Proc. Nat. Acad. Sci. USA Vol. 68, No. 11, pp. 2846-2851, November 1971

Anti- Activity of a Monoclonal Macroglobulin (Waldenstroim's macroglobulinemia/IgM/flagellin/antigen-antibody complexes)

NOEL L. WARNER,* MALCOLM R. MACKENZIEI AND H. HUGH FUDENBERG The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia; and the Division of Hematology and Immunology, University of California, Medical Center, San Francisco, Calif. 94122 Communicated by Sir Macfarlane Burnet, August 2, 1971

ABSTRACT A monoclonal macroglobulin from a MATERIALS AND METHODS patient with Waldenstrom macroglobulinemia has been shown to have antiglobulin activity of a new type, namely, Waldenstrbm Macroglobulins (IgM). IgM from that of a true anti-antibody. The reacts with sera of patients with macroglobulinemia were isolated; human, primate, and rabbit IgM and IgG,-only when the monomer subunits, heavy and light chains were produced as target immunoglobulin is complexed to an antigen. The implications of the general findings of antiglobulin described (20). activity of monoclonal proteins is discussed in relation to Fragmentation of Immunoglobulins. Papain digestion of the possible etiology of the disease. IgMBrd was performed as per the method of Deutsch et al. Monoclonal immunoglobulins derived from malignant- (21). Adequacy of the separation of fragments was tested by lymphoid or plasma-cytoid cells have been firmly established electrophoresis versus antisera to ps and K chains, in rabbits. to be analogous to their nonmalignant counterparts in Assay for Anti-Antibody Activity. The basic assay system is possessing both constant and variable regions of the immuno- a modification of the Herzenberg and Warner technique (22) polypeptide chains (1-3). Various antibody activities for precipitation of mouse immunoglobulins with antiallotype have been reported for these proteins, including an anti- sera. The assay involves the use of a liquid precipitation globulin specificity like that of the rheumatoid factor (4-7), system with an lu2I-labeled antigen combined with a specific and binding to lipoproteins (8), cold agglutinins (9, 10), antibody in different dilutions, such that soluble complexes nucleic acid derivatives (11), streptolysin (12), phosphoryl are formed, which do not sediment under the conditions used. cholinet, §, some bacterial antigens (13), and several haptens, The complexes can, however, be precipitated by the addition such as dinitrophenol (14-17). of a second antibody directed against the antibody involved In the consideration of the specific case of monoclonal in the soluble complex. Briefly, 125I-labeled proteins were proteins (macroglobulins) produced from Waldenstr6m-type prepared by the method of Greenwood et al. (23), with tumors, it appears that a rather high incidence of antiglobulin Chloramine T as oxidant. Labeled antigens were diluted in specificities have been observed. In previous reports of this 3% bovine-serum (BSA) in 0.05 MI Tris buffer type, the Waldenstr6m macroglobulin was shown to bind to (pH 7.5); about 3- to 10-ng samples were placed in 6 X 50 mm native autologous IgG globulin (6, 18) and in a few cases, to tubes in a volume of 50 jul. Antisera were similarly diluted in also crossreact with the dinitrophenol hapten (19). As this BSA-buffer, and an appropriate dilution determined by high association of antiglobulin activity with Waldenstr6m prior titration, was then added to the tubes, also in a volume proteins may not be through mere chance, but rather reflects of 50 Al. Samples of macroglobulin or myeloma sera, or purified some basic autoimmune process in the etiology of the disease, proteins to be tested for anti-antibody activity, were then it is of considerable importance to examine for other types of added to the tubes in 10 Al volumes, again diluted in BSA- possible antiglobulin specificity in Waldenstr6m proteins. buffer. After mixing, the tubes were incubated at 370C for This report documents the first example of a Waldenstrom 2 hr, chilled at 4°C for 2-18 hr, and then centrifuged at macroglobulin with the antiglobulin specificity of a true 10,000 rpm for 10 min; 50 ;&l samples of supernatant were anti-antibody, namely, the reaction with gammaglobulin then removed for radioactivity counting. only when the gammaglobulin is itself complexed to another antigen. Antigen-Antibody Assays Used. (i) (kindly provided by Dr. A. C. Wang); rabbit antitransferrin. (ii) Chicken IgG (isolated by starch block electrophoresis and Abbreviation: BSA, bovine-; Dnp, dinitrophenyl. Sephadex gel filtration of adult chicken serum) and a poly- * Address for reprints: Dr. N. L. Warner, The Walter and Eliza valent rabbit antiserum to chicken IgG. (iii) Monomer Hall Institute of Medical Research, R.M.H. Post Office, Park- preparation of flagellin of Salmonella adelaide strain 1338, ville, Victoria, Australia, 3050. Mr. John with various antisera t Present address: Department of Internal Medicine, School of kindly provided by Pye, Medicine, University of California, Davis, Calif. to flagellin of S. adelaide, prepared in either rabbit, mouse, t Leon, -M. A., and N. M. Young, Fed. Proc., 29, 437 Abstr. rat, or man. Several antisera to S. adelaide were fractionated (1970). by Sephadex G-200 gel filtration, to give an IgM and IgG § Potter, M., R. Lieberman, L. Wood, and D. S. McKean, Fed. antiflagellin fraction. These antiflagellin sera were kindly Proc., 29, 437 Abstr. (1970). provided by Drs. Richard Wistar and Ian Mackay. (iv) 2846 Downloaded by guest on September 24, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Anti-Antibody Activity of IgM 2847 TABLE 1. Precipitation of antigen-antibody complex by TABLE 2. Precipitation of antigen-antibody Waldenstrim macroglobulins complexes by IgMBrd

