It is proposed to establish a molecular biologic core that will perform analyses of the genes encoding the MHC and TCR molecules that are intrinsic to the objectives of each of the five projects in the Autoimmune Disease Center. The DNA sequence and T-cell repertoire analysis that are directed to characterizing the central steps of cognitive autoimmune recognition and its regulation should facilitate study of the magnitude of autoreactive and other T-cell clonal expansion in these diseases, analysis of mechanisms used by T-cells to regulate the emergence of autoreactive T-cells and provide improved delineation of the genetic basis of susceptibility too autoimmune disease. The TCR repertoire analysis will be studied either through isolation of T-cell subpopulations through their expression of particular differentiation or activation markers or through the establishment and clonal analysis of the in vitro response of T-cell populations to candidate autoantigens. In this way, the core will specifically enhance the ability of the Center's investigators to address questions involving the presence, number, activity and specificity of autoreactive T-cells and how they are affected by novel therapeutic agents. Similarly, the core will facilitate the transfer of the insights gained in murine research into human work, and vice verse, especially those that have developed in murine systems in the Columbia program project """"""""Pathogenic Mechanisms in Autoimmunity"""""""". The TCR repertoire will be delineated using the combined approach of CDR3 length distribution analysis and direct sequencing of the TCR chains. Additionally, the core will provide sequence-based typing of class I and class II MHC alleles of patients and other experimental populations for the purpose of delineating the molecular basis of susceptibility that can be used to stratify cases into similar subsets or identifying the structures likely to present candidate autoantigens in fundamental immune recognition events. Where relevant the core will provide additional genotyping information considered important to disease susceptibility, such as in SLE and TIDM. This attempt to characterize the genetic basis of autoimmunity parallels the clinical effort to provide precise clinical and immunological phenotypes in an effort to increase the precision of the studies. In addition, the core will also provide a variety of support services, such as providing clones and constructs of certain genes, such as MHC genes used in transfection experiments. The core is strongly organized along a thematic scientific principle with a considerable amount of experimental design and interpretation of results occurring within the core. However, the overarching purpose of this core is to enable each of the projects to approach the understanding of autoimmunity at a clonal level.
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