Proc. Nat. Acad. Sci. USAVol. 72, No. 5, pp. 1707-1711, May 1975

Production of Antibodies of Identical Idiotype but Diverse ImmunoglobulinClasses by Cells Derived from a Single Stimulated B Cell

(V gene sharing/simultaneous class production/monoclonal antibody)

PATRICIA J. GEARHART, NOLAN H. SIGAL, AND NORMAN R. KLINMAN

Department of Pathology, School of Medicine, University of Pennsylvania, Philadelphia, Pa. 19174

Communicated by Herman N. Eisen, February 14, 1976

ABSTRACT The availability of anti-phosphocholineantibody of the TEPC 15 idiotype from the clonal progenyof a single precursor cell, stimulated in vitro, permittedthe demonstration of monoclonal antibodies with as manyas three immunoglobulin classes with identical variableregions. This demonstration was dependent on sensitiveradioimmunoassays which showed a one to one relation-ship between the total anti-phosphocholine antibodyproduced by a clone and the sum of anti-phosphocholineantibody of the different classes as well as the amount ofantibody of the TEPC 15 idiotype. The class distributionwas confirmed by isoelectric focusing identification ofIgM, IgA, and IgGI antibodies of the TEPC 15 idiotypeproduced by single clones which showed characteristic plvalues for each immunoglobulin class. Thus, within thegenerative phase of a single antibody-producing cell clone,various heavy chain constant regions can be linked to thesame variable region, and single precursor cells have thecapacity to generate progeny expressing at least three dis-tinct immunoglobulin classes.

The heterogeneity of immunological responses to mostantigens represents the composite antibody product of manymonospecific precursor cells responsive to those antigens (1).In addition to heterogeneity of the variable domains and theirrespective antigen-binding sites, heterogeneity also exists inthe constant region of the heavy chain, since antibodies ofsimilar specificity are produced by individual animals inseveral immunoglobulin classes (2). Considerable evidence isavailable in support of the hypothesis that the variable andconstant regions are under separate genetic control and arejoined to form a heavy or light chain (3-5). Inherent in this"two gene-one polypeptide chain" hypothesis is the conceptthat any constant region gene may be associated with anyvariable region gene (6). The most compelling evidence forthis is derived from the study of sera from myeloma patients,which contain monoclonal IgM and monoclonal IgG2 proteinsidentical in the heavy chain variable region by partial sequenc-ing and idiotype (5). The sharing of idiotypic markers byIgM and IgG immunoglobulin has been reported in othersystems (7, 8), and recently several investigators havedemonstrated idiotypic cross reactions between a mousemyeloma protein of the IgA class with specificity for phospho-choline (PC) and anti-PC antibody of the IgM class fromBalb/c mice (9, 10). Whether immunoglobulins containing thesame variable region on different heavy chains are producedby progeny of the same precursor cell or different precursor

Abbreviations: PC, phosphocholine (frequently termed phos-phorylcholine); Dnp, 2,4-dinitrophenyl; B cell, bone-marrow-derived antibody-forming cell precursor; PPC-TGG-Hy, 3-(p-azophenylphosphocholine) - N-acetyl- L- tyrosylglycylglycine-he-mocyanin; PC-BSA-BAC, phosphocholine-bovine serum albu-min-bromoacetyl cellulose.

1707

cells, however, is still not certain. Definitive evidence wouldrequire the analysis of the variable region on different im-munoglobulin classes produced by a single cell or a clone ofcells derived from a single stimulated precursor cell.

