FUNCTION OF YOTCR+ CELLS FROM EPIDERMIS & MAMMARY GLAND

表皮 YOTCR 细胞的功能

基本信息

批准号：
2077414
负责人：
Christopher Lee Reardon
金额：
$ 8.78万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-08-01 至 1998-07-31
项目状态：
已结题

项目摘要

The overall objective of the proposed research is to contribute to the better understanding of the function of lymphocytes expressing the gammadelta T-cell receptor (TCR), particularly subsets found in ectodermally-derived tissues, the epidermis and the adjacent mammary glands which are exposed to the external environment. How these T cells function in relation to cell-mediated immunity as a whole is unknown. The specific aims of this proposal are: (1) To study the reactivity of murine gammadelta TCR+ epidermal T cells (ETC) and mammary gland T cells (MTC) to various normal and tumor cells with and without cell stress. Taking advantage of collections of non-clonal and clonal cell lines and hybridomas derived from the murine epidermis and lactating mammary gland and transgenic mouse spleen cells, expressing TCR identical to ETC, we will optimize the conditions necessary for these cells to be activated by normal cells, e.g. keratinocytes, melanocytes, and mammary stromal cells, and by tumor cells, e.g. squamous cell carcinoma, melanoma, mammary carcinoma, and embryonic cell lines. Attempts to increase the lymphocyte reactivity to these target cells will be performed by stressing the cells with heat shock, ultraviolet light, and heavy metals. (2) To isolate and characterize in molecular terms naturally processed endogenous self antigens that are recognized specifically by epidermal and mammary gland gammadelta TCR+ cells. We plan to purify endogenous small molecular weight (<3000 D) acid-eluted cell material from a variety of cell sources that specifically stimulate ETC and MTC and obtain general characteristics using mass spectrometry and obtain sequence information using microsequencing methods should the material prove to be a peptide. (3) To determine if MHC class I, class I-related, or class II molecules present endogenous self antigens to epidermal and mammary gland gammadelta TCR+ cells. The application of several methods, such as blocking class I and class II molecules with monoclonal antibodies, assessing reactivity to cells from various mouse strains expressing different sets of presenting molecules, and using cell lines transgenic for various antigen presenting molecules, will provide the means to characterize the purported class I or class II molecules recognized by ETC and MTC. (4) To develop an assay system for epidermal and mammary gammadelta T cell function using partially reconstituted severe combined immunodeficiency (scid) mice. Taking advantage of the availability of large numbers of relatively homogeneous gammadelta T cells from T cell clones and from gammadelta TCR+ transgenic mice, we will attempt to generate mice with defined gammadelta reconstitution patterns and then attempt to study immune responses of gammadelta T cell subsets to specific antigens in vivo as well as to analyze the possible effects to gammadelta T cells on other cell populations.

拟议研究的总体目标是促进更好地了解表达淋巴细胞的功能 γδ T 细胞受体 (TCR)，特别是在外胚层衍生的组织、表皮和邻近的乳腺暴露于外部环境的腺体。这些T细胞如何与细胞介导的免疫相关的整体功能尚不清楚。本提案的具体目标是： (1) 研究鼠 gammadelta TCR+ 表皮 T 细胞 (ETC) 和乳腺 T 细胞（MTC）对有或没有细胞应激的各种正常细胞和肿瘤细胞。利用非克隆和克隆细胞系的集合以及源自小鼠表皮和哺乳期乳腺的杂交瘤和转基因小鼠脾细胞，表达与 ETC 相同的 TCR，我们将优化这些细胞被激活所需的条件由正常细胞，例如角质形成细胞、黑素细胞和乳腺基质细胞，以及肿瘤细胞，例如鳞状细胞癌、黑色素瘤、乳腺癌和胚胎细胞系。尝试增加淋巴细胞对这些靶细胞的反应将通过用热休克、紫外线和重金属对细胞施加压力。 (2) 以自然加工的分子术语进行分离和表征表皮特异识别的内源性自身抗原和乳腺 gammadelta TCR+ 细胞。我们计划净化内源性来自多种的小分子量（<3000 D）酸洗脱细胞材料特异性刺激 ETC 和 MTC 的细胞来源并获得使用质谱分析的一般特征并获得序列如果材料证明是使用微测序方法的信息是一种肽。 (3) 确定 MHC 是否为 I 类、I 类相关或 I 类 II 分子将内源性自身抗原呈递至表皮和乳腺腺gammadelta TCR+细胞。多种方法的应用，例如作为用单克隆抗体阻断 I 类和 II 类分子，评估对表达不同小鼠品系的细胞的反应性不同组的呈递分子，并使用转基因细胞系对于各种抗原呈递分子，将提供方法描述所声称的 I 类或 II 类分子的特征 ETC 和 MTC。 (4) 开发表皮和乳腺检测系统使用部分重建的严重组合的γδT细胞功能免疫缺陷（scid）小鼠。利用可用的来自 T 细胞的大量相对同质的 γδ T 细胞克隆和 gammadelta TCR+ 转基因小鼠，我们将尝试生成具有定义的 gammadelta 重建模式的小鼠，然后尝试研究 γδ T 细胞亚群的免疫反应体内特定抗原以及分析可能的影响其他细胞群上的 gammadelta T 细胞。