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

Abstract

SummaryIdentifying 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 .D4+ and CD8 + T cells recognize peptide/MHC complexes on the surface ofAPCs and are normally required for protective immunity. The peptide antigens presented by MHC class 11 and recognized by CD4+ T cells are ofparticular interest in that CD4+ T cells have been shown to play a central role in immunity to bacterial pathogens as well as in autoimmune diseases (1-3) . The existence of thousands of different peptides bound to the MHC class II molecules on the cell surface, however, makes it very difficult to identify the unique antigenic peptide that is recognized by any one particular T cell (4-6) .In contrast to successful identification of several antigen/ MHC class I complexes recognized by CD8 + T cells (7-11), available methods for identifying CD4 + T cell-stimulating antigens have had little success . The difficultie result primarily from conventional methods for generating peptide/ MHC class II complexes in APCs and for detecting these APCs by T cell activation assays . In conventional bulk assays, CD4+ T cell responses are usually detected only when APCs are incubated with purified proteins in the nano-to micromolar concentration range . With the exception of the abundantly expressed ribosomal L9 protein that was recently identified as the source ofa peptide/Ek complex recognized by tumor-specific CD4 + T cells (12), antigenic proteins from complex extracts have been difficult to obtain in amounts and purity sufficient for their identification . Likewise, antigenic peptides eluted from MHC class II are difficult to purify to homogeneity because only small amounts are expressed by the APCs and they are heterogenous with respect to size (amino acids 13-25) (references 4-6, 13) . Alternative antibody-based strategies suffer from the additional disadvantage that only proteins that share B and T cell epitopes can be identified, and it is possible that the proteins that elicit protective T cell immunity are different from those that induce a humoral immune response.We describe here a novel and simple strategy for gene...

A recombinant E. coli vaccine to promote MHC class I-dependent antigen presentation: application to cancer immunotherapy

“…[24][25][26] In addition to utilising this system as a vaccination strategy for tumour immunotherapy, infecting DCs with recombinant E. coli/LLO may represent an efficient method for identifying new tumour antigens from either potential cDNA candidates specifically expressed on tumours that could be identified by microarray analysis, or by screening tumour cDNA libraries against tumourspecific CTL. A similar approach has already been utilised to identify novel human CD4 + -restricted antigens from Mycobacterium tuberculosis, 27,28 and the addition of LLO would allow the identification of antigens recognised by CTLs.…”

The Toxoplasma gondii Peptide AS15 Elicits CD4 T Cells That Can Control Parasite Burden

“…Identification of the cognate antigen recognized by BTg01Z.A hybridoma by expression cloning. We have previously shown that recombinant Escherichia coli expressing a variety of proteins can serve as an exogenous antigen source for MHC class II presentation pathway in bone marrow-derived macrophages or DCs (BMDCs) and that detection of these antigens with the appropriate T cell hybridoma can be used to identify the relevant antigen (8,50,51). We used this approach to screen a T. gondii cDNA library (see Materials and Methods for details).…”

T Cell Expression Cloning of aMycobacterium tuberculosisGene Encoding a Protective Antigen Associated with the Early Control of Infection

“…The use of a proven protective T cell line as the readout of the T cell expression cloning approach is an attractive alternative. This approach, initially developed for the identification of a leishmanial and a listerial gene encoding T cell Ags (45,46), was recently described as a powerful strategy for the direct screening and cloning of genes from a M. tuberculosis genomic expression library (36) using human M. tuberculosis reactive T cell lines as readouts. Here, the use of this technology, employing a proven protective murine CD4 ϩ T cell line, resulted in the cloning of several potentially protective genes.…”