Analysis of the expression of cloned HLA class I genes in mouse transfected L cells by quantitative flow cytometry

Abstract

By using a calibrated cell sorter and monoclonal antibodies conjugated to fluorochromes, a quantitative analysis of the levels of expression of HLA class I molecules at the surface of cloned murine L cells transfected with purified A3, B7, or C W 3 genes was performed and compared with radioimmunoassay data. We selected clones of heterogenous levels of HLA class I expression, which were shown to remain constant over a period of 4 mo in absence of HAT selection and not to be correlated to the DNA copy number of the corresponding integrated gene.Key terms: DNA mediated gene transfer, monoclonal antibodies, HLA class I gene, flow cytometry.Recent technological developments in DNA-mediated gene transfer and flow cytometry (FCM) have made possible novel approaches to the analysis of the expression and regulation of gene products (10,311 such as those of the major histocompatibility complex (6,8,18,32). When total DNA is used for transfection, populations expressing very rare cell surface antigens can be isolated by repeated sortings (lO,ll), which gives valuable material to cloning of corresponding genes.When cloned genes are used, a qualitative analysis of their products expressed at the surface of transfected cells is generally performed (2,15,20,25). Beyond this qualitative analysis there is a need for quantitative data giving the number of molecules expressed, particularly for the study of the regulation of expression. Several methods using flow cytometry that allow such a quantitative analysis have been described (3,9,17,26,34).The aims of this paper are: 1) to define, with a Calibrated cell sorter and monoclonal antibodies (mAb) conjugated to fluorochrome (171, the quantitative levels of expression of HLA class I at the surface of selected cloned transfected L-A3, B7, CW3 cells; 2) to analyse the stability of this expression; and 3) to define whether a correlation existed between the levels of expression and the number of integrated HLA genes. MATERIALS AND METHODS Monoclonal Antibodies (mAb) and Their Conjugation to FluorochromeThe origin and characteristics of the mAb used are listed in Table 1. B9.12.1, B1.23.2, BlG6, 11-4.1, and 10.3.6 mAb were conjugated to fluorescein isothiocyanate (FITC) according to the method described in reference (17). Molar fluorochrome/protein (FP) ratios of FITC-conjugated mAb, determined after correction for quenching (17), were: B9.12.1,2.0; B1.232, 2.0; Bl.lG6, 1.2; 11.4.1,2.3; and 10.3.6,l.O. Transformation of Ltk-cellsCloned Ltk-murine fibroblasts (H-2kf, Iak-) were transfected by the calcium phosphate technique (35), either with cosmids 44 or 64 (HLA-A3) (19), cosmid 42 (HLA-CW3) (19), or cosmid 37 (HLA-B7) (12), all of which contained the herpes virus thymidine kinase (tk) gene. Control HLA-t k + cells were obtained by transfection with pAG0 DNA which contains the tk gene (20). TKt transfectants were selected in HAT medium (35). Transfected cells were cloned once or twice by limiting dilution (average 0.2 cell/well) and maintained in RPMI 1640 medium supplemented with 5% f...