ANALYSIS OF T CELL RECEPTOR FUNCTION BY GENE TRANSFER

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Activation of helper/inducer T lymphocytes initiates the immune response to foreign pathogens, and triggers the processes of transplant rejection and the onset of autoimmune disease. Activation occurs when these cells recognise foreign antigen in association with histocompatibility proteins on the surface of antigen-presenting cells. To be able to clinically manipulate the immune response, it is important to understand the mechanisms of antigen/MHC recognition and activation. The long-term objectives of this research are to analyse the detailed structure of antigen/MHC receptors from selected T cell clones. Arsonate-reactive inducer T cell clones will be used as a model system, since the functions of their receptors can be assayed both with antigen-binding assays and with assays for activation. Receptor genes from these clones will be transferred into other T cells using retroviral vectors, and the recipient cells will be tested for arsonate responsiveness. In future studies site-directed mutagenesis will be used to define receptor residues involved in antigen/MHC recognition and subsequent activation. The specific aims of this proposal are to subclone receptor genes of the arsonate-reactive inducer T cell clone Ar-5 into pZIP retroviral vectors, transfect them into Psi-2 and Psi-AM packaging cell lines, infect recipient T cells with recombinant retroviruses using a cocultivation procedure, and test the infected cells for acquisition of arsonate responsiveness. Two kinds of recipient cells will be used: individual genes will be transferred into antigen-unresponsive variants of clone Ar-5, which are likely to have lost expression or function of one receptor chain; and both receptor chains will be transferred into the unrelated inducer T cell clone BCC 11, which shares neither antigen nor MHC responsiveness with clone Ar-5. Acquisition of a functional clone Ar-5 receptor will be tested by arsonate binding, activation by arsonate plus I-Ad, binding and activation by clone-specific antibodies, and expression of receptor polypeptides with the righy electrophoretic characteristics.