talmage - immunological specificity - 1959

7/29/2019 Talmage - Immunological Specificity - 1959

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19 June 1959, Volume 129, Number 3364 S C I E N C E 1 -

CURRENT PROBLEMS IN RESEARCH

Immunological SpecificityUnique combinations of selected natural globulins

provide an alternative to the classical concept.David W. Talmage

The elucidation at a molecular levelof the nature of specific biological inter-actions constitutes one of the most im-portant and challenging problems ofbiology today. Specificity was defined byLandsteiner (1, p. 6) as the "dispropor-tional action of a number of similaragents on a variety of related substrata."Specificity is a property of a wide rangeof biological agents such as antibodies,antibiotics, toxins, viruses, enzymes, ge-netic material, and plant agglutinins. Ofthese, only antibodies have the propertythat their production by the host can bestimulated by the injection of an almostunlimited range of substances. Further-more, an antiserum reacts specificallywith the antigen which stimulated itsproduction, thereby providing a power-ful tool for the detection and identifica-tion of biological materials.

It is probable that by the use of ap-propriate antisera any of the millions ofspecies of plants, animals, and micro-organismscan be distinguished from oneanother. Landsteiner, to whose nameand work the subject of immunologicalspecificity is most closely attached, ex-tended the demonstration of the rangeof this specificity to a wide variety ofsimple synthetic compounds. It wouldthus appear that the number of sub-stances which may be distinguished andidentified is nearly infinite if it includesall substances which have been and are

yet to be synthesized. This is, indeed,distinctive behavior.

In view of the uniqueness of antibodyspecificity, it is not surprising that im-munologists have always considered thatthe relationship of an antibody to itsantigen is something special. The termanti-egg albumin which designates anantibody to egg albumin carries with itthe concept of special formation alongwith the concept of specificity. Whereasother types of specific interactions couldbe attributed to chance structural com-plementariness between independentlyfabricated molecules, in the case ofantibodies this idea was generally re-jected because of the infinitely largenumber of different molecules thoughtto be required. Because of these con-siderations, Landsteiner stated, "thereremains hardly any other conclusionthan . . . to assume that under the in-fluence of antigens the formation ofcertain globulins (and perhaps normalantibodies) is modified in such a man-ner that the resulting globulins areclosely adapted to the immunizing sub-stance" (1, p. 148). This concept of aspecial relationship between antigen andantibody has been a cornerstone of im-munological thinking for half a century.Widely accepted before Landsteiner'simportant work, it has not been seriouslychallenged since.

The classical line of reasoning whichleads to the conclusion quoted abovemay be divided into the following steps.

*1) In many vertebrates, the responseto the injection of any one of a verylarge number of substances is the for-mation of antibodies capable of identi-fying the substance injected and thusdistinguishing it from all other sub-stances.

2) The antibodies so formed must bedifferent and unique for each of themany substances to which a distinctiveresponse can be made.

3) An animal could not possess ofitself the information necessary to syn-thesize all of the different types of anti-bodies it is capable of forming in re-sponse to the varied environmentalstimuli.

4) Antibodies must represent a uniquemodification in the synthesis of naturalprotein for which information is sup-plied by the antigen injected.

WeaknessesWhile it is understandable that thisline of reasoning seemed inescapable

only 20 years ago, several weaknesseshave developed in the argument as theresult of the progressof knowledge in im-munology and related fields of biology.

An increasing awareness of the diver-sity of antibodies in a single antiserumhas emphasized the distinction betweenan antiserum and the antibodies it con-tains (for reviews, see 2-4). Although itis necessary that a different antiserumbe formed for each antigen that may bedistinguished, it is possible that the largenumber of different antisera contain amuch smaller number of different anti-bodies in different combinations. For ex-ample, a system consisting of only fivedifferent antibodies, A, B, C, D, and E,could distinguish nine antigens which re-acted with three of the five in differentcombinations: ABC, ABD, ABE, ACD,ACE, BCD, BCE, BDE, and CDE. Thedistinguishing ability of antisera is simi-lar to that present in this simplified sys-tem, for the injected antigen is distin-guished largely by the fact that it reactswith the maximum number of anti-he author is associate professor of medicine atthe University of Chicago, Chicago, Ill.

