Creating CTL Targets with Epitope-Linked β2-Microglobulin Constructs

Abstract

AbstractEliciting a strong CTL response is dependent upon displaying suitably high levels of specific class I MHC/peptide complexes at the cell surface. In an effort to enhance the presentation of defined CTL target structures, two unique peptide-linked β2-microglobulin (β2m) molecules were constructed. The first, designated NP(366–374)-L8-hβ2m, links the carboxyl terminus of the H-2Db-restricted influenza nucleoprotein (NP) epitope NP366–374 to the amino terminus of hβ2m through an eight-amino acid glycine/serine linker. The second molecule, designated NP(147–155)-L12-hβ2m, similarly couples the H-2Kd-restricted influenza NP epitope NP147–155 to hβ2m via a 12-residue polypeptide linker. Transfection of the NP(366–374)-L8-hβ2m vector into H-2b-expressing cell lines sensitized these cells for lysis by NP366–374-specific CTLs. Free NP peptide could not be detected when class I bound peptides were acid-extracted from the surface of NP(366–374)-L8-hβ2m transfectants, indicating that CTL killing was mediated by recognition of the peptide linked to hβ2m and not by a degradation by-product. CTL target structure formation was also achieved by an exogenous presentation pathway. H-2d-expressing target cells were sensitized for lysis when pulsed with NP(147–155)-L12-hβ2m protein derived from an Escherichia coli cell lysate. The effect of recombinant NP(147–155)-L12-hβ2m was inhibited by competitor wild-type hβ2m, indicating that the active peptide-hβ2m fusion protein remained intact. The observation that β2m with covalently attached peptide can effectively create CTL target structures in vitro offers new possibilities for the in vivo induction of epitope-specific CTL responses by either DNA immunization or injection of the purified epitope-linked β2m.