Proc. Natl. Acad. Sci. USA Vol. 85, pp. 3990-3994, June 1988 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 /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 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 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 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 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 . 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 Protein 5G10. the regulatory cell receptor. To determine if the antibody- The carbohydrate side chains of protein M104E and hybrid- combining site and the M104E idiotypic determinant have oma protein 5G10 were oxidized with 0.001 M sodium remained intact following exposure to oxidation and reduc- periodate at pH 6.5 for 1 hr at 4°C and were dialyzed at 4°C tion, the antigen binding and idiotypic determinant of protein to remove the excess periodate. araC (16 mg per mg of M104E were evaluated in RIAs. To assure maximum cyto- oxidized protein) was allowed to couple to the oxidized toxicity, procedures were investigated that permitted large antibodies at pH 8.0 for 1 hr at 4°C, and then the adducts were amounts of the drug to be incorporated into the ligand. The stabilized by addition of 0.25 ml of 2 M sodium borohydride optimum concentration of sodium periodate that resulted in to reduce the Schiff base to an amine linkage. The araC was minimal damage to the antibody molecule while at the same labeled with [3H]araC to allow one to determine the molar time permitted effective coupling of araC was 0.001 M. The ratio of araC attached to the antibodies. amount of araC incorporated into the toxogen was calculated In Vivo Elimination of Idiotype-Binding Cells. One group of to be 322 mol of drug per mol of protein M104E. The mice was injected i.v. with 200 ul of phosphate buffered dextran-binding activity of protein M104E-araC322 conju- saline (PBS) containing 50 jig of protein M104E-araC322 (i.e., gates prepared under these conditions was unchanged. Fig. 2 a toxogen composed of 322 mol of araC per mol of protein shows that the M104E IdI has only been slightly affected by M104E), and a control group of mice was injected i.v. with slight change in binding should 200 ,ul of PBS containing 50 ,tg of hybridoma protein the coupling procedure. This (i.e., a conjugate composed of 103 mol of araC 5G10-araC103 100 0. 0 0 per mol of protein 5G10). Two days after administering the r idiotype toxogen or control antibody-drug conjugate, the 0 0 mice were immunized i.p. with 200 ,ul of PBS containing 100 80 F jig of dextran B1355S emulsified with an equal volume of complete Freund's adjuvant. The mice were bled 7, 14, and 21 days after immunization, and their sera were assayed both 60 - for total dextran-binding antibodies by a Farr binding RIA and for expression of the M104E IdI by an RIA using the . J558 inhibition curve for protein M104E as the standard. 40K o M104E Similar experiments using three groups of BALB/c nu/nu mice, four mice in each group, were used to evaluate the role of mature T cells. One group was injected with 200 ,u of PBS 20 containing 50 ,ug of protein M104E-AraC322 toxogen i.v.; a second group received a control hybridoma protein-drug conjugate, 5G10-AraC103; and a third group received no treatment. These animals were immunized and assayed as 10-4 lo-, lo-2 lo-' 100 10' described above. Inhibitor, ug/ml RIA. The amount of anti-dextran antibodies was evaluated FIG. 1. Specificity ofthe rabbit anti-M104E IdI antibody reagent in a Farr binding assay as modified by Skom and Talmage as determined by the RIA used to quantitate the proportion of (14). Briefly, 50 Al of ['25I]iodotyramine-dextran B1355S (13) anti-dextran antibodies that express the M104E IdI. The ability of containing =100,000 cpm was incubated with 50 pl of affinity-purified M104E protein to inhibit the binding of rabbit dilutions of antisera or standard for 1 hr, and the immune anti-M104E-IdI antibody to 1251-labeled M104E was compared to that complexes were precipitated with 200 pl of appropriately of protein J558. The highly homologous protein J558 that does not titrated goat-anti-mouse-Ig antisera. The standard for these express the M104E IdI shows no inhibition in this assay. Downloaded by guest on September 29, 2021 3992 Immunology: Abu-hadid et al. Proc. Natl. Acad Sci. USA 85 (1988)

