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Identifying the immunogenic proteins that elicit pathogen-specific T cell responses is key to rational vaccine design. While several approaches have succeeded in identifying major histocompatibility complex (MHC) class I bound peptides that stimulate CD8+ T cells, these approaches have been difficult to extend to peptides presented by MHC class II molecules that stimulate CD4+ T cells. We describe here a novel strategy for identifying CD4+ T cell-stimulating antigen genes. Using Listeria monocytogenes-specific, lacZ-inducible T cells as single-cell probes, we screened a Listeria monocytogenes genomic library as recombinant Escherichia coli that were fed to macrophages. The antigen gene was isolated from the E. coli clone that, when ingested by the macrophages, allowed generation of the appropriate peptide/MHC class II complex and T cell activation. We show that the antigenic peptide is derived from a previously unknown listeria gene product with characteristics of a membrane-bound protein.

Identification of a CD4+ T cell-stimulating antigen of pathogenic bacteria by expression cloning.