The purpose of our research is to explain antigen presentation by class II MHC molecules having a strong biochemical, quantitative and mechanistic foundation in order to avoid the many empiricisms that dominate this complex area. We investigated how the model protein antigen hen egg-white lysozyme (HEL) was processed by antigen presenting cells (APC); and established the chemical basis for the processing, selection by, and binding of, its various peptides to I-Ak molecules. This now places us in a situation where these parameters can be strictly related to the biology of the T cell response, i.e. to the specificities and frequencies of HEL clones.
The first aim of this renewal is to examine post translational modification of the HEL peptides induced when APC are activated by cytokines. Evidence is presented that activated APC can modify HEL to generate specific T cells to the modified peptides. The initial focus of examination is on HEL peptides in which their tyrosines and tryptophans are nitrated (or oxidized). The plans are to characterize the specificities of the T cells, the biology of the APC that induces the changes, and the biochemical nature of the changes using mass spectrometry approaches. The biology of the T cells to modified peptides will be examined in mice bearing a T cell receptor as a transgene; we intend to identify the possible role of the T cells in inflammation and tissue pathology focusing on beta cells expressing HEL. In the second aim, an explanation is sought for why the response to the various HEL epitopes does not relate to the number of peptide-MHC complex presented by APC. We can quantitate the density of the various peptide-MHC complexes from HEL: those represented at a high level induce about the same number of T cells as those presented at about one- or two-hundred fold less. We consider a possible number of explanations such as molecular competition, competition or cooperativity of the complexes on APC surface, the time of persistence of the complexes, and finally the modulating presence of the murine lysozyme, a strong cross-reactive protein expressed normally on APC. By knowing these important variables, we should be able to have a much fuller understanding of the CD4 T cell responses. The experiments are based on; i) using HEL molecules with relevant biochemical changes that alter either epitope expressions or the persistence of an epitope in APC, ii) tracing the cellular response with TCR receptor transgenic mice to more than one epitope, iii) using APC in transfer systems; and, iv) genetically ablating the murine lysozyme gene.
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