An approach to the study of structure-function relationships of MHC class I molecules: isolation and serologic characterization of H-2Kb somatic cell variants.

Abstract

Somatic cell variants expressing an altered antigenic form of the H-2Kb molecule were isolated for the purpose of performing structure-function analysis of a class I MHC molecule. Over 25 independently isolated variants were derived from an Abelson virus transformed pre- B cell line (R8) by mutagenesis with ethyl methane sulfonate or ethyl nitrosourea. Negative selection was performed by complement-dependent cytotoxicity with anti-H-2Kb monoclonal antibodies subsequently followed by positive selection to separate the H-2Kb surface negative variants from structural variants. Biochemical characterization of a random selection of three independent variants indicated that the variant H-2Kb molecule was present in normal amounts in lysates, and was unchanged in size. Cytofluorometric analysis with the use of a panel of seven monoclonal antibodies against H-2Kb indicated that all of the variants had lost one or more alloantigenic determinants (monoclonal antibody binding sites). For these variants, the pattern of monoclonal antibody loss of recognition suggested that antibody defined alloantigenic determinants appear to be discretely localized to a single domain, either the alpha 1 or the alpha 2 domain, of the H-2Kb molecule. In contrast, CTL recognition of the Kb molecule of these variants depends on involvement of both alpha 1 and alpha 2 domains as shown in the companion paper.