SOMATIC GENETICS OF T CELL IMMUNITY

为了研究免疫反应发生时的结构和动力学， 克隆性限制性免疫的表型和基因特征 反应可用于识别和表征抗原特异性 冰冻脾组织切片中的淋巴细胞群。我们 应利用克隆性限制的T和B细胞反应 (4-羟基-3-羟基-4-羟基-3-羟基-4-羟基-3-羟基-4-羟基-3-羟基乙酸乙酯)偶联 (硝基苯基)乙酰基(NP)用于鉴定和表征抗原特异性 淋巴细胞群在原位。酶和荧光素标记 抗体和凝集素将被用来跟踪种群和空间 与脾解剖相关的反应性T细胞的动力学， 与抗原提呈细胞的关联和与特异性的相互作用 B淋巴细胞。将使用BrdUrd和TdT标记来跟踪T细胞 增殖和程序性原位死亡。小T细胞的显微解剖 来自组织切片的细胞群体或单个细胞将用于 结合规范V(D)Jα-和β-的PCR扩增 链重排，研究反应细胞的体细胞遗传学。 克隆V(D)J片段在报告细胞中的转染和表达 LINE将允许重建和研究单个TCR表型 显微解剖的细胞。 这些技术使T细胞的第一次原位研究成为可能 对抗原的竞争和选择，对细胞的洞察 脾T区和B区的协同作用及其特点 显性TCR表型。总之，这些研究奠定了以下基础 为T和B淋巴细胞创造真正的群体遗传学 一种免疫反应。

To study the architecture and dynamics of immune responses as they occur, the phenotypic and genotypic hallmarks of clonally restricted immune responses may be used to identify and characterize antigen-specific lymphocyte populations in histological sections of frozen spleen. We shall take advantage of the clonally restricted T- and B cell response of B10.A mice to pigeon cytochrome c (PCC) conjugated with (4-hydroxy-3- nitrophenyl) acetyl (NP) to identify and characterize antigen-specific lymphocyte populations in situ. Enzyme- and fluorochrome labeled antibodies and lectins will be used to follow the population and spatial dynamics of responding T cells in reference to the splenic anatomy, association with antigen-presenting cells, and interaction with specific B lymphocytes. BrdUrd- and Tdt-labeling will be used to follow T cell proliferation and programmed death in situ. Microdissection of small T cell populations or single cells from histologic sections will be used in conjunction with the PCR amplification of canonical V(D)J alpha- and beta- chain rearrangements to study the somatic genetics of responding cells. Transfection and expression of cloned V(D)J fragments in a reporter cell liner will allow the reconstruction and study of TCR phenotypes of single microdissected cells. These techniques have made possible the first in situ studies of T cell competition and selection in response to antigen, insight into cellular collaboration in the T- and B zones of the spleen, and the character of dominant TCR phenotypes. Together, these studies lay the foundation for creating a true population genetics for the T and B lymphocytes that mount an immune response.