100 r anti-dextran antibodies expressed the M104E IdI. Control mice treated with a toxogen prepared from the hybridoma protein 5G10 conjugated to araC produced normal concen- 80 trations ofanti-dextran antibodies with normal proportions of * M104E-araC conjugate the M104E IdI. These effects are evident in sera 1, 2, or 3 o M104E 601 weeks after immunization. Another striking difference that was observed between the control and experimental groups was the finding that the toxogen-treated mice produced 40 significant quantities of immunoglobulins that expressed the M104E IdI but did not recognize the antigen dextran (M104E IdI + Dex ). + 20 Although the absolute amount of IdI protein was in- creased (as much as 16-fold) in the experimental group, there was no significant difference between experimental and it control groups in the absolute amount of anti-dextran anti- 1o-4 lo-, 10-2 14o-1 100 1o' bodies present in the sera. The amount of anti-dextran Inhibitor, a.g//ml antibodies and the proportion of anti-dextran antibodies that are M104E IdI + in untreated mice were essentially identical FIG. 2. RIA to evaluate the integtrity of the M104E-araC to the values reported in Table 1 for the mice that received the conjugate. The ability of the M104E-ara(C conjugate to inhibit the control toxogen. IdI + Dex molecules were never detected binding ofrabbit anti-M1O4E-IdI antibody to 251I-labeled M104E was in the immune sera of the control or untreated (no toxogen) compared to that of affinity-purified M1M4E protein. mice. Failure of Toxogen to Affect the Level of M104E IdI in Nude not limit its ability to bind to the recep tor on idiotype-specific Mice. In an attempt to establish whether or not mature T cells

regulatory cells. This toxogen was us ed to treat mice prior to were involved in the augmentation of the IdI + Dex + anti- antigenic stimulation. dextran antibodies and the generation of IdI + Dex - immu- A control toxogen was prepared by using the same proce- noglobulin molecules, athymic nude BALB/c mice were dure to couple araC to the hybridomla protein 5G10, which given either the experimental or control toxogen prior to has the same heavy-chain as protein M104E but has immunization. A third group of nude mice received no specificity for the nucleic acid base thymine. Experiments treatment prior to immunization. The data presented in Table with labeled araC indicated that we had attached 103 araC 2 indicate that T cells are required for the enhanced idiotype residues per molecule of hybridoma Iprotein 5G10. expression observed in euthymic BALB/c mice since athy- Effect of Toxogen on the Anti-Dextrran Antibody Response. mic mice, which lack mature T cells, do not show enhanced The role of idiotype-binding cells in rcegulating the amount of expression of the M104E IdI when treated with the toxogen. idiotype expressed during an immune response was tested by The BALB/c nu/nu mice were treated with the same proto- in vivo administration of the toxogen. An assumption is made col as the euthymic mice using the idiotype toxogen, and that the experimental toxogen protein M104E-araC322 will there was no difference between the toxogen-treated athymic bind to idiotype-specific immunocomipetent cells in vivo (B mice and the control