(field Nos. 88 and 104). They are part of the 28. Notizie degli scavl (1923), p. 273.collection of Pompeian flora that I have 29. Corpus inscriptionum Latinarum, vol. 4, in-deposited in the U.S. National Arboretuz. scriptions 6886-6887.herbarium. 30. Pliny, Naturalls historla, book 18, sect. 110.

19. The Romans knew both the white poplar 31. For a discussion of the "dry farming" tech-(Populus alba L.) and the black poplar nique ;_anean area, see(Populus nigra L.). I collected Populus nig Stevens, in The Cambridge EconomicL. (field No. 87). History of Europe, M. M. Postan, Ed. (Cam-

20. The individual coins are identified elsewhere bridge Univ. Press, Cambridge, England, ed.(3, p. 69). 2. 1969), vol. 1, pp. 96-104.

21. Trees were also found in the informally 32. For a description of this press, see A. Maiuri,planted vineyard in the garden restaurant of La Villa del Misteri (Istituto PoligraficoEuxinus (region I, insula 11, entrance 10), dello Stato, Rome, ed. 3, 1960), pp. 41-44.wihich I excavated in 1964 [W. F. Jashemski, 33. For the marked effect of temperature onArchaeology 20, 36 (1967)]. wine production, when expressed as heat

22. See L. Wittmack, Beibi. Bot. Jahrb. No. 73 summation ("sum of the mean daily tempera-(1903), p. 38 for a study of the carbonized ture above 50' F" from April through Octo-fruits and nuts that had been found in the ber), see A. J. Winkler, General Viticultureexcavations up to that time. (Univ. of California Press, Berkeley and Los

23. A study of all the plants known from the Angeles, 1962), p. 58: "The base line is setwall paintings, sculpture, mosaics, carbonized at 50° [Fahrenheit] because there is almostremains, and graffiti in the area destroyed by no shoot growth below this temperature." TheVesuvius will be included in an appendix of summation is expressed as degree-days. Naplesa book on the gardens of the Pompeii area has "4010 degree-days above 50° F" (p. 61,(W. F. Jashemski, in preparation; F. G. table 3), which would place it in climaticMeyer is collaborating on the appendix). region 5 of Amerine's and Winkler's classifica-

24. Pliny, Naturalis historia, book 17, sects. 164- tion scheme of five climatic regions for wine166, 199-200. production. Region 5 includes locations thai

25. See the report of the 1970 season (4) for a have 4001 or more degree-days. Pompeiidescription and criticism of previous explana- would be in the same region as Naples.tions of the vitis compluviata. 34. Martial, Epigrams, book 5, sect. 70, 1. 3.

26. Varro, De re rustica, book 1, chap. 8, sect. 35. Notizie degli scavi (1900), p. 31.2. 36. Corpus inscriptionem Latinarum, vol. 10,

27. Columella, De re rustica, book 4, chap. 17, inscription 1074; F. Mazois, Les ruines desects. 3-6. Pompe'i (Firmin-Didot, Paris, 1812), vol. 1;

Antibody Structure andMolecular Immunology

Gerald M. Edelman

Some sciences are exci.ting becauseof their generality and some becauseof their predictive power. Immunologyis particularly exciting, however, be-cause it provokes unusual ideas, someof which are not easily come uponthrough other fields of study. Indeed,many immunologists believe that forthis reason, immunology will have agreat impact on other branches of biol-ogy and medicine. On an occasion suchas this in which a very great honor isbeing bestowed, I feel all the moreprivileged to be able to talk about someof the fundamental ideas in immunol-

Copyright i 1973 by the Nobel Foundation. Theauthor is professor of biochemistry at RockefellerUniversity, New York 10021. This article is thelecture he delivered in Stockholm, Sweden, on 11December 1972 when he received the Nobel Prizein Physiology or Medicine, a prize he sharedwith Professor Rodney R. Porter. It is publishedhere with the permission of the Nobel Foundationand will also be included in the complete volumeof Les Prix Nobel en 1972 as wen as in the seriesNobel Lectures (in English) published by theElsevier Publishing Company, Amsterdam andNew York. Professor Porter's lecture appearedin the 18 May 1973 issue, page 713.

830

ogy and particularly about their rela-tionship to the structure of antibodies.Work on the structure of antibodies

has allied immunology to molecularbiology in much the same way as previ-ous work on hapten antigens alliedimmunology to chemistry. This struc-tural work can be considered the firstof the projects of molecular immunol-ogy, the task of which is to interpretthe properties of the immune system interms of molecular structures. In thislecture, I should like to discuss someof the implications of the structuralanalysis of antibodies. Rather than re-view the subject, which has been amplydone (1-3), I shall emphasize severalideas that have emerged from the struc-

tural approach. Within the context ofthese ideas, I shall then consider therelated but less well explored subjectof antibodies on the surfaces of lym-phoid cells, and describe some recentlydeveloped experimental efforts of mycolleagues and myself to understand the

pp. 47, 51; p1. 30, fig. 1; pi. 31, fig. 3; pi. 32,fig. 3.

37. Pliny, Naturalis historia, book 8, sect. 210;Juvenal, Satires, book 1, 11. 140-141.

38. Pliny (Naturalls historia, book 28, sect. 265)gives horse flesh thoroughly boiled and takenin drink as a specific for the diseases of pigs.

39. The discovery that this large insula wasplanted makes me believe that other largeopen areas in the city may also have beenplanted and not used for commercial orother purposes. Several important areas wereexcavated in 1972, and in each I was ableto recover the planting pattern and determineland use.

40. The excavations were conducted with thepermission and generous cooperation of Pro-fessor Alfonso de Franciscis, superintendentof antiquities in Campania. Nicola Sicignanowas foreman. I am also grateful to John R.McGrew, research plant pathologist, U.S.Department of Agriculture, Plant ScienceResearch Division, Beltsville, Md., for invalu-able counsel regarding the technical aspectsof viticulture; to Frederick G. Meyer, researchbotanist in charge of the U.S. NationalArboretum, for his generous help in identify-ing carbonized specimens and comparing themwith contemporary specimens; and to HenrySetzer, mammalogist in charge of the Africansection at the Smithsonian Institution, throughwhose kindness the bones found in our ex-cavations were examined and identified. Allphotographs, drawings, and statistical studieswere made by Stanley A. Jashemski.

molecular mechanisms by which thebinding of antigens induces clonal pro-liferation of these cells.

Antibodies occupy a central place inthe science of immunology for anobvious reason: they are the proteinmolecules responsible for the recogni-tion of foreign molecules or antigens.It is, therefore, perhaps not a verypenetrating insight to suppose that astudy of their structure would be valu-able to an understanding of immunity.But what has emerged from that studyhas resulted in both surprises and con-ceptual reformulations.

These reformulations provided amolecular basis for the selective theoriesof immunity first expounded by NielsJerne (4) and MacFarlane Burnet (5)and therefore helped to bring about avirtual revolution of immunologicalthought. The fundamental idea of thesetheories is now the central dogma ofmodern immunology: molecular recog-nition of antigens occurs by selectionamong clones of cells already com-mitted to producing the appropriateantibodies, each of different specificity(Fig. 1).The results of many studies by

cellular immunologists (1) stronglysuggest that each cell makes antibodiesof only one kind, that stimulation ofcell division and antibody synthesis oc-curs after interaction of an antigenwith receptor antibodies at the cellsurface, and that the specificity of theseantibodies is the same as that of theantibodies produced by daughter cells.

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Several fundamental questions areraised by these conclusions and by thetheory of clonal selection. How can asufficient diversity of antibodies be syn-thesized by the lymphoid system? Whatis the mechanism by which the lympho-cyte is stimulated after interaction withan antigen?

