和异种抗血清试剂 定义后，将检查胸腺内亚单位的表达。 具体地说，将确定表面和胞质内 钛的Alpha和Beta亚基的存在形式及其出现的位置 胸腺内T细胞相对于彼此和已知T细胞的分化 表面分子包括T3、T4、T8、T11和T6。此外， 将定义表达一个或两个亚基的细胞的定位。 最后，这些抗肽试剂将被用于研究一些 三钛分子络合物的结构特征。

Human T cell receptors have now been defined on inducer, suppressor and class I and class II specific cytotoxic T lymphocytes as 90KD T3-associated clonotypic molecules. These are comprised of one 49-51KD Alpha and 43KD Beta subunit which are disulfide linked. Peptide map analysis studies suggested that each subunit is comprised of constant and variable domains. Moreover, the precise molecular basis for this variability in the Beta subunit has been defined by DNA cloning studies, showing that similar to immunoglobulin, specific V, D, J and C segments fuse to form an active Ti Beta gene. In the thymus, such disulfide linked heterodimers are restricted in expression to a minor population of cells which are surface T3+. However, the precise molecular details regarding synthesis of Alpha and Beta subunits and the existence of cytoplasmic and surface forms has not been defined due to lack of appropriate reagents capable of reacting with the individual isolated subunits. To understand in greater detail the nature of human T cell receptor expression during intrathymic ontogeny, we will address three areas: 1) production and characterization of anti-peptide antisera to constant regions of the Ti Alpha and Beta subunit employing both heterologous immunization and monoclonal antibody techniques; 2) analysis of intrathymic ontogeny of Ti Alpha and Beta subunits with specific probes; and 3) utilization of anti-peptide antisera to further delineate the structure of the T3-Ti molecular complex. Firstly, peptides corresponding to regions of individual subunits likely to be immunogenic based on Kyte-Doolittle and Hopps and Wood predictions will be synthesized and utilized for production of heteroantisera and monoclonal antibodies. Reagents will then be screened against the immunizing peptide as well as denatured isolated subunits and subsequently assessed for reactivity with the native molecules on the surface of thymocytes and T lymphocytes. Secondly, once monoclonal and heteroantisera reagents are defined, the intrathymic expression of the subunits will be examined. Specifically, it will be determined whether surface and intracytoplasmic forms of the Ti Alpha and Beta subunits exist and where they appear in intrathymic T cell differentiation relative to one another and known T cell surface molecules including T3, T4, T8, T11 and T6. In addition, the localization of cells expressing one or both subunits will be defined. Finally, these anti-peptide reagents will be utilized to investigate some of the structural features of the T3-Ti molecular complex.