Differentiation Antigens, Hlaand .82-Microglobulin
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Proc. NatL Acad. Sci. USA Vol. 80, pp. 524-528, January 1983 Immunology Stable transformation of mouse L cells for human membrane T-cell differentiation antigens, HLA and .82-microglobulin: Selection by fluorescence-activated cell sorting (gene cloning/Leu-1, Leu-2 transformants) PAULA KAVATHAS AND LEONARD A. HERZENBERG Department of Genetics, Stanford Univeristy School of Medicine, Stanford, California 94305 Contributed by Leonard A. Herzenberg, October 12, 1982 ABSTRACT We isolated stable transformants ofmouse L cells To enrichfor transformants in general, we cotransformed mouse expressing human cell surface differentiation antigens by using L cells with total cellular DNA and with the herpes simplex immunofluorescence with monoclonal antibodies and selection thymidine kinase (TK) gene and selected for TK' transformed with a fluorescence-activated cell sorter (FACS). Mouse L cells cellsbygrowthinhypoxanthine/aminopterin/thymidine (Hyp/ (TK-) were cotransformed with human cellular DNA and the Apt/Thd) medium. Initially, HLA class I and human p2-mi- herpes simplex virus thymidine kinase (TK) gene. TKV transfor- croglobulin (132m) transformants were selected to establish pro- mants were first selected. The TKV populations were stained with cedures for selecting rare transformants from populations of various fluorescent antibodies to membrane antigens, and positive TKV transformed cells. Later, selection for transformants ex- cells were sorted and cloned by using a FACS. Transformants for T-cell differentiation was The fre- HLA class I antigens, for f32-microglobulin, and for the T-cell dif- pressing antigens imposed. ferentiation antigens Leu-1 and Leu-2 were isolated. The fre- quency of antigen transformants for both Leu-1 and Leu-2 an- quency of antigen transformants among the TKV transformants tigens among the TKV transformants was about 0.5 x 10-3. This was about 0.5 x 10-3. The sizes of the HLA, Leu-1, and Leu-2 frequency is high enough so that appropriately stained Leu molecules expressed by the transformants were the same as those transformants can be detected by the FACS and directly cloned of the proteins present on DNA-donor cells. into microtiter wells. FACS selection of transformants for cell surface antigens should be generally applicable. Gene transformation of mammalian cells has been a useful tool Because the transformants are stable, the differentiation an- for cloning certain genes (1, 2) and for analysis of transferred tigens produced by them can be analyzed biochemically and genes and their products (3, 4). However, an appropriate se- compared with the corresponding molecules in T cells. The lection system is needed to transfer genes with total cellular Leu-1 molecule is a 70-kilodalton (kDal) polypeptide and the DNA because the frequency of transfer of single-copy genes Leu-2 molecule is a disulfide-linked heterodimer of 75-80 kDal is quite low, 1-10 X 10-7 (5, 6). Biochemical and morphological (12). Molecules specifically immunoprecipitated from several selection systems have been widely used to identify and clone ofthe Leu-1 and Leu-2 transformants had the same polypeptide oncogenes and genes coding for certain enzymes (7-10). chain compositions and apparent molecular masses as the cor- Recently, Chang et al. (11) showed that transient expression responding molecules on the human donor cells. Two chains oftwo human differentiation antigens can be detected in mouse of the Leu-2 molecule were present. Thus, these antigens ap- L cells (a transformed fibroblast line) after transformation with pear to be synthesized by the mouse L cells identically to the human cellular DNA. The antigens My-i (granulocyte specific) DNA-donor T cells. and OKT3 (T-cell specific) were detected with fluorescent monoclonal antibodies and a fluorescence microscope. How- METHODS ever, expression was lost after 72 hr so that established pro- Cell Lines. TK- mouse L cells were originally established cedures for gene cloning oftransfected genes and certain kinds by Kit et aL (17). They were grown in Dulbecco's modified Ea- ofgenetic analysis are not feasible because stable transformants gle's medium/10% fetal calf serum containing penicillin (100 are required. units/ml) and streptomycin (100 ,g/ml). Cells were collected In this paper, we report the isolation of stable transformants either by incubating them at 37°C for 5 min in 0.25% trypsin of mouse L cells expressing the human T-cell differentiation (GIBCO) or by rinsing the dishes with phosphate-buffered sa- line (Pi/NaCl) and adding warm (37°C) P1/NaCl/0.6 mM antigens Leu-1 and Leu-2 after transformation with human T- Na2EDTA for 5 min at 37°C. Cells were dislodged by vigorous cell DNA. Leu-1, also called OKT1, is expressed on all T lym- pipetting. phocytes (12) and a subpopulation of B lymphocytes (13) while The JM line was established from a patient with acute lym- Leu-2, also called OKT5 (-T8), is expressed