The Control of Humoral and Associative Antibody Synthesis

Transplant. Rev. (\972),\o]. 11 217-267Published by Munksgaard, Copenhagen, DenmarkNo part may be reproduced by any process without written permission from the author(s)

The Control of Humoral

and Associative Antibody SynthesisPETER BRETSCHER, The Salk Institute, P.O. Box 1809, San Diego, California 92112.

Contents

Introduction 218

Theories of Self-nonself Discrimination 218A particular Historical Theory: the Principle of Associative Recognition 220Other Historical Theories 222

The Regulation of the Synthesis of Associative Antibody 225

The Three Basic Laws 229

The Cellular Interpretation of the Theory 230

Feedback' Regulation of the Response 233

Quantitative Aspects of the Theory 234

A Minimal Approach 234

Inadequacies of the Minimal Approach 239

Other Views of T Cell Function 243

Thymus-independent Antigens' 245Polyvinylpyrrolidone or PVP 245E-coli lipopolysaccharide or LPS 247Pneumococcal Polysaccharide Type HI or SIII 247Polymeric Form of Flagellin from Salmonella Adelaide or POL 248High-zone Paralysis by 'Thymus-independent Antigens' 250

Stable 19s Humoral Antibody Synthesis 250

Abnormal Induction 252

The Asymmetry in the Requirement for Specific B and t cells for the

Induction of Humoral Antibody Synthesis 254

Other Views of Paralysis 256

'In Vitro Paralysis' 257Evidence for the Associative Recognition of Antigen During the Induction

of t cells 259

Reconstitution of the Response of T Cell-Depleted Spleen Cells by VariousSubstances 260

Summary 261

218 PETER BRETSCHER

INTRODUCTION

A theory for the paralysis and induction of antibody synthesis by antigenhas been proposed (Bretscher & Cohn 1968, 1970). The theory providesan explanation of how, under normal circumstances, an animal can respondto foreign antigens while remaining tolerant of his own antigens. I will notdiscuss here the majority of those experiments that led to the proposal ofthe theory, as this has been done elsewhere (Bretscher •& Cohn 1968, 1970).Instead I will analyze what other theories of self-nonself discrimination arepossible, and give reasons for favouring the one we originally proposed. Iwill then examine various experimental observations which have not beenpreviously discussed in any detail in terms of the theory. I shall deal explicitlyonly with questions related to the induction and paralysis af humoral anti-body synthesis.

THEORIES OE SELE-NONSELF DISCRIMINATION

I list below the assumptions on which the analysis of possible theories ofself-nonself discrimination is based:

1) The clonal selection theory is correct. This theory states that for anyantibody whose synthesis is specifically inducible or paralysable by antigenthere exists a precursor cell which bears antibody receptors for the antigenon its surface. Each precursor cell bears antibody receptors of only onespecificity, which is identical to that of the antibody whose synthesis isinduced if induction of this cell occurs. An interaction of antigen with thesereceptors is required for the paralysis or induction of the precursor cell.

2) As tacitly assumed in 1), paralysis by antigen is a result of inactivatingprecursor cells such that they are no longer inducible. Paralysis does notinvolve a suppression of the effector mechanism (i.e. rendering the inducedantibodies ineffective). In other words, paralysis is central and not peripheral.

3) Precursor cells with specificity for both foreign and self antigens beginto be generated at some stage during foetal development, and are thereaftergenerated throughout the life of the animal.

4) An understanding of the antigen-specific steps in the induction andparalysis of precursor cells must lead to an explanation of self-nonself dis-crimination. This assumption is essentially a statement of the belief that thephenomenon of paralysis is relevant to an understanding of why antibodiesare usually not induced to self components.

CONTROL OF ANTIBODY SYNTHESIS 219

5) A precursor cell which is inducible is also paralysable and vice versa.This assumption is the most contentious and will he discussed later.

Before discussing any mechanism for self-nonself discrimination, it is worth-while to consider whether there is any characteristic that self componentshave in common and that distinguishes them from foreign antigens. Theimmune system must rely upon such a characteristic in discriminating be-tween self and foreign antigens. It is not plausible, for example, that all selfcomponents share a chemical structure that enables the immune system torecognize them as self components. The concentration of different self com-ponents must vary widely in the envirorunent of the precursor cell, and ittherefore does not seem likely that a precursor cell could discriminate be-tween self and foreign antigens by being inducible at only certain concen-trations of antigen. The one common characteristic of self components thatdistinguishes them from foreign antigens appears to be that they are con-tinuously present in the animal. That it is this property of self antigens thatthe immune system relics upon in distinguishing self components from foreignantigens is supported by many experiments. For example, if an adult isparalysed to a foreign antigen, he will recover from his unresponsive stateunless the antigen is continually administered. Moreover, the amount ofantigen required to maintain an unresponsive state can vary upwards froma lower level, at which recovery takes place,, to include levels which, in anormal animal, would give rise to an antibody response (Dresser & Mitchison1968).

