Early events in peptide antigen handling by antigen presenting cells.

Abstract

The precise details of the processes of protein antigen degradation, intracellular trafficking of peptide epitopes and eventual association with self MHC molecules are unknown. In the first part of the research described in this thesis, the hypothesis that a previously identified putative accessory molecule, SARS (Specific Antigen Retention Structure), was involved in one or more of these processes was tested. These experiments indicated that the formation of the radiolabeled apparent peptide-protein complex that had previously been termed SARS was due to the rapid degradation of radiolabeled peptide antigens and the subsequent reincorporation of the radiolabeled amino acids into various cellular proteins even in the presence of cycloheximide. In addition, the putative SARS complex failed in a number of antigen systems in two different species to reproducibly cause peptide antigen-specific T cell stimulation. An analysis of the mechanism of peptide antigen uptake by APC showed that peptide antigens become cell associated via a mechanism that is consistent with fluid phase endocytosis (FPE). This was determined by analyzing data obtained from radiolabeled peptide uptake time course assays using a previously published mathematical model of FPE. An accurate estimate of peptide association with APC was obtained only after the radiolabeled peptide degradation and subsequent reincorporation of labeled amino acids into various cellular proteins was prevented. The unexpectedly rapid and essentially complete degradation of peptide antigens detected in these studies was investigated further. The intracellular degradation of the peptide antigens angiotensin II and PB2(146-159) was determined to be rapid and complete to the level of single amino acids. The extracellular degradation of peptide antigens was found to be selectively inhibited by the angiotensin converting enzyme (ACE) inhibitor, captopril. This inhibition may involve a number of carboxy- and aminopeptidases, including ACE. However, the presence of captopril and a mixture of other protease inhibitors was shown to be ineffective in increasing T cell stimulation in several different T cell stimulation assays, again pointing to the exquisite sensitivity of the T cell in detecting very small quantities of antigen associated with MHC molecules on antigen presenting cells.