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Phosphatidyl inositol-linked forms of a murine class I MHC molecule expressed on Chinese hamster ovary cells retain
M L Fahnestock1, J M Dadgari, M McMillan
1Howard Hughes Medical Institute, California Institute of Technology, Pasadena 91125.
A gene encoding a phosphatidyl inositol-linked form of the murine class I MHC molecule H-2Kd was constructed and the protein expressed in Chinese hamster ovary cells together with murine or human beta 2-microglobulin (beta 2m). The resulting lipid-linked class I heterodimers can be efficiently converted into a soluble form by treatment of transfected cells with a phospholipase. Cells expressing Kd heterodimers were characterized with respect to heavy chain levels at the cell surface, peptide binding, and recognition by Kd-specific antibodies and alloreactive cytotoxic T cells. All transfectants bound a 3H-labeled Kd-restricted nonamer peptide, although more peptide bound to cells expressing the Kd/human beta 2m combination, perhaps because of a greater number of empty molecules at the cell surface. A dissociation constant of 5 x 10(-8) M derived by Scatchard analysis is within the range expected for interactions of peptides with class I MHC molecules. Alloreactive cytotoxic T cells which recognize wild-type Kd on murine cells lysed the hamster cells expressing lipid-linked Kd without regard to the species of the beta 2m light chain. These results indicated that the engineered lipid-linked Kd molecule is expressed at the cell surface, is recognized by antibodies and T cells, and functions to bind peptide.
A gene encoding a phosphatidyl inositol-linked form of the murine class I MHC molecule H-2Kd was constructed and the protein expressed in Chinese hamster ovary cells together with murine or human beta 2-microglobulin (beta 2m). The resulting lipid-linked class I heterodimers can be efficiently converted into a soluble form by treatment of transfected cells with a phospholipase. Cells expressing Kd heterodimers were characterized with respect to heavy chain levels at the cell surface, peptide binding, and recognition by Kd-specific antibodies and alloreactive cytotoxic T cells. All transfectants bound a 3H-labeled Kd-restricted nonamer peptide, although more peptide bound to cells expressing the Kd/human beta 2m combination, perhaps because of a greater number of empty molecules at the cell surface. A dissociation constant of 5 x 10(-8) M derived by Scatchard analysis is within the range expected for interactions of peptides with class I MHC molecules. Alloreactive cytotoxic T cells which recognize wild-type Kd on murine cells lysed the hamster cells expressing lipid-linked Kd without regard to the species of the beta 2m light chain. These results indicated that the engineered lipid-linked Kd molecule is expressed at the cell surface, is recognized by antibodies and T cells, and functions to bind peptide.