Consider an adult animal which generates two precursor cells. One pre-cursor cell has receptors with specificity for a self-component S, and theother precursor cell receptors with specificity for a foreign antigen F whichdoes not possess any determinants which occur on self components. Both Fand S are present in the animal at concentrations at which they can interactwith some of the specific receptors on their corresponding precursor cells.If there is to be self-nonself discrimination the anti-S precursor cell must beparalysed by S, while the anti-F precursor cell must, in general, be inducedby F. The only way of ensuring that this will occur is that there be someantigen-specific molecules in the animal which cause S and F to interactin different manners with their corresponding precursor cells. We couldimagine, for example, that there are molecules specific for F which allowF to interact with its precursor cell in a manner different from that in whichS interacts with its precursor cell. The way in which an antigen interactswith its precursor cell would depend on the presence or absence of thesespecific molecules. The presence of these molecules would have to dependon the past history of the animal such that only molecules specific for

220 PETER BRETSCHER

foreign antigens were present. This theory is 'historical' in that the decisionof whether to induce or paralyse a precursor cell depends on the past historyof the animal.

A Particular Historical Theory: the Principle ofAssociative Recognition

It is known that, in order for some antigens to induce specific antibody, atleast two determinants on the antigen must be recognized (Duton et al. 1970,Mitchison 1967, Raff 1970, Rajewsky & Rottander 1967, Rajewsky et al.1969). Other evidence, as I shall diseuss later, is consistent with the postulatethat only one determinant on the antigen need be recognized, by the antigenbinding to the receptors on the precursor cell, in order to paralyse the cell.Suppose that it is obligatory for the induction of a precursor cell that anotherdeterminant on the antigen, besides the determinant bound to the receptoron the precursor cell, be bound by a specific molecule. Then we have a wayof ensuring that the anti-S precursor cell is paralysed by S and that the anti-Fprecursor cell can be induced by F. Th& animal, according to this view, hassome molecules specific for F that allow F to interact with its precursor cellin a manner different from that in which S interacts with its precursor cell.The molecule which specifically binds to the second determinant of F isassumed to be an antibody and to be derived from another cell. Thepostulated requirement for the recognition of two detenninants on an antigenfor the induction of specific antibody is referred to as the Principle of Asso-ciative Recognition. The molecule which recognizes the second determinanton the antigen is called associative antibody.* We can present the inductionand paralysis of a precursor cell as shown in Figure 1.

Paralysis occurs when antigen binds to the receptors on the precursorcell ,thus delivering signal CD as a result of a conformational change in itsbound receptors. Induction involves the precursor cell receiving both signals® and (D, the latter as a result of associative antibody binding to the antigen.The precise mechanism by which the precursor cell receives signal © is notcrucial to the analysis at this stage; it is only important to stress that signal(D is postulated to be delivered to the precursor cell as a result of specificassociative antibody interacting with the antigen. I shall briefly discuss thenature of signal © later (see Reconstitution of the Response of T Cell-Depleted Spleen Cells by Various Substances).

* In previous discussions (Bretscher & Cohn 1968, 1970) this molecule has beenreferred lo as carrier antibody. This name caused some confusion and so thismolecule has been given the new name of associative antibody.


It will be necessary to consider how the synthesis of associative antibodyis regulated in order to give a complete description of how this theory ex-plains the maintenance of tolerance to self components. It follows, however,from the proposed mechanisms for the induction and paralysis of precursorcells that, as associative antibody specific for only foreign antigens is present,precursor cells specific for a self component S will be paralysed by S, whereasa precursor cell specific for a foreign antigen F can be induced in thepresence of F.

Although the administration of foreign antigens to an animal usually re-sults in the induction of specific antibody, this is not invariably the case.Even when precursor cells are present they may not be induced undercertain circumstances. If there is insufficient associative antibody present,or if it is unable to interact in the appropriate way as depicted in Figure 1,then it would be expected that a foreign antigen would not induce specificantibody but would paralyse its specific precursor cells. The suggestedmechanisms for the paralysis and induction of precursor cells account forseveral observations:

a) The amount of associative antibody specific for an antigen will dependon the number of foreign sites on the antigen. There will be more associativeantibody specific for an antigen with many foreign sites than for an antigenwith few foreign sites, and the former antigen will be more immunogenic.This expectation is consistent with the observation that a hapten, if coupledto a self component, is not immunogenic, but is paralytic (Borel 1971, Golan& Borel 1971). For this to be true the structure of the self component mustnot be grossly altered by the hapten being coupled to it, thereby revealingnon-self detenninants.

b) The expectation that antigens with many foreign sites are more immuno-genic than antigens with a few foreign sites is consistent with other observa-tions. A conjugate of a non-immunogenic molecule, N, and an immunogenic

Figure I.

