The goal of this project is to study how a novel class of cancer therapeutics impact lymphocyte function. These compounds are active-site inhibitors of the bserine/threonine kinase mTOR (mammalian target of rapamycin). The mTOR kinase is present in two complexes, termed mTORC1 and mTORC2, with distinct substrates and function. The natural compound rapamycin is a selective mTOR inhibitor that potently suppresses lymphocyte proliferation, and is used clinically for immunosuppression. However, rapamycin is an allosteric mTORC1 inhibitor that does not acutely inhibit mTORC2. Novel, active-site (ATP-competitive) mTOR inhibitors block all functions of both mTOR complexes and have more potent cytostatic and cytotoxic effects than rapamycin in cancer cell lines. Consequently, there are worldwide efforts to develop active-site mTOR inhibitors for cancer therapy. The immunoregulatory properties of active-site mTOR inhibitors, however, have not been reported. Defining the effects of these agents on lymphocyte activation will reveal their potential as novel immunosuppressants, while also addressing concerns about host defense and anti- tumor immunity in the cancer therapy setting. Our preliminary data suggest that active-site mTOR inhibitors are selectively toxic to leukemia cells compared to nontransformed lymphocytes. In this project we will use a carefully selected set of experimental approaches to address one specific aim: to compare the effects of rapamycin and active-site mTOR inhibitors on lymphocyte activation and differentiation. We will use both in vitro and in vivo systems, and study both T cells and B cells. Inhibitors will be studied over a concentration range to facilitate comparisons of potency. For T cells, we will use T cell receptor-transgenic systems to measure proliferation in response to cognate antigen. We will employ adoptive transfer approaches to assess T cell expansion in vivo. We will also measure cytokine-mediated survival and T helper differentiation. For B cells, we will measure proliferation and survival in response to different mitogens and cytokines. We will compare the differentiation of B cells into antibody-secreting cells and their ability to produce antibodies in immunized mice. We will correlate the functional responses of lymphocytes with signal transduction events, to determine the molecular basis for inhibitor effects. PUBLIC HEALTH RELEVANCE: This project will study a newly discovered class of anti-cancer drugs and determine how they affect the immune response. This work will help us understand and predict potential side effects of these drugs on the immune system of cancer patients. In addition, we might find evidence that the drugs will be useful to treat immune-related diseases.