title: A single-chain H-2Db molecule presenting an influenza virus nucleoprotein epitope shows enhanced ability at stimulating CD8+ T cell responses in vivo.
abstract: We have generated a construct encoding a single-chain H-2D(b) mouse MHC class I molecule in which an influenza virus nucleoprotein (NP) epitope, amino acid sequence ASNENMDAM, is fused to mouse beta(2)-microglobulin and the D(b) H chain via flexible linker sequences. This single-chain trimer (SCT) was efficiently expressed at the cell surface independently of TAP and endogenous beta(2)-microglobulin, and it was recognized directly and efficiently by specific T cells in vitro. A recombinant vaccinia virus encoding the D(b) NP SCT primed a CD8(+) T cell response in C57BL/6 mice 4-fold greater than an equivalent virus expressing the NP epitope as a minigene, as shown by tetramer staining, whether or not the minigene was directed into the endoplasmic reticulum by a signal sequence. This response was functional as shown by in vivo lysis assays with peptide-pulsed target cells, and it was greatly expanded following secondary challenge in vivo with influenza virus. The SCT was also significantly more immunostimulatory for CD8(+) cells than the NP minigene in adoptive transfer experiments using F5 TCR transgenic spleen cells, in which the magnitude of the T cell response was much greater. Our results extend previous DNA vaccination studies using SCTs, which demonstrated that such molecules are capable of generating functional CD8(+) T cell responses. We have shown that class I SCTs are more immunogenic than even preprocessed Ag in the form of an epitope minigene, and they therefore should be considered for use when the generation of optimal CD8(+) T cell responses is required.
authors.affiliation: Tumour Immunology Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom;
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Palmowski
authors.forename: Mathew
authors.initials: M
authors.lastname: Parker
authors.forename: Kaushik
authors.initials: K
authors.lastname: Choudhuri
authors.forename: Christopher
authors.initials: C
authors.lastname: Chiu
authors.forename: Margaret F C
authors.initials: MF
authors.lastname: Callan
authors.forename: P Anton
authors.initials: PA
authors.lastname: van der Merwe
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Cerundolo
authors.forename: Keith G
authors.initials: KG
authors.lastname: Gould