Studies by several investigators have suggested that onecell or its clonal progeny can produce both IgM and IgGantibody. Pernis et al. (11) have shown that the same rabbitallotype is associated with a cell's surface IgM and cyto-plasmic IgG; however, the genetic regulation of allotype in thevariable region may be distinct from that which controls thehypervariable region (12). Nossal et al. (13) have demon-strated that single antibody-forming cells can release IgMand IgG; furthermore, Press and Klinman have shown thatmonoclonal anti-2,4-dinitrophenyl (Dnp) antibody fromsplenic foci generated in vitro can contain both IgM andIgG1 (14). These observations, while indicating that one cellor a clone of cells can produce antibody of two classes withsimilar antigen specificity, do not necessarily establish theidentity of the variable region of the two immunoglobulinclasses. Since such a demonstration would provide crucialproof for the stated postulate, we have examined antibodyproduced by isolated clones, derived from single stimulatedprecursors to antibody-forming cells specific for PC andderived from bone marrow (B cells) for specificity, class,idiotype, and isoelectric spectrum. Evidence is presented thatsingle clones can produce antibody with apparently identicalvariable regions but different heavy chain constant regions;therefore, regardless of the class, the idiotype of the variableregion is conserved at the clonal level.

MATERIALS AND METHODS

Fragment Culture. The splenic focus technique of antigeni-cally stimulating individual B cells in fragment cultures hasbeen described previously (15). Limiting doses of spleen cellsfrom unimmunized donor Balb/c mice were injected intra-venously into lethally irradiated hemocyanin-primed syn-geneic recipients. Sixteen hours later, the recipient's spleenswere diced and the fragments were stimulated in vitro with3-(p-azophenylphosphocholine) -N-acetyl-L-tyrosylglycylgly-cine-hemocyanin (PPC-TGG-Hy) (16). The culture super-natants collected 8-13 days after stimulation were analyzedby radioimmunoassay for the presence of anti-PC antibodyas well-as the immunoglobulin class and idiotype of thatantibody.

Radioimmunoassays for Fab, Immunoglobulin Class, andIdiotype. Specific antibody bound to PC-bovine serum album-in-bromoacetyl cellulose (PC-BSA-BAC) immunoadsorbant(17) was quantified with rabbit 1i5I-labeled antibody to mouse

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1708 Immunology: Gearhart et al.

0

2

a:CD0

2

20 *17

,12/i 8,,-X

'16-oo

4 -

6~~ ~ ~ ~ ~ ~ ~ ~~1

0~~~~

2 -

2 4 6 8 10 12 14 16NANOGRAMS I DIOTYPE

I

is 20

FIG. 1. Linear relationship of multi-heavy chain class mono-clonal antibody for Fab and TEPC 15 idiotype content. Samplesfrom culture fluids or TEPC 15 ascites were tested for Fab, idio-type, IgM, IgGl, and IgA antibody as described in Materials andMethods. The standard curves for the Fab antibody and idiotypeassays were derived using known amounts of purified, reduced andalkylated TIPC 15 protein. Crosses represent two dilutions of a

45% (NH4)2S04 preparation of TEPC 15 ascites. Numberedclosed circles denote monoclonal anti-PC antibody containingthe following heavy chain classes: 1l-1, Syl; 2-pA, y1; 3-iu, a;

4-u, yl, a; S-A, -yl, a; 6-mu, yl, a; 7-Kyl, a, 8-Aj, yl, a;9P-g, a; l0A, Lyl, a; 11-,, a, 12-1a, yl 13-1, a; l4-, -yl,

a; i-lu, a; l6-u, a; 17-- , yl, a.

Fab (15). Heavy chain classes were quantified by the same

procedure, only employing "2'I-labeled goat antibody tomouse IgG1, IgM, and IgA as the detecting reagent (14, 17).The TEPC 15 plasma cell tumor was obtained from Michael