In the late 1950's, at the beginningof the intensive work on antibody struc-ture, these questions were not so welldefined. The classic work of Land-steiner on hapten antigens (6) had pro-vided strong evidence that immuno-logical specificity resulted from molecu-lar complementarity between the de-terminant groups of the antigen mole-cule and the antigen-combining site ofthe antibody molecule. In addition,there was good evidence that most anti-bodies were bivalent (7) as well assome indication that antibodies of dif-ferent classes existed (8). The physico-chemical studies of Tiselius (9) hadestablished that antibodies were pro-teins that were extraordinarily hetero-geneous in charge. Moreover, a numberof workers had shown the existence ofheterogeneity in the binding constantsof antibodies capable of binding a sin-gle hapten antigen (10). Despite thevalue of all of this information, how-ever, little was known of the detailedchemical structure of antibodies or ofwhat are now called the immunoglobu-lins.

Multichain Structure of Antibodies:Problems of Size and Heterogeneity

If the need for a structural analysisof antibodies was great, so were theexperimental difficulties. Antibodies arevery large proteins (molecular weightof 150,000 or greater), and they areextraordinarily heterogeneous. Twomeans were adopted around 1958 in aneffort to avoid the first difficulty. Fol-lowing the work of Petermann (11)and others, Rodney Porter (12) ap-plied proteolytic enzymes, notably pa-pain, to achieve a limited cleavage ofthe gamma globulin fraction of seruminto fragments. He then successfullyfractionated the digest, obtaining anti-gen-binding (Fab) and crystallizable(Fc) fragments. Subsequently, otherenzymes such as pepsin were used ina similar fashion by Nisonoff et al.(13). I took another approach, in anattempt to cleave molecules of im-munoglobulin G and immunoglobulinM into polypeptide chains by reduc-tion of their disulfide bonds and ex-

25 MAY 1973

Clone of cells oll moking identicolimmunoglobulin

Fig. 1. A diagram illustrating the basicfeatures of the clonal selection theory.The stippling and shading indicate thatdifferent cells have antibody receptors ofdifferent specificities, although the speci-ficity of all receptors on a given cell isthe same. Stimulation by an antigen re-sults in clonal expansion (maturation, mi-tosis, and antibody production) of thosecells having receptors complementary tothe antigen.

posure to dissociating solvents such as

6M urea (14). This procedure resultedin a significant drop in molecularweighi, demonstrating that the im-munoglobulin G molecule was a multi-chain structure rather than a singlechain as had been believed before.Moreover, the chains obtained fromboth immunoglobulins had about thesame size. The polypeptide chains (15)were of two kinds (now called lightand heavy chains) but were obviouslynot the same as the fragments obtainedby proteolytic cleavage, and thereforethe results of the two cleavage proce-dures complemented each other. Ultra-centrifugal analyses indicated that one

of the polypeptide chains had a molecu-lar weight in the vicinity of 20,000, a

reasonable size for determination of theamino acid sequence by the methodsavailable in the early 1960's.

Nevertheless, the main obstruction toa direct analysis of antibody structurewas the chemical heterogeneity of anti-bodies and their antigen-binding frag-ments. Two challenging questions con-

fronted those attempting chemicalanalyses of antibody molecules at thattime. First, did the observed hetero-geneity of antibodies reside only in theconformation of their polypeptidechains, as was then widely assumed, ordid this heterogeneity reflect differencesin the primary structures of thesechains, as required implicitly by theclonal selection theory? Second, if theheterogeneity did imply a large popu-

lation of molecules with different pri-mary structures, how could one obtainthe homogenous material needed forcarrying out a detailed structural analy-sis?

These challenges were met simul-taneously by taking advantage of anaccident of nature rather than by directphysicochemical assault. It had beenknown that tumors of lymphoid cellscalled myelomas produced homogene-ous serum proteins that resembled thenormal heterogeneous immunoglobu-lins. In 1961, M. D. Poulik and Ishowed that the homogeneity of theseproteins was reflected in the starch-gelelectrophoretic patterns of their dis-sociated chains (15). Some patientswith multiple myeloma excrete urinaryproteins that are antigenically relatedto immunoglobulins but whose naturehad remained obscure since their firstdescription by Henry Bence Jones in1847. These Bence Jones proteins weremost interesting, for they could bereadily obtained from the urine in largequantities, were homogeneous, and hadlow molecular weights. It seemed rea-sonable to suggest (15) that BenceJones proteins represented one of thechains of the immunoglobulin moleculethat was synthesized by the myelomatumor but not incorporated into thehomogeneous myeloma protein and wastherefore excreted into the urine.

This hypothesis was corroboratedone exciting afternoon when my studentJoseph Gally and I (16) heated solu-tions of light chains isolated from ourown serum immunoglobulins in theclassical test for Bence Jones protein-uria. They behaved as Bence Jonesproteins, the solution first becomingturbid, then clearing upon further heat-ing. A comparison of light chains ofmyeloma proteins with Bence Jonesproteins by starch-gel electrophoresisin urea (16) and by peptide mapping(17) confirmed the hypothesis (Fig. 2).Indeed, Berggard and I later found(18) that in normal urine there werecounterparts to Bence Jones proteinsthat shared their properties but werechemically heterogeneous.No physical means was known at

the time that was capable of fraction-ating antibodies to yield homogenousproteins. It was possible, however, toprepare specifically reactive antibodiesby using the antigen to form antigen-antibody aggregates and then dissociat-ing the complex with free hapten. Al-though we knew that these specificallyprepared antibodies were still hetero-geneous in their electrophoretic proper-

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Table 1. Human immunoglobulin (Ig) classes.

Class Physiological pr*perties Heavy Light Molecular Molecular weight CarbohydrateClass Physiological prGperties chain* chain formulat (mX 10ti) andcsed content (%)mentation constant ntt(%

IgG Complement fixation; placental transfer K or X ('y2K2) or (Y2X2) 143-149; 6.7S 2.5IgA Localized protection in external secretions a K orX (a2K2) or (a2X2) 158-162; 6.8S-11.4S 5-10IgM Complement fixation; early immune response K or X (j,9K2) or (u2)X2)s 800-950; 19.OS 5-10IgD Unkhown a K Or X (62K2) or (82X2) 175-180; 6.S 10IgE Reagin activity; mast cell fixation c K or X (e,K2) or (eA2) 185-190; 8.OS 12* The class distinctive features of these chains are in their constant regions. t IgA can have additional unrelated chains called SC and J; J chains arealso found in IgM.

ties, it seemed possible that antibodiesto different haptens might show dif-ferences in their polypeptide chains.Baruj Benacerraf had prepared a col-lection of these antibodies, and togetherwith our colleagues (19) we decidedto compare their chains, using the samemethods that we had used for BenceJones proteins. The results were strik-ing: purified antibodies showed fromthree to five sharp bands in the BenceJones or light chain region and anti-bodies of different specificities showeddifferent patterns. In sharp contrast,normal immunoglobulin showed a dif-fuse zone extending over the entirerange of mobilities of these bands.These experiments showed not onlythat antibodies of different specificitieswere structurally different but also thattheir heterogeneity was limited.The results of the experiments on

Bence Jones proteins and purified anti-bodies had a number of significantimplications. Because different BenceJones proteins had different amino acidcompositions, it was clear that immuno-globulins must vary in their primarystructures. This deduction, confirmedlater by Koshland (20) for specifically

I~~~~~~~~~~~~~~I:;

1 2 3 4

purified antibodies, lent strong supportto selective theories of antibody forma-tion. Moreover, it opened the possibilityof beginning a direct analysis of theprimary structure of an immunoglobulinmolecule; for not only were the BenceJones proteins composed of homoge-neous light chains, but their subunitmolecular weight was only 23,000. Thefirst report by Hilschmann and Craig(21) on partial sequences of severaldifferent Bence Jones proteins indicatedthat the structural heterogeneity of thelight chains was confined to the amino-terminal (variable) region, whereas thecarboxyl-terminal half of the chain(the constant region) was the same inall chains of the same type. This findingwas soon extended by studies of otherBence Jones proteins (22).

