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Creating CTL Targets with Epitope-Linked ß₂-Microglobulin Constructs¹

Department of Immunology, University of Toronto, Toronto, Canada

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 ß₂-microglobulin (ß₂m) molecules were constructed. The first, designated NP(366–374)-L8-hß₂m, links the carboxyl terminus of the H-2D^b-restricted influenza nucleoprotein (NP) epitope NP_366–374 to the amino terminus of hß₂m through an eight-amino acid glycine/serine linker. The second molecule, designated NP(147–155)-L12-hß₂m, similarly couples the H-2K^d-restricted influenza NP epitope NP_147–155 to hß₂m via a 12-residue polypeptide linker. Transfection of the NP(366–374)-L8-hß₂m 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-hß₂m transfectants, indicating that CTL killing was mediated by recognition of the peptide linked to hß₂m 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-hß₂m protein derived from an Escherichia coli cell lysate. The effect of recombinant NP(147–155)-L12-hß₂m was inhibited by competitor wild-type hß₂m, indicating that the active peptide-hß₂m fusion protein remained intact. The observation that ß₂m 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 ß₂m.

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