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bodies. The concept that a differentantibody is made for each antigen isinherent in the conclusion of step 4 inthe classical line of reasoning givenabove. Its insertion in step 2 is an as-sumption which makes the final con-clusion almost inevitable.An almost unlimited number of dif-ferent antibodies is not only unnecessaryto explain immunological specificity, butmay be an impossible assumption. Thefinding that the combining site on theantibody molecule is relatively small (5)has raised the question of whether it ispossible to construct a unique antibodymolecule for each of the almost infinitenumber of antigens. This question isparticularly relevant if it is consideredthat not one but a whole family of dif-ferent antibody molecules with differentcombining constants is made in responseto a single antigenic determinant, andthat all of these molecules appear nearlyidentical to the other gamma globulinsof that animal in physicochemical prop-erties, in amino acid composition andsequence, and in antigenic properties.Another problem which has raiseddoubts concerning the uniqueness ofantibodies is the difficulty in definingthe term. Antibodies are so diverse intheir physical properties and in their re-actions with antigens and blend so grad-ually into obviously nonantibody sub-stances that any definition is necessarilyarbitrary (6).While the preceding considerationscast doubt on the uniqueness of therelationship between an antibody andits antigen, other developments havebrought into question the concept thatantibodies are necessarily modificationsof a normal protein. The analogy of in-duced enzyme synthesis in bacteria hasdemonstrated that a substance may ap-pear where nothing was previously de-tectable without necessarily implying theproduction of a new substance (7). Theinduction by environmental stimuli ofan increased production of naturally oc-curring animal proteins such as prop-erdin (8) or ferritin (9) has indicatedthe existence of induced protein syn-thesis in animals. The relatively largenumber of genetically determined en-zymes which can be synthesized by abacterium has increased the estimatesof the number of different natural pro-teins which can be synthesized by a ver-tebrate. It has been estimated that thereis enough deoxyribonucleic acid (DNA)in a single human cell to encode 1000large textbooks 10) .

Perhaps the most compelling reasonsfor questioning the concept that anti-bodies are globulins modified by anti-gens are the recent observationsrelativeto immunological tolerance-that is, thefinding that except under unusual cir-cumstances an adult animal does notsynthesize antibodies to his own sub-stances which are antigenic to other in-dividuals. The mechanism of this obvi-ously advantageous characteristic is notknown, nor it is known to what extent,if any, this is due to restrictionsimposedby heredity. That it is at least partiallyacquired during development might bededuced from the fact that with the ex-ception of highly inbred animals, an in-dividual can make antibodies against theantigens of both his parents. The moststriking experimental evidence that im-munological tolerance can be acquiredis its establishment by the injection offoreign antigens during an immunolog-ically unresponsiveperiod-for example,during the fetal or immediate newbornperiods (11), or after whole body x-radi-ation (12).If after the initial injection into anewborn animal the concentration ofantigen is maintained by repeated in-jections or by use of a viable, replicatingcellular antigen, tolerance may last in-definitely. If the concentration of anti-gen falls below a critical level for a shortperiod, subsequent injection of the sameantigen may lead to antibody production(13). Unlike the accelerated secondaryantibody response to an antigen, toler-ance to an antigen in the absence of thatantigen is short-lived. Probably relatedto immune tolerance are recent observa-tions which indicate that antibody-pro-ducing cells are highly specialized. In astudy of 456 single lymph node cellsfrom a rabbit immunized to two differ-ent salmonellae, Nossal and Lederberg(14) found 33 cells that synthesized im-mobilizing antibody to one organism, 29cells that synthesized antibody to theother, and no cells that synthesized anti-body to both. This confirms a similarconclusion that Coons (15) drew fromstudies of doubly immunized animalswith fluorescein-labeled antibody mark-ers.It is impossible to conclude from bothof these experiments that cells do notmake more than one type of protein,but only that the number of types anycell can make is greatly restricted com-pared with the capacity of the organismas a whole. In this sense they are highlyspecialized. There is a strong implica-

tion in other work that the immediateprecursors of antibody-producing cellsare also highly specialized. When cellsfrom immunized animals are transferredto normal, tolerant, or x-radiated recipi-ents, a prompt anamnestic response isobtained from an injection of the orig-inal antigen (16), but not from an in-jection of another antigen. The differ-ence between the tolerant and sensitizedanimals would seem to lie in the posses-sion by the latter of a large number ofdifferentiated precursorsof antibody-pro-ducing cells. Therefore, in some way theprocess of immune tolerance must berelated to the process of cell differenti-ation and replication.