Although some work had also beendone on the heavy chains of immuno-globulins, there was much less informa-tion on their structure. For instance,it was suspected but not known thatthey also had variable regions resem-bling those of light chains. Comparisonsof heavy chains and light chains evenat this early stage did, however, clarifythe nature of another source of anti-

Heavychain

Ughtchain

body heterogeneity, the existence ofimmunoglobulin classes (23).

Antibodies within a particular classhave similar molecular weight, carbo-hydrate content, amino acid composi-tion, and physiological functions (Table1) but still are heterogeneous in netcharge and antigen-binding affinity.Studies of classes in various animalspecies indicated that both the multi-chain structure and the heterogeneityare ubiquitous properties of immuno-globulins. The different classes apparent-ly emerged during evolution (24) tocarry out various physiologically im-portant activities that have been namedeffector functions in order to distinguishthem from the antigen-binding or recog-nition function. The various manifesta-tions of humoral immune responses aswell as their prophylactic, therapeutic,and pathological consequences can nowbe generally explained in terms of theproperties of the particular class ofantibody mediating that response. Asa result of comparing their chain struc-ture, it became clear that although im-munoglobulins of all classes containsimilar kinds of light chains (Table 1),the distinctive class character (23) is

Fab (t)Fab(t)

.. ..

a ~~~~~~~b Fc(t)

Fig. 2 (left). Comparisons of light chains isolated from serum immunoglobulin G myeloma proteins with urinary Bence Jones pro-teins from the same patient. (a) Starch-gel electrophoretic patterns in urea are shown for (1) serum myeloma globulin, (2) uri-nary Bence Jones protein, (3) Bence Jones protein reduced and alkylated, and (4) myeloma protein reduced and alkylated; L, lightchain; H, heavy chain. (b) Tryptic hydrolyzates were analyzed by two-dimensional high-voltage electrophoresis. Pattern on left isurinary Bence Jones protein; that on right is of light chain isolated from the serum myeloma protein of the same patient. Fig.3 (right). Overall arrangement of chains and disulfide bonds of the human yG, immunoglobulin, Eu. Half-cystinyl residues arenumbered I to XI; I to V designate corresponding half-cystinyl residues in light and heavy chains; PCA, pyrrolidonecarboxylic acid;CHO, carbohydrate. Fab(t) and Fc(t) refer to fragments produced by trypsin, which cleaves the heavy chain as indicated by dashedlines above half-cystinyl residues VI. Variable regions, VH and VL, are homologous. The constant region of the heavy chain (CH)is divided into three regions, CHI, CH2, and C,,3, that are homologous to each other and to the C region of the light chain. Thevariable regions carry out antigen-binding functions and the constant regions the effector function of the molecule.

', ,m..:!,

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1

ASP ILE GLN MET THR GLN SER PROPCA VAL GLN LEU VAL GLN SER GLY

10SER THR- ALA

20 30LEU SER ALA SER VAL GLY ASP ARG VAL THR ILE THR CYS ARG ALA SER GLN SER ILE ASNGLU VAL LYS LYS PRO GLY SER SER VAL LYS VAL SER CYS LYS ALA SER GLY GLY THR PHE

40THR - - TRP LEU ALA TRP TYR GLN GLN LYS PRO GLY LYS ALA PRO LYS LEU LEU METSER ARG SER ALA ILE ILE TRP VAL ARG GLN ALA PRO GLY GLN GLY LEU GLU TRP MET GLY

50TYR LYS ALAGLY ILE VAL

70GLY THR GLUTHR ILE THR

SER SER - LEU CLU SER GLY VAL PROPRO MET PHE GLY PRO PRO ASN TYR ALA

60SER ARGGLN LYS

PHE: ILE C:GLY SER GLY SER

PIE: CLN CLV - ARG VAL

PHE THR LEU THR ILE SER SER ::LEUO::GLNALA ASP GLU SER THR ASN THR ALA TYR MET GLU LEU SER S0iiERk0 LEU- ARG

90ASP ASP PHE ALA THR TYR TYR CYs GLN GLNGLU ASP THR ALA PHE TYR PHE CYS., ALA GLY

80PROSER

- TYR ASN SER ASP SER LYS MET PHE GLYGLY TYR GLY ILE TYR SER PRO GLU GLU TYR

100GLN GLY THR LYS VALL GLU VAL LYS GLYASN GML GLY LEU VALt: THR

Fig. 4. Comparison of the amino acid sequences of the V,, and VL regions of pro-tein Eu. Identical residues are shaded. Deletions indicated by dashes are introducedto maximize the homology.

EU CL (RESIDUES 109-214'EU CH1 (RESIDUES 119-220)EU CH2 *RESIDUES 234- 341 )

EU CH3 (RESIDUES 342-446'

ASP _M GLN -

SER LYS SER

PRO LYS ASP THR

ARG MWGLU

THR

SERLEU

GLN

110VAL

THR

LEUPRO

ALA ALA

LYS WGLY m

ARG GL U

-.LEO LYS

- THR SER CLY

- X THR LYS ASN

140 1 SOARC GLU ALA LYS GLN LYS ASN

~~~~~PROVAL THR SER ASN SERW-SER HI PRO GLN LYS PHE ASN TYR VAL VAL HIS

.SER 4ILE ALA - - GLU GLU SER ASN ASP - GLY CLU PRO GLU

160 170n SER GLN GLU SER VAL THR LYS

VAL HIS PHE ALA VAL LEW SER C;LYV. LEO

ALA LYS ARCGUTYR THR PRO SER PHE PHE TYR

180 190

TH-SR SER ALA_l20

~~~~~~~~ALAASP TYR CLU LYS HISAL GLU THR

IIIIIIIIIIVAL PRO E5R LEO CLY TH GLN THR ILE ASN ASN

LEO HIS CLN ASN LEO ASP L Y Y ELYS ASP ARC GLN CLU PHE SER SER MET

200 210

GLN CLY SER SER -PHE - ASN ARC CLUPRO SER ASN THR LYS ASP VAL - GLU LYS SER

ASN | | ISPRO ALA H ILE GLU THR ILE LYS ALAL

GLO HIS ASN HIS TYR CN LN LEO LEO SER

conferred by structural differences inthe heavy chains, specifically in theirconstant regions, as I shall discuss later.With the clarification of the nature

of the heterogeneity of immunoglobulinchains and classes, attention could beturned to the problem of relating thestructure and evolution of antibodieswithin a given class to their antigen-binding and effector functions. Wechose to concentrate on immunoglobulinG, because this was the most prevalentclass in mammals and the work onchain structure suggested that it wouldbe sufficiently representative.

The Complete Covalent Structure

and the Domain Hypothesis

An understanding of the chain struc-ture and its relation to the proteolyticfragments (25) made feasible an at-tempt to determine the complete struc-ture of an immunoglobulin G molecule.My colleagues and I started this projectin 1965, and before it was completed in1969 (26) seven of us had spent agood portion of our waking hours onthe technical details. One of our mainobjectives was to provide a completeand definitive reference structureagainst which partial structures of otherimmunoglobulins could be compared. Inparticular, we wished to compare thedetailed structure of a heavy chain anda light chain from the same molecule.

Another objective was to examinein detail the regional differentiation ofthe structure that had been evolved tocarry out different physiological func-tions in the immune response. The workof Porter (12) had shown that theFab fragment of immunoglobulin Gwas univalent and bound antigenswhereas the Fc fragment did not. Thisprovided an early hint that immuno-globulin molecules were organized intoseparate regions, each mediating dif-ferent functions. In accord with selec-tive theories of immunity, it was logicalto suppose that variable regions fromboth the light and the heavy chainsmediated the antigen-binding functions.Early evidence that some of the constantregions were concerned with physiologi-cally significant effector functions wasobtained by showing that Fc fragmentswould bind components of the comple-ment system (27), a complex groupof proteins responsible for immuno-logically induced cell lysis. A moredetailed assignment of structure to func-tion required a knowledge of the totalstructure.

