This proposal will follow two sets of goals which represent continuation of studies initiated in the previous granting period, namely the propagation and cloning of T lymphocytes with specific function and the elucidation of the mechanisms inherent in the epitope restricted unresponsiveness exhibited by A.TL mice to beef cytochrome c. The first of these goals will take advantage of the capacity of primed T cells to proliferate in vitro using defined culture conditions and focus on such questions as whether F1 primed T cells which exhibit T helper function can be selected into individual populations which recognize antigen in the context of F1, parental A or parental B haplotype, whether such cells can be cloned and demonstrate functional activity and whether T suppressor lymphocytes can be similarly propagated and cloned. Inherent in these approaches will be the development and refinement of cellular assays capable of assessing T lymphocyte functions. The second principal goal will be to delineate the cellular basis underlying the observations that the unresponsive phenotype exhibited to beef cytochrome c by mice of the A.TL haplotype can be dissected into the capacity to respond to determinant(s) located on one peptide of the cytochrome c molecule but only when that peptide is presented away from the context of yet another peptide residue. The thesis to be tested is whether it is possible to demonstrate that, within the context of the A.TL haplotype, one set of determinants located in one part of beef cytochrome c induces suppressor T lymphocytes whose activity is dominant over T proliferating (? T helper) lymphocytes induced by determinants located in another part of the molecule. Mapping specific amino acid residues involved in the dominant suppressor effect will be attempted using specie variants of cytochrome c, peptide fragments and hybrid molecules which can be reconstituted from the appropriate peptides of different species of cytochrome c.