Proc. NatL Acad. Sci. USA Vol. 78, No. 2, pp. 1138-1142, February 1981 Immunology Switch from hapten-specific immunoglobulin M to immunoglobulin D secretion in a hybrid mouse cell line (immunoglobulin genes/fluorescence-activated cell sorting) MICHAEL S. NEUBERGER AND KLAUS RAJEWSKY Institute for Genetics, University ofCologne, D-5000 Cologne 41, Federal Republic ofGermany Communicated by Herman N. Eisen, October 13, 1980 ABSTRACT From a hybrid mouse cell' line (B1-8) that se- all monoclonal anti-Bl-8.IgM idiotopic antibodies except for creted an IgM,Al anti-(4-hydroxy-3-nitrophenyl)acetyl antibody A6-24 (unpublished data) were gifts from M. Reth (Cologne); but that had no detectable surface IgM, selection for avariant with rabbit anti-Bl-8.IgM idiotypic antiserum was a gift from T. Im- Al chains on the surface resulted in the isolation ofa line that had anishi-Kari (Cologne). Immunosorbents were made by coupling switched from la to 8 expression. The surface and secreted Igs of this line were typed as IgD with two monoclonal antibodies, and purified antibodies to CNBr-activated Sepharose (7). the parental IgM and variant IgD molecules carried the same vari- Serology, Immunofluorescence, and Cell Sorting. Radioim- able regions as judged by hapten-binding and idiotypic analysis. munoassays were performed as described (8). Purified myeloma The surface and secreted. 6 chains of the IgD variant have appar- proteins and purified fluorescein- or rhodamine-coupled goat ent molecular weights of 64,000 and 61,000, respectively. How- ever, the unglycosylated secreted 8polypeptide chain has a molec- anti-mouse class antisera were gifts from H. Dorff, B. Liese- ular weight ofonly 44,000. The secreted IgD exists-predominantly gang, and A. Radbruch (Cologne). Preparation of samples for in the 2A2 form, does not contain J protein, is relatively stable in the examination ofsurface or cytoplasmic Ig in the fluorescence serum, and does not fix complement. microscope and cell sorting and analysis by using a fluores- cence-activated cell sorter (FACS-1; Becton Dickinson) have IgD was first identified as a new class ofhuman myeloma pro- been described (5). tein (1); subsequently, IgD myelomas have been isolated from Purification ofCell Surface Ig. Cells were surface labeled by the rat (2) and the mouse.* The level of IgD in normal serum lactoperoxidase catalyzed radioiodination (9) and the l25I-la- from all species studied is extremely lowalthough IgD is present beled Ig was purified from a Nonidet P-40 lysate ofthe cells on as a surface Ig on a large proportion ofB cells (3). anti-8 sorbents, anti-Al sorbent, or NIP-Acap Sepharose. The function of IgD remains unclear and in part this can be Biosynthetic Labeling of 1g. Cells were preincubated for 30 ascribed to the lack ofaserum containing or acell line producing min in RPMI-1640 medium lacking methionine and then la- an IgD ofknown antigen binding specificity. Here we describe beled by the addition of 120 ,uai ofL-[LS]methionine (specific the isolation ofa variant cell line that has switched from ,ua to 8 activity, 1100 Ci/mmol; 1 Ci = 3.7 X 10's becquerels). If re- expression and produces both surface and secreted IgD with quired, tunicamycin (gift ofR. L. Davie, Eli Lilly) was included specificity for the 4-hydroxy-3-nitrophenylacetyl (NP) hapten. in the medium (8 uag/ml) throughout. Cytoplasmic Ig (from a Nonidet P-40 lysate of-the cells after a 20-min labeling) and se- MATERIALSAND METHODS creted Ig (from. the culture supernatant after a 6-hr labeling) were purified by adsorption on NIP-Acap Sepharose. Treat- Animals. Mice were obtained from the Zentralinstitut fur ment of [3SS]IgD with endo-(3-N-acetylglucosaminidase H (Sei- Versuchstierzucht (Hannover, Fed. Rep. Germany). kagaku Kogyo, Tokyo, Japan) was carried out by incubation for Cell Lines. B1-8.64 (IgM,Al, anti-NP) was kindly donated 6 hr at 37°C with 1.5 units of enzyme per ml in 100 mM Na ci- by C. Muller (Cologne) and 11-6.3.1 cells (4) were obtained from trate at pH 5.5. the Salk Institute (San Diego, CA). B1-8.64. 1 (IgM,A1, anti-NP) Gel Electrophoresis. Samples were subjected to polyacryla- is a subelone of B1-8.64. BALB/c-derived hybrid cell line mide gel electrophoresis as described (10), and gels containing 267.7, which secretes an IgM,Al anti-NP antibody was a gift [3S]methionine were soaked in EN3HANCE (New England from M. White-Scharf (Cologne). Cells were either cultured as Fed. and dried to described (5), except that supplemented RPMI 1640 medium Nuclear, Dreieich, Rep. Germany) prior (Seromed, Munich, Fed. Rep. Germany) was used; or were autoradiography. maintained intraperitoneally in Pristane-primed mice. Cells were cloned by limiting dilution. RESULTS Antisera and Monoclonal Antibodies. Ascites fluid from H6/ 31 anti-8" tumor-bearing mice (6) was obtained from Sera-lab Isolation ofB1-8.61. B1-8 is a hybrid cell line resulting from (Sussex, England) and the IgM anti-8" antibodies were purified fusion between a spleen cell from an NP-immunized C57BL/6 by gel filtration. 