833

EU VL ( RESIDUES 1 108 )

EU VH RESIDUES 1- 114 )

Fig. 5. Comparison of the amino acid sequences of CL, CH 1, C,12, and CH3 regions.Deletions, indicated by dashes, have been introduced to maximize homologies. Iden-tical residues are darkly shaded; both light and dark shadings are used to indicateidentities which occur in pairs in the same position.

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Amino acid sequence analysis of theFc region of normal rabbit 'y chains(Table 1) by Hill et al. (28) demon-strated that the carboxyl-terminal por-tion of heavy chains was homogeneous.On the basis of internal homologiesin this region, Hill et al. (28) andSinger and Doolittle (29) proposed thehypothesis that the genes for immuno-globulin chains evolved by duplicationof a gene of sufficient size to specifya precursor protein of about 100 aminoacids in length. Although direct con-firmation of this hypothesis is obviouslynot possible, it was strongly supportedby the results of our analysis (26) ofthe complete amino acid sequence andarrangement of the disulfide bonds ofan entire immunoglobulin G myelomaprotein.

Comparisons of the amino acid se-quences of the heavy chain of thisprotein with others studied in Porter'slaboratory (30) and by Bruce Cunning-ham and his colleagues in our labora-tory (31) showed that heavy chainshad variable (VI,) regions, that is,regions that differed from one anotherin the sequence of the 110 to 120 resi-dues beginning with the amino terminus(Fig. 3).

Examination of the amino acid se-quences (Figs. 4 and 5) allowed us todraw the following additional conclu-sions:

1) The variable (V) regions of lightand heavy chains are homologous to

each other, but they are not obviouslyhomologous to the constant regions ofthese chains. Variable regions from thesame molecule appear to be no moreclosely related than V regions fromdifferent molecules.

2) The constant (C) region of ychains consists of three homology re-

gions, CH1I, CH2, and CH3, each ofwhich is closely homologous to theothers and to the constant regions ofthe light chains.

3) Each V region and each C homol-ogy region contains one disulfide bond,with the result that the intrachaindisulfide bonds are linearly and peri-odically distributed in the structure.

4) The region containing all of theinterchain disulfide bonds is at thecenter of the linear sequence of theheavy chain and has no homologouscounterpart in other portions of theheavy or light chains.

These conclusions prompted us tosuggest that the molecule is folded ina congeries of compact domains (26,31), each formed by separate V homol-ogy regions or C homology regions

834

Fig. 6. The domain hypothesis. Diagram-matic arrangement of domains in the freeimmunoglobulin G molecule is shown.The arrow refers to a dyad axis of sym-

metry. Homology regions (Figs. 3 to 5)that constitute each domain are indi-cated: VL and V,1 are domains made up

of variable homology regions; CL, CH1,CH2, and CH3 are domains made up ofconstant homology regions. Within eachof these groups, domains are assumed to

have similar three-dimensional structures,

and each is assumed to contribute to an

active site. The V domain sites contributeto antigen recognition functions and theC domain sites to effector functions.

(Fig. 6). In such an arrangement, eachdomain is stabilized by a single intra-chain disulfide bond and is linked to

neighboring domains by less tightlyfolded stretches of the polypeptidechains. A twofold pseudosymmetryaxis relates the VLCL to the VUCH 1

domains, and a true dyad axis throughthe disulfide bonds connecting theheavy chains relates the CH2 and CH3domains. The tertiary structure withineach of the homologous domains is as-

sumed to be quite similar. Moreover,each domain is assumed to contributeto at least one active site mediating a

function of the immunoglobulin mole-cule.

The last supposition is nicely demon-strated by the interaction of V regiondomains. The reconstitution of activeantibody molecules by recombiningtheir isolated heavy and light chains(32) as well as affinity-labeling experi-ments (29) confirmed our early hy-pothesis that the V regions of both heavyand light chains contributed to the an-

tigen-combining sites. Moreover, theexperiments of Haber (33) providedthe first indication that Fab fragmentsof specific antibodies could be unfoldedafter reduction of their disulfide bondsand refolded in the absence of antigento regain most of their antigen-bindingactivity. This clearly indicated that theinformation for the combining site was

contained entirely in the amino acidsequences of the chains. That this in-formation is contained completely inthe variable regions is strikingly shownby the recent isolation of antigen-bind-ing fragments consisting only of VI,

and VH (34). The chain recombinationexperiments suggested a hypothesis toaccount in part for antibody diversity:The various combinations of differentheavy and light chains expressed in dif-ferent lymphocytes allow the formationof a large number of different antigen-combining sites from a relatively smallnumber of V regions.One of the remaining structural tasks

of molecular immunology is to obtaina direct picture of antigen-binding sitesby x-ray crystallography of V domains'at atomic resolution. Although crystalsof the appropriate molecule or fragmentyielding diffraction patterns that extendbeyond Bragg spacings of 3.0 A havenot yet been obtained, it is likely thatcontinued searching will provide them.The details of a particular antigen-antibody interaction revealed by such astudy will be of enormous interest. Forexample, certain sequence positions ofV regions are hypervariable (35) andare good candidates for direct contribu-tion to the site. It will be particularlyimportant to understand how the basicthree-dimensional structure can accom-modate so many amino acid substi-tutions. X-ray crystallographic workmay also show in detail how the disul-fide bonds in each of the V domainsprovide essential stability to the site(26, 31, 36).The proposed similarities in tertiary

structures among C domains have notbeen established, nor have the functionsof the various C domains been fully de-termined. There is a suggestion thatC112 may play a role in complementfixation (37). A good candidate forbinding to the lymphocyte cell mem,brane is CH3, the function of whichmay be concerned with the mechanismof lymphocyte-triggering after antigenis bound by V domains. The CH3 do-main has already been shown to bind tomacrophage membranes (38), and thereis now some evidence that lymphocytescan synthesize isolated domains (39)similar to CH3 as separate molecules.

Although many details are still lack-ing, the gross structural -aspects of thedomain hypothesis have received directsupport from x-ray crystallographicanalyses of Fab fragments (40) andwhole molecules (41), in which sepa-rate domains were clearly discerned.Indirect support for the hypothesis hasalso come from experiments on proteo-lytic cleavage of regions between do-mains (34, 42).

It is not completely obvious why thedomain structure was so strictly pre-served during evolution. One reasonable

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hypothesis is that although there was afunctional need for association of Vand C domains in the same molecule,there was also a need to prevent allo-steric interactions among the domains.Whatever the selective advantages ofthis arrangement, it is clear that immu-noglobulin evolution by gene duplica-permitted the possibility of modularalteration of immunological function byaddition or deletion of domains.

Translocons: Proposed Units of

Evolution and Genetic Function

The evolution by gene duplication ofboth the domain structure and the im-munoglobulin classes raises several ques-tions about the number and arrange-ment of the structural genes specifyingimmunoglobulins. Although time doesnot permit me to discuss this complexsubject in detail, I should like to sug-gest how structural work has sharpenedthese questions.

According to the theory of clonalselection, it is necessary that there pre-exist in each individual a large numbetof different antibodies with the capacityto bind different antigens. One of themost satisfying conclusions that emergedfrom structural analysis is that the di-versity of the V regions of antibodychains is sufficient to satisfy this re-quirement. This diversity arises at threelevels of structural or genetic organiza-tion, two of which are now reasonablywell understood:

1) Variable regions from both heavyand light chains contribute to the anti-gen-binding site, and therefore the num-ber of possible antibodies may be asgreat as the product of the number ofdifferent VI, and VI, regions.

2) Analyses of the amino acid se-quences of V regions of light chains byHood et al. (43) and Milstein (44)and later of heavy chains from mye-loma proteins (30, 31) indicated thatV regions fall into subgroups of se-quences, which must be specified byseparate genes or groups of genes.Within a subgroup, the amino acidreplacements at a particular positionare of a conservative type consist-ent with single base changes in co-dons of the structural genes. Variableregions of different subgroups differmuch more from each other than dovariable regions within a subgroup.