mice (treated with the hybridoma and/or T cells) and that the toxogen Nwill be internalized and protein 5G10-araC conjugate) either in the proportion of selectively eliminate idiotype-binding cells in the mouse. If idiotype-bearing anti-dextran antibodies or in the production the above is correct, then one woulcd expect to observe an of M104E IdI+ Dex- immunoglobulins. These results sug- alteration in the level of M104E IdI expressed in mice given gest the possibility that an idiotype-binding is the target the experimental toxogen but not in mice given the control of the toxogen and that the purposes of these idiotype- toxogen prior to immunization with aantigen. specific T cells are to regulate the level of idiotype expressed The normal amount of anti-dextran antibodies in BALB/c by B cells and to prevent production of immunoglobulin mice immunized with the bacterial dex:tran B1355S is 200-300 molecules that express the M104E IdI but do not bind ,ug/ml of serum. A significant but varriable proportion of the dextran. anti-dextran antibodies expresses the M104E IdI (5-60%o). The data presented in Table 1 show that BALB/c mice DISCUSSION treated with 50 ug of the toxogen (and, two days later, immunized with dextran) produced normal amounts of anti- In our previous studies, we evaluated the effects ofremoving dextran antibody, but, unlike the control mice, all the idiotype-binding cells by using two different in vitro protocols Table 1. Anti-dextran antibody response in BALB/c mice treated with protein M104E-araC322 toxogen: Quantitation of the amount of anti-dextran antibodies and the amount of immunoglobulins expressing the M104E IdI Days M104E-araC treatment* 5G10-araC treatmentt after M104E M104E immuni- Anti-dextran IdIt, Anti-dextran IdIt, zation Ab, jig/ml Ag/ml Ratio§ Ab, Ag/ml Ag/ml Ratio§ 7 287 38 431 ± 96 156 194 ± 13 99 ± 10 45 14 243 31 423 ± 95 173 298 ± 72 26 ± 7 11 21 249 64 375 ± 38 174 263 ± 77 135 ± 65 47 Values represent the mean ± SEM. Ab, antibody. *Mice were treated with 200 ,Au of PBS containing 50 ,ug of the protein M104E-araC322 toxogen. tMice were treated with 200 ,ul of PBS containing 50 ,ug of protein 5G10-araC103. (Protein 5G10 is an IgM anti-thymidine .) tlmmunoglobulins expressing the M104E IdI. §(Immunoglobulins expressing the M104E IdI/anti-dextran antibodies) x 100%o. Downloaded by guest on September 29, 2021 Immunology: Abu-hadid et al. Proc. Natl. Acad. Sci. USA 85 (1988) 3993

Table 2. Anti-dextran antibody response in BALB/c nu/nu mice treated with protein M104E-araC322 toxogen: Quantitation of the amount of anti-dextran antibodies and the amount of immunoglobulins expressing the M104E IdI Days M104E-araC treatment* 5G10-araC treatmentt No treatment after M104E M104E immuni- Anti-dextran M104E Anti-dextran IdIt, Anti-dextran IdIt, zation Ab, gg/ml IdIt, tig/ml Ratio§ Ab, tig/ml ,ug/ml Ratio§ Ab, tLg/ml Ag/ml Ratio§ 7 292 ± 107 137 ± 54 51 ± 14 113 ± 29 57 ± 18 56 ± 17 235 ± 92 82 ± 15 51 ± 14 14 588 ± 121 146 ± 37 41 ± 17 179 ± 52 76 ± 13 43 ± 6 309 ± 149 146 ± 45 60 ± 11 21 366 ± 48 265 ± 122 67 ± 22 227 ± 59 117 ± 21 71 ± 3 440 ± 114 273 ± 95 60 ± 11 Values represent the mean ± SEM. Ab, antibody. *Mice were treated with 200 ,ul of PBS containing 50 ,ug of M104E-araC322 toxogen. tMice were treated with 200 ,ul of PBS containing 50 ,g of 5G10-araC103 toxogen. tImmunoglobulins that express the M104E IdI. §(Immunoglobulins expressing the M104E IdI/anti-dextran antibodies) x 100%. (9). We present here a protocol that has permitted us to also internalize the antigen-drug conjugate, and the deplete idiotype-specific cells in vivo by receptor-specific drug is activated in a rapidly dividing cell, where it is directly targeting of a drug-idiotype conjugate. araC was selected for incorporated into replicating genes. Drug