A novel experimental animal model of drug-induced autoimmunity has been produced by injecting procainamide-hydroxylamine (PAHA) into the thymus of normal mice. The earliest sign of humoral autoimmunity was the appearance of antibodies to denatured DNA, followed by IgG anti-chromatin antibodies when a second intrathymic injection of PAHA was performed. It is proposed that self-tolerance normally accompanies positive selection of T cells in the thymus, and generation of chromatin-reactive T cells is the first or most sensitive indicator of disruption of this process by PAHA. The current application will evaluate the hypothesis that chromatin-reactive T cells provide helper function for denatured DNA-specific B cells, driving them by somatic mutation to anti-chromatin specificity. The T cell dependency of the autoantibody response will be tested by introducing anti-CD4 and anti-gp39 antibodies in vivo, which are known to interfere with immune responses mediated by T-helper cells. The capacity of denatured DNA-specific B cells, primed in vivo with a denatured DNA-protein complex, to present antigen to naive and activated peripheral T cells expressing a transgenic T cell receptor specific for an epitope in the protein will be measured by T cell proliferation and autoantibody secretion assays. The proposed evolution of the autoantibody response from anti-dDNA to anti-chromatin specificity driven by chromatin-reactive T cells and endogenous chromatin will be traced by sequencing the immunoglobulin genes of the expressed autoantibody repertoire immortalized in B cell hybridomas. Determination of the sequence of the alpha and beta chains of the T cell receptor in chromatin-reactive T cell clones and hybridomas immortalized before and after in vivo expansion will provide insight into the importance of endogenous self-antigen in the periphery. Finally the role of activation-induced cell death in the B and T cell compartments in limiting the autoantibody response will be examined in mice carrying a defective Fas gene. Findings from these studies will produce a fundamental understanding of one way autoimmunity can develop and hold promise for revealing which tolerance mechanisms are defective in human lupus-like disease.