Role of Antigen in Cell DifferentiationThe key issue relevant to this discussionis the role of antigen in the differentiationof antibody-forming cells. Although anti-

gen-induced differentiation might seeman obvious analogy to substrate-inducedpermease synthesis in bacteria (17), dif-ferences arise because specific capabili-ties in the latter phenomenon are strictlyinherited and are not associated withanything analogous to acquired immunetolerance. In order to explain acquiredimmune tolerance in animals, it is nec-essary to postulate a recognition systemcapable of distinguishing between theantigen and the tolerated substance(18). If one chooses the hypothesis thatthe antigen induces differentiation, it isnecessary to postulate that a system forrecognizing tolerated antigens resides inthe undifferentiated cell. Furthermore,the recognition system must be completein every cell for every autogenous sub-stance and must have at least three ad-ditional properties: (i) a specificityequal to that of antibodies, (ii) an in-ability to be overloaded or competitivelyinhibited by an excess of "self," and(iii) a flexibility sufficient to add andsubtract elements for the recognition offoreign substances depending on the lat-ter's presence or absence in the cell'senvironment.

The alternative to antigen-induced dif-ferentiation is a progressive differentia-tion which is either spontaneous or in-duced by inherited mechanisms similarto those which cause other forms ofdifferentiation in multicellular organ-isms. This inherently requires that anti-bodies be considered naturally occurringproteins. The advantage of this hypothe-sis is that it permits the control of dif-

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GLOBULIN A GLOBULIN B,t %',

'r,.

' T IGe ", UGS.| -\MJTIGEW AHTIGEN4

GLOBULIN C GLOBULINS A,B,AWDC

ANIGE /~"4'

Fig. 1. Two-dimensional diagram illustrating the concept that the information and netspecificity possessed by a combination of three different globulin molecules is greater thanthat possessed by one.

ferentiation, which must be present inany case, to substitute for the complexrecognition system required by the firsthypothesis. For if the combination ofantigen with the first antibody formedin the early stages of differentiation hasthe effect of inhibiting this differentia-tion, the specificity and other character-istics of immune tolerance can be ex-plained without postulating a secondrecognition system. As suggested by Bur-net (19) and by Lederberg (20), the in-hibition of partially differentiated cellsmight be considered a specialized ex-ample of hypersensitivity which resultsin selective destruction of these cells.The same result would be obtained ifthe "hypersensitivity"was a stimulus toanother channel of differentiation. Ananalogy to the latter phenomenon is theinhibition of antigen development inparamecia gro'wn in the presence ofspecific antisera (21). The action ofantigen in suppressing differentiationwould explain' the requirement of itscontinued presence to maintain acquiredimmune tolerance. The persistence ofantigen would be required if differentia-tion of antibody-producing cells was in-itiated approximately at birth and wascontinued throughout the life of theanimal.

The concept of autogenously con-trolled differentiation of antibody-pro-ducing cells provides a highly satisfac-tory explanation for the large increasein mitoses observed in lymphatic tissuefollowing an antigenic stimulus. If celldifferentiation occurs without antigen,each individual cell type will accountinitially for a small fraction of the totalcell population. Only by a replication ofselected differentiated cell types can thecapacity to produce the correspondingantibody be increased. Replication with-out prior differentiation is not adaptive.Replication of a cell differentiated byantigen is an unnecessaryadaptive effort.The only alternative to replication ofpredifferentiated cells would seem to bethe concept that replication is a neces-sary part of antigen induced differen-tiation.One of the most important problemsin experimental immunology today isthe design of experiments to distinguishbetween the alternativesdescribed above-that is, to determine the role of anti-gen in cell differentiation. An extensionof the work of Nossal and Lederberg totwo or more antigenic determinants onthe same antigen or to two differentantibody molecules to the same deter-minant would yield important informa-

tion. An alternative approach is thestudy of immune tolerance to cross-re-acting antigens and of the selective in-hibition of only a fraction of the many,different antibodies made to the same.antigen. From the latter experiments itmight be possible to determine whetheracquired tolerance is the placing of aparticular determinant within a self-recognition system or the selective sup-pression of pre-existing molecular typesof natural globulin.

