Phosphatidyl inositol-linked forms of a murine class I MHC molecule expressed on Chinese hamster ovary cells retain peptide binding capability and alloreactivity

Abstract

A gene encoding a phosphatidyl inosltoHlnked form of the murine class I MHC molecule H-2K^d was constructed and the protein expressed in Chinese hamster ovary cells together with murine or human β2-mlcroglobulln (β2m). The resulting llpld-llnked class I heterodlmers can be efficiently converted into a soluble form by treatment of transfected cells with a phosphollpase. Cells expressing K^d heterodlmers were characterized with respect to heavy chain levels at the cell surface, peptide binding, and recognition by K^d-speclflc antibodies and alloreactlve cytotoxlc T cells. All transfectants bound a ^3H-labeled K^d-restr1cted nonamer peptide, although more peptide bound to cells expressing the K^d/human βm combination, perhaps becafse of a greater number of empty molecules at the cell surface. A dissociation constant of 5 × 10^(−8) M derived by Scatchard analysis Is within the range expected for interactions of peptldes with class I MHC molecules. Alloreactlve cytotoxlc T cells which recognize wild-type K^d on murine cells lysed the hamster cells expressing llpld-llnked K^d without regard to the species of the βm light chain. These results indicated that the engineered llpld-llnked K^d molecule Is expressed at the cell surface, Is recognized by antibodies and T cells, and functions to bind peptide.

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