Although different V region sub-groups are specified by a number ofnonallelic genes (44), the analysis ofgenetic or allotypic markers suggests

25 MAY 1973

VKlC; VK ,VKm, CKI

V>I v;<=Vx mV\/x,iM ,Vxy1,CAIC ,CA2 iCX3 i

VHI VHH ,VHM. C/L1, CY2 C C3ItcY2 c Cr4 itCcaI Ca21, CB Ce,

Fig. 7. A diagrammatic representation of the proposed arrangement in mammaliangerm cells of antibody genes in three unlinked clusters termed translocons. Light chainsK and X are each specified by different translocons, and heavy chains are specified by athird translocon. The exact number and arrangement of V and C genes within atranslocon is not known. Each variable region subgroup (designated by a subscriptcorresponding to chain group and subgroup) must be coded by at least one separategerm-line V gene. The number of V genes within each subgroup is unknown, however,as is the origin of intrasubgroup diversity of V regions. A special event is required tolink the information from a particular V gene to that of a given C gene. The propertiesof the classes and subclasses (Table l) are conferred on the constant regions by C.genes.

that C regions of a given immunoglob-ulin class are specified by no more thanone or two genes. These allotypicmarkers, first described by Grubb (45)and Oudin (46), provide a means inaddition to sequence analysis for under-standing the genetic basis of immuno-globulin synthesis (3). Variable regionsspecified by a number of different genescan occur in chains each of which mayhave the same C region specified by asingle gene. It therefore appears thateach immunoglobulin chain is specifiedby two genes, a V gene and a C gene(3, 43, 44).Work in a number of laboratories

[reviewed in (3)] has shown that thegenetic markers on the two types oflight chains are not linked to those ofthe heavy chains or to each other. Thesefindings and the conclusion that thereare separate V and C genes led Gallyand me to suggest (3) that immuno-globulins are specified by three unlinkedgene clusters (Fig. 7). The clustershave been named translocons (3) toemphasize the fact that some mechanismmust be provided to combine geneticinformation from V region loci withinformation from C region loci to makecomplete V-C structural genes. Accord-ing to this hypothesis, the transloconis the basic unit of immunoglobulinevolution, different groups of immuno-globulin chains having arisen by dupli-cation and various chromosomal rear-rangements of a precursor gene cluster.Presumably, gene duplication duringevolution also led to the appearance ofV region subgroups within each trans-locon.The key problem of the generation of

immunoglobulin diversity has been con-verted by the work on chains and sub-groups to the problem of the origin ofsequence variations within each V region

subgroup. It is still not known whetherthere is a germ-line gene for each Vregion within a subgroup, or whethereach subgroup contains only a fewgenes (Fig. 7) and intrasubgroupvariation arises by somatic genetic re-arrangements of translocons withinprecursors of antibody-forming cells.At this time, therefore, we can concludethat only the basis but not the origin ofdiversity has been adequately explainedby the work on structure. Althoughstructural analysis of various immuno-globulin classes will continue to be im-portant, it does not in itself seem likelyto lead to an explanation of the originof antibody diversity. What will prob-ably be required are imaginative experi-ments on DNA, RNA, and their associ-ated enzymes obtained from lymphoidcells at the proper stage of development.

In this abbreviated and necessarilyincomplete account, I have attemptedto show how structural work on im-munoglobulins has provided a molecularbasis for a number of central featuresof the theory of clonal selection. Thework on humoral antibodies is just abeginning, however, for two great prob-lems of molecular and cellular immu-nology remain to be solved. The firstproblem, the origin of intrasubgroupdiversity, will undoubtedly receive greatattention in the next few years. Thesecond problem is concerned withtriggering of the clonal expansion oflymphocytes after combination of theirreceptor antibodies with antigens andthe quantitative description of the pop-ulation dynamics of the respondingcells. An adequate solution to this prob-lem must also account for the phenom-enon of specific immune tolerance asdescribed by the original work ofMedawar and his associates (47).For the remainder of this lecture, I

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shall turn my attention to some recentattempts that my colleagues and I havemade to see whether these problemscan be profitably studied using molecu-lar approaches.

Lymphocyte Stimulation by

MIeans of Lectins

The mechanisms of the cellularevents underlying immune responsesand immune tolerance remain a majorchallenge to theoretical and practicalimmunology (47, 48). How does agiven antigen induce clonal prolifera-tion or immune tolerance in certainsubpopulations of cells?

Cells reactive to a given antigen con-stitute a very small portion of thelymphocyte population and are difficultto study directly. Two means have beenused to circumvent this difficulty: theapplication of molecules that can stimu-

late lymphocytes independent of theirantigen-binding specificity, and frac-tionation of specific antigen-bindinglymphocytes for studies of stimulationby antigens of known structure. Al-though the problem of lymphocytestimulation is far from being solved,both of these approaches are valuable,particularly when used together.

Antigens are not the only means bywhich lymphocytes may be stimulated.Certain plant proteins called lectins canbind to glycoprotein receptors on thelymphocyte surface and induce blasttransformation, mitosis, and immuno-globulin production [reviewed in (49)].Different lectins have different specifici-ties for cell surface glycoproteins anddifferent molecular structures, althoughtheir mitogenic properties can be quitesimilar. In addition, they have a varietyof effects on cell metabolism and trans-port. Such effects are independent ofthe antigen-binding specificity of the

a

Fig. 8. Three-dimensional structure of con-canavalin A, lectin mitogenic for lym-phocytes. (a) Schematic representation of

the tetrameric structure of Con A vieweddown the z axis. The proposed bindingsites for transition metals, calcium, andsaccharides are indicated by Mn, Ca, andC, respectively. The monomers on top(solid lines) are related by a twofoldaxis, as are those below. The two dimersare paired across an axis of D2 symmetryto form the tetramer. (b) Wire model ofthe polypeptide backbone of-the Con Amonomer oriented approximately to cor-

respond to the monomer on the upperright of the diagram in (a). The two ballsat the top represent the Ca and Mn atomsand the ball in the center is the positionof an iodine atom in the sugar derivative,8-iodophenylglucoside, which is bound to

the aotive site. Four such monomers are

joined to form the tetramer as shown in(a). (c) A view of the Kendrew model of the Con A monomer rotated to show thedeep pocket formed by the carbohydrate-binding site. The white ball at the bottom

is at the position of the iodine atom of p-iodophenylglucoside. The two white ballsat the top represent the metal atoms.

836

cell, and they may therefore be studiedprior to specific cell fractionation.The fact that antigens and lectins of

different specificity and structure maystimulate lymphocytes suggests that theinduction of mitosis is a property ofmembrane-associated structures thatcan respond to a variety of receptors.Triggering appears to be independentof the specificity of these receptors fortheir various ligands. To understandmitogenesis, it is therefore necessaryto solve two problems. The first is todetermine in molecular detail how thelectin binds to the cell surface and tocompare it to the binding of antigens.The second is to determine how thebinding induces metabolic changes ned-essary for the initiation of cell division.These changes are likely to include theproduction or release of a messenger,which is a final common pathway forthe stimulation of the cell by a particu-lar lectin or antigen.One of the important requirements for

solving these problems is to know thecomplete structure of several differentmitogenic lectins. This structural infor-mation is particularly useful in tryingto understand the molecular transfor-mation at the lymphocyte surface re-quired for stimulation. With the knowl-edge of the three-dimensional structureof a lectin, various amino acid sidechains at the surface of the moleculemay be modified by group reagentsthat may also be used to change thevalence of the molecule. The activitiesof the modified lectin derivatives maythen be observed in various assays oftheir effects on cell surfaces and cellfunctions.My colleagues and I (50) have re-

cently determined both the amino acidsequence and three-dimensional struc-ture of the lectin, concanavalin A (ConA) (Fig. 8). This lectin has specificity forglucopyranosides, mannopyranosides,and fructofuranosides and binds toglycoproteins and possibly glycolipidsat a variety of cell surfaces. The purposeof our studies was to know the exactsize and shape of the molecule, itsvalence, and the structure -and distribu-tion of its binding sites.With this knowledge in hand, we have

been attempting to modify the structureand determine the effects of that modi-fication on various biological activitiesof the lymphocyte. So far, there areseveral findings suggesting that suchalterations of the structure have dis-tinct effects. Con A in free solutionstimulates thymus-derived lymphocytes(T cells) but not bone marrow-derived

