We propose to carry out a definitive study on peptide selection by the class II MHC molecules, I-Ak and I-Ek. What are the chemical and cellular rules that the APC uses to select for peptides? What are the features of the protein antigen and/or the selected peptides that influence their display and their biological response? We focus on the antigenic protein hen-egg white lysozyme [HEL] - we have a large armamentarium of reagents for studying this molecule from various perspectives. The first goal is the technical exercise of identifying and quantitating the whole spectrum of HEL peptides from a B lymphoma APC line exposed to HEL. The infrastructure to carry out this goal is in place and already some peptides have been extensively examined. We have a state of the art mass spectrometry operation [M. Gross] to analyze, quantitate and sequence small amounts of peptides from class II extracts. Moreover, we have developed an improved methods for identifying peptides based on anti-peptide antibodies to capture them. The key goal here is not just to identify the spectrum of peptides, but to get at the fundamentals of their selection. Thus, each peptide will be examined for their binding features to I-A/I-E molecules - we expect to establish peptide motifs that are responsible for anchoring the peptide as well as flanking residues. Importantly we will search the residues responsible for their display by mutagenizing the HEL genes at their anchor or flanking sites and will then examine the selection of epitopes. Thus, our approaches involve i) identification of peptides, ii) mutagenesis experiments, combined with iii) chemical binding analysis with purified class II molecules. We plan to examine the role of exposed peptide segments and protein folding, whether there is intramolecular competition among peptides and competition of peptides between the class II isotypes. We also plan to examine peptides from HEL segments that are not selected ex vivo with the purpose of explaining their lack of presentation. These studies will lay the ground work that will then enable us to examine peptide selection by each of the three professional APC despite the limitations in their number. We also propose to examine and quantitate peptides from APC that lack the auxilliary molecules Invariant chain and H-2M. In summary, we expect to obtain quantitative and chemical data on HEL peptides and the explanation for their selection under a number of variables. This approach should offer a rational explanation for immunogenicity that avoids the empiricisms of results based only on T cell assays.