222 PETER BRETSCHER

molecule, I, will induce anti-N antibody if given to an animal (Green et al1968, Katz et al. 1971, Landsteiner 1962, Rajewsky & Rottlander 1967).The fragmentation of an antigen, resulting in fewer foreign sites being presenton a single physical entity, renders the antigen less immunogenic and moreparalytic (Ada et al. 1967, Katz et al. 1971). An aggregate of an antigenis more immunogenic than the antigen itself (Ada et al. 1967, Dresser 1962,Golub & Weigle 1969). (These observations arc discussed later in a semi-quantitative fashion, see Quantitative Aspects of the Theory.)

c) Large doses of antigen, which would be expected to saturate the receptorson the precursor cell and the associative antibody, are known to be paralytic(Dresser & Mitchison 1968). It is known that monomeric antigens, whichdo not possess identical determinants on their surface, paralyse at extra-vascular concentrations of antigen of about 10''M (Mitchison 1968). Thisconcentration of antigen, which is above the binding constant of most anti-bodies, will saturate most of the receptors and the associative antibody suchthat associative recognition cannot occur. The precursor cells are paralysed.

d) It is known that an animal paralysed to an antigen A can be reconstitutedwith normal spleen cells from an unparalysed syngeneic donor (Mitchison1962, Weigle & Dixon 1959). This reconstitution is effective so long asparalysing amounts of A are not present in the recipient. This observationis consistent with paralysis being due to the absence of the ability to respondrather than to the presence of molecules in the paralysed animal which eitherprevent induction or favour paralysis.

Other Historical Theories

It was concluded above that, given the five basic assumptions on which theanalysis was based, there must in genera! be specific molecules that enablea self-component S and a foreign antigen F to interact with their respectiveprecursor cells differently. A particular possibility was considered: that inan animal there is associative antibody specific for foreign antigens, andthat the presence of specific associative antibody is obligatory for an antigento be able to induce its precursor cells. However, there are two other theo-retical possibilities:

i) There could be molecules specific for S which ensure that S paralyses itsprecursor cell and F, by simply binding to the receptors on its precursorcell, could induce its precursor cell. These molecules would be specific fordeterminants occurring on self-components and would have to ensure that


an antigen, sharing several determinants with self-components, would para-lyse its corresponding precursor cells.ii) Both paralysis and induction could require the recognition of more thanoae determinant on an antigen. In this case there would be one set ofmolecules, m , specific for determinants occurring on self-components, whichwould have to bind to the antigen at a second site in order to paralyse aprecursor eel!. Another set of molecules, mf, of a different class and specificfor determinants occurring only on foreign antigens, would have to bind tothe antigen at a second site in order to induce a precursor cell.

These two possibilities share the common features that i) more than onedeterminant on an antigen must be recognized in order for an antigen toparalyse a precursor cell, and ii) a paralysed animal should have moleculesspecific for the antigen with which it was paralysed. The latter featurefollows from the assumption that paralysis is relevant to self-nonself discrimi-nation, and that there are molecules specific for self-components which arerequired for the paralysis of anti-self precursor cells. These two featuresare inconsistent with many observations which find a ready explanation interms of the theory originally discussed. For example, the paralytic potencyof an antigen would depend on the number of sites it shared with self compo-nents, and fragmentation of antigen should not generally favour paralysis,nor should aggregation generally lead to increased immunogenicity. Thesetwo theories do not readily explain the observation that high doses of antigenparalyse. As a paralysed state is characterized by the presence of moleculesspecific for the antigen with which the animal was paralysed, and whosepresence is required for the paralysis of precursor cells, a paralysed animalis not expected to be reconstituted on the injection of syngeneic unparaiysedspleen cells.

It thus appears highly plausible that, if the five basic assumptions arecorrect, the theory that requires associative recognition of antigen for theinduction of antibody synthesis and requires the recognition of only onedeterminant on the antigen for the paralysis of antibody synthesis is true.However, the evidence for the fifth and most contentious assumption, namelythat precursor cells are always both paralysable and inducible, has not beendiscussed and if violated can lead to alternative theories of self-nonselfdiscrimination.