incorporation in- these studies because extensive literature shows that the drug activates the cell and relieves the immune response to is an excellent cytotoxic agent for (15) and dextran from this regulatory pressure. because Hurwitz et al. (16) have reported that attachment to Although the observed loss of regulation of the M104E IdI carbohydrates through Schiff base formation and reduction seen in euthymic mice seems to be correlated with the with sodium borohydride does not affect its biological activ- presence of mature T cells, the anti-dextran response in ity. Although sodium borohydride is a strong reducing agent athymic mice appears to be well regulated in the absence of that could reduce disulfide bonds, previous studies have T cells. This would indicate that the increased expression of shown that the disulfide bonds in immunoglobulin molecules the M104E IdI in euthymic mice involves regulatory net- are resistant to this reduction under the reaction conditions works above and beyond those available in athymic mice. employed in these studies (17). Drug attachment was made to Part of these complex interconnecting networks includes a the carbohydrate side chains to avoid possible modification balance of T-helper and T-suppressor cells for controlling of determinants on the protein that could be important for expression of the antibody response through anti-idiotypic binding to the idiotype-specific receptors on the regulatory circuits. Removal of the suppressor arm would allow unreg- cells. The presence of intact idiotype determinants and the ulated expansion of T-helper cells and B cells producing antigen binding site on the toxogen was verified by RIA. The idiotype-positive immunoglobulins. This expansion cannot results presented in Fig. 1 and the failure to detect any loss occur in athymic mice, which lack the necessary T-helper cell in antigen-binding activity demonstrate that oxidation with population. sodium periodate, attachment of 322 araC substituents, and The idiotype-specific regulatory cells that are targeted by reduction with sodium borohydride all had minimal effects on our toxogen may be the normal counterpart of the immuno- the important structural components ofthe protein portion of globulin-specific suppressor T cells described by Lynch (28) the toxogen. that suppress immunoglobulin secretion by murine plasmacy- The target of our drug delivery protocol could be one of tomas as well as block B-cell proliferation. In these studies three lymphocyte cell types: (i) Lyt-1-positive B cells (18, they have shown that the target ofthe suppressor T cell is the 19), a major proportion of which secrete anti-idiotypic anti- plasmacytoma and not helper T cells (29, 30). Since bodies; (ii) Lyt-1-negative anti-idiotypic antibody-producing dextran B1355S is a T-independent antigen, the putative cells that secrete appropriate amounts of anti-idiotypic anti- suppressor T cell targeted by our toxogen might be expected bodies; or (iii) the extensively studied idiotype-specific to regulate the B-cell repertoire directly. Several earlier suppressor T cells that are capable of binding and internal- studies have also indicated that suppressor T cells can act izing the idiotype-araC toxogen directly. The failure to directly on B cells to suppress antibody secretion (reviewed observe any augmentation of the idiotype in toxogen-treated in ref. 28). In our system, we would expect the toxogen to athymic mice suggests that anti-idiotypic Lyt-1-positive B remove the idiotype-specific suppressor cells, thereby re- cells are not the target of our toxogen since they have been leasing the immune system from an idiotype-specific regula- observed in athymic nude mice (20). In addition, since we tory pressure. We suggest that idiotype regulatory cells serve have