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lymphocytes (B cells), and leads to in-creased uptake of thymidine and blasttransformation. The curve of stimula-tion of T cells by native Con A showsa rising limb representing stimulationand a falling limb (Fig. 9), probablythe result of cell death. The fact thatthe mitogenic effect and killing effectare dose-dependent suggests an analogyto stimulation and tolerance inductionby antigens. When Con A is succin-ylated, it dissociates from a tetramer toa dimer without alteration of its carbo-hydrate-binding specificity. Althoughsuccinylated Con A is just as mitogenicas native Con A, the falling limb isnot seen until much higher doses arereached.

Succinylation of Con A also altersanother property of the lectin. At cer-tain concentrations, the binding of ConA to the cell surface restricts the move-ment of immunoglobulin receptors (51).This suggests that it somehow changesthe fluidity of the cell membrane, re-sulting in reduction of the relative mo-bility of these receptors. In contrast,succinylated Con A has no such effect,although it binds to lymphocytes to thesame extent as does the native mole-cule. Both the abolition of the killingeffect in mitogenic assays and the fail-ure to alter immunoglobulin receptormobility in B cells after succinylationof Con A may be the result of changein valence or of alteration in the sur-face charge of the molecule. Examina-tion of other derivatives and localizationof the substituted side chains in thethree-dimensional structure will help toestablish which is the major factor.Recent experiments suggest that thevalence is probably the major factor,for addition of divalent antibodiesagainst Con A to cells that had boundsuccinylated Con A resulted again inrestriction of immunoglobulin receptormobility.Con A may also be modified by

cross-linking several molecules. A strik-ing effect is seen if the surface densityof the Con A molecules presented tothe lymphocyte is increased by cross-linking it at solid surfaces (52). ConA in free solution stimulates mouse Tcells to an increased incorporation ofradioactive thymidine but has no effecton B cells. When cross-linked at a solidsurface, however, it stimulates mainlymouse B cells, although both T andB cells have approximately the samenumber of Con A receptors (52).Similar results have been obtained withother lectins (53). A reasonable inter-pretation of these phenomena (although25 MAY 1973

50 100 150 200Dose (pg/mi)

Fig. 9. Stimulation of uptake of radio-active thymidine by mouse spleen cellsafter addition of concanavalin A and suc-cinylated concanavalin A in increasingdoses.

not the only one) is that the lectin actsat the cell surface rather than insidethe cell, that the presence of a high sur-face density of the mitogen is an im-portant variable in exceeding the thresh-old for the lymphocyte stimulation,and that the threshold differs in the twokinds of lymphocytes.

Alteration of the structure and func-tion of various lectins appears to be apromising means of analyzing themechanism of lymphocyte stimulation.One intriguing hypothesis is that cross-linkage of the proper subsets of glyco-protein receptors by lectins is essentiallyequivalent in inducing cell transforma-tion to cross-linkage of immunoglobulinreceptors in the lymphocyte membraneby multivalent antigens. The centraleffector function of receptor antibodies,triggering of clonal proliferation, mayturn out to be specifically related tothe mode of anchorage of the antibody

Zygote

Antigen-independentsomatic proliferation andexpression of Ig genes

Immunocompetentsmall lymphocytes

Clonal growthtriggered byaporticular antigen}

Ig-secreting ormemory cells

molecule to the cell membrane. Themode of attachment of antibody andlectin receptors to membrane-associatedstructures and their perturbation bycross-linkage at the cell surface may besimilar and have similar effects despitethe difference in their specificities andmolecular structures.

Antibodies on the Surfaces of

Antigen-Binding Cells

The most direct attack on the prob-lem of lymphocyte stimulation is to ex-plore the effects of antigens of knownmolecular geometry on specifically puri-fied populations of lymphocytes. Forthis and other reasons, it is necessaryto develop methods for the specificfractionation of antigen-binding cells.

In carrying out this task it is impor-tant both theoretically and operationallyto discriminate between antigen-bindingand antigen-reactive cells. In clonal se-lection, the phenotypic expression ofthe immunoglobulin genes is mediatedin the animal by somatic division ofprecommitted cells (Fig. 10). The pio-neering work of Nossal and Makelaand later of Ada and Nossal [reviewedin (48)] clearly showed that each cellmakes antibodies of a single specificityand that there are different populationsof specific antigen-binding cells. Ananimal is capable of responding spe-cifically to an enormous number ofantigens to which it is usually neverexposed, and it therefore must contain

Different Iggenes(Genotype)

Different Ig's produced;one specificity per cell(Primotype)

Different rg's detectablein plasma or on expandedclones(Clonotype)

Fig. 10. A model of the somatic differentiation of antibody-producing cells accordingto the clonal selection theory. The number of immunoglobulin genes may increaseduring somatic growth so that in the immunologically mature animal, different lym-phoid cells are formed, each committed to the synthesis of a structurally distinctreceptor antibody (indicated by an Arabic numeral). A small proportion of these cellsproliferate upon antigenic stimulation to form different clones of cells, each clonep-oducing a different antibody. This model represents bone marrow-derived (B) cellsbut with minor modifications it is also applicable to thymus-derived (T) cells.

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cells from the mouse

spleen bound bytheir antigen-specificreceptors to a ny-

lon fiber to whichdinitrophenyl bovineserum albumin hasbeen coupled. Treat-ment of bound cellsin (a) with antise- b

rum to the T cellsurface antigen 0

and with serum com-

plement destroys theT cells, leaving Bcells in (b) still vi-able and attached(Table 2) (X 175).

genetic information for synthesizing a

much larger number of different immu-noglobulin molecules than actually ap-

pear in detectable amounts in thebloodstream. In other words, the immu-noglobulin molecules whose propertieswe can examine may represent only a

minor fraction of those for whichgenetic information is available.One may distinguish two levels of

expresssion in the synthesis of immu-noglobulins that I have termed for con-

venience the primotype and the clono-type (3). The primotype consists ofthe sum total of structurally differentimmunoglobulin molecules or receptor

antibodies generated within an orga-

nism during its lifetime. The number ofdifferent molecules in the primotype isprobably orders of magnitude greaterthan the number of different effectiveantigenic determinants to which theanimal is ever exposed (Fig. 10). Theclonotype consists of those differentimmunoglobulin molecules synthesizedas a result of antigenic stimulation andclonal expansion. These molecules can

be detected and classified according to

antigen-binding specificity, class, anti-

genie determinants, primary structure,

allotype, or a variety of other experi-mentally measurable molecular proper-

ties. As a class, the clonotype is smallerthan the primotype and is wholly con-

tained within it (Fig. 10).Although a view of the clonotype is

afforded by the analysis of humoralantibodies, we know very little aboutthe primotype. It is therefore impor-tant to attempt to fractionate the cellsof the immune system according to thespecificity of their antigen-binding re-

ceptors (54). We have been attemptingto approach this problem of the specificfractionation of lymphocytes using ny-

lon fibers to which antigens have beencovalently coupled (55, 56). The deriva-tized fibers are strung tautly in a tissueculture dish so that cells shaken insuspension may collide with them. Someof the cells colliding with the fibers are

specifically bound to the covalentlycoupled antigens by means of their sur-

face receptors. Bound cells may becounted microscopically in situ by fo-cusing on the edge of the fiber (Fig.11). After unbound cells are washedaway, the specifically bound cells may

Table 2. Characterization of mouse lymphoid cells fractionated according to their antigen-binding specificities. Nylon fibers were derivatized with hapten conjugates of bovine serum

albumin, and mice were immunized with either the dinitrophenyl (DNP) or tosyl haptencoupled to hemocyanin. Inhibition of binding was achieved by addition of hapten-proteinconjugates (250 /Ag/m1) or rabbit antiserum against mouse immunoglobulin (Anti-1g) (250

jg/ml) to the cell suspension. High-avidity cells are defined as those which are preventedfrom binding by concentrations of DNP-bovine serum albumin of less than 4 jug/ml in the

cell suspensions. Cells inhibited by higher concentrations are defined as low-avidity cells.

