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Induction and Properties of Syngeneic Murine Anti-Immunoglobulin D

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Induction and Properties of Syngeneic Murine Anti-Immunoglobulin D Proc. Natl. Acad. Sci. USA Vol. 85, pp. 2293-2297, April 1988 Immunology Induction and properties of syngeneic murine anti-immunoglobulin D SEIJI HABA*t, G. JEANETTE THORBECKEt, AND ALFRED NISONOFF* *Department of Biology, Rosenstiel Research Center, Brandeis University, Waltham, MA 02254; and tDepartment of Pathology, New York University Medical Center, New York, NY 10016 Contributed by Alfred Nisonoff, December 21, 1987 ABSTRACT High titers of autoantibodies directed to ever, the greatly reduced numbers of remaining 4+, 5- B isotypic determinants of IgD were produced by inoculation of cells in such mice were shown to be actually hyperresponsive syngeneic monoclonal IgD, conjugated covalently to keyhole to antigens injected i.v. (6, 8). limpet hemocyanin, into adult or neonatal inbred mice. Anti- idiotypic antibodies were induced at the same time. The MATERIALS AND METHODS average affinity of the mouse antibodies (Ka 1079 M-1) is similar to that of rabbit anti-IgD and of syngeneic anti-IgE Mice. BALB/c mice were obtained from The Jackson induced by the same procedure. Results indicate that B cells of Laboratory. Mice were 5-8 weeks old at the start of each the mice are not tolerant to serum IgD and that tolerance is experiment. Neonatal mice were 2 days old at the first maintained at the level of T cells. Direct interaction of the antigen inoculation. syngeneic anti-IgD with cell-surface IgD was minimal, and Monoclonal IgD. Two IgDK preparations were used; both there was no convincing evidence that cell-surface IgD was were derived from BALB/c myeloma cell lines (TEPC-1033 down-regulated in the anti-IgD-producing mice. Further stud- and TEPC-1017) developed by F. Finkelman, M. Potter, and ies, preferably employing monoclonal anti-IgD, are required collaborators (11). The cell lines were grown as ascites cells to determine whether epitopes on cell-surface IgD can be in pristane-primed BALB/c mice. Purification of IgD was recognized by syngeneic anti-IgD. The ability to generate in done by modifying slightly the method of Finkelman et al. vivo high titers of anti-IgD should facilitate the production of (11). Our procedure includes two precipitations with ammo- such monoclonal antibodies. nium sulfate followed by chromatography on DEAE- cellulose in 0.03 M sodium phosphate buffer, pH 8.3. The Recently we reported the induction of high titers (to 1 mg/ IgD was obtained together with IgG in the fall-through ml) of syngeneic anti-IgE with isotypic specificity in adult fraction; IgG was removed by repeated passage through a A/J mice (1). The immunogen used was a copolymer of protein A-Sepharose column (Sigma) in a high-salt buffer keyhole limpet hemocyanin (KLH) with an A/J monoclonal (0.1 M sodium phosphate/i M sodium chloride, pH 8.3). antibody (mAb) of the IgE class. Results indicated that mice Finally, gel filtration was done on Sephacryl S-300 (Phar- are not tolerant at the level of B cells to their own IgE. The macia). Purification was monitored by double diffusion in fact that the copolymer was immunogenic, whereas mono- agarose gel, immunoelectrophoresis, and NaDodSO4/ meric IgE was not, indicated that tolerance is mediated by T PAGE. In a nonreducing gel the final product migrated as a cells and can be broken by the use of a foreign carrier single major band with an apparent Mr of 125,000 (cf. ref. molecule, KLH. These observations permitted the produc- 12); gel filtration indicated a Mr for both TEPC-1033 and tion of syngeneic anti-IgE mAbs (2), which we are using in TEPC-1017 somewhat higher than that of IgE, a result studies of regulation of IgE synthesis. consistent with the presence of a dimer of IgD (11). A The capacity to produce high titers of syngeneic anti-IgE portion of each purified IgD preparation was trace-labeled suggested that the same methods might be applied to the with 1251 by the chloramine-T method (13). Samples were induction of syngeneic anti-IgD because IgD and IgE both tested for degradation by gel filtration shortly before using exist at very low concentrations in normal mouse serum. In the material in RIAs. Small amounts of breakdown products fact, existence of circulating autoantibodies to human IgD in were detected, but the major product was of high molecular rheumatic diseases has been reported (3). However, because weight. IgD is expressed on the cell surface of >90% of B cells, it Conjugation of IgD with KLH. Conjugates were prepared seemed possible that self-tolerance to IgD might be more (14) by mixing 10-mg quantities of purified IgD and KLH in firmly established than self-tolerance to IgE. We here de- a final volume of 2 ml in 0.1 M sodium phosphate buffer, pH scribe the induction of high titers of