Amount Percent Percent precipitation of antigen*- Protein (ug) precipitation* Amount antibody complex Nil 5 Protein (mg) I II III Normal human IgM 2.5 5 1gMBrd 6.0 68 Nil 1 5 1 1.5 65 1gMBrd 3.0 16 48 49 41 different 0. 8 12 40 37 Waldenstr6m IgM 0.2 19 18 proteins 1.5-6.0 5 JgMBi 8.2' 3 .5 3 4.1 1 5 3 *Of BC-IgGl myeloma protein with its anti-idiotype anti- * Antigen was labeled with 1251. serum. I. Transferrin-rabbit antiserum to transferrin. II. Chicken gammaglobulin-rabbit antiserum to chicken gammaglobulin. Human IgG3 myeloma protein Vi, with a rhesus monkey III. Flagellin of S. adelaide strain 1338-rabbit antiserum to antiserum to another IgG3 myeloma protein. (v) Human flagellin. IgG1 myeloma protein BC, with an anti-idiotype serum prepared in a rabbit that was immunised with BC protein and then absorbed with normal human IgG. (ti) Human 16 ,Ag of each of two other IgM proteins gave no enhanced IgG1 myeloma protein BC, with a goat antiserum to human precipitation. Fresh normal rabbit serum in a final concen- IgG, and (vii) Mouse IgG2a myeloma protein GPC-7, with tration of 2.5% also gave significant precipitation of the a mouse antiallotype serum (C57 anti-NZB [New Zealand labeled antigen-antibody complex, but did so in all four Black ]) . assays, in distinction to IgMBrd achieving this in only two cases. RESULTS To further test species specificity, four other antigen- Precipitation of a Soluble Antigen-Antibody Complex by antibody combinations were used, involving rabbit, monkey, lValdenstrim Macroglobulins. 42 purified Waldenstr6m macro- mouse, and goat antisera (Table 4). Normal rabbit serum were tested for their ability to precipitate a soluble gave significant enhanced precipitation in all cases, whereas antigen-antibody complex involving '251-labeled IgG1 my- IgMBrd failed to react with mouse antibody, did not react eloma protein BC with its anti-idiotype serum. Whereas no with the goat antibody, but did give marked enhanced pre- precipitation was observed with any of the 41 macroglobulins, cipitation with both rabbit and monkey antibody. Control nor with normal human IgM, by the use of between 1.5-6.0 IgMBj failed to react in any assay. ug of protein, Waldenstrom protein BRD gave 65% precipi- Isospecificity of Ig4Brd. IgM was shown in the previous tation with 1.5 Ag (Table 1). assays to react against a human antiflagellin antiserum, This protein (IgMBrd) and one of the other inactive Walden- therefore demonstrating its isospecificity. To fuither examine strfm proteins (IgMBi), were then similarly tested in three this, primary and secondary human antisera to flagellin other antigen-antibody assay systems, all involving rabbit were fractionated into IgM- and IgG-containing fractions. antibody, but with entirely different antigens and their appropriate antisera. Again, IgMBrd gave significant pre- cipitation in all three assays (Table 2), with as little as 0.2 TABLE 3. Species specificity of IgMBrd against antigen- ug of protein. The different degrees of precipitation of the antibody complexes with flagellin 1338 antigen labeled antigen in the three assays is only a reflection of the amount of its specific antiserum used. In all cases, IgMBd Percent of 12I-labeled flagellin- was tested for its ability to precipitate the labeled antigen antibody complex precipitated without the presence of the appropriate antiserum, and in all Protein Amount Rabbit* Humant Ratt Mouse§ cases no precipitate was observed. Control IgMBi gave no 0 significant precipitation in any of the assay systems. Nil 0 8 9 By the latex-particle agglutination test with human Normal rabbit gammaglobulin-coated particles, purified IgMBrd at a con- serum 2. a5 ,l 35 16 23 22 centration of 12.0 mg/ml gave positive agglutination to a IgMBrd 12.0 jug 56 54 2 7 dilution of '/80. 3.0 /ug 48 50 2 7 Species Specificity of IgMIIBrd Anti-Antibody. Flagellin O. 8jug 37 39 2 7 IgMBi 16.0 jug 0 8 1 monomer 1338 was labeled with 1251 and used with antisera IgMK 16. 0 ug 1 0 to flagellin, prepared in the rabbit, rat, man, or mouse. When these sera were used at a dilution such that less than 10% * Hyperimmune rabbit antiserum to flagelliD. precipitation occurred, IgMBrd gave marked enhanced pre- t Hyperimmune (secondary) human antiserum (containing cipitation with both the rabbit and human antisera, but not IgM and IgG ) to flagellin. with the rat or mouse antisera (Table 3). Significant pre- I Purified IgG from antiserum to flagellin, prepared in the rat. cil)itation was observed with 0.8 jxg of IgMBMd whereas § Hyperimmune mouse (BALB/c) antiserum to flagellin. Downloaded by guest on September 24, 2021 2848 Immunology: Warner et al. Proc. Nat. Acad. Sci. USA 68 (1971) TABLE 4. Species specificity of IgMBrd against antigen-antibody complexes with gammaglobulin antigens