Lederberg's theory of self-nonself discrimination was based on the firstfour assumptions but violated the fifth (Ledenberg 1959). He proposed thatevery precursor ceil exists in a paralysable state before it differentiates intoan inducible state, as shown in Figure 2.

The paralysabte cell, and the inducihle cell it gives rise to, bear receptorsof the same specificity on their surface, and an interaction of antigen results

224 PETER BRETSCHER

in the inactivation or induction of the cell depending on which state the cellis in. Inducible cells specific for a self component, which is continuallypresent, cannot arise, but inducible cells specific for a foreign antigen F canaccumulate in the absence of F. This theory must be modified in view ofthe fact that there is competition between the paralysis and induction of aprecursor cell. This fact is most clearly seen in the following kind of experi-ment.

An animal is rendered unresponsive to an antigen A. Suppose A has thedetenninants ai-a2o on its surface. The animal's response to various antigensis examined subsequently, at a time when he has not recovered from hisunresponsive state, i.e. when a normally inducing dose of A does not resultin the induction of antibody specific for A. If the animal is administered anantigen B, which cross-reacts with A and has detenninants ai-asbi-bis,specific antibody is raised against both the determinants bi-bi--, and ai-aa.This result shows that the animal possesses precursor cells specific for thedeterminants a^-as which are common to A and B. The theory explains theseresults on the basis that the amount of associative antibody specific for A isinsufficient for associative recognition to occur on administration of A, butthat the amount of associative antibody specific for the determinants bj-bi^is sufficient to induce the precursor cells specific for the determinants aj-a^,on administration of B. If, instead of B being administered alone, A and Bare administered together, only anti-(bi-b,5) antibodies are raised. Thisresult shows that A can paralyse the anti-(ai-a5) precursor cells in thepresence of B which, if acting alone, would induce these cells. This showsthat the anti-(ai-a5) precursor cells which are inducible are also paralysable(Humphrey 1964, Weigle 1964).

Lederberg's theory could be modified to explain this result by postulatingthat a paralysable cell gives rise to one which is both paralysable andinducible: as shown in Figure 3.

This theory predicts that there are paralysable cells which are not in-ducible. The truth of this prediction is very difficult to ascertain experi-

Figure 2.


mentally in a direct manner. However, it is known that if a normal mouseis given foreign thymus cells, antibody with specificity for both the donorand recipient thymus cells is induced (Boyse et al. 1970). This result isdifficult to reconcile with the modified Lederberg theory, as all precursorcells specific for self-components should be paralysed and thus inactivated,and inducible anti-self precursor cells should not arise. Thus an inducibleanti-thymus precursor cell should not arise and antibody specific for hostand donor thymus cells should not be induced. On the other hand, thisresult ean be explained in terms of the theory of self-nonself discriminationthat I favour, as described elsewhere (Bretscher, submitted for publication).

The above analysis has attempted to show that there is only one viabletheory of self-nonself discrimination within the limits imposed by the fivebasic assumptions. This theory states that the associative recognition of twodetenninants on an antigen is required for the induction of humoral antibodysynthesis by the antigen, and that antigen binding directiy to the receptorson the precursor ceil can paralyse the precursor cell.

THE REGULATION OF THE SYNTHESIS

OF ASSOCIATIVE ANTIBODY

It has been argued above that an animal possesses associative antibodyspecific for foreign antigens, but does not possess associative antibody spe-cific for self components. If the phenomenon of paralysis is really relevantto self-nonself discrimination one must expect that an animal, paralysed toa foreign antigen A, has no associative antibody specific for A. This meansthat the synthesis of associative antibody must be paralysable. According tothe clonal selection theory this requires that there are unispecific precursorcells which are paralysable. I shall call this cell the associative precursor cell,and the precursor cell which, if induced, divides and whose descendantssecrete humoral antibody, the humoral precursor cell.

TIHE

Figure 3.

Transplant. Rev. (1972) 11 15

226 PETER BRETSCHER

Evidence that the synthesis of associative antibody is paralysable has beenobtained in many experimental systems. For example, the molecule DNP-PLL (dinitrophenyl-poly-L-lysine) is a hapten in some animals, and is notitself immunogenic. This molecule can be coupled to BSA (bovine serumalbumin), an immunogenic molecule in these animals, to form the conjugateDNP-PLL-BSA. When DNP-PLL-BSA is administered to these animals ananti-DNP response occurs. This result shows that there are anti-DNPhumoral precursor cells in these animals. The inability of DNP-PLL toinduce the synthesis of anti-DNP antibody suggests that there is insufficientassociative antibody specific for PLL to induce the anti-DNP humoralprecursor cells. When the complete conjugate DNP-PLL-BSA is given tothese animals the associative antibody specific for BSA enables the completeconjugate to induce the anti-DNP humoral precursor cells. If the animalsare specifically paralysed to BSA, before the complete conjugate DNP-PLL-BSA is administered, no response against DNP occurs (Green et al. 1968).This result shows that the synthesis of associative antibody specific for BSAis paralysable.

