THYMIC ENVIRONMENT AND T-CELL DIFFERENTIATION

构成胸腺环境的基质细胞影响许多 T淋巴细胞生成的重要方面。其中包括表达非- 克隆型、谱系受限的细胞表面分子和抗原 受体与未成熟T细胞的阳性和阴性选择 基于它们的抗原受体特异性。尽管这一点很重要 胸腺环境对这些过程是既定的，作用(S) 由构成该环境的非淋巴样细胞发挥作用 人们对此知之甚少。胸腺基质作用的研究进展 由于缺乏知识，T细胞分化一直受到阻碍 关于胸腺上皮(TE)。拟议的工作将产生 小鼠TE细胞株和单抗检测TE细胞表面分子和 使用这些试剂来表征TE支持多个 T细胞体外分化的几个方面。第一，社会责任的性质 影响胸腺细胞早期发育的基质细胞分子 发展，从CD4-CD8(Lo)表型到CD4CD8的进展 人口将被确定。这将通过筛查来实现 抗基质细胞单抗影响这种表型改变的能力 在短期内与胸腺细胞和间质细胞系共培养 调整这一过程。具有抑制活性的抗体将用于 对它们识别的分子进行生化表征，并分离出 编码这些基质细胞表面分子的DNA使用直接的 表达克隆--方法。第二，代表大脑皮层细胞系 将生成TE。这个实验室取得的最新技术进步 (流式细胞仪鉴定存活的皮质TE)将有助于 最适培养条件的快速系统分析 这种细胞类型的繁殖。分离的皮质TE细胞将用于 产生针对皮质TE细胞表面分子的单抗，以识别 皮层TE产生的细胞因子，并检测其功能 皮质醇的体外活性。第三，Z210R.1胸腺基质细胞 由该实验室分离的品系，先前显示支持一种新的 B淋巴细胞在体外的生成模式将根据它的 支持T淋巴细胞生成的能力。基质细胞表面分子 参与这些过程的人将被确定为使用反 基质细胞单抗是针对该细胞系而产生的。此外，还有一部小说 从该基质细胞系克隆的细胞因子将在以下方面进行评估 对胸腺细胞成熟和功能的影响。建议的工作是 重要的是，它将有助于从以下方面定义胸腺环境 间质细胞异质性，由TE表达的细胞表面分子 细胞，它们阐述的细胞因子，以及这些分子和细胞因子是如何 影响T细胞分化。此信息应导致 更好地了解胸腺基质细胞对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.