Phosphatidyl inositol-linked forms of a murine class I MHC molecule expressed on Chinese hamster ovary cells retain peptide binding capability and alloreactivity.
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.
Fahnestock
M L
ML
Howard Hughes Medical Institute, California Institute of Technology, Pasadena 91125.
Dadgari
J M
JM
McMillan
M
M
Bjorkman
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003601:Cytotoxicity Tests, Immunologic; D005434:Flow Cytometry; D006183:H-2 Antigens / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D010716:Phosphatidylinositols / Q000737:chemistry*; D011994:Recombinant Proteins / Q000276:immunology; D014407:Tumor Cells, Cultured
D006183:H-2 Antigens; C024755:H-2K(K) antigen; D010455:Peptides; D010716:Phosphatidylinositols; D011994:Recombinant Proteins