Other evidence shows that the formation of associative antibody is in-ducible. To achieve a good secondary response to a hapten, h, the haptenmust be conjugated to a molecule against which the animal has been immu-nized. If the animal is sensitized with a conjugate h-X, and challenged witha conjugate h-Y, the anti-h response is generally much weaker than if theanimal is challenged with h-X. When an animal is sensitized with both h-X andY, and then challenged with h-Y, the anti-h response is strong. This evidencesuggests that the synthesis of associative antibody is inducible (Mitchison1968, Rajewsky et al. 1969). An alternative interpretation of these observa-tions is that humoral antibody, specific for Y and induced on the animalbeing sensitized with Y, enables a better response to be mounted against thehapten on the animal being challenged with h-Y. Other evidence (Duttonet al. 1970, Mitchison et al. 1970, Raff 1970), however, which I do nothave the> space to describe here, shows that the strong response to the haptenis due to the induction of associative antibody specific for the carrier.

It is known that an animal paralysed to an antigen A can, in the absenceof A, recover from its unresponsive state. This shows that, if the paralysisof an associative precuror cell is irreversible (as I shall argue later, seeAlternative Views of Paralysis), new associative precursor cells are generatedin an adult animal.

Humoral and associative antibody synthesis are thus similar in that bothare inducible and paralysable. It seems very likely, as concluded above,that all humoral precursor cells which are inducible are paralysable, andvice versa. However, it is not known whether all associative precursor cells


which are inducible are paralysable and vice versa. 1 shall assume thathumoral and associative precursor cells are similar in this respect. I shallnow consider what the requirements are for the specific recognition of anti-gen during the induction and paralysis of associative precursor cells.

We can apply the same kind of analysis used above in discussing therequirement for the specific recognition of antigen during the induction andparalysis of humoral precursor cells, to discuss the induction and paral-ysis of associative precursor cells. Consider an animal which generatestwo associative precursor cells, one specific for a self component S, and theother specific for a foreign antigen F, which shares no determinants withself components. If self-nonself discrimination is to occur, there must bespecific molecules in the animal which allow S and F to interact in differentways with their corresponding associative precursor cells; these specific mole-cules must ensure that the anti-S precursor cell is paralysed by S and that theanti-F precursor can, in general, be induced by F. A particular possibilityis that S, on binding to the receptors on its associative precursor eell, para-lyses the cell, whereas the induction by F of its associative precursor cellrequires a second determinant on the antigen, besides the determinant boundby the receptor of the precursor cell, to be bound hy a molecule specific forF. This is the possibility I favour. The molecule binding to the seconddeterminant of F is assumed to be an antibody molecule. It is reasonableto postulate that this antibody is associative antibody. (The consequencesof postulating that this second molecule is not associative antibody is thatthe theory becomes considerably more complicated.) If it is obligatory forthe induction of an associative precursor eell that a second determinant onthe antigen be bound by associative antibody, then S will not induce an anti-Sassociative precursor cell in the absence of associative antibody specific for S.The postulated mechanisms for the induction and paralysis of humoral andassociative precursor cells are shown in Figure 4.

The theory accounts for the maintenance of a tolerant state in. an adultanimal in the following way. The animal, if tolerant of S, will not possessany associative antibody specific for S. As new humoral and associativeprecursor cells are generated, one at a time, they will be paralysed as there

' *S50CliTIVfl*HT[B0OI IINBUCIIVE

-^ SISUL

Figure 4.

228 PETER BRETSCHER

is no associative antibody specific for S. The animal will maintain a state inwhich he possesses no associative antibody specific for S.

The theory explains how an adult animal maintains a tolerant state to hisown antigens as opposed to how he establishes a tolerant state. For most, ifnot all, self components the problem of the establishment of a tolerant statedoes not exist. Consider a self component S which is present before theimmune system develops. Precursor cells specific for S will be paralysed oneat a time as they arise. At no stage will the animal be able to respondagainst S. According to the theory the problem of the establishment of atolerant state does not arise for an antigen present before the immunesystem develops.

