In certain strains of inbred mice immune responses are dominated by antibodies that share common variable region structures (idiotypes) detected serologically by anti-idiotypic reagents. Idiotypic determinants characterizing certain antibody specificities have proven valuable structural and genetic markers in studies of antibody diversity and regulation. Jerne proposed that interactions between idiotype and anti-idiotype regulate immune responses through recognition of idiotypic determinants, rather than by regulation of specific antigen combining sites. The major cross-reacting idiotype (IdCR) in strain A/J mice immunized with p-azophenylarsonate (Ars) is heritable and encoded by germline genes. The Ars system is a model for examining regulations defining the structural epitopes responsible for idiotypy and antibody structure changes involved in enhanced antigen affinity occurring temporally during the immune response (affinity maturation). A second idiotype family (Id36-60) among anti-Ars antibodies shared in A/J and BALB/c strains is serologically and structurally distinct from IdCR, representing an independent marker for studies of regulation. The germline VH genes for both IdCR and Id36-60 were cloned and sequenced. By appropriate selection methods, the feasibility of isolating molecules in which idiotype and antigen binding are dissociated, as well as obtaining variants with enhanced affinity was demonstrated. Further the 3-dimensional structure of an IdCR Fab fragment is at hand. Therefore, we will 1) Define the molecular site of IdCR by determining V region structures of anti-Ars molecules which lack idiotype despite use of IdCR associated gene segments, 2) Characterize the site and temporal pattern of somatic mutation and its contribution to affinity changes among IdCR+ HP, utilizing a) IdCR+ Ars-binding HP obtained during primary and secondary immune responses, b) Spontaneous mutants in cell culture which do not bind Ars or demonstrate enhanced binding. 3) Characterize the pattern of structural changes seen during affinity maturation among Id36-60 antibodies to assess the role of antigen driven selection in explaining why IdCR dominates Id36-60, 4) Examine the contribution of gene junctional diversity to Ars-binding by selecting of unmutated primary response antibodies with junctional changes. 5) These studies are correlated with alterations in idiotype and Ars-binding induced by site-directed mutagenesis and with x-ray crystallographic analysis.
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