The major difference between the twohypotheses is that in the first instanceantibody production is considered aunique biological phenomenon for whichunique mechanisms may be postulated,and in the second it is considered ahighly specialized example of certaingeneral cellular processes. The ability ofantibodies to distinguish an almost un-limited number of different antigens hasalways seemed ample justification forthe first view. However, the intrinsicunlikelihood of a truly unique biologicalprocess and the difficulties presented ineven defining an antibody are justifica-tion for an attempt to develop the al-ternative. Because an understanding ofthe alternative hypothesis requires analternative concept of immunologicalspecificity, the remaining portion of thisarticle is an attempt to explain immu-nological specificity on the basis of alimited number of different naturallyoccurring globulin molecules.

Unique Combinations ofMaster Molecules

As a basis for immunological speci-ficity the alternative to a unique anti-body for each distinguishable antigen isa combination of naturally occurringglobulin molecules which is unique foreach antigen. The latter concept is basedon the following premises.1) A relative stable complex betweena globulin molecule and some other sub-stance can occur whenever the configu-rations of the two molecules permit thedevelopment of short-rangeintermolecu-lar forces in excess of some criticalamount. These forces have been dis-cussed in detail by Pauling (22) and byPressman (23).

2) The formation of a stable complexdoes not require a perfect fit betweentwo complementary configurations (23).The heterogeneity of the antibodies com-bining with a single haptene and the re-actions of the same antibody with differ-19 JUNE 1959 1645


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FC2EE ENE12GY OF COMBINAITION IN V. CAL PE12 MOLE0 -I -Z -5 -4 -5 -G -7 -8 -q -10 -II -IZ

W THPE514OLD CU2VES10 l o\ \. - P2ECIPITAT701

a ........ 70XIM NEU7QALIZAN-IOWui 5 r .. S \ SAGGLUTI4AX101-4ai i0-5 \. EQUILIBQI1UM DAYId) _w

2 0 0 F X~~~~00lC-00

10 loz 10 104 1o0 1G, 107 108 109 1010EQUILIEB2IUM CONSTANTFig. 2. The relationship between antibody concentration, the energy of combination ofantigen and antibody, and the threshold required for detection. The threshold calcula-tions are based on the Goldberg theory and the following assumptions: Agglutinationinvolves a suspension of 108 particles per milliliter, each particle containing 6 x 10 sites;precipitation and toxin neutralization involve antigen molecules with a valence of 6 andantibody molecules with a valence of 2, and an optimal ratio of antigen to antibody;equilibrium dialysis requires the binding of at least 20 percent of the haptene in theantiserum compartment.

ent haptenes (1) provide experimentalevidence for this premise.

3) A single globulin molecule maycombine with a large number of differ-ent substances in the same sense that amaster key may open a large number ofdifferent locks. There should be manydifferent antigenic configurations struc-turally suited to combine with the sameglobulin if a lack of perfection in thefit in one area may be compensated byan increased binding energy elsewhere.

4) In a mixture of a large number ofdifferent globulin molecules, the domi-nant reactivity will be that common tothe largest number of the moleculespresent. This is because the reactions be-tween antigens and antisera are stronglydependent on concentration and have aswell sharp thresholds below which noreaction can be detected.5) The specificity of an antiserumcontaining a mixture of different globu-lin molecules is likely to be very muchgreater than that of an antiserum inwhich all the molecules are exactlyalike. (See Fig. 1.) This follows fromthe probability that the number of reac-tivities common to all of the differentmolecules will be greatly restricted com-pared with the number of reactivitiespossessed by a single molecule.

