Biochemical and functional characterization of soluble multivalent MHC L(d)/Fcγ1 and L(d)/Fcμ chimeric proteins loaded with specific peptides

Central to the specificity of the immune system is the interaction between the T cell receptor and the major histocompatibility complex (MHC)- peptide ligand complex. To better understand the nature of this interaction, and to investigate possible avenues for specific therapeutic intervention, we have produced soluble recombinant molecules that can modulate antigen- specific T cells. Our approach involved the construction of recombinant murine genes composed of the MHC class I gene H.2L(d) and the Fc portion of immunoglobulin (Ig) heavy chain genes μ or γ1. Stable transfectants of these L(d)/Fcγ1 and L(d)/Fcμ genes generated correctly spliced transcripts and were capable of secreting chimeric protein. Immunoprecipitation analyses demonstrated the presence of chimeric L(d)/Fcγ1 and L(d)/Fcμ monomers of approximately 69 kDa and 90 kDa, respectively, as well as chimeric dimers under nonreducing conditions. The capacity of L(d)/Ig molecules to bind specific peptide ligands was demonstrated using radiolabeled peptides or with monoclonal reagents that specifically identify peptide-induced conformational changes in the L(d) ligand binding site. Soluble divalent L(d)/Fcγ1 molecules were loaded with the murine cytomegalovirus-derived peptide and other L(d)-specific peptide ligands and subsequently isolated and purified. Peptide-loaded L(d)/Fcγ1 molecules were capable of inhibiting the response of class I-restricted T cells in vitro in a peptide-specific fashion. The development of soluble multivalent chimeric proteins that possess unique properties of both the MHC class I and Ig molecules provides a valuable reagent for the study of potential mechanisms of in vitro and in vivo immune modulation.