The functions of the highly polymorphic classical transplantation antigens in graft rejection and in defense against viral infections have long been well recognized. In contrast, the properties and the physiological effects of the much larger family of nonpolymorphic class I-like MHC molecules are still unknown. The structural similarities of the two classes of polypeptides suggest that the nonclassical MHC molecules may bind self or nonself peptides and may interact with T-cell receptors. The identification of the proteins from which the putative peptides are derived and the recognition of the effects that they may have on cells with which they interact represent major challenges as they may lead to the formulation of new concepts in immunology. Qa-2 molecules are among the best characterized nonclassical transplantation antigens in the mouse. They exist in two forms: 1) glycophosphatidyl-inositol (GPI) linked membrane antigens which are predominantly expressed on lymphoid derived cells and can serve as targets for cytotoxic cells as well as transducers of activation signals in T cells and 2) soluble Qa-2 molecules which are released into the serum and have been hypothesized to induce activation or anergy states. The "switching" from membrane to soluble form is inducible in vitro by cell activation and involves alternative splicing of exon 5 of the Qa-2 gene. The specific aims of this proposal are: 1. To identify the cis-acting elements of the Qa-2 genes that control alternative splicing of exon 5. 2. To test a hypothesis that various regimens and signals inducing activation of immune system (i.e., lead to the alternative splicing of Qa-2 mRNA and secretion of soluble Qa-2 molecules in vivo, and to identify the major Qa-2 expressing cell subsets participating in different responses. 3. To sequence the peptides bound to the secreted and GPI-linked Qa-2 molecules in order to identify the self-antigens presented by the two Qa-2 isoforms, and to perform crystallography on the highly purified Qa-2 proteins in order to solve a 3D structure of the nonclassical MHC antigen. The studies proposed in Aims 1 and 2 will advance the understanding of the general mechanisms of inducible alternative splicing of eukaryotic genes and will provide information on specific conditions and cell subsets during immune responses that correlate with preferential production of soluble or GPI-linked Qa-2 products. These data may allow us to define the physiological functions of the Qa-2 isoforms. Studies in Aim 3 will examine directly the peptides bound to these proteins and will assess structural similarity of Qa-2 polypeptides to classical transplantation antigens. The results will be used to deduce the antigens presented by Qa-2 molecules and the parameters required for binding of protein fragments by MHC molecules. In the long term the proposed research may help to design vaccines, prevent autoimmunity and...