The capacity to distinguish self from non-self depends on the ability to generate a diverse, yet balanced, repertoire of antigen-binding receptors. The mechanisms that enhance, as well as those that constrain, diversity are thus of fundamental interest. The primary aim of this study is to define the constraints imposed upon the third complementarity determining region of the immunoglobulin heavy chain (HCDR3). HCDR3 lies at the center of the antigen-binding site and typically determines antibody specificity. At the genetic level, HCDR3 represents the focal point of diversification of the pre-immune B-cell repertoire since it is the direct product of V(D)J recombination. The DH (diversity) gene segments compose the core of HCDR3 and are potentially translatable in six reading frames, each with a characteristic hydropathicity - charged, hydrophobic, or hydrophilic. The sequence composition of DH gene segments is conserved from shark to mouse to man, and diverse mechanisms are used in these species to ensure that the final HCDR3 repertoire is enriched for a neutral, hydrophilic sequence. Utilization of charged or hydrophobic HCDR3 domains is common only in exceptional circumstances (e.g., pathogenic anti-DNA autoantibodies). The hypothesis to be tested holds that use of alternative reading frames (or hydropathicities) is deleterious due either to production of unstable H chains, to reduced generation of antibodies of beneficial specificity, or to formation of antibodies with improper specificities (e.g., autoantibodies). Cre-loxP gene targeting will be used to generate mice wherein the antibody repertoire has been globally altered to produce HCDR3 intervals encoded by alternative DH reading frames. The DH locus will be reduced to a single, frame-shifted DH gene segment. These alterations will prejudice lymphotype precursors to produce germline-encoded charged or hydrophobic HCDR3s intervals, while maintaining a normal developmental pattern of V(D)J rearrangement. The effect of this redirection on the neonatal and adult response to antigen, and on the predisposition of these mice to diseases of altered immune function, will be examined. B-cell development will be monitored in neonatal and adult mice, and in adult TdT-/- mice that lack the ability to adjust hydropathicity by N-region addition. As a functional measure of the capacity to respond normally to antigen, mutant mice will be challenged with T-independent and T-dependent antigens, and with Strep. pneumoniae and influenza virus, to determine whether alteration of the repertoire can prove lethal to the organism. Finally, the effect that alteration of HCDR3 has on the development of the natural autoantibody repertoire and on the progression of autoimmune disease in the MRL-lpr/lpr mouse will be examined.