syngeneic anti-IgD and 6.0. Glutaraldehyde was added in an amount equal to 20 mol report on the affinities and specificity of such antibodies. per mol of IgD. After approximately one-half hour of incu- The results are relevant to questions of self-tolerance and bation at room temperature, the mixture became cloudy; the autoimmunity and may permit the eventual production of reaction was then quenched with 1 M L-lysine, and the syngeneic anti-IgD mAbs for use in studies of regulation of mixture was then dialyzed against neutral phosphate-buf- the immune system. fered saline. The significance of surface IgD in regulation has been Immunization of Mice. Neonatal or adult BALB/c mice demonstrated in many investigations (4-10). For example, it were immunized i.p. two or three times with IgD (TEPC- has been shown that treatment of mice from birth with 1017 or TEPC-1033) or with a conjugate of KLH with IgD; allogeneic anti-IgD suppresses cell-surface IgD expression complete Freund's adjuvant (CFA) was used in each case. and down-regulates the production of antibodies of all Schedules of immunization and amounts inoculated are spec- isotypes to antigens administered s.c. or i.p. (4, 5, 8). How- Abbreviations: CFA, complete Freund's adjuvant; FITC, fluores- The publication costs of this article were defrayed in part by page charge cein isothiocyanate; KLH, keyhole limpet hemocyanin; mAb, payment. This article must therefore be hereby marked "advertisement" monoclonal antibody; Fc, Fc fragment of IgG. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. Downloaded by guest on October 4, 2021 2293 2294 Immunology: Haba et al. Proc. Natl. Acad. Sci. USA 85 (1988) ified in table footnotes. Equal volumes ofantigen solution and to varying dilutions of serum. Development was done with CFA were used to prepare emulsions. '25I-labeled affinity-purified rabbit anti-IgD (40 ng in 0.1 ml). Antisera. Rabbit anti-mouse IgD antiserum was prepared As a standard we used purified IgD (TEPC-1033). against unconjugated TEPC-1017. Rabbit and goat anti-mouse Analysis of Cell Surface Immunoglobulin Phenotypes. These Fc (of IgG), used for double-diffusion analyses and for immu- analyses were done with a fluorescence-activated cell sorter noelectrophoresis, were prepared in our laboratory. For use (Orthocytofluorograph Spectrum II; Ortho Diagnostic Sys- in the solid-phase assay for anti-IgD, the goat anti-mouse Fc tems, Westwood, MA). Spleen cells or axillary and brachial was affinity-purified and trace-labeled with 125I by the chlo- lymph node cells were washed and suspended in phosphate- ramine-T method; the antibody reacted with mouse IgG but buffered saline/1% bovine serum albumin/0.1% sodium not with IgM. azide. The percentage of cells expressing surface IgM or IgD Assays for Anti-IgD. Two types of assay were used. In a was determined by indirect staining of 1 x 106 cells in 50 .ul solid-phase assay (15), wells of polyvinylchloride microtiter of medium using optimal concentrations of goat anti-mouse plates were coated with a purified IgD mAb (not the immu- IgM or goat anti-mouse IgD (provided by F. D. Finkelman, nogen). The coating solution contained 1 ;Lg of IgD in 0.1 ml. Uniformed Services University of the Health Sciences, Be- Horse serum (2.5%) was used to saturate surfaces. Coated thesda, MD), followed by fluorescein isothiocyanate (FITC)- wells were exposed to varying dilutions of antisera for 6 hr, labeled rabbit anti-goat immunoglobulin (Miles). The percent- and wells were developed with 50 ng of 125I-labeled affinity- age of cells expressing cell-surface Thyl.2 antigen was deter- purified goat anti-mouse Fc fragment. As a standard we used mined by direct staining with FITC-anti-Thyl.2 (ICN pooled anti-IgD serum, the titer of which was determined by Immunobiologicals, Lisle, IL). Labeled cells were analyzed a liquid-phase assay. in the cell sorter at 1 x 106 cells per ml. To determine whether Assays in the liquid phase were done by mixing varying mouse anti-IgD interacted with surface IgD, cells were incu- amounts of 1251I-labeled purified IgD (TEPC-1017) with a bated first with affinity-purified syngeneic mouse anti-IgD fixed dilution ofantiserum. Five microliters of normal mouse (100 ug/ml), or with dilutions of mouse serum initially con- serum was added as carrier. Complexes were precipitated taining 70 ,ug of anti-IgD per ml, and then stained with with rabbit antiserum directed against the Fc fragment of FITC-labeled goat anti-mouse IgG (American Qualex, Lami- mouse IgG. In the absence of anti-IgD a precipitate was rada, CA). In some experiments cells were exposed to mouse obtained, but it contained <1% of the added radioactivity. anti-IgD for 10 min before addition of goat anti-IgD or F(ab')2 Measurement of IgD-Binding Capacities and Average Ka fragments of rabbit anti-IgD to determine whether mouse Values of Anti-IgD (TEPC-1033) Antibodies.
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