Percent precipitation of antigen*-antibodyt complex Human IgG, (BC)- ' Human IgG, (BC)- Human IgG3- rabbit antibody to Mouse GPC-7IgG2.- goat antibody to monkey (rhesus) human BC idiotype mouse antiallotype human gamma- Protein Amount antibody to IgG3 specific (C57 anti-NZB) globulin Nil 20 0 30 7 Normal rabbit serum 5. 0 1Al 50 62 57 46 IgMBrd 12.0 jug - 28 7 6.0 pg 48 52 30 7 0.8 /g 37 30 7 IgMsi 8.0 lig 19 0 30 7 * All antigens are purified myelomas labelled with 125J. t Antibodies are prepared in different species against gammaglobulin antigens.

These were then separately tested with iodinated monomer BC-idiotype serum complex was obtained with the rabbit preparation of S. adelaide flagellin, and the results in Fig. 1, IgG anti-BSA-BSA complex, but not with rabbit IgG alone show that IgMBrd gives strong precipitating activity against (Fig. 2). As little as 100 ng of the complex resulted in signifi- both IgM and IgG fractions when combined with the flagellin cant inhibition, whereas 10,000 ng of soluble rabbit IlG monomer. In a separate assay, another Waldenstr6m IgMBe failed to inhibit. was complexed with '25I-labeled Dnp28 BSA under dilution Activity of BRD Subunits. The ability of papain-digested conditions such that no precipitation occurred. In the presence fragments of IgMBsd, of isolated L and H chains, and of the of IgMIBrd, marked precipitation was observed (Fig. 1). It 7S monomer of IgMBrd, to show precipitating activity was should be noted that as in all other assays, the BSA diluent assayed in the IgGI myeloma BC-anti-idiotype assay. The was used which contained 30 mg/ml of BSA, thus indicating that the IgMBei complexed to the Dnp haptenic determinant, results shown in Fig. 3 demonstrate that the 7S monomeric subunit of IgMBrd is fully active in precipitating the complex. rather than the BSA carrier. Independent studies have con- firmed that IgMBei does show anti-Dnp activity. In this No precipitating activity was detected in either the isolated chains or fragments. present context, the assay further confirms that IgMBrd will bind to both IgM- and IgG-complexed antibodies. Failure of I9MBrd to React with Fab Fragments of IgG. In Inhibition ofIgMBrd Activity by Antigen-Antibody Complexes. order to determine the submolecular localization of the anti- genic site on IgG to which IgMBrd reacts, a papain and pepsin IgMBrd was used to precipitate the soluble complex of 125J_ labeled IgG1 myeloma BC and its idiotype-specific antiserum. digest of a rabbit IgG antibody were prepared. The target IgG used was from a rabbit antiserum to a chain, with an A series of tubes were prepared, containing in addition to the 125I-labeled human IgA myeloma protein. Three assay above reagents, normal rabbit IgG, or the resuspended systems were studied, each with the use of the labeled IgA, complex of a rabbit anti-BSA serum with BSA. Marked inhibition of precipitation of the labeled IgG1 myeloma with the intact IgG of the antiserum to a chain, Fab (papain digestion, or F(ab')2 (pepsin digestion) of the antibody. Protein IgMBrd did not react with the free labeled myeloma

