The CD8 molecule on the surface of class I reactive T cells was initially believed to function as an accessory molecule, increasing the affinity of the TCR for class I MHC. Recent evidence suggests that CD8 molecules may associate with the TCR for antigen and thereby function in signal transduction and activation. It appears now that the CD8 receptor is also involved in both positive and negative selection of class I reactive T cells during thymic maturation. The recent observation by the principal investigator the CD8 binds to residues in the class I alpha3 domain was the first identification of the monomorphic determinant recognized by CD8. Of paramount importance in these studies was the use of the Dd alpha 3 domain mutant, Ddlys, that had lost the ability to be recognized by CD8. The primary objectives of this proposal are to probe questions concerning the role of CD8 in T cell recognition and to extend the observation that the CD8 molecule o class I-specific CTL: recognized residues in the class I alpha3 domain. Whereas the previous studies involved heterogeneous populations of CTL the new investigations will employ clonal populations of CD8-dependent and CD8-independent CTL. These clones will be analyzed for their ability to lyse cells expressing Ddlys and other alpha3 domain mutants and for the ability of antibodies to the alpha3 domain and CD8 to inhibit their function. They will also be used in cell:cell binding assays to determine their dependency on CD8 for cell binding and the effect of alpha3 domain mutants of cell binding. Additional alpha3 domain mutants obtained by site directed mutagenesis are being provided by Dr. T. Potter. These new mutants will be compared in a variety of assays and will define the extent of the class I ligand recognized by CD8. In addition, the CTL response against the class I alpha3 domain mutants will be analyzed to determine if additional residues are involved in recognition by CD8. Finally, the involvement of CD8 in the generation and effector function of MHC-restricted CTL responses will be analyzed. Thus several salient properties of CD8 will be investigated using a variety of immunologic assays and a unique collection of site-specific class I mutants.