11-6.3.1 anti-6b was purified from ascites fluid mouse and a cell of the myeloma line X63.Ag8 (11). B1-8 cells by (NH4)2504 fractionation. and chromatography on DE-52. secrete both an IgM, Al anti-NP antibody and the yl and K Anti-NP IgM from Bl-8.64. 1 ascites fluid and anti-NP IgD from chains ofthe X63 fusion partner. However, a B1-8 variant, Bl- B1-8.81 ascites fluid (Bl-8.IgD) were purified by binding on to 8.64, has been isolated that has lost expression ofthe X63 yl and (4-hydroxy-5-iodo-3 nitrophenyl)acetyl-aminocaproic acid (NIP- Acap)-Sepharose and elution with 1 mM NIP-Acap in phos- Abbreviations: B1-8. IgD, secreted Ig ofB1-8.81; B1-8.IgM, secreted Ig ofB1-8.64. 1; Acap, 6-aminocaproic acid; FACS, fluorescence-activated phate-buffered saline. Monoclonal anti-Al antibody (Ls136) and cell sorter; NIP, (4-hydroxy-5-iodo-3-nitrophenyl)acetyl; NP, (4-hy- droxy-3-nitrophenyl)acetyl.V region, variable region. The publication costs ofthis article were defrayed in part by page charge * Finkleman, F. D., Kessler, S. W., Mushinski, J. F. & Potter, M. payment. This article must therefore be hereby marked "advertise- (1980) Abstracts of the 4th International Congress of Immunology, ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Paris, 6.5.02. 1138 Downloaded by guest on September 27, 2021 Immunology: Neuberger and Rajewsky Proc. NatL Acad. Sci. USA 78 (1981) 1139 23 10 a~~~~ 24-0 X LtA2 C.) .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~..... .1o3~~ ~ ~ ~ ~ ~ ~ ~ a Relativefluorescence~~~~S,chne o FIG. 1. Fluorescence profiles of B1-8.64.1 (curve 1), B1-8.81 (curve 2), and 267.7 (curve 3) cells stained with fluorescent goat anti-/. antiserum (a), 11-6.3.1 anti-8" antiserum (b),H6/31 anti-c antiserum (c), and Ls136 anti-A antiserum (d). The fluorescence profiles of B1-8.81 cells stained with fluorescent 11-6.3.1 anti-8b' or Ls136 anti-A in the presence of B1-8.IgM protein (3 mg/mI) are also shown (curve 4). K chains (C. Muller, personal communication). been enriched six times with anti-Al revealed no cells that had Although both B1-8 and B1-8.64 cells secrete IgM, neither switched to y3, yl, y2a, y2b, or a expression. Among 106 cells line possesses surface IgM as detected by FACS analysis ofcells screened, we also found no revertants of B1-8.61 that stained stained with goat anti-s antisera. That this is not a general defect for cytoplasmic p chains. in the synthesis or membrane incorporation ofsurface Ig is dem- Surface Ig ofBl-8.6i1. FACS analysis demonstrated that B1- onstrated by the fact that B1-8 cells do stain for surface IgGi. In 8.61 stained brightly with fluorescent anti-Al (Ls136) and anti- the hope of isolating B1-8.64 variants containing surface IgM, 6b (H6/31 and 11-6.3.1) antisera but not with goat anti-p. anti- we used the FACS to enrich for B1-8.64 variant cells staining serum (Fig. 1). Furthermore, whereas the binding ofanti-Al to with a fluorescein-conjugated monoclonal anti-Al antiserum the surface Ig could be competitively inhibited by soluble Bl- (Ls136). After four successive sortings, each time taking the 8.IgM protein, the binding ofH6/31 anti-6 could not. To deter- brightest 1% ofthe population, analysis in the fluorescence mi- mine the Mr ofthe surface 6 chains, Ig was purified on anti-6 or croscope revealed the presence of about 1 in 100 cells which anti-Al immunosorbents or on NIP-Acap-Sepharose from cells could be stained for A on the surface but not A; the rest of the cells remained surface-Ig negative. Analysis with fluorescein- conjugated monoclonal, antisera directed against the b-allotype 1 2 3 4 5 6 chain (H6/31 or 11-6.3.1 anti-a) suggested that these variants -92 had switched from IgM to IgD expression. The cells were there- fore further sorted twice by using either theLsl36 anti-Al or the _68 FIG. 2. Analysis of radioiodin- H6/31 anti-81 antiserum. Both procedures yielded populations _1Il ated and reduced Ig samples in a in which more than 50% ofthe cells stained for surface 6 and in 10%NaDodSO4/polyacrylamide gel. Y Lanes: 1 and 5, secreted Ig of Bl- which a similar proportion of the cells typed in the cytoplasm 8.8; 2, spleen cell surface Ig puri- as &68+.