The primary objective of the proposed studies is to determine whether the Lyt-2 and Lyt-3 molecules play a role in the function and specificity of class I-specific cytotoxic T lymphocytes (CTLs) in the mouse. Long term CTL lines will be established from mice heterozygous at both the Lyt-2 and Lyt-3 loci and characterized with respect to major and cross-reacting specificity and inhibition of function by anti-Lyt-2 and anti-Lyt-3 antibodies. Lyt-2- negative mutants and Lyt-3-negative mutants of the same parent CTL line will each be derived in two steps. For example, for the Lyt-2-negative mutant, we would first select for a cell line which lost expression of Lyt-2.2 but still expressed Lyt-2.1. Then, in a second round of mutagenesis and selection, we would select for loss of Lyt-2.1 expression. This process should help us obtain structural gene rather than pleiotropic mutants. Single and double mutant cell lines will be characterized extensively for surface phenotype and Lyt-2 and Lyt-3 gene expression, and their cytolytic function and specificity will be compared with each other and with the parent line. If Lyt-2 and/or Lyt-3 play a role in increasing the affinity of low affinity CTLs for their target, some alteration in function should be observed. Comparison of Lyt-2-negative mutants (which will probably lack surface Lyt-3 as well) and Lyt-3-negative mutants (which may express Lyt-2 homodimers) of the same parental CTL line should provide information on the relative contributions of Lyt-2 and Lyt-3 to function. The cloned, expressable Lyt-2a gene (already in hand) and Lyt-3a gene (to be isolated in the present study) will then be stably transfected into appropriate mutant cell lines. We will determine whether re-expression of a functional Lyt-2 or Lyt-3 gene in the double mutant cell lines restores the function or specificity characteristic of the original parent CTL line. Expression of the protein and mRNA products of the transfected gene and of the host cell Lyt-2 and Lyt-3 genes will be analyzed to study the stoichiometry of and look for possible co-regulation of Lyt-2 and Lyt-3 antigen and gene expression.
