Creating CTL targets with epitope-linked β2-microglobulin constructs

RA Uger, BH Barber - The Journal of Immunology, 1998 - journals.aai.org
RA Uger, BH Barber
The Journal of Immunology, 1998journals.aai.org
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 …
Abstract
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.
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