Lab Research Focus
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. Based on the presumption that processes central to the development of the immune response will be conserved across evolution, our approach has been to define and contrast development of the antibody repertoire in the human to other species in order to identify conserved elements that are likely to define essential structure/function relationships. These studies have led us to focus on 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). In order to test the hypothesis 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), we are using Cre-loxP gene targeting to generate mice wherein the DH locus has been reduced to a single, frameshifted DH gene segment. In these mice, mutations have been introduced into the remaining DH gene segment, forcing lymphocytes 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, is being examined. A second focus in the laboratory is the use of classic reverse genetics techniques to study human immune deficiency disorders.
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