SOMATIC GENETICS OF T CELL IMMUNITY

为了研究免疫反应的结构和动力学， 克隆限制性免疫的表型和基因型特征 应答可用于鉴定和表征抗原特异性 冷冻脾组织切片中的淋巴细胞群。 我们 应利用克隆限制的T和B细胞反应， B10.A小鼠对鸽细胞色素c（PCC）与（4-羟基-3-羟基-1，2-二氧杂环戊烯）缀合， 硝基苯基）乙酰基（NP），以鉴定和表征抗原特异性 原位淋巴细胞群。 酶标记和荧光标记 抗体和凝集素将被用来跟踪人口和空间 与脾解剖学相关的响应T细胞的动力学， 与抗原递呈细胞的结合以及与特定细胞的相互作用 B淋巴细胞。 BrdUrd和Tdt标记将用于跟踪T细胞 原位增殖和程序性死亡。 小T细胞的显微解剖 来自组织切片的细胞群或单细胞将用于 结合典型V（D）J α和β的PCR扩增， 链重排来研究应答细胞的体细胞遗传学。 克隆的V（D）J片段在报告细胞中的转染和表达 内衬将允许重建和研究TCR表型的单一 显微解剖的细胞 这些技术使得首次原位研究T细胞 对抗原的竞争和选择反应，对细胞的洞察 脾T区和B区的协同作用，以及 显性TCR表型。 总之，这些研究奠定了基础， 为T和B淋巴细胞创造了一个真正的群体遗传学， 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.