Virtually complete inhibition occurs at concentrations of homologous hapten greater than

100 ,ug/ml.

Inhibition of binding (%) by

Cells DNP Tosyl High- Low-Immuni- bound NO2 CH, avidity avidity cells cells

zation to fiber sAnti-Ig cell cells (c%l) (c%e)(cm-')

Ant-Ig(cm-') (cm-')

SO.

None 1200 90 1 85 < 100 1200 41 59

DNP 4000 95 2 93 2800 1200 39 56

None 800 5 75 73 43 54

Tosyl 2000 10 87 90

838

De removea Dy pIUCKilng Lnte iuci aiiu

shearing the cells quantitatively fromtheir sites of attachment. The removedcells retain their viability provided that.the tissue culture medium containsserum.

Derivatized nylon fibers have the ca-.pacity to bind both T cells and B cells(57) according to the specificity oftheir receptors for a given antigen (58)(Fig. 11 and Table 2). About 60 per-cent of spleen cells specifically isolatedare B cells, and the remainder are Tcells. By the use of appropriate anti-sera to cell surface receptors (Table2), the cells of each type can be countedon the fibers, and most of the cells ofone type or the other may then be de-stroyed by the subsequent addition ofserum complement. In this way, onecan obtain populations of either T or Bcells that are highly enriched in theircapacity to bind a given antigen (Fig.11).

Cells of either kind may be furtherfractionated according to the relativeaffinity of their receptors. This can beaccomplished by prior addition of a

chosen concentration of free antigen,which serves to inhibit specific attach-ment of subpopulations of cells to theantigen-derivatized fibers by binding totheir receptors. As defined by this tech-nique, cells capable of binding specifi-cally to a particular antigen constituteas much as 1 percent of a mouse spleencell population. Very few of these origi-nal antigen-binding cells appear to in-crease in number after immunization,however, and the cells that do respondare those having receptors of higherrelative affinities (55).

Whether these populations correspondto the primotype and clonotype re-mains to be determined. It is significant,however, that fiber-binding cells do not

include plaque-forming (59) cells, andit is therefore possible to fractionateantigen-binding cells from cells that arealready actively secreting antibodies.Recent experiments indicate that theantigen-binding cells isolated by thismethod may be transferred to irradiatedanimals to reconstitute a response to

the antigen used to isolate them. Thissuggests that the antigen-specific popu-lation of cells removed from the fiberscontains precursors of plaque-formingcells.We have been rather encouraged by

these findings, for the various methodsof cell fractionation appear to havepromise not only in determining thespecificity and range of T and B cellreceptors for antigens but also in analyz-ing the population dynamics of T and

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B cells in both adult and developinganimals. Now that fractionated popula-tions of ly,-nphocytes specific for par--ticular antigens are available, it shouldbe possible to determine the connectionbetween lectin-induced and antigen-*induce%A changes by comparing re-Spon!es to both agents on the samecejXs.

I..

Although many experiments remainto be done in this area of the molecularimmunology of the cell surface, con-tinued analysis of the mitogenic mech-anism should undoubtedly clarify theproblems of immune induction andtolerance. The results obtained withlymphocytes may also have generalsignificance, however, and bear uponthe nature of cell division in normaland tumor cells as well as upon growthcontrol and cell-cell interactions in de-velopmental biology. Immunology canbe expected to play a double role inthese areas of study, for it will be atool as well as a model system of cen-tral importance.

Conclusion

Immunology has been and is a curi-ously reflexive science, generating itsown tools for understanding, such asantibodies to antibody molecules them-selves. While this approach is a power-ful one, a fundamental understandingof immunological problems requireschemical analysis. The determination ofthe molecular structure of antibodiesis a persuasive example and its virtualcompletion has allied immunology tomolecular biology in a very satisfyingway:

1) The heterogeneity of antibodiesand complexity of immunoglobulinclasses have been rationalized in afashion consistent with selective theoriesof immunity.

2) The structural basis for differentia-tion of the biological activity of anti-bodies into antigen-binding and effectorfunctions has been made clear.

3) The detailed analysis of antibodyprimary structure has provided a basisfor studying the molecular genetics ofthe immune response, particularly theorigin of diversity and the commitmentof each cell to the synthesis of one kindof antibody.

4) A general framework has beenprovided for studying antibodies at thecell surface, opening several molecularapproaches for analyzing stimulationand cell triggering.

5) Finally, it is perhaps not tooextravagant to suggest that the exten-

25 MAY 1973

sions of the ideas and methods ofmolecular immunology to fields such asdevelopmental biology has been facili-tated. In this sense, immunology pro-vides an essential tool as well as a modelwith distinct advantages: dissociablecells with unique gene products ofknown structure; the capacity to inducespecific cloned cell lines for in vitroanalysis; the means to fractionate cellsaccording to their state of differentiationand binding specificity, allowing quanti-tative studies of their selection, inter-action, and population dynamics.Whether or not the immune response

turns out to be a uniquely useful model,we can expect that continued work bymolecular and cellular immunologistswill solve the major problems of theorigin of diversity and the induction ofantibody synthesis and tolerance. Inview of the intimate connection of theseproblems with problems of gene expres-sion and cellular regulation, their solu-tion should bring valuable insights toother important areas of eukaryoticbiology and again transform immunol-ogy both as a discipline and as anincreasingly important branch of medi-cine.

References and Notes

1. Cold Spring Harbor Symp. Quant. Biol. 32,(1967), entire issue; Gamma Globulins: Struc-ture and Control of Biosynthesis, Nobel sym-posium, 3rd, Sodergarn, J. Killander, Ed.(Almqvist & Wiksell, Stockholm, 1967).

2. G. M. Edelman and W. E. Gall, Annu. Rev.Biochem 38, 415 (1969).

3. J. A. Gally and G. M. Edelman, Annu. Rev.Genet. 6, 1 (1972).

4. N. K. Jerne, Proc. Nat. Acad. Sci. U.S.A.41, 849 (1955).

5. F. M. Burnet, The Clonal Selection Theoryof Acquired Immunity (Vanderbilt Univ. Press,Nashville, Tenn., 1959).

6. K. Landsteiner, The Specificity of SerologicalReactions (Harvard Univ. Press, Cambridge,Mass., ed. 2, 1945).

7. J. R. Marrack, The Chemistry of Antigensand Antibodies: No. 230, Medical ResearchCouncil Special Report Series (His Majesty'sStationery Office, London, ed. 2, 1938).