previously established that toxogens targeted to anti- to prevent proliferation and clonal expansion of a portion of gen-specific B cells effectively eliminate those specific for the idiotype-positive dextran-binding B cells since we have T-independent antigens but not those that are specific for demonstrated here that the removal of the regulatory cells T-dependent antigens (10), it seems unlikely that the target of leads to an enhanced expression of the reference idiotype. the idiotype-araC toxogen is a T-dependent B cell. Accord- Elimination of the idiotype-specific regulatory cells also ingly, the idiotype-specific suppressor T cell represents the resulted in the expression of large amounts of immunoglob- most likely target ofthe idiotype-araC toxogen. Many studies ulins that express the M104E IdI but do not bind the antigen have demonstrated that suppressor T cells are capable of dextran (M104E IdI+ Dex- immunoglobulin). Similar ob- binding antigen directly (21-25) and that upon binding, servations were made when idiotype-binding cells were re- antigen are often stimulated to secrete antigen-specific sup- moved in vitro (9). Although the M104E IdI+ Dex- immu- pressor factors (24-27). This cell, since it expresses an noglobulin failed to bind to a dextran affinity column, it did antigen-specific receptor, could also be removed by idiotype- coelute with IgM in gel permeation chromatography on a specific panning on idiotype-coated Petri dishes or be killed Sephadex G-200 column (9). Antibodies ofthis type have been by idiotype-specific radioactive ligands (9). Our previous previously observed when mice were treated with anti- studies have shown that antigen-drug conjugates were only idiotypic antibody prior to immunization with antigen (31, 32). effective on cells that are capable of internalizing the antigen M104E IdI+ Dex- immunoglobulin could arise as a result of that is capable of stimulating proliferation. It appears that unregulated proliferation of the antibody-producing cells that suppressor T cells that are capable of directly binding occurs in response to antigen stimulation in the absence ofthe Downloaded by guest on September 29, 2021 3994 Immunology: Abu-hadid et al. Proc. Natl. Acad. Sci. USA 85 (1988) corresponding regulatory cells. Thus, the M104E IdI-bearing M. I. & Germain, R. N. (1981) J. Exp. Med. 153, 450-463. B cells proliferating without regulation could lead to the 7. Miller, J. F. A. P. (1961) Lancet ii, 748-749. generation of somatic variants that have lost their antigen- 8. Ada, G. L. & Byrt, P. (1969) Nature (London) 222, 1291-1292. binding capacity (33). On the other hand, the absence of 9. Schepart, B. S., Abu-hadid, M. M., Mayers, G. L. & Bankert, regulatory cells could permit the expression of silent B-cell R. B. (1985) J. Immunol. 135, 1690-1697. clones (34, 35) that are normally suppressed by the idiotype 10. Abu-hadid, M. M., Bankert, R. B. & Mayers, G. L. (1987) cells. In Proc. Natl. Acad. Sci. USA 84, 7232-7236. regulatory both cases, it would explain why dextran 11. Hiramoto, R., Ghanta, V. K., McGhee, J. R., Schrohenloher, stimulation is necessary for production ofthe idiotype-positive R. & Hamlin, N. M. (1972) Immunochemistry 9, 1251-1253. variant antibodies observed in the toxogen-treated mice. 12. Raza, A., Preisler, H. D., Mayers, G. L. & Bankert, R. B. Our observation that the removal of idiotype-specific cells (1984) N. Engl. J. Med. 310, 991. alters the proportion of idiotype-positive antibodies but has 13. Schepart, B. S., Mayers, G. L. & Bankert, R. B. (1985) J. little or no effect upon the absolute amount of anti-dextran Immunol. 135, 1683-1689. antibody produced during the immune response to dextran 14. Skom, J. H. & Talmage, D. W. (1958) J. Clin. Invest. 