These studies are designed to examine how antigen presenting cells (APC) are able to efficiently process lemA, a bacterial polypeptide produced by Listeria monocytogenes (LM), and present it to CD8 T cells. We also will test the feasibility of using functionally important elements borrowed from lemA to enhance the antigenicity of other class I-restricted peptide antigens. LemA, unlike most peptide antigens that are presented to murine CD8 cells by the MHC molecules H2K, D, and L, is presented by a novel class Ib MHC molecule designated H2M3. LemA, a hydrophobic 185-amino-acid polypeptide of unknown function, and its 6 N-terminal amino acids have been identified as the immunogenic component recognized by H2M3-restricted CD8 effectors. Mice infected with LM regularly generate a significant lemA-specific CD8 response, and these cells can protect mice against infection confirming the physiologic relevance of this antigen in vivo.In prior studies, we have documented that native lemA has several highly unusual properties. The immunogenic core of the molecule is profoundly resistant to degradation by a variety of proteolytic enzymes including proteinase K. Yet, unlike most class I-dependent antigens, which can not be processed and presented to CD8 as exogenous antigens, lemA is processed and presented by a range of APC via a novel brefeldin- and pepstatin-sensitive, TAP1/2-independent pathway.To gain insight into the mechanisms underlying processing, we have, during the past year, constructed and expressed a truncated lemA product, containing only the immunogenic amino-terminus and adjacent transmembrane portion of lemA (31 amino acids) linked to a histidine tag to facilitate purification. Our studies to date have established that the resulting small (46 amino acid) construct retains all the distinctive functional properties of native lemA described above. Using site-directed mutagenesis, we have placed other amino acid sequences in the N-terminal location, and demonstrated that these chimeric molecules retain the specificity conferred by their aminoterminal peptide. They are processed and presented, however, via the distinctive pepstatin and brefeldin-sensitive pathway described above. Preliminary studies suggest that lemA constructs are highly effective in generating a CD8 immune response in mice, even in the absence of exogenous adjuvants.During the coming year, we hope: 1.) to define with more precision the cellular biology of lemA processing and presentation; and 2.) to test in greater detail the efficacy of these products in inducing immunity in mice against CD8 epitopes. We are particularly interested in assessing the relative efficacy of lemA-chimer products in stimulating mucosal immunity at sites such as the GI tract, where local proteases would be expected to rapidly degrade more conventional peptide antigens. If these products can be shown to have high immunogenicity in vivo, they may ultimately have a variety of practical applications in enhancing host CD8 immunity.