The hypothesis that the Principle of Associative Recognition applies tothe induction of associative precursor cells, and that the paralysis^ of thesecells occurs when naked antigen binds to the receptors of the precursor cells,is consistent with several observations.

a) Large doses of antigen, which saturate both the associative antibody andthe receptors on the associative precursor cell, are expected and known toparalyse the associative precursor cell (Green et al. 1968, Mitchison 1971b).

b) As discussed above, the induction of humoral precursor cells is expectedand known to be particularly efficient for antigens which bear many foreignsites, and those antigens which bear few foreign sites tend to be paralytic.In general, although the relevant observations are not very extensive, it isfound that those antigens which are good inducers of humoral antibodysynthesis are good inducers of associative antibody synthesis, and those mole-cules which paralyse humoral precursor cells also paralyse associative pre-cursor cells (Mitchison et al. 1970, Weigle et al. 1971). This is expected ifassociative recognition is obligatory for the induction of hoth humoral andassociative precursor cells, and if the paralysis of both kinds of precursorcell requires only the recognition of one determinant on an antigen by thereceptors on the precursor cell binding to the antigen.

c) It is known that a paralysed animal can be reconstituted with normalspleen cells from an unparalysed syngeneic animal (Mitchison 1962, Weigle& Dixon 1959). This observation is consistent with paralysis being due tothe absence of the ability to respond rather than the presence of moleculeswhich either prevent induction or favour paralysis.

It was concluded above that in general there must be specific molecules inan animal which enable a self-component S and a foreign antigen F to inter-act with their respective associative precursor cells in different ways. The


particular possibility has been considered above that the associative recogni-tion of antigen is required for the induction of associative precursor cellsbut not for their paralysis. As the analysis can be extended in a manneridentical to that employed above when the induction and paralysis of hu-moral precursor cells was considered, I shall only summarize the arguments.The two other theoretical possibilities, which ensure that S will paralyse itsassociative precursor cell and allow F to induce its associative precursor cell,have features which are difficult to reconcile with what is known about theparalysis and induction of associative precursor cells. In particular, they donot provide an explanation for those three sets of observations which werediscussed above and which the theory I favour readily accounts for. The onlyreasonable escape from this analysis is to deny that associative precursorcells are always both paralysable and inducible. This assumption is crucialto the theory, but has not been tested experimentally.

THE THREE BASIC LAWS

Before discussing the interpretation of the theory in cellular terms it will beuseful to summarize the theory i nthe form of three basic laws (Bretscher& Cohn 1970):

1) The induction of a humoral precursor cell by antigen involves the obliga-tory recognition of two determinants on the antigen; one determinant isbound by the receptors of the precursor eel!, thus delivering signal ® to thecell, and the other determinant is bound by associative antibody, thus leadingto the cell receiving signal ®. Paralysis occurs when antigen binds to thereceptors of the precursor cell thus delivering signal ® to the cell.

2) The induction and paralysis of associative precursor cells is similar tothe induction and paralysis of humoral precursor cells in that inductionrequires the precursor cells to receive signals ® and ®, whereas paralysisrequires the cell to receive only signal O.

3) A stable tolerant state to a self component S requires the absence of bothhumoral and associative precursor cells specific for S (Bretscher & Cohn1970). If an animal possessed humoral precursor cells specific for S, then aforeign antigen F, which had some determinants in common with S, couldinduce antibodies to these shared determinants in the following way. SupposeS has determinants Si-Sm, and F determinants fi-fio S1-S3. As there will ingeneral be associative antibody specific for the determinants fj-fio, a humoralprecursor cell with receptors specific for So could be induced by F as shownin Figure 5.

If an animal has associative precursor cells specific for S then his tolerant

230 PETER BRETSCHER

State to S will not be stable. For example, if some of these associative pre-cursor cells were induced by a foreign antigen F, which had some determi-nants in common with S, the induced associative antibody specific for Scould lead to the induction of a single, newly arising, anti-S humoral pre-cursor cell.

THE CELLULAR INTERPRETATION OF THE THEORY

It is known that there is a requirement for both bone marrow, or B, cellsand thymus derived, or T, eells for an antibody response to occur againstsome antigens. Much evidence has been adduced to show that the B cellis the precursor cell of the humoral antibody-producing cell, and that specificT cells are necessary for a response to occur (Chiller et al. 1970, Duttonet al. 1970, Mitchell & Miller 1968, Nossal et al. 1968). The B cell is thusidentified as the humoral precursor cell, and it seems most plausible that theT cell is the producer, or bearer, of associative antibody. This identificationmakes the requirement for the presence of T cells, or the associative antibodythey may secrete, obligatory for the induction of B cells.