507 IgA. The results are given in Table 5, and show that with

o _G

CL.0 a: x TABLE 5. Reaction of IgMBrd with IgG antibody or fragments XJ am CL Co Percent precipitation of complex o z [l2I]IgA 2c "iz IV C Precipitator of IgG of Fab of IgG F(ab')2 of antigen-antibody anti-a anti-a IgG anti-a z complex serum* serumt serumt Sheep anti-rabbit OF PROTEIN Fab 66 52 32 FIG. 1. Precipitation of soluble antigen-antibody complexes IgMBrd 52 0 0 with IgMBrd (@ -). All assays use 12I-labeled antigens, flagellin monomer 1338 in (a) and (b), and Dnp2s-BSA in (c). All assays All assay tubes contain [la2I]human IgA with either, no added use human antibody; an IgM antiflagellin, an IgG antiflagellin, antiserum (control, all values = 0); the IgG fraction of a specific and IgMsei, in (a), (b), and (c), resepetively. Control macro- anti-a serum*; the papain Fab fragment of the IgG antibodyt; globulins IgMBI and IgMK (X X) and IgGIBC (O-O) or the pepsin F(ab')2 fragment of the IgG antibodyt. In cases were tested for ability to precipitate the complex. Control value where no precipitator or IgMwh was added, there was no pre- without any added protein is indicated, (- -). cipitation of ["25I]IgA complex. Downloaded by guest on September 24, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Anti-Antibody Activity of IgM 2849

