The purpose of this proposal is to investigate human T cell activation in normal and autoimmune states by using antibodies to the T3 and antigen receptor molecules as probes. We have evidence to suggest that treatment of T lymphocytes with anti-T3 antibodies induces the expression of receptors for IL-2 and thus renders the cells sensitive to the growth promoting effects of this lymphokine. Using the synthetic peptide approach, we have prepared heteroantisera and monoclonal antibodies to the beta chain of the antigen receptor. In preliminary studies, the heteroantisera recognize by surface immunofluorescence 20% of peripheral blood lymphocytes and T cell tumor lines, but are unreactive with B lymphocytes. In T cell lysates, they recognize a molecule of 44 Kd size, consistent with the molecular weight of the beta chain of the antigen receptor. Finally, similar to the anti-T3 antibodies, they can render T cells sensitive to the growth promoting effects of IL-2. In other studies, various samples from monoclonal antibody cultures stained 24-66% of the leukemic T cell line MOLT-3 from which the sequence of the beta chain was obtained. Furthermore, one of the supernatant cultures was able to induce peripheral T cell proliferation in the presence of exogenous recombinant IL-2. We propose to further characterize the anti-beta chain reagents by analyzing their functional effects on T cells and thymocytes, their reactivities with various cell types and the identity of their reactive molecule(s) on cell surfaces. We will further assess the effects of glycosylation on the expression of their reactive molecules. Finally, we will study their T cell subset specificity. We shall study metabolic and molecular events induced by stimulating T cells by anti-beta chain and anti-T3 antibodies. We shall examine the intracellular Ca++ mobilization as it relates to the expression of receptors for the Ca++ regulating hormone 1,25 Dihydroxyvitamin D3, as well as membrane phosphorylation events. Furthermore, we shall test for the transcriptional activation of the IL-2 and IL-2-receptor genes by hybridization with the appropriate cDNA probes. We shall test for the presence and association of the antigen receptor and T3 molecules on the surfaces and cytoplasms of early and late thymocytes, peripheral T cells and T cells from the synovial cavities of rheumatoid arthritis (RA) patients as representatives of various stages of the T cell ontogenetic development.