The theory does not specify whether the associative antibody is free orcell-bound during the inducing event. The most obvious possibilities for itslocation are: in the membrane of the T cell itself, on the surface of a cellfor which it is cytophilic, or not bound to any eell (Bretscher & Cohn 1970).Of these three alternatives the first requires that the induction of a B cellinvolves an interaction between two rare unispecific cells. Although this ispossible, the other two alternatives seem much more plausible. If associativeantibody is secreted and passively absorbed onto a commonly occurring cell

HUMORALPRECURSOR

CELLANTI -

ASSOCIATIVE-ANTIBODY

A N T I - fe

Figure 5.


it could interact with a precursor cell in the presence of antigen withoutdifficulty. A rough calculation shows that a cell with a diameter of 10microns could easily absorb 10® IgG antibody molecules, and hence that acollision between such a cell and a precursor cell would have a good chanceof leading to the induction of the precursor cell in the presence of sufficientantigen (Bretscher & Cohn 1970). The possibility in which associative anti-body is cytophilic is the one I favour (Bretscher & Cohn 1968, 1970).

The main features known about B cell induction are shown in Figure 6.The precursor cell, which divides on induction, is X-ray sensitive. The

plasma cell does not divide frequently, if at all, and its production of humoralantibody is X-ray resistant. Memory B eells are also produced if a B cell isinduced. The generation of memory cells ensures that the induction of B cellsdoes not result in a depletion of B ceils. The exact genealogy of the memorycell is unknown, but it is an X-ray sensitive cell. Neither is it known whethera 'virgin' B cell is different from a "memory' B cell.

There are cells in the thymus which can be induced by an immunogen tomultiply, giving rise to specific T cells (Shearer & Cudkowicz 1969). Thesespecific T cells can interact in the presence of antigen with specific B cellsto induce these B cells (Dutton et al. 1970, Mitchison et al. 1970). Thethymus cells are X-ray sensitive (Miller & Mitchell 1968), and the Tcells are X-ray resistant (Hirst & Dutton 1970, Katz et al. 1970). It isnatural to identify these thymus or t cells with the associative precursorcell which, if induced, multiplies and gives rise to specific T cells. This identi-fication of t cells as associative precursor cells is reinforced by the finding

PLASMA CELL. X-RAY INSEWSITIVE;

PRODUCES HUMORAL ANTIBODY

AND IS SHORT-LIVEO

Figure 6.

232 PETER BRETSCHER

that in an animal paralysed to an antigen A there are no t cells specific forA (Chiller et al. 1970). It was concluded above that the associative precursorcell is paralysable, and the identification of this cell as a t cell requires thatt eells are paralysable.

It is also known that there are cells in the thoracic duct which cancooperate with B cells, and that these cells are X-ray sensitive (Miller &Mitchell 1968), which suggests that they are precursor cells. I would suggestthat these cells are the analogue of those B cells which are found outsidethe bone marrow, for example in the spleen, and which are presumably amixture of virgin and memory B cells. The main features of the inductionof thymus cells that I suggest are represented in Figure 7.

This scheme has the virtue of making t cell and B cell induction verysimilar (compare Figure 6 with Figure 7), and of resolving the paradox thatthose cells outside the thymus and able to induce B cells, in the presenceof antigen, are found to be sometimes X-ray sensitive and sometimes X-rayresistant (Hirst & Dutton 1970, Katz et al. 1970, Miller & Mitchell 1968).This scheme would suggest that the X-ray resistant T eells are the result ofsome recent induction of t cells by antigen. My nomenclature for these celltypes is different from that usually used in the literature. It is not an unusualpractice to refer to both the X-ray resistant cell and the X-ray sensitive ceilas a T cell. I have tried to change the usual nomenclature as little as possible

BEARS OR SECRETESASSOCIATIVE ANTIBODY;X-RAY RESISTANT ANDIS SHORT-LIVED

Figure 7.


to describe in a logically consistent fashion the cell types that I believe exist.An interesting paradox arises if one postulates that T cells must in

general be present for the induction of precursor cells, either by secretingassociative antibody or by being the only cell which can deliver signal @.Consider a self component S present before the immune system develops.The B cells and t cells specific for S will be paralysed, one at a time, asthey arise. When an animal has accumulated B and t cells specific for aforeign antigen F, F is given. As no T cells specific for F are present, neitherthe B nor the t cells specific for F can be induced. There are at least threeways of resolving this paradox:

1) Associative antibody molecules, or T cells, are passed from the motherto the foetus. The danger involved in this suggestion is that the mother mayhave associative antibody and T cells specific for paternal and foetal antigens

2) Thymus cells secrete a bit of associative antibody, and this associativeantibody can prime the system. This suggestion is most plausible if thesecretion by a T cell of associative antibody is normally required for theinduction of precursor cells.

3) t cells can directly interact with B and t cells, in the presence of antigen,to induce the B and t cells, but probably much less efficiently than T cells.This suggestion is most plausible if a direct B-T and t-T interaction is nor-mally required for the induction of a B and t cell respectively.