the amounts of anti-a protein or fragments used, no direct LL x 100 precipitation of the IgA myeloma occurred. That a complex 0 ZLLE with the IgA myeloma was formed in each case is however z 0. confirmed by the precipitation of the complex with a sheep pX Foof antiserum to rabbit Fab. The addition of IgMIBrd resulted in 0a P: precipitation of the complex only in the case of the intact zZ1 oo IgG antibody. No precipitation was observed in any assay z after the addition of control protein IgMwh. This result infers that IgMBrd reacts with the Fc fragment of the IgG I= antibody in the complex. 0. Z 1.0 2-0 10.0 Binding ofIodinated IgMIBrd to Antigen-Antibody Complexes. A1g OF PROTEIN In attempting to show whether the IgMBd preparation is homogeneous in its ability to bind to antigen-antibody FIG. 2. Inhibition of precipitation of [12I] IgGI-BC-anti- complexes, a sample of the protein was labeled with "3II idiotype serum complex by either normal rabbit IgG (0- - -4), or and then used to precipitate a soluble complex of '251-labeled a complex of rabbit anti-BSA IgG and BSA (X )X). Protein flagellin monomer with a rabbit antiserum to flagellin. IgMIBrd is present in all tubes to form precipitable complexes with Extremely mild conditions of chloramine-T treatment were the labeled antigen-antibody complex. necessary in the labeling of IgMBrd, as under the standard conditions, all antibody activity was lost. With the milder antigen-antibody complex involving the human myelomia conditions used, antibody activity was retained, and 50% protein BC-idiotype assay was used with either normal of the [1251]flagellin monomer was precipitated. A maximum rabbit serum or IgMBrd in the presence or absence of IgAlst. of 51% of I3'l activity was also found in the precipitate. This Whereas 1.0 ug of IgMst almost completely inhibited the result clearly shows that the precipitating activity of IgMBrd precipitating activity of normal rabbit serum (Table 6), no is not due to a minor component, and is resident in at least effect on IgMBrd activity was observed. 51% of the protein. In view of the marked lability of IgMBrd DISCUSSION to iodination, it may well be that some destruction of activity occurred even under the milder conditions used, resulting in That monoclonal macroglobulins derived from malignant only 51%, rather than 100% precipitation of IgMBrd. cells can have a defined antibody specificity, is by now well established (24). That many of these proteins show anti- Inhibition with Complement-Fixing IAg Proteins. In globulin specificity has also been frequently alluded to in the previous Tables, it has been shown that normal rabbit serum past few years (4-7). This present report further documents contains a component capable of precipitating antigen- this general observation and describes a hitherto unreported antibody complexes in a similar manner to IgMBd, with the type of antiglobulin specificity in the Waldenstr6m proteins. exception that no species specificity was evident in the rabbit Whereas most previously reported monoclonal antiglobulins serum. Previous studies have indicated that this is largely studied in detail have been shown to directly bind to native due to complement lq in the rabbit serum (20). To further 7S IgG globulin, IgMBrd does not react with 7S IgG, unless show that IgMBrd behaves differently from this activity in the target IgG is itself complexed to an antigen. rabbit serum, a complement-binding IgM protein (IgMst) Of 42 Waldenstr6m proteins studied, IgMBrd is the only was tested for its ability to inhibit IgMBrd or the enhanced one found to show this activity. In studies with antigen- precipitation, that is mediated by rabbit serum. A soluble antibody complexes involving antibodies made in different species, it is quite clear that IgMBrd shows marked species TABLE 6. Effect of complement binding I1gM on ability of normal rabbit serum or IgMBrd to precipitate antigen-antibody complex 60-1 __O Percent precipitation LU 5J

of antigen-antibody o PrecipitatorPrecipitator ofof* AL antigen-antibody Amount complex* I °- 40- complex added Controlt 1 lug IgAIst$ SC.- >-a.4 0 Nil - 3 2 CC m°3 Normal rabbit C . -3,> serum 10.0 JAl 54 11 0 5.0 1AI 38 0 X Z 1 2.5u1 19 0 P- IgMsrd 6.0 lug 55 59 1.5,ug 53 49 II ,, O. 7jug 47 46 NIL 1.0 2.0 3.0 40 5.0 12.0 Aig OF PROTEIN * Soluble complex of 15I-labeled BC IgGi myeloma protein, with anti-idiotype specific antibody. FIG. 3. Precipitation of soluble antigen-antibody complex of t Precipitation in presence of precipitator alone. [1211]IgGi-BC with anti-idiotype serum, by IgMBrd intact protein I Precipitation in presence of precipitator and 1.0 jug of a (0 0), its 7S monomer (0-- -0), papain digest fragments complement-binding IgM Waldenst5m macroglobulin, IgAIst. (A A), or H or L chains (0-0). Downloaded by guest on September 24, 2021 2850 Immunology: Warner et al. Proc. Nat. Acad. Sci. USA 68 (191-1)

