The a/fl T-cell receptor recognies a complex ligand formed by the association ofantigenic peptides with molecules ofthe major histocompatibility complex (MHC). The inherent limitations of the conventional T-cell activation assays used to detect these peptide/MHC ligands have, until now, hampered the development of expression cloning systems for T-cell antigens. To overcome these limitations, we have recently introduced a method for detecting ligand-induced activation of individual T cells. This assay, which makes use of a lacZ reporter construct, differs from conventional ligadinduced activation assays in that it allows the detection of single, activated T cells in large pools of resting cells. We applied the IacZ assay to the problem of screening expression libraries, which requires the ability to detect ligand-bearing antigen-presenting cells when they are present at very low frequency. We show here that ligand-expressing antigenpresenting cells can be detected at frequencies of 1:103-104, a level of sensitivity compatible with the screening of cDNA libraries. Furthermore, by using as antigen-presenting cells COS-7 cells stably transfected with the murine Kb class I MHC molecule, we demonstrate that transiently expressed ovalbumin is efficiently processed and presented to an ovalbumin/Kb_ specific T-cell hybridoma. IacZ expression is induced in a detectable number of cocultured T cells, even when the ovalbumin cDNA consists of only 1:104 of the total DNA used to transfect the COS cells. These results suggest that unknown T-cell antigens may be identified by screening cDNA libraries in MHC-expressing COS cells using lacZ-inducible T cells as indicators of peptide antigen expression.The a/X3 T-cell receptor (TCR) recognizes small antigenic peptides bound to major histocompatibility complex (MHC)

The nuclear factor of activated T cells (NFAT) enhancer element of the IL-2 gene can regulate expression of the Escherichia coli lacZ reporter gene in activated T cells. Based upon this observation, we showed that this inducible NFAT--lacZ construct could be used to measure TCR mediated, ligand-specific activation in single T cells. Here we describe a general approach to obtaining lacZ inducible, T cell hybrids by generating two new fusion partners BWZ.36 and BWZ.36 CD8 alpha derived by transfecting alpha-beta-BW5147 cells with the NFAT--lacZ construct. Using these fusion partners and normal T cells from immunized mice, we obtained T cell hybrids in which lacZ activity is specifically induced in response to antigen/MHC class II or MHC class I complexes. We show that measuring ligand induced T cell activation by the non-radioactive lacZ assay is simpler, faster, and most cost-effective relative to conventional IL-2 assays. Most importantly, the unique ability to detect activation of single T cells by the lacZ assays permits detection of rare antigen presenting cells and thus provides the basis for developing expression cloning strategies for identifying unknown T cell antigens.

CD4+ T cells recognize major histocompatibility complex (MHC) class II-bound peptides that are primarily obtained from extracellular sources. Endogenously synthesized proteins that readily enter the MHC class I presentation pathway are generally excluded from the MHC class II presentation pathway. We show here that endogenously synthesized ovalbumin or hen egg lysozyme can be efficiently presented as peptide-MHC class II complexes when they are expressed as fusion proteins with the invariant chain (Ii). Similar to the wild-type Ii, the Ii-antigen fusion proteins were associated intracellularly with MHC molecules.Most efficient expression of endogenous peptide-MHC complex was obtained with fusion proteins that contained the endosomal targeting signal within the N-terminal cytoplasmic Ii residues but did not require the luminal residues of Ii that are known to bind MHC molecules. These results suggest that signals within the Ii can allow endogenously synthesized proteins to efficiently enter the MHC class II presentation pathway. They also suggest a strategy for identifying unknown antigens presented by MHC class II molecules.T cells recognize antigens as peptides bound to major histocompatibility complex (MHC) molecules on the surface of antigenpresenting cells (APCs). CD4+ and CD8+ T cells recognize antigens bound to MHC class II and MHC class I, respectively. In addition to providing immunity against foreign pathogens, both CD4+ and CD8+ T cells are implicated in the destruction of self tissues that leads to autoimmune disorders (1). Identification of these peptides and their donor proteins that elicit T-cell immunity is essential for development of vaccines and understanding the role of these antigens in autoimmunity.Antigens that stimulate T cells can in principle be identified by sequencing the purified MHC-bound antigenic peptide. This biochemical approach is, however, made difficult by the fact that many different peptides are bound to MHC on the APC surface, and individual peptides represent only a very small fraction of the total (2-4). We have proposed (5) an alternative genetic strategy for identifying T-cell-stimulating antigens that does not depend upon biochemical purification of peptides but instead, relies upon expression of peptide-MHC complexes in APCs transfected with the antigen genes. The generation of peptide-MHC class I complexes from transiently expressed genes and the ability to detect rare APCs with the single-cell lacZ T-cell assay suggested that these methods could serve as an expression cloning strategy to identify antigens recognized by CD8+ T cells (5,6). Indeed this strategy has allowed the identification of several antigens that are presented by MHC class I molecules (ref. 7 and S. Malarkannan, M. Afkarian, and N.S., unpublished data).The efficiency of peptide-MHC expression after gene transfer is central to expression cloning strategies. Not only should the genes transferred cause expression of peptide-MHC complexes, but this should occur in a large fraction of APCs to pe...

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...

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