Potter, National Cancer Institute, National Institutes ofHealth, Bethesda, Md., and maintained by serial passage inBalb/c mice. Protein was purified from ascites fluid by ad-sorption to and elution from PC-glycyltyrosine-Sepharose 4B(Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) as

described previously (17). The idiotypic determinant of theTEPC 15 protein, which has binding specificity for PC (18),is found on the majority of antibody to PC in the Balb/cstrain (9). Anti-idiotypic serum was raised in A/He mice bymultiple injections of TEPC 15 protein (19). The serum was

used in a solid phase radioimmunoassay in which the culturefluid containing antibody sharing the TEPC 15 idiotype was

detected by inhibition of 12"L-labeled TEPC protein bindingto the anti-idiotype antibody immobilized on plastic tubes(17).Radioimmunoassays for Fab, idiotype, and a heavy chain

class were standardized using purified, reduced and alkylatedTEPC 15 protein. The quantity of antibody of the IgM andIgG1 class was determined by comparing the amount of

antibody detected by anti-Fab from foci containing solelyIgM or IgG1 and the corresponding counts detected by anti-por anti--yl. The specificity of assays for heavy chain class andidiotype has been previously shown (17).

Isoelectric Focusing. Isoelectric focusing analyses of culturesupernatants were performed by a micro-adaptation using a

Proc. Nat. Acad. Sci. USA 72 (1976)

TABLE 1. Quantitation of monoclonal anti-PC antibody byClass and idiotype

Nanograms antibody in sample*

Heavy chain class

Clone Idiotype Is 71 a

PC17A19 5.1 0.3 4.5 0.1PC17C65 4.7 1.1 1.9 0.2PC17D42 0.6 0.2 0.1 0.3PC17G14 2.3 0.5 1.2 0.1PC17H38 4.2 1.1 1.7 0.2

* Twenty-microliter aliquots from culture fluids collected 8-10days after in vitro stimulation were quantified for idiotype andheavy chain class as described in Materials and Methods.

discontinuous sucrose density gradient containing 5% Am-pholine (LKB Instruments, Rockville, Md.) as described byPress and Klinman (20). Ampholine mixtures containing 50%pH 4-6 and 50% pH 5-7 provided a linear gradient from pH4 to pH 6.5 and were used to focus culture fluids containingsolely IgM, and IgM plus IgA. IgA was focused in an Ampho".line mixture containing 66% pH 5-8, 17% pH 4-6, and 17%pH 7-9, which yielded a linear gradient from pH 5 to pH 7.An Ampholine mixture of 50% pH 6-8 and 50% pH 7-9 gavea linear gradient from pH 6 to pH 8 and was used to focusthe IgG1 antibody as well as fluids containing IgG1 and IgA.Two-tenths milliliter of culture fluid was distributed among0.25 ml volumes of 20%, 30%, 40%, and 50% solutions ofsucrose containing the appropriate Ampholine; or 0.1 jul ofTEPC 15 ascites fluid was added to the 30% layer. The solu-tions were then successively layered into a 1 ml disposablesyringe closed at one end with dialysis tubing held in placeby a rubber gasket. The tubes were focused for 6-8 hr at 40at 160 V, after which approximately 100 drops were indi-vidually collected into wells of a collection tray containing 0.3ml of 0.15 M NaCl- After the pH was read, one-half of eachsample was assayed for the presence of the idiotype by in-hibition in the anti-idiotype assay, and the other half wastested for anti-PC antibody by binding to PC-BSA-BAC.Bound antibody was detected with "15I-labeled anti-p, andagain relabeled with iodinated anti-yl, and then with iodinatedanti-a. Radioactivity from a previous labeling was subtractedfrom each new labeling, and increments exceeding the back-ground adsorption- Were denoted as heavy chain positive.

RESULTS

Simultaneous Expression of Several Immunoglobulin Classesby Clones. Fig. l presents a comparison of the amount ofanti-PC antibody on a weight basis detected by the anti-idiotype assay versus that detected by analysis of Fabdeterminants. In this figure, all of the data points were ob-tained from clones producing antibody of more than oneimmunoglobulin class, and in every case, the experimentalvalues fall on a theoretical line constructed for a one to one

correlation. Thus, all of the anti-PC antibodies in these mixedclass clones are of the TEPC 15 idiotype. Table 1 demonstratesthat in these fragment cultures readily detectable amounts ofanti-PC antibody of each of the immunoglobulin classes are

being synthesized-and that the sum of the antibody detectedfor each imtnunoglobulin class approximates and does notexceed the values obtained by analysis of the idiotype.

