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J. Biol. Chem., Vol. 278, Issue 29, 27105-27111, July 18, 2003

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Enhanced Immune Presentation of a Single-chain Major Histocompatibility Complex Class I Molecule Engineered to Optimize Linkage of a C-terminally Extended Peptide^*

Lonnie Lybarger , Y. Y. Lawrence Yu  , Michael J. Miley ¶, Daved H. Fremont ¶, Nancy Myers , Tina Primeau , Steven M. Truscott , Janet M. Connolly  and Ted H. Hansen  ||

From the Departments of Genetics and ^¶Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110

Major histocompatibility complex class I molecules can be expressed as single polypeptides wherein the antigenic peptide, ₂-microglobulin, and heavy chain are attached by flexible linkers. These molecules, single-chain trimers (SCTs), are remarkably stable at the cell surface compared with native (noncovalently attached) class I molecules. In this study, we used a structure-based approach to engineer an F pocket variant SCT of the murine class I molecule K^b that presents the SIINFEKL epitope of ovalbumin. Mutation of heavy chain residue Tyr⁸⁴ (Y84A) in the SCT resulted in enhanced serological and cytolytic CD8 T cell recognition of the covalently linked peptide due to better accommodation of the linker extending from the C terminus of the peptide. These SCTs exhibit significant cell-surface stability, which we hypothesize is rendered by their ability to continuously and efficiently rebind the covalently attached peptide. In addition, we demonstrate that SCT technology can be applied to tetramer construction using recombinant SCTs expressed in Escherichia coli. SCT-based tetramers could have applications for the enumeration of T and natural killer cells that recognize peptide·class I complexes prone to dissociation.

Received for publication, April 9, 2003 , and in revised form, May 5, 2003.

^* This work was supported by National Institutes of Health Grants AI19687, AI42793, and AI27568 (to T. H. H.), Grant T32AI07163 (to L. L.), and Grant CA86803-04 (to D. H. F.) and by the Burroughs Wellcome Fund (to D. H. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Surgery Branch, NIH, Bldg. 10, Rm. 2B02, 9000 Rockville Pike, Bethesda, MD 20892.

^|| To whom correspondence should be addressed: Dept. of Genetics, Washington University School of Medicine, P. O. Box 8232, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-2716; Fax: 314-362-4137; E-mail: hansen{at}genetics.wustl.edu.

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