6) The number of different dominantreactivities and hence the number ofantigens which may be distinguished bya system of different globulin moleculesis the number of different combina-tions in which these molecules can bemixed.

On the basis of the premises listedabove, the requirements for producingan almost unlimited variety of immunesera are a moderately large number ofdifferent natural globulins with overlap-ping reactivities and a mechanism forselectively increasing the production ofthose globulins with high affinity for theantigen injected. The total number ofdifferent globulin molecules required isdetermined by three factors: (i) thenumber a of combinations required toaccount for the almost unlimited dis-tinguishing ability of immune sera; (ii)the fraction b of all possible antigenicconfigurations with which the averageglobulin molecule can combine, whichshouild be approximately equal to thefraction of different globulin moleculeswith which the average antigen can com-bine; and (iii) the number c of differentglobulin molecules in the average mono-specific antiserum. Unfortunately, verylittle is known about these factors ex-cept that a is very large. It is implicit

in this formulation that b is much largerthan l/a and probably lies between0.001 and 0.01; it is postulated that clies between 10 and 100. A large num-ber of different types of globulin in anantiserum has the effect of reducing theconcentration of each individual typeso that the concentration reacting bychance with an unrelated antigen is be-low the threshold of detection. How-ever, a structurally related antigen mightreact with a sufficiently high percentageof the different types to exceed thethreshold and be detected in a cross-re-action.If each globulin is so constructed thatits reactions at some arbitrarily ow levelof affinity are restricted to approxi-mately 1 percent of all possible anti-genic configurations,and there are 5000molecular types, each antigen will reactwith approximately 50 different globulinmolecules. In this case there would be500050/50! or approximately 3 x 10120different combinations and an equalnumber of different antigens whichcould be distinguished. Since this islarger than the number of electrons theuniverse is thought to contain, it is asatisfactorily large number. If the frac-tion of antigens which can combine witha single type of globulin molecule is lessthan 0.01, somewhat more than 5000different natural globulins are required.In Fig. 2, the process of immunizationis pictured as accelerating the produc-tion of a selected fraction of normalglobulins until the concentration of theseglobulins exceeds the threshold requiredfor detection. Threshold curves for pre-cipitation and agglutination can be cal-culated from the Goldberg theory (24)if certain assumptions are made con-cerning the valence of the antigen andthe ratio of antigen to antibody used inthe test procedure (4). The meanequilibrium constant for antibody is thatobtained experimentally by Nisonoff andPressman for p-iodobenzoate (25). Theexact shape of the distribution curve fornormal globulins is unknown and un-doubtedly varies according to the anti-gen used and the genetic and environ-mental history of the individual. Withmany antigens the distribution curve fornormal globulin crosses the threshold re-quired for agglutination but only rarelythe threshold required for precipitation.The selective increase in rate of produc-tion during the process of immunizationrepresented in Fig. 2 is analogous to theinduction of enzyme synthesis in bac-teria. The factor by which the rate of

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production is increased by the inductiveaction of the antigen might be expectedto be proportional to the energy of bind-ing. The small numbers in the middleof Fig. 2 represent the factor of increaserequired to convert the normal globulincurve into the antibody curve.The possession by protein moleculesof affinities for a large number of sub-stances is well recognized. Serum albu-min has been shown to bind a largenumber of natural and synthetic sub-stances (26). The number of substanceswhich bind effectively to individualglobulin molecules appears to be muchmore restricted, due perhaps to agreater rigidity of the molecule, as sug-gested by Karush (27). This results ina greater specificity of that bindingwhich does occur. A similar degree ofspecificity was found by Boyd (28) ina study of a large number of plant pro-teins. Some possessed a highly specificagglutinating ability for red cells con-taining antigens A, AN, H, and N. Boydcalled these plant proteins lectins fromthe Latin word legere, "to select," andsuggested that the name might be usedto include "those normal antibodies ofanimal serum thought not to result fromantigenic stimulus." In the case of serumglobulins, the restricted number of sub-stances an individual globulin will bindis compensated for by the much greaterheterogeneity of the globulins. Thus, itis not surprisingthat normal serum con-tains in its globulin fraction agglutininsfor the red blood cells of other speciesand for many bacteria. When the speci-ficity of these agglutinins was demon-strated by his absorption experiments, asdepicted in Table 1, Malkoff (29)reached the conclusion that a normalserum contains as many specific agglu-tinins as there are sorts of cells that areagglutinated by the serum. Because ofthe large number of different substanceswhich could be specifically agglutinated,and because each of them amounted toseveral micrograms per milliliter ofserum, Landsteiner rejected this hy-pothesis. He was able to purify the ag-glutinins by absorption and elution fromred cells and to show that the purifiedagglutinins acted most strongly on thered cells used for absorption, but alsoagglutinated other sorts of blood. Heconcluded that "if one assumes thatnormal serum contains a sufficient num-ber of agglutinins, each reacting witha certain proportion of all. bloods, agiven sort of blood will absorb from aserum all those agglutinins for which it

