A novel single chain I-A(b) molecule can stimulate and stain antigen-specific T cells.

Multimers of soluble major histocompatibility complex class I and II molecules have proven to be useful reagents in quantifying and following specific T cell populations. This study describes the design, generation, and characterization of a novel, single chain I-A(b) molecule which utilizes a unique linker derived from the murine invariant chain. A fragment of the invariant chain, residues 58-85, binds to a region proximal to the class II peptide binding groove and stabilizes occupancy of the class II invariant chain-associated peptide. We have utilized this fragment, replacing CLIP with the Ealpha peptide sequence, to lock the attached peptide into the class II binding groove. The single chain I-A(b) molecule was recognized by a panel of conformation-sensitive, I-A(b)-specific, monoclonal antibodies. Membrane-bound and soluble forms of the single chain I-A(b) stimulated an antigen-specific T cell hybridoma, and tetramers made from soluble monomers stained these cells. The unique features of this molecule may be useful in the design of recombinant T cell receptor ligands containing peptides with low affinity for MHC.