Rapid epitope identification from complex class-II-restricted T-cell antigens.

The identification of CD4 + + T-cell epitopes is a laborious and tedious process. Various computer-assisted algorithms have been designed to assist in this process by predicting potential MHC-binding epitopes. Recently, the novel approach of using soluble MHC class II tetramers as a tool to identify T-cell epitopes has been developed successfully. We show that the T-cell epitopes identified using MHC tetramers are also predicted by a computer-assisted algorithm to bind to the same MHC molecule. We propose that epitope mapping using the MHC-tetramer-guided approach, in conjunction with predictive computer algorithms, will allow the rapid identification of T-cell epitopes from large proteins. The trimolecular interaction between MHC, peptide and T-cell receptor (TCR) provides the structural basis for the antigen (Ag) specificity of T-cell recognition. There are two key energetic determinants of this specificity: the binding interaction between MHC and peptide; and the binding of the TCR to the combined MHC–peptide complex. The former interaction has been studied well using peptide-binding assays to delineate the structural determinants of a peptide that allow energetically favorable binding to particular MHC molecules. Data from these studies have led to the development of computer-assisted algorithms, by which the sequences of large proteins can be scanned to predict individual peptides that might bind to distinct MHC molecules 1–5. The interaction of the TCR with MHC–peptide complexes has also been studied extensively, and has led to the recent development of MHC-tetramer technologies, in which soluble MHC–peptide complexes are used to bind to Ag-specific T cells present in heterogeneous lymphocyte populations 6–10. In the past, studies relied on a working knowledge of a particular peptide that binds to a specific MHC. Recently, soluble-MHC technology has been used to identify novel Ag-specific T cells and their restricted peptides in the absence of any knowledge of MHC-binding epitopes from the protein. MHC–mixed-peptide complexes were generated from a range of short, overlapping peptide sequences encompassing the whole protein, such that each MHC–mixed-peptide complex contains only a subset of all peptides. These complexes were used to identify and clone Ag-specific reactive T cells 11. Success in using this approach relies on the assumption that only a small fraction of the peptide sequences from any given protein is capable of binding to a particular MHC. This method of identifying Ag-specific T cells and their previously unknown epitopes has been named tetramer-guided epitope mapping (TGEM). In the light of these developments, we have examined …