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Idiotypic Identity of Monoclonal Immunoglobulins 1709

TABLE 2. Frequency offoci releasing anti-PC antibodycontaining more than one immunoglobulin class

Heavy chain class Number of foci*

A only 4yl only 4a only 3A + -yl 7 (0.58)tJ + a 4 (0.51)t'4 + a 5 (0.53)t+ yl + a 9 (0.01)t

* Total fragments analyzed = 1048. The fragments analyzedwere from hemocyanin-primed recipients injected with smallnumbers of donor spleen cells, which yielded a low multiplicity ofPC precursors when stimulated with 0.5 1AM PPC-TGG-Hy.

t Number of foci predicted for the random occurrence of two(or three) simultaneous, independent events. The predicted valueswere obtained by multiplying the frequency of total foci contain-ing one immunoglobulin class by the frequency of foci containingother classes.

It has been previously shown that the frequency of fociproducing more than one antibody specificity is close to thatpredicted by the random distribution of precursors (14).The results of Table 2 indicate that the frequency of clonesproducing anti-PC antibody of more than one immunoglobu-lin class is significantly greater than the number predictedby the random occurrence of more than one precursor in thesame fragment. Thus, the vast majority of fragment culturesproducing anti-PC antibody even of two or more immuno-globulin classes are derived from a single precursor cell.

Analysis of Clonal Antibody Separated by Isoelectric Focus-ing. The isoelectric spectrum of each immunoglobulin class of

TABLE 3. Isoelectric focusing analysis of idiotype positivemonoclonal antibody to phosphocholine*

Nanogramsantibody in-arepresentative

aliquotchain

Clone class pI range Fabt Idiotypet

PC14F16 '4 7.8-7.4 1.1 1.1a 6.1-5.8 2.1 2.4

PC16E34 '4y 7.9-7.5 . 0.6 0.5a 6.2-6.0 0.4 0.6

PC14G16 '1 7.7-7.3 0.8 0.9a 6.2-5.8 4.3 1.6

PC14F50 a 6.2-5.9 0.6 0.5;& 5.2-4.6 0.6 0.7

PC14G46 a 6.2-5.9 1.6 2.2JA 4.7-4.4 0.2 0.2

GD3D62 -a 7.4t 1.5 1.7,u 4.7-4.4 0.5 1.5

* Culture fluids were focused.in pH gradients covering pH 6-9for IgGi and IgA antibody and pH 4-7 for-IgA and IgM. Samplesfrom the collected gradients were assayed for immunoglobulinclass, Fab, and idiotype as described in Materials and Methods.

t In some instances, accurate quantitation was precluded byinsufficient material for analysis.$pH determinations higher than this value were beyond the

