This project will characterize T lymphocytes infiltrating the myocardium in human heart allografts and idiopathic myocarditis. We have demonstrated that allograft infiltrating lymphocytes are highly enriched for reactivity against donor HLA antigens. In both allografts and myocarditis tissues """"""""dominant"""""""" T cell clones have been identified in Southern blot analyses using a probe for the Cbeta regions to identify gene rearrangement of the T cell receptor (TCR). We propose to determine the function and specificity of these dominant clones and attempt to demonstrate their role in the pathogenesis of myocardial injury. Since cardiac allograft patients are biopsied frequently, this system represents an almost unique opportunity to monitor the evolution of human inflammatory processes; analyzing sequential alterations during graft rejection and tolerance. Myocarditis patients are likewise re-biopsied to monitor cardiac inflammation. Populations of T lymphocytes will be selected from endomyocardial biopsies and propagated in interleukin 2 to obtain sufficient numbers of T cells from sites of heart muscle destruction to evaluate their phenotype, specificity and TCR gene rearrangement. The polymerase chain reaction (PCR) will be used to amplify cDNA of the alpha and beta genes of the TCR to determine whether there is selective V-region gene expression among the infiltrating cells and if there is any correlation with specificity for certain HLA antigens. In addition, monoclonal antibodies against the TCR, including anti-idiotypic and anti-variable region antibodies, will be produced as a means for identifying the dominant clones in the original tissues, and for detection of cross-reactive idiotypes indicative of shared TCR usage among cells with similar or different specificities. IN parallel studies on myocarditis- derived lymphocyte populations, analysis of """"""""dominant"""""""" clones of T lymphocytes at sites of inflammation may lead to identification of common antigen and selective TCR gene usage (such as Valpha or Vbeta) in idiopathic myocarditis. Monoclonal antibodies to these dominant clones could provide diagnostic and future therapeutic reagents targeted to these disease-related lymphocytes. These studies are central to questions of in vivo selection of the immune repertoire in inflammatory processes involved in the pathogenesis of cardiac injury.