37, suggests that mechanisms other than idiotype recognition are 787-793. involved in regulating the quantity of antibody produced. 15. Valeriote, F. (1982) Med. Pediatr. Oncol. Suppl. 1, 5-26. Clearly many such regulatory mechanisms have been pro- 16. Hurwitz, E., Kashi, R., Amnon, R., Wilchek, M. & Sela, M. posed. One early hypothesis that needs to be further explored (1985) J. Med. Chem. 28, 137-140. is that antibody quantity is controlled by a dynamic equilib- 17. Murayama, A., Shimada, K. & Yamamoto, T. (1978) Immu- rium among nochemistry 15, 523-528. circulating antibody, antigen, and antigen-anti- 18. Hardy, R. R. & Hayakawa, K. (1986) Immunol. Rev. 93,53-79. body complexes (36). 19. Herzenberg, L. A., Stall, A. M., Lalor, P. A., Sidman, C., These experiments and our previous studies with a T- Moore, W. A., Parks, D. R. & Herzenberg, L. A. (1986) independent antigen (10) demonstrate that ligands can be Immunol. Rev. 93, 81-102. used to deliver a cytotoxic drug to a specific cell population 20. Hayakawa, K., Hardy, R. R., Parks, D. R. & Herzenberg, in vivo without perturbing other immunocompetent cells. Our L. A. (1983) J. Exp. Med. 157, 202-218. ability to deliver cytotoxic agents selectively to cell popula- 21. Higgins, G. & Choi, Y. S. (1979) J. Immunol. 123, 2068-2075. tions in vivo has many obvious potential applications in 22. Kumagai, Y., Okumura, K. & Tada, T. (1984) Mol. Immunol. designing new approaches both to cancer therapy and to the 21, 545-559. manipulation of the immune response for addressing ques- 23. Lee, M. S., Rosenspire, A. J., Higgins, G. C., Pollak, S. V. & tions related to (i) T- and B-cell cooperation, (ii) kinetics and Choi, Y. S. (1986) Mol. Immunol. 23, 97-109. 24. Dorf, M. E. & Benacerraf, B. (1984) Annu. Rev. Immunol. 2, thresholds of lymphocyte activation, and (iii) immune regu- 127-158. lation. 25. Healy, C. T., Kapp, J. A. & Webb, D. R. (1983) J. Immunol. 131, 2843-2847. We wish to thank Dr. R. Lynch for helpful discussion and 26. Fresno, M., Nabel, G., McVay-Boudreau, L., Furthmayer, H. suggestions concerning these studies and Art Trott and Richard & Cantor, H. (1981) J. Exp. Med. 153, 1246-1259. Maturski for technical assistance. M.M.A. is a recipient of a 27. Fresno, M., McVay-Boudreau, L., Nabel, G. & Cantor, H. Fulbright Scholarship through AMIDEAST. A major portion of the (1981) J. Exp. Med. 153, 1260-1274. data was taken from the dissertation submitted by M.M.A. to the 28. Lynch, R. G. (1987) Adv. Immunol. 40, 135-151. State University of New York at Buffalo in partial fulfillment of the 29. Rohrer, J. W. & Lynch, R. G. (1979) J. Immunol. 123, requirements for the degree of Doctor of Philosophy. This work was 1083-1087. supported by National Institutes of Health Grants CA41285, 30. Milburn, G. L. & Lynch, R. G. (1982) J. Exp. Med. 155, CA25253, CA33462, and CA22786. 852-861. 31. Miller, G. G. P., Nadler, P. I., Hodes, R. J. & Sachs, D. H. (1982) J. Exp. Med. 155, 190-200. 1. Burnet, F. M. (1959) The Clonal Theory ofAcquired 32. Janeway, C. A. (1984) Prog. Clin. Biol. Res. 150, 143-154. (Cambridge Univ. Press, Cambridge, U.K.). 33. Gearhart, P. J. (1983) Ann. N.Y. Acad. Sci. 418, 171-176. 2. Cosenza, H. (1976) Eur. J. Immunol. 6, 114-116. 34. Primi, D., Juy, D. & Cazenave, P.-A. (1981) Eur. J. Immunol. 3. Bankert, R. B. & Pressman, D. (1976) J. Immunol. 117, 11, 393-398. 457-462. 35. Rubinstein, L. J. & Bona, C. A. (1983) Ann. N. Y. Acad. Sci. 4. Eichmann, K. (1978) Adv. Immunol. 26, 195-254. 418, 97-108. 5. Owen, F. L., Ju, S.-T. & Nisonoff, A. (1977) J. Exp. Med. 145, 36. Bystryn, J.-C., Schenkein, J. & Uhr, J. W. (1971) in Progress 1559-1566. in Immunology: First International Congress ofImmunology, 6. Dietz, M. H., Sy, M.-S., Benacerraf, B., Nisonoff, A., Green, ed. Amos, B. (Academic, New York), pp. 627-636. Downloaded by guest on September 29, 2021