Proc. Natl. Acad. Sci. USA Vol. 85, pp. 3990-3994, June 1988 Immunology Selective elimination of idiotype-binding cells in vivo by a drug-idiotype conjugate demonstrates the functional significance of these cells in immune regulation (ligand/drug targeting/suppressor T celis/T-independent antigen/dextran B1355S) M. M. ABU-HADID*, R. B. BANKERTt, AND G. L. MAYERStt *Department of Microbiology, State University of New York at Buffalo, Buffalo, NY 14214; and tDepartment of Molecular Immunology, Roswell Park Memorial Institute (a unit of New York State Department of Health), 666 Elm Street, Buffalo, NY 14263 Communicated by Niels K. Jerne, February 1, 1988 ABSTRACT A receptor-specific cytotoxic drug delivery tain control over the production of non-dextran-binding system has been used to eliminate idiotype-binding cells in vivo immunoglobulins that express the M104E individual idiotype to ascertain the possible functional significance of these cells in (M104E IdI). The same results were observed using two regulating the humoral immune response to dextran. Protein different methods ofdepleting the idiotype-binding cells [i.e., M104E, a mouse myeloma protein that binds dextran, ex- by adherence of the cells to idiotype bound to a solid matrix presses a private idiotope that is present on a significant or by pulsing spleen cells with soluble idiotype-expressing proportion of the normal dextran-specific antibody repertoire. protein M104E (a mouse myeloma protein that binds dextran) Immunocompetent cells that bind and internalize M104E that had been radiolabeled to a high specific activity with idiotype-bearing molecules were eliminated by the intravenous 125I]. Unfortunately, both of these approaches were limited administration of a single dose of cytosine arabinonucleoside by the need for in vitro manipulation of the responder conjugated to purified M104E protein. The administration of lymphocyte populations and the use of irradiated recipient this cytotoxic drug-idiotype conjugate had a profound effect mice to assess the effects of the cell depletion upon the upon the expression of the M104E idiotype in euthymic but not dextran response. We have designed a protocol, based upon in athymic BALB/c mice following immunization with dex- the receptor-specific delivery of a cytotoxic drug, that is tran. In euthymic mice, the depletion of the idiotype-binding capable of eliminating a selected population of cells in vivo. cells resulted in a marked elevation in the level of M104E This protocol does not require any in vitro manipulation of idiotype present in the immune sera. Moreover, treated but not the immunocompetent cells and eliminates the need for control mice developed idiotype-positive molecules that did not adoptive cell transfer of these cells to evaluate the effect of bind dextran. These results demonstrate the functional signif- depletion. The success of this strictly in vivo approach is icance of idiotype-binding cells in the regulation of individual demonstrated by our recent finding that antigen-binding cells clonotypes during an immune response. specific for the T-independent antigen dextran B1355S were eliminated by administering a molecule composed of the The heterogeneous collection of antibodies produced during antigen and the drug cytarabine (cytosine arabinonucleoside; a typical immune response is a reflection ofan equally diverse araC) (10). After treatment with this drug-antigen conjugate set of antibody-producing cell clones (1). In addition to (which we have called "toxogen"), the anti-dextran antibody expanding antigen-specific cells, immunization has been response in BALB/c mice is suppressed for at least 9 months. shown to provoke an increase in immunocompetent cells that The suppression is specific since toxogen treatment had no are specific for the idiotype associated with the immunoglob- effect upon other antibody responses. The administration of ulin that is secreted or displayed on the surface of antigen- such a toxogen with limited amounts of drug would be specific cells (2, 3). These anti-idiotypic antibodies were expected to have a minimal effect on the microenvironment initially postulated to regulate individual antibody responses of the immune system. We have now constructed a toxogen at the humoral level but, later, idiotype regulation was consisting ofthe myeloma protein M104E and araC to exploit suggested to involve cellular networks composed of a variety this in vivo drug delivery protocol to deplete BALB/c mice of T-cell subsets (4-6). of cells capable of binding to the idiotopes displayed on One classical approach that has been used to establish the M104E protein. The effect of this in vivo depletion of role of an organ, tissue, or cell type is to remove