a

1.0 . CLONE GD 3D53

0;Of/CD .bX91LZ_ CLONE PC 14F16

IVAz C-

~~~~~~~~~CLONEPC1380793.0

TEPC 15 ASCITESZ 1.0

i , . I

4.0 5.0 6.0 7.0 8.0pH

FIG. 2. Identification by isoelectric focusing of IgM, IgGI,and IgA proteins containing the TEPC 15 idiotype. Culture fluidscontaining monoclonal antibody or TEPC 15 ascites fluid were

focused and the collected fractions were assayed for pH, idiotype,and heavy chain class.

idiotype positive monoclonal antibody was determined byisoelectric focusing analyses of culture supernatants using a

micromethod described by Press and Klinman (20). Since thesupporting medium in this technique is sucrose, focusing ofIgM and IgA as well as IgG in an Ampholine pH gradient can

be accomplished. After the pH gradient was analyzed, theeluted samples were divided and one-half was assayed foranti-PC antibody and heavy chain class while the other halfwas assayed for idiotype content.The focusing patterns presented in Fig. 2 show that anti-

body containing the TEPC 15 idiotype of the IgM class has a

pI range of 4.5-5.1 with a peak at 4.6-4.7, IgGi antibodybearing the idiotype focuses with a pI of 7.4-7.8, and IgAantibody of the idiotype focuses in a pI range of 5.9-6.2 witha peak at 6.1. The broadness in the focusing patterns of themonoclonal antibodies may reflect in vitro post-synthetic mod-ification. In vivo modifications have been previously reported(21) and seem to be present in the TEPC 15 myeloma proteinfound in ascites fluid, which focuses in the same pI range as

monoclonal IgA antibody but with a less distinct peak.The most direct demonstration of monoclonal production of

several immunoglobulin classes with identical idiotypes ispresented in Table 3. Here the immunoglobulins from frag-ment culture supernatants containing more than one classwere separated by isoelectric focusing pH gradients. As seen

in Table 3, antibody from a clone that produced any combina-tion of IgM, IgGi, and IgA also had the idiotype region as-

sociated with each class. Since the amount of antibody de-tected by the anti-idiotype assay usually approximated thatobserved with anti-Fab, each peak represents antibody withonly the TEPC 15 idiotype. In addition, antibody containingthe different heavy chain products focused consistently withinthe pH range delineated for each class in Fig. 2; thus the pIvalues served as invariant markers for the TEPC 15 variableregion and appropriate heavy chain classes. The focusinganalysis is further strengthened by preliminary observationsthat non-TEPC 15 idiotype clonal antibodies focus at severalpI values, distinct from those found for the three classes shar-

range of Ampholine used.

Proc. Nat. Acad. Sci. USA 720 (1975)

ing the TEPC 15 idiotype.

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1710 Immunology: Gearhart et al.

DISCUSSIONA previous report from this laboratory indicated that 16% ofprimary Dnp-specific Balb/c B cells and 14% of secondaryDrp-specific B cells yielded clonal progeny producing anti-Dnp antibody of both the u and 'y1 heavy chain classes (14).Absolute evidence for two immunoglobulins sharing the samevariable heavy region in these clones was lacking, however,since the idiotype of the antibody product was not identified.The experiments reported here extend this analysis and serveas an unambiguous demonstration of the production of anti-body with differentheavy chain constant regionsbutapparentlyidentical variable domains by the clonalprogeny ofa single B cellresponding to antigenic stimulation. The experimental designnot only insured the single cell origin of the antibody-produc-ing cell population, but also provided sufficient antibody froma single clone to permit establishing its immunoglobulin classas well as the identity of the variable regions, both by isoelec-tric focusing and anti-idiotype antibody analysis. Althoughother workers have indicated that the TEPC 15 idiotype canonly be found on IgM antibody after antigenic stimulation ofBalb/c mice (10, 22, 23), we have previously demonstratedthat maximizing carrier recognition allows the production ofother immunoglobulin classes both in vivo and in vitro (17).Much evidence is available indicating the single cell origin