specificity. It is a true autoantibody (or more strictly iso- be suggested that I1MBrd is reacting against a shared IgNI antibody) in that it reacts with human antibody-antigen and IgG conformational site, created after the antigen- complexes. Its specificity also includes other primate immuno- antibody interaction. Although heterologous immunization globulin (monkey), rabbit immunoglobulin, but not goat, studies do not readily demonstrate such a shared As and oy rat, or mouse antibody. That these latter assays did indeed Fe site, a precedent may be found in the site to which com- contain potentially precipitable soluble complexes was plement binds. verified by precipitation with the complement lq activity in In considering the general antiglobulin specificities of normal rabbit serum. In studies with fractionated human Waldenstr6m macroglobulins, it appears that there are antisera to flagellin, it was shown that IgMBrd reacts with both several distinctly different specificities represented. These human IgM and IgG antibodies. Reaction with an IgM might be at present listed as three main types (i) reactivity antibody was also shown in the interaction of two Walden- with Fe of human or primate 7S IgG, such as IgMj8y (6); strom proteins, IgMI~srd and IgMBei. By conventional latex (ii) reactivity with both human IgG and with the Dnp agglutination tests, both these IgM proteins have strong hapten (19); (iii) anti-antibody specificity shown by IgMBrd- antiglobulin activity. IgMBei, however, also precipitates If this is indeed a fair representation of a significant proportion dinitrophenylated proteins, such as Dnp28 BSA. This is not of Waldenstrbm proteins, at least three questions must be shown by IgMBrd. This anti-Dnp reactivity appears to be very asked. (a) Do the other Waldenstr6m proteins have some type similar to that described by Hannestad et al. (19) in that the of similar auto-antibody activity? (b) Do myeloma proteins anti-Dnp reactivity can be inhibited by IgG globulin. A also show this? (c) Does this have some central significance similar antiglobulin-anti-Dnp reactivity has also been for the etiology of the malignant clone of cells? found with a mouse IgG2a myeloma protein (Warner, N. L., (a) Several other reports of antibody activity in Walden- unpublished observations). When soluble complexes of a str6m proteins have appeared in the literature, notably Dnp-protein conjugate and IgMBei are prepared, these can involving cold agglutinins (9, 10), antibodies to stored then be precipitated by IgMBrd, again showing anti-antibody erythrocytes (26) and complement-fixing antibodies detected activity in IgMBrd to complexed IgM antibody. with human tissue extracts (27). The finding that some Although normal rabbit serum will also precipitate soluble anti-Dnp reactions may represent crossreactions with either antigen-antibody complexes, previous studies (20) have gammaglobulin (19) (Warner N. L., unpublished observa- suggested that this activity is predominantly due to comple- tions) or nucleic acid components (11) also raises the possi- ment lq binding to complexes. This was further indicated by bility that other anti-Dnp reactivities (28) may indeed the results in Table 6, in which the addition of IgMst, a represent crossreactions to some unknown autoantigen, i.e., Waldenstr6m macroglobulin of the complement fixing sub- the proliferation of the original malignant cell precursor may class, completely inhibited normal rabbit serum precipitating have been immunologically stimulated by contact of its activity. That it did not do so with IgMBrd also shows that receptors with autoantigen, rather than representing a IgMBrd is not capable of binding to uncomplexed IgM, random mutation to malignancy. It is also possible that some although it can do so with complexed IgM as in Fig. 1. antibody activities may have been overlooked through That IgMBrd cannot bind to native 7S IgG is shown by the nonoptimal conditions for antibody binding, as for example, inhibition test in which rabbit IgG alone cannot inhibit the antiglobulin activity of a Waldenstr6m macroglobulin; Ig1MBrd activity, whereas a nonspecific rabbit IgG-antibody described by Kritzman et al. (4) shows a striking and unusual complex (BSA-anti-BSA) can do so. (Fig. 2). pH optimum for binding activity. IgMBrd which has antiglobulin specificity therefore differs (b) Many antibody activities have now been described for from the other reported antiglobulin specificities of Walden- myeloma proteins of either human or mouse origin; some of str6m proteins in showing specificity for human, primate, these have shown autoantibody specificity. Antiglobulin and rabbit immunoglobulins; in reacting with both IgM and activity in seven IgG myelomas was shown by Grey et al. (29), IgG, and in reacting only with an immunoglobulin when it is antilipoprotein activity has been found (12) and in at least complexed with an antigen. This is in distinction to the well- three cases, the anti-Diip activity of myeloma proteins has documented protein of Metzgar and Stone (6, 18), which been shown to crossreact with a self component, namely, reacts with Fc fragments and 7S IgG molecules of human nucleic acid components (11), gammaglobulin (Warner, and primate, but not of rabbit origin. Most other reports of N. L., unpublished observations), and vitamin K (30). antiglobulin activity in Waldenstr6m proteins also show Several other antibody specifications have also been found reactivity with nonaggregated immunoglobulin fragments (5) for myeloma proteins which are not apparently related to or at least reactivity with 7S immunoglobulin which, in some autoantigens [antistreptolysin and antistapylysin (12), cases, is increased by the use of aggregated IgG (25). In various other antibacterial specificities (13), and antidextran some of the earliest reports on antiglobulin activity (4), activity (31, 32)]. Several of the antibacterial myelomas reactivity was shown with aggregated IgG; it is possible that were, however, found to react with phosphoryl cholinet; these cases may represent a similar specificity to IgMBrd. it is, therefore, at present a matter of some debate as to As IgMBrd reacts with both IgM and IgG antibody- whether these and the other anticarbohydrate reactions are antigen complexes, it might be expected that it reacts against really antibacterial or antiautoantigen (cell membrane com- an Fab site on the target antibody. However, the studies with ponent) in origin. papain- or pepsin-digested antibody indicate the lack of (c) If we therefore start from the premise that at least a reactivity to Fab or F(ab')2 fragments. This, therefore, high proportion of Waldenstr6m proteins, and possibly a suggests that IgMBrd is reacting against an Fc antigen that significant proportion of myeloma proteins, have auto- is present on both the ,u and -y heavy chains. As this antigenic antibody activity, then we might consider two basic alterna- site is apparently absent from the native molecule, it might tives for the origin of these tumors: (i) That there is a high Downloaded by guest on September 24, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Anti-Antibody Activity of IgM 2851