it and idiotype-binding cells is reported here. These results support evaluate the effect of its removal upon the host's ability to the theory that idiotype-binding cells play a crucial role in function. Two elegant examples of this approach in immu- regulating the expression of cells secreting idiotype-positive nology are the surgical removal of the thymus (7) and the anti-dextran antibodies and cells secreting an idiotype- selective elimination of antigen-binding populations by in positive immunoglobulin that does not bind dextran. vitro manipulation of cells followed by adoptive transfer of the cells to an irradiated syngeneic recipient (8). We have used a modification ofthe latter approach to evaluate the role MATERIALS AND METHODS of idiotype-binding cells and have found that the depletion of Reagents. Dextran B1355S was a gift from M. E. Slodki these cells results in a significant augmentation in the expres- (Northern Regional Research Laboratory, Peoria, IL). araC sion of the reference idiotype subsequent to immunization was obtained from Upjohn and the radiolabeled drug (9). These studies indicated that idiotype-specific cells reg- [3H]araC was obtained from Amersham. Mineral oil plasma- ulate idiotype expression during an immune response against cytoma M104E, which secretes an IgM, A-1 myeloma protein dextran and suggested that the idiotype-binding cells main- with a-1,3 dextran-binding activity, was a gift from M. Potter The publication costs of this article were defrayed in part by page charge Abbreviations: araC, cytosine arabinonucleoside; IdI, individual payment. This article must therefore be hereby marked "advertisement" idiotype. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. Downloaded by guest on September 29, 2021 3990 Immunology: Abu-hadid et al. Proc. Natl. Acad. Sci. USA 85 (1988) 3991 (National Institutes of Health, Bethesda, MD) and was RIAs was protein M104E, a mouse myeloma protein that maintained in our animal colony for the preparation ofprotein binds dextran B1355S. M104E. Mineral oil plasmacytoma J558, which secretes a The amount of immunoglobulins expressing the M104E IdI similar dextran binding myeloma protein that is an IgA, was was determined from an RIA using the purified rabbit obtained from the American Type Culture Collection and was anti-M1O4E IdI antibody preparation and '25I-labeled protein maintained in our animal colony for the preparation of protein M104E in a double-antibody assay as previously described J558. These myeloma proteins were purified by affinity (9). chromatography on a dextran-bovine serum albumin- Sepharose 4B column as described by Hiramoto et al. (11). Hybridoma protein 5G10 is an IgM with specificity for RESULTS thymidine and was prepared as described for monoclonal Preparation of the Anti-Idiotypic Antibody Reagent. The anti-5-bromodeoxyuridine antibody (12), and hybridoma pro- anti-idiotypic antibody reagent was prepared in rabbits as tein 1F4-F2 is an IgM with specificity for 4-azophthalate and previously described (13). After purification, its sensitivity was used to prepare an affinity column as described previ- and specificity were evaluated by RIA. Rabbit 3027 anti- ously (13). Rabbit anti-M104E IdI antibodies were prepared M104E IdI antibody reagent is highly sensitive (i.e., it can as previously described (9). Most of the assays for these detect less than 1 ng of protein M104E per ml) and is highly studies used antibodies isolated from rabbit 3027. specific since protein J558, whose variable regions differ by Animals. BALB/c mice were obtained from West Seneca only two amino acids in the diversity region of the heavy Laboratory (West Seneca, NY). BALB/c nu/nu mice were chain, does not show any inhibition even at 158 Ag/ml, as obtained from National Institutes of Health. New Zealand shown in Fig. 1. White rabbits were obtained from Hazleton Research Ani- Preparation of Idiotype Toxogens. The toxogen was pre- mals (Denver, PA). Rabbits were immunized with 1 mg of pared by attaching the drug araC to the carbohydrate portion affinity-purified protein M104E emulsified in complete of the myeloma protein M104E to avoid chemical modifica- Freund's adjuvant and boosted after 3 weeks and then tion of amino acid residues that might be important to the monthly with 0.5 mg of affinity-purified protein M104E structure ofthe idiotypic determinant. However, exposure of emulsified in incomplete Freund's adjuvant. After the first the protein to oxidation by periodate and reduction by boost, the rabbits were bled weekly by venous puncture of borohydride could cause chemical modification of sensitive the lateral ear vein. amino acid residues that are important for its recognition by Attachment of araC to Protein M104E and