8. K. 0. Pedersen, Ultracentrifugal Studies onSerum and Serum Fractions (Almqvist &Wiksell, Stockholm, 1945).

9. A. Tiselius, Biochem. J. 31, 313 (1937); ibid.,p. 1464.

10. F. Karush Advan. Immunot. 2, 1 (1962).11. M. L. Petermann, J. Biol. Chem. 144, 607

(1942).12. R. R. Porter, Biochem. J. 73, 119 (1959).13. A. Nisonoff, F. C. Wissler, L. N. Lipman,

D. L. Woernley, Arch. Biochem. Biophys. 89,230 (1960).

14. G. M. Edelman, J. Amer. Chem. Soc. 81, 3155(1959).

15. G. M. Edelman and M. D. Poulik, J. Exp.Med. 113, 861 (1961).

16. G. M. Edelman and J. A. Gally, ibid. 116,207 (1962).

17. J. Schwartz and G. M. Edelman, ibid. 118,41 (1963).

18. I. Berggard and G. M. Edelman, Proc. Nat.Acad. Sci. U.S.A. 49, 330 (1963).

19. G. M. Edelman, B. Benacerraf, Z. Ovary,M. D. Poulik, ibid. 47, 1751 (1961).

20. M. E. Koshland and F. M. Englberger, ibid.S0, 61 (1963).

21. N. Hilschmann and L. C. Craig, ibid. 53,1403 (1965).

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25. J. B. Fleishman, R. H. Pain, R. R. Porter,Arch. Biochem. Biophys. (Suppl. 1) (1962), p.174; J. B. Fleishman, R. R. Porter, E. M.Press, Biochem. J. 88, 220 (1963); M.Fougereau and G. M. Edelman, J. Exp. Med.121, 373 (1965).

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27. K. Amiraian and E. J. Leikhim, Proc. Soc.Exp. Biol. Med. 108, 454 (1961); A. Tarantaand E. C. Franklin, Science 134, 1981 (1961).

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30. E. M. Press and N. M. Hogg, Nature 223,807 (1969).

31. B. A. Cunningham, P. D. Gottlieb, M. N.Pflumm, G. M. Edelman, in Progress in Im-munology, B. Amos, Ed. (Academic Press,New York, 1971), pp. 3-24; B. A. Cunning-ham, M. N. Pflumm, U. Rutishauser, G. M.Edelman, Proc. Nat. Acad. Sci. U.S.A. 64,997 (1969).

32. F. Franek and R. S. Nezlin, Biokhimiya 28,193 (1963); G. M. Edelman, D. E. Olins, J. A.Gaily, N. D. Zinder, Proc. Nat. Acad. Sci.U.S.A. 50, 753 (1963); D. E. Olins and G. M.Edelman, J. Exp. Med. 119, 789 (1964).

33. E. Haber, Proc. Nat. Acad. Sci. U.S.A. 52,1099 (1964).

34. D. Inbar, J. Hachman, D. Givol, ibid. 69,2659 (1972).

35. T. T. Wu and E. A. Kabat, J. Exp. Med. 132,211 (1970).

36. G. M. Edelman, Ann. N.Y. Acad. Scd. 190,5 (1971).

37. J. M. Kehoe and M. Fougereau, Nature 224,1212 (1969).

38. D. Yasmeen, J. R. Ellerson, K. J. Dorrington,R. H. Painter, J. Immunol., in press.

39. I. Berggard and A. G. Beam, J. Biol. Chem.243, 4095 (1968); 0. Smithies and M. D.Poulik, Science 175, 187 (1972); P. A.Peterson, B. A. Cunningham, I. Berggard,G. M. Edelman, Proc. Nat. Acad. Sci. U.S.A.69, 1967 (1972).

40. R. J. Poljak, L. M. Amzel, H. P. Avey,L. N. Becka, A. Nisonoff, Nature New Biol.235, 137 (1972).

41. D. R. Davies, V. R. Sarma, L. W. Labaw,E. W. Silverton, W. D. Terry, in Progress inImmunology, B. Amos, Ed. (Academic Press,New York, 1971), pp. 25-32.

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DePetris, Nature New Blol. 23X, 225 (1971);R. Loor, L. Forni, B. Pernis, Eur. J. Immunol.2, 203 (1972).

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60. By its very nature, science is a communalenterprise. I am deeply aware of the essentialcontributions to this work made by my manycolleagues and friends throughout the last 15years. This occasion recalls the daily life we

have shared wtwawellas the personal deb I owethem. I am equallyc thatthe knowledge of anti de-,veloped by many labor rsthroughout the world. * f rkhas been cited, for spec - reruns the risk of an unin sreference may be made toIn addition to the fundamentl dt of *Rockefeller University, the 4"colleagues and myself was supp ,from the National Institutes ofNational Science Foundation.

Advocacy is a time-honored conceptthat originated in the law, permeatedmedicine by way of the physician-advocate, and is now accepted as anexplicit function of social work (1).Within social work, the terms "advo-cacy" and "advocate" ["one who pleads,intercedes, or speaks for another" (2)]are used to denote the actions and rolethat social workers are committed towhen the human, moral, civil, and legalrights of their clients are transgressedby individuals, groups, or social institu-tions. This article presents some facetsof advocacy that are now confrontingsocial workers as a result of recentdramatic advances in the medical sci-ences and the impact these advanceshave upon the lives of individuals-inthis instance, upon the mentally handi-capped and their families. I attempt toexplore major points at issue that canarise between social workers and re-search scientists, especially those work-ing in the biological sciences. I also sug-gest areas of common concern that canbe exploited to develop a constructivedialogue between the two professionalgroups instead of the mutual disparage-ment, suspicion, and even paranoia thatsometimes color the thinking of both,to the detriment of cooperative effortand a more sophisticated understandingof the complex nature of the prob-

840

lems of mental handicap. To illustratesimply: the research scientist must keepin mind that an anomaly in, say, thechemical behavior of a neurone termi-nates in a badly damaged child whobelongs to a distraught family; equally,the social worker, who is dealing withtheir immediate distress and future anx-iety, must realize that this chemicalmisbehavior may derive from an aber-rant gene, which could manifest itselfin;the tragedy of a second affected fetusunless there is scientific intervention inthe shape of amniocentesis and geneticcounseling (3).

Research and Social Priorities

The crucial areas in which scienceand social work are apt to overlap andwork at cross-purposes are (i) futuregains versus immediate relief, (ii) pre-vention versus supportive help, (iii) com-mon good versus individual good, allof which impinge upon most of socialwork's cherished tenets and firmly en-trenched methods of working. Consider,first, the different perspectives on thetime factor-namely, reasonably cer-tain, prompt relief as against predictablefuture gains. Social workers, with theirorientation toward problem-solving,crisis intervention, and the pressing

problems of the individual's adjustmentto his social milieu, sometimes find ithard to accept the value of experimentsthat can do nothing for the damagedchild and besieged family, even thoughthey may save future families from thetragedy that their clients are experi-encing. But if social workers are tokeep pace with the march of crucial de-velopments and retain the professionalrespect of their scientific colleagues,they must try to identify, at least inpart, with these long-term goals, eventhough their primary allegiance is to thepresent client.

This area of concern is very closelytied in with another-public healthversus individual treatment-whichraises many issues. For example, giventhat it is desirable to.reduce the inci-dence of defective children and that re-search technology has provided mecha-nisms for identifying at-risk parents,how should we react to a proposal forscreening for Tay-Sachs disease thepopulation known to be at high risk-namely, Jewish men and women of Ash-kenazi origin? Although this is a physi-cally harmless public health measure ofunquestionably benign intent, it alsocontains a psychologically disruptiveelement: anxiety about ethnic discrim-ination. Because of their long historyof persecution, all members of thisgroup, particularly recent immigrantsfrom Europe who carry inherited andfirsthand memories of genocide, are

potentially sensitive to discrimination.For people of African descent, screen-ing for sickle cell anemia may havesimilar implications, which are rein-forced by realistic fears of adverse dis-crimination in respect to employment,life insurance, and so forth (4).The third issue, personal welfare-

versus the common good, presents a

constant conflict to social workers,The author is a consultant in social work to the

interdisciplinary training program at the EuniceKennedy Shriver Center, Walter E. Fernald StateSchool, Waverley, Massachusetts 02178. This articleis adapted from a paper presented to the socialwork division of the American Association onMental Deficiency at Minneapolis, 18 May 1972.

SCIENCE, VOL. 180

Science, Technology, andSome Dilemmas of Advocacy

Social implications of biological research indevelopmental disabilities are considered.

Margaret Adams

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