on a subset ofT cells phoblastic leukemia ofT-cell origin and was made available by that have suppressor or cytotoxic function. Leu-2 appears to J. Minowada (Roswell Park Memorial Institute, Buffalo, NY). play a role in recognition offoreign antigen by cytotoxic T cells Its HLA type is A3, A25, B7, B37, C4, and it also expresses the (14). T-cell antigens Leu-1-Leu-6 but does not have detectable sur- To identify and select transformants for cell surface antigens, face HLA-DR. we used fluorescein-conjugated monoclonal antibodies and a Reagents. The fluorescein-conjugated mouse anti-human fluorescence-activated cell sorter (FACS). The sorter has been Leu reagents were obtainedfrom Becton Dickinson Monoclonal used previously to isolate cells at frequencies of 10-7 (15, 16). Abbreviations: FACS, fluorescence-activated cell sorter; TK, thymi- The publication costs ofthis article were defrayed in part by page charge dine kinase; Hyp/Apt/Thd, hypoxanthine/aminopterin/thymidine; payment. This article must therefore be hereby marked "advertise- R32m, /3%-microglobulin; kDal, kilodalton(s); Pi/NaCI, phosphate-buff- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. ered saline; kb, kilobase(s). 524 Downloaded by guest on September 28, 2021 Immunology: Kavathas and Herzenberg Proc. Natd Acad. Sci. USA 80 (1983) 525 Antibody Center (Mountain View, CA). Monoclonal antibodies TK gene on plasmid pBR322. TK' transformants were selected W6/32 and BBM1 were provided by Frances Brodsky and Pe- by growth in Hyp/Apt/Thd medium. After 3 wk, there were ter Parham (Stanford University). Reagents were centrifuged roughly 2 x 103 colonies per dish per 106 cells initially plated. at 100,000 x g for 10 min in an Airfuge before use. HLA and fi2m Transformant Isolation. To test our ability Immunofluorescence Staining and FACS Analysis. Cells to isolate transformants coding for cell surface antigens by using were collected in Pi/NaCl/EDTA and chilled on ice. For sort- the FACS, we first screened for transformants expressing HLA ing, one million cells were stained with saturating amounts of class I molecules and human f32m. We expected these antigens antibody for 30 min at 0C in 100 ,4 ofstaining medium [biotin- to be expressed in L cells because the homologous antigens H- free RMPI 1640 medium (Irvine Scientific)/10 mM Hepes, pH 2 and f32m are expressed by the L cells. In addition, monoclonal 7.4/0.1% NaN3/2% heat-inactivated serum]. Cells were washed antibodies such as W6/32 and BBM1, which detect aframewor! with staining medium and stained with fluorescein-conjugated (monomorphic) determinant on the HLA heavy chain (28) or on goat anti-mouse Ig for 30 min. Propidium iodide (Calbiochem- human f32m (29), respectively, are not affected by association Behring, La Jolla, CA) was added to a final concentration of 1 of the human HLA heavy chain with mouse 32m or the mouse ,uM. After 5 min, the cells were centrifuged and washed with H-2 heavy chain with human f32m, as shown by work with so- staining medium. For routine analysis, round-bottomed poly- matic cell hybrids (30). Samples of 106 cells from nine dishes propylene microtiter plates (Dynatech Laboratory, Alexandria, were allowed to react with W6/32 or with BBM1 for the first VA) containing 5 X 105 cells per 0. 1-ml well were used. step and with fluorescein-conjugated goat anti-mouse antibody Cells were analyzed and sorted on a FACS II (Becton Dickin- as the second step. Viable cells were then analyzed with the son FACS Systems, Mountain View, CA) modified to include FACS. Because dead cells pick up fluorescent proteins non- a logarithmic amplifier and a direct cloning attachment (18). A specifically, we eliminated all such cells from analysis by in- 540-nm shortpass filter (Ditric Optics, Marlboro, MA) was used cluding (in the last wash of the cells prior to passage through to reduce autofluorescence ofthe L cells relative to fluorescein the FACS) the red nucleic-acid-intercalating dye propidium io- fluorescence. A second detector receiving light above 580 nm dide, which penetrates only cells with damaged membranes and was used to detect propidium iodide. The signals from this de- thus stains dead cells red (31). Red propidium iodide-stained tector were electronically balanced to subtract any signal due cells were then electronically gated out. It is important to ex- to fluorescein. clude the unavoidable few percent ofdead cells when detecting For fluorescence microscope work, 1 to 2 X 105 cells were and sorting stained cells occurring at a low frequency. plated in a 30-mm dish containing a glass coverslip. After 2 to The distributions ofstaining intensities observed with the L- 3 days ofgrowth, the dishes were rinsed with staining medium, cell and the TK' transformant populations were the same; how- 10 A.l ofantibody solution was added, a coverslip was placed on ever, a lowfrequency ofcells that stained above background was top, and the dish was covered with moist filter paper to prevent seen in the transformed populations (Fig.