Creating CTL targets with epitope-linked beta 2-microglobulin constructs.

Eliciting 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 β 2 -microglobulin (β 2 m) molecules were constructed. The first, designated NP(366–374)-L8-hβ 2 m, links the carboxyl terminus of the H-2D b -restricted influenza nucleoprotein (NP) epitope NP 366–374 to the amino terminus of hβ 2 m through an eight-amino acid glycine/serine linker. The second molecule, designated NP(147–155)-L12-hβ 2 m, similarly couples the H-2K d -restricted influenza NP epitope NP 147–155 to hβ 2 m via a 12-residue polypeptide linker. Transfection of the NP(366–374)-L8-hβ 2 m vector into H-2 b -expressing cell lines sensitized these cells for lysis by NP 366–374 -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-hβ 2 m transfectants, indicating that CTL killing was mediated by recognition of the peptide linked to hβ 2 m and not by a degradation by-product. CTL target structure formation was also achieved by an exogenous presentation pathway. H-2 d -expressing target cells were sensitized for lysis when pulsed with NP(147–155)-L12-hβ 2 m protein derived from an Escherichia coli cell lysate. The effect of recombinant NP(147–155)-L12-hβ 2 m was inhibited by competitor wild-type hβ 2 m, indicating that the active peptide-hβ 2 m fusion protein remained intact. The observation that β 2 m 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 β 2 m.