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.