proportion of cells in the normal individual making auto- 8. Beaumont, J. L., B. Jocotot, C. Vilain, and V. Beaumont, antibodies, but that they have a similar chance of becoming C. R. H. Acad. Sci., 260, 5960 (1965). 9. Fudenberg, H. H., and H. G. Kunkel, J. Exp. Med., 106, 689 malignant as do nonautoantibody-producing cells, or (ii) that (1957). the initiation of an autoimmune clone is a necessary first 10. Deutsch, H. F., Biopolymers, 7, 21 (1969). event, which is then followed by this clone becoming malig- 11. Schubert, D., A. Roman, and M. Cohn, Nature, 225, 154 nant. There is little evidence for the first possibility other than (1970). 12. Zettervall, O., Gammaglobulins, Structure and Control of to say that there does seem to be a normal low level of some Biosynthesis (Interscience, New York, 1967). autoantibodies in serum, particularly in old age (33, 34). 13. Potter, M., Fed. Proc., 29, 85 (1970). Recent preliminary studies (Dwyer, J., unpublished ob- 14. Eisen, H. N., J. R. Little, C. K. Osterland, and E. S. Simms, servations) have also demonstrated the presence in low Cold Spring Harbor Symp. Quant. Biol., 32, 75 (1967). numbers, of lymphoid cells in peripheral blood capable of 15. Eisen, H. N., E. S. Simms, and M. Potter, Biochemistry, 7, 4126 (1968). binding autoantigens that are labeled with 1251, namely IgG 16. Schubert, D., A. Jobe, and M. Cohn, Nature, 220, 882 and the human encephalitogenic protein. Further examination (1968). of this type may be of great relevance to the question of 17. Ashman, R. F., and H. Metzgar, J. Biol. Chem., 244, 3405 origin of the malignant clone, particularly for Waldenstrdms (1969). 18. Stone, M. J., and H. Metzgar, Cold Spring Harb. Symp. macroglobulinemia, as most antigen-binding cells have Quant. Biol., 32, 83 (1967). IgM surface immunoglobulin (35). 19. Hannestad, K., Clin. Exp. Immunol., 4, 555 (1969). Evidence for induction of an autoimmune clone before 20. MacKenzie, M. R., N. L. Warner, W. D. Linscott, and a malignant change in the clone, is also sparse. There is H. H. Fudenberg, J. Immunol., 103, 607 (1969). strong suggestive evidence that in both mice and man, (at 21. Deutsch, H. G., E. R. Steihm, and J. I. Morton, J. Biol. Chem., 236, 2216 (1961). least in the latter case for Waldenstrbm macroglobulinemia, 22. Herzenberg, L. A., and N. L. Warner, in Regulation of the (36)), there is a marked genetic susceptibility to plasma cell Antibody Response, ed. B. Cinader (C. C Thomas, Spring- tumor development and this aspect is discussed fully else- field, 1968), p. 322. where (Warner et al., submitted for publication). 23. Greenwood, F. C., W. M. Hunter, and J. S. Glover, Bio- chem. J., 89, 114 (1963). Further evidence for or against either of these latter 24. Metzgar, H., M. Potter, and W. Terry, Immunochemistry, possibilities is definitely required, and is being sought for by 6,831 (1969). experimental studies at the cellular and protein level. 25. Balazs, V., and M. M. 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