SOMATIC GENETICS OF T CELL IMMUNITY

Duke University, Durham NC

Investigators

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Abstract

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.