To study the architecture and dynamics of immune responses as they take place within the spleen, the phenotypic and genetic hallmarks of genetically restricted immune responses may be used to characterize B cell populations in histological sections of frozen spleen. Enzyme- or fluorochrome-labeled antigen, anti-idiotope and class specific antibodies, and lectins identify the responding B cells. Individual populations of antigen- activated lymphocytes may be microdissected from labeled sections of spleen and the recovered VDJ DNA amplified by a two-step PCR using a series of nested primers. Amplified DNA can be cloned into plasmid vectors for sequencing. VDJ inserts may then be transferred to a compatible expression vector for transfection and rescue of the endogenous antibody phenotype. For the first time, a detailed anatomic, phenotypic, and molecular genetic characterization of the immune response in situ is possible. These methods will be used to study the affinity requirements for in vivo activation of antigen-specific B cells and to characterize the paratopic phenotypes that are selected in the course of thymus-dependent responses. The role of antigen concentration in the process of affinity maturation will be studied using surgically implanted osmotic pumps to deliver non-limiting amounts of antigen. These studies will test directly the paradigms of clonal selection. The observation that B cell proliferation within germinal centers may not result in Ig hypermutation will be investigated. The progeny of mutating B cell clones will be screened for the generation of novel autoimmune reactivates that arise as a consequence of mutation. Together, these studies lay the foundation for creating a true population genetics of an immune response.
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