THYMIC ENVIRONMENT AND T-CELL DIFFERENTIATION

构成胸腺环境的基质细胞影响许多胸腺细胞， T淋巴细胞生成的重要方面。其中包括非- 克隆型、谱系限制性细胞表面分子和抗原 受体，以及未成熟T细胞的阳性和阴性选择 基于它们的抗原受体特异性。虽然重要的是 这些过程的胸腺环境已经确立， 由构成该环境的非淋巴细胞发挥作用 不太了解。胸腺基质细胞作用的研究进展 T细胞分化中的细胞由于缺乏知识而受到阻碍， 关于胸腺上皮（TE）。拟议的工作将产生 鼠TE细胞系和检测TE细胞表面分子的mAb， 使用这些试剂来表征TE支持几种 体外T细胞分化的方面。第一， 影响早期胸腺细胞基质细胞分子 发展，从CD 4-CD 8（lo）表型到CD 4 + CD 8+表型的进展。 人口将得到确认。这将通过筛选来实现 抗基质细胞mAb影响这种表型变化的能力 在与胸腺细胞和基质细胞系短期共培养中， 调整这个过程。具有抑制活性的抗体将用于 对它们识别的分子进行生物化学表征， 使用直接测序法， 表达式克隆方法。第二，代表皮质细胞的细胞系 将生成TE。本实验室最近取得的技术进步 (flow活皮质TE的细胞计数鉴定）将有助于 快速和系统的分析最佳培养条件， 这种细胞类型的繁殖。分离的皮质TE细胞将用于 产生特异于皮质TE细胞表面分子的mAb，以鉴定 皮质TE产生的细胞因子，并检查功能性 体外皮质TE活性。第三，Z210R.1胸腺基质细胞 该实验室分离出的线，先前显示支持一种新的 体外B淋巴细胞生成模式将根据其 支持T淋巴细胞生成的能力基质细胞表面分子 在这些过程中所涉及的将被确定与使用反- 针对该细胞系产生的基质细胞mAb。此外，一部小说 从该基质细胞系克隆的细胞因子将根据 对胸腺细胞成熟和功能影响。拟议的工作是 重要的是，它将有助于定义胸腺环境方面， 基质细胞异质性，TE表达的细胞表面分子 细胞，它们产生的细胞因子，以及这些分子和细胞因子 影响T细胞分化。此信息应导致 更好地理解胸腺基质细胞对T细胞的贡献 分化，并可能导致细胞因子和/或 具有治疗价值的细胞相互作用分子。

The stromal cells comprising the thymic environment influence a number of important aspects of T-lymphopoiesis. These include the expression of non- clonotypic, lineage restricted cell surface molecules and antigen receptors, and the positive and negative selection of immature T cells based on their antigen receptor specificity. Although the importance of the thymic environment to these processes is well established, the role(s) played by the non-lymphoid cells comprising that environment remains poorly understood. Progress in understanding the role of thymic stromal cells in T cell differention has been hampered by a lack of knowledge regarding the thymic epithelium (TE). The proposed work will generate murine TE cell lines and mAbs detecting TE cell surface molecules and employ these reagents to characterize the ability of TE to support several aspects of T cell differentiation in vitro. First, the nature of the stromal cell molecules that influence an early stage of thymocyte development, the progression from a CD4-CD8(lo) phenotype to a CD4+CD8+ population will be identified. This will be accomplished by screening anti-stromal cell mAbs for their ability to affect this phenotypic change in short term co cultures with thymocytes and a stromal cell line shown to modulate the process. Antibodies with inhibitory activity will be used to biochemically characterize the molecules they recognize and to isolate the cDNA encoding these stromal cell surface molecules using a direct expression cloning-approach. Second, cell lines representative of cortical TE will be generated. Recent technical advances made in this laboratory (flow cytometric identification of viable cortical TE) will facilitate a rapid and systematic analysis of optimal culture conditions for the propagation of this cell type. Isolated cortical TE cells will be used to generate mAbs specific for cortical TE cell surface molecules, to identify the cytokines produced by cortical TE,and to examine the functional activity of cortical TE in vitro. Third, the Z210R.1 thymic stromal cell line isolated by this laboratory and previously shown to support a novel pattern of B-lymphopoiesis in vitro will be assessed in terms of it's ability to support T-lymphopoiesis. Stromal cell surface molecules involved in these processes will be identified with the use of anti- stromal cell mAbs raised against this cell line. In addition, a novel cytokine cloned from this stromal cell line will be assessed in terms of it's effect on thymocyte maturation and function. The proposed work is significant in that it will help define the thymic environment in terms of stromal cell heterogeneity, the cell surface molecules expressed by TE cells,the cytokines they elaborate, and how these molecules and cytokines influence T cell differentiation. This information should result in a better understanding of thymic stromal cell contributions to T cell differentiation and may lead to the identification of cytokines and/or cell interaction molecules with therapeutic value.