Table 1. Malkoff's results (29).Goat serum absorbed with

Unab- Pigeon PigeonBlood sorbed Pigeon Rabbit Human and andserum blood blood blood rabbit humanblood blood

Pigeon blood + 0 + + 0 0Rabbit blood + + 0 + 0 +Human blood + + + 0 + 0

has affinity and there will remain afterabsorption some that react with freshlyadded blood of other species. . . . Onemay conjecture that there exists a muchgreater variety of globulin molecules ina serum than would appear from physi-cochemical examination, some of whichby virtue of accidental affinityto certainsubstrates are picked out as antibodies"(1, pp. 129, 133).The preceding conclusion of Land-steiner attributed the specificity of nat-ural antibodies to unique combinationsof natural globulins. An extension of thisconcept to include immune antibodiesmight have seemed likely, because ofthe similarity of Landsteiner's own re-sults with synthetic haptenes to those ofMalkoff with natural agglutinins (seeTables 1 and 2). However, Landsteinerrejected this concept as an explanationof the specificity of immune antibodies,apparently because of a firm convictionthat immune antibodies were differentfrom natural antibodies. More recently,because of the failure to find differencesbetween immune and natural antibodies,all natural antibodies have been con-sidered to be immune antibodies pro-duced in response to the antigens offood or intestinal bacteria. The other

alternative, that all immune antibodiesare unmodified natural globulins, is thethesis of this article.Landsteiner was one of the first todemonstrate the heterogeneity of anti-bodies produced in response to a singleantigenic determinant. A classical ex-periment is reproduced in Table 2 (30).This experiment demonstrated that anti-bodies to a single determinant were het-erogeneous with respect to their abilityto cross-react with related chemicalgroupings. Heterogeneity in another di-mension-that is, with respect to com-bining constants-has been amply dem-onstrated by the more quantitative tech-niques of precipitation inhibition andequilibrium dialysis. More recently, Nis-onoff and Pressman (31) have been ableto confirm with equilibrium dialysis theexperiments of Landsteiner and van derScheer. Using a radioactively labeledp-iodobenzoate, Nisonoff and Pressmanshowed that an antiserum to this deter-minant was heterogeneous with respectto the ratio of combining constants totwo related substances.In general, a determination of thecombining constants between purifiedantibodies and various haptenes hasshown that the haptene used in the

Table 2. Results obtained by Landsteiner and van der Scheer (30). Since the test anti-gens contained the same proteins, unrelated to the horse serum used for immunization,the protein component could not be responsible for the differential reactions.Azoproteins made from chicken serum and

Immune sera for o-Amino- n-Amino- r-Amino-m-aminobenzene sulfonic nm-Amino-acid after absorption with sulfonic sulfonic arsenzec benzoicacid acid acid acid

o-Aminobenzene sulfonic acid* 0 + +o-Aminobenzene sulfonic acidt 0 q + +m-Aminobenzene arsenic acid* e 1-1 0 +m-Aminobenzenearsenic acidt 4+ 0 -1-4-m-Aminobenzoic acid* ++ 0Om-Aminobenzoic acidt ++ + + +Unabsorbed immune serum* ++ + + +?Unabsorbed immune serumt 44+ ++-I +4-* After standing 1 hour at room temperature.t After standing overnight in the icebox.

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talmage - immunological specificity - 1959

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