of antibody-forming cells in fragment cultures. This includesthe linear dose-response relationship between the number ofcells transferred and the resulting number of positive fragmentcultures found for responses to a variety of haptens (15). Theantibody produced by such fragment cultures is homogeneousby the criteria of isoelectric focusing spectrum (20) and equi-librium dialysis analysis of hapten binding characteristics be-fore and after polypeptide chain recombination (1, 24). Fur-thermore, previous analyses have shown that in spite of apotentially heterogeneous response to PC in Balb/c mice,fragments producing antibody of the TEPC 15 idiotype do notproduce anti-PC antibody bearing another idiotype (17).The presence of two clones in a single fragment culture hasbeen reported to be a statistically random event (14). How-ever, the frequency of clones producing anti-PC antibody ofmore than one class is significantly higher than that predictedby the random distribution of more than one precursor in afragment, and this has also been observed in double-class-producing anti-Dnp foci (14). Thus it would seem from thisand the linear relationship shown between Fab and idiotype inFig. 1 that the vast majority of fragment cultures producinganti-PC antibody even of two or more immunoglobulin classesare derived from a single precursor cell. This is most apparentin the frequency of cultures producing all three immunoglobu-lin classes and demonstrates that the lineage of cells producingIgM, IgG, and IgA can derive from a single immunocompetentcell.The most definitive evidence in support of monoclonal

production of several immunoglobulin classes with identicalidiotypes has been obtained by isoelectric focusing analysiswhere it is shown in Fig. 2 and Table 3 that antibody of theTEPC 15 idiotype and a given heavy chain class will alwaysfocus in the same pI range. Accordingly, the immunoglobulinpopulations from clones producing more than one heavy chainclass were separated by isoelectric focusing and each peak wasindependently analyzed for idiotype and class. It is clearlydemonstrated from these experiments that regardless of im-

clones could be identified as being of the TEPC 15 idiotype byboth anti-idiotype analysis and isoelectric focusing pattern.The validity of the focusing technique is confirmed by thedemonstration of similarity between the pI value for TEPC 15protein, the product of a plasmacytoma, and induced mono-

clonal antibody to PC. In this regard, the identity in pI as wellas heavy chain class and idiotype provides strong evidencethat myeloma proteins represent products of normal precursor

cells (25).While analyses of myeloma proteins have demonstrated that

identical variable regions can be associated with distinct im-munoglobulin classes (5, 8, 9), the single cell origin and physio-logical relevance of these immunoglobulin-producing cells isuncertain. The studies reported here demonstrate the sharingof idiotypic specificity among immunoglobulin classes pro-

duced in response to antigenic stimulation of a single precursor

cell and thus indicate that the genetic information coding forthe variable heavy-chain region can be joined to any of severalconstant heavy chain genes within the generative phase of a

single antibody cell clone.

The authors thank Mses. Judy Owen, Geraldine Ball, andAlicia Scott for fine technical assistance. This investigation was

supported by U.S. Public Health Service Grant A1-08778, CA-15822, Training Grants A1-00419 (P.G.), and MSTP GM-02046(N.S.), and a Career Development Award 1-K04-A1-33983 toN.K.

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4. Frangione, B. & Milstein, C. (1969) "Partial deletion in theheavy chain disease protein ZUC," Nature 224, 597-599.

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8. Penn, G., Kunkel, H. & Grey, H. (1970) "Sharing of in-dividual antigenic determinants between a TG and a -yMprotein in the same myeloma serum," Proc. Soc. Exp. Biol.Med. 135, 660-665.

9. Cosenza, H. & K6hler, H. (1972) "Specific inhibition ofplaque formation to phosphorylcholine by antibody againstantibody," Science 176) 1027-1029.

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Proc. Nat. Acad. Sci. USA 72 (1976)

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16. Sigal, N., Gearhart, P. & Klinman, N. (1975) "The frequencyof phosphorylcholine-specific B cells in conventional andgermfree Balb/c mice," J. Immunol., in press.

17. Gearhart, P., Sigal, N. & Klinman, N. (1975) "Heterogene-ity of the Balb/c anti-phosphorylcholine antibody responseat the precursor cell level," J. Exp. Med. 141, 56-71.

18. Potter, M. & Lieberman, R. (1970) "Common individualantigenic determinants in five of eight Balb/c IgA myelomaproteins that bind phosphorylcholine," J. Exp. Med. 132,737-751.

19. Lieberman, R. & Humphrey, W. (1971) "Association of H-2types with genetic control of immune responsiveness to IgAallotypes in the mouse," Proc. Nat. Acad. Sci. USA 68,2510-2513.

20. Press, J. & Klinman, N. (1973) "Isoelectric analysis of neo-

Idiotypic Identity of Monoclonal Immunoglobulins 1711

natal monofocal antibody," Immunochemistry 10, 621-627.21. Williamson, A., Salaman, M. & Kreth, H. (1973) "Micro-

heterogeneity and allomorphism of proteins," Ann. N.Y.Acad. Sci. 209, 210-224.

22. Lee, W., Cosenza, H. & Kohier, H. (1974) "Clonal restric-tion of the immune response to phosphorylcholine," Nature247, 55-57.

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