As I favour the alternative that associative antibody must be secreted bya T cell in order tO' be involved in inducing precursor cells, I favour thesecond possibility that thymus cells secrete a bit of associative antibody.

•FEEDBACK" REGULATION OF THE RESPONSE

It has been shown that the administration of specific humoral antibody to ananimal inhibits its humoral antibody response to the antigen and that theremoval of specific antibody results in a remarkable increase in the animal'sresponse to the antigen (Bystryn et al. 1971). The suggestion has beenmade that antibody specific for the antigen either clears the antigen fromthe environment of precursor cells or that it smothers the antigen withantibody such that the antigen can no longer bind to the receptors of theprecursor cells. The fact that it is difficult to raise specific antibody againstsimple monomeric antigens to levels much greater than ! mg/ml is consistentwith this suggestion. 1 mg/ml of antibody corresponds to a molarity of about

234 PETER BRETSCHER

10 "M, which is a bit higher than a typical binding constant of a monomericantigen for its antibody; hence, if the antigen is present at a concentrationof less than IO"^M, one would expect the determinants of the antigen to becovered by antibody.

This 'feed-back inhibition' by humoral antibody on the induction of pre-cursor cells would be expected to control the induction of both B and t cells.The 'feed-back inhibition' is particularly important for t cells, for the induc-tion of t cells results in the production of T cells which favour, in thepresence of antigen, the induction of further t cells. The effect of 'feed-backinhibition' can be fitted into the picture we have of B and t cell inductionas shown in Figure 8.

QUANTITATIVE ASPECTS OE THE THEORY

A Minimal Approach

Although it is difficult to give the theory a detailed quantitative formulation,certain semi-quantitative considerations can be useful. The most obviousassumption to make in treating the interaction between receptors, antigenand associative antibody, is to assume that they exist under equilibrium con-ditions. However, although such considerations give us valuable insightswhen comparing various situations, they may lead us astray in certain cases.For example, for polyvalent receptors interacting with polymeric antigens,the half-life of the receptor-antigen complex may be of the order of hoursor even days. This means that it will take considerably longer than severalhours for an equilibrium state to be reached.

(•) erPROVIDING

ASSOCIATIVEANTiaOOT

Figure 8.


Precursor cells will often receive both inductive and paralytic signals. Forexample, BSA induces a response at an extravascular concentration ofantigen of about 10 'M (Mitchison 1969a); this molarity must be fairly closeto the binding constant of the receptor for the antigen, and if all the receptorsare not receiving an inductive signal, as seems likely, some must be receivinga paralytic signal. An experiment has been described above in which it wasshown that there was competition between the induction and paralysis ofprecursor cells. It therefore seems reasonable to assume that the decision ofwhether a cell is induced or paralysed depends in part on the ratio of thenumber of inductive and paralytic signals it receives averaged over a shortlength of time (i.e. about one day). However, a precursor cell, in order tobe induced, probably requires a minimum number of inductive signals, sayone, averaged over this time. Hence two precursor cells which receive thesame ratio of inductive to paralytic signals may respond to the antigendifferently. For example, a precursor cell receiving 2 inductive signals and10 paralytic signals may be induced, whereas a precursor cell which receivesonly 0.02 inductive signals and O.I paralytic signals averaged over this timemay be neither induced nor paralysed. 1 shall assume in the calculationsbelow that the induction of a precursor cell is dependent on the ratio of thenumber of inductive to paralytic signals and on the absolute number ofinductive signals it receives.

For simplicity, I shall initially assume that the antigen is monomeric inthe sense that it does not possess identical sites on its surface, that thereceptors and associative antibody are univalent, and that the receptors onthe cell surface bind to antigen independently. The situation I shall consideris represented in Figure 9.

RECEPTORS

ASSOCIATIVEANTIBODY

ANTIGEN

Figure 9.

236 PETER BRETSCHER

Let the binding constant of the receptor for antigen be (K^),the binding constant of the associative antibody for antigen, be (K^),the concentration of unbound receptors be (r),the concentration of unbound associative antibody be (a),the concentration of free antigen be (p),the total concentration of receptors be (R),and the total concentration of associative antibody be (A).

r - i - p ^ r p , and K^ ^ (r)• (p) ,.^K, (rp) ^'^

p + a : ^ pa, and Ka ^ (p)'(^) (2)K. (pa)

a ^ r p a , and Ky =K. (rpa) ^ -'

(A) - (a) + (pa) + (rpa), (4)

(R) -= (r) + (rp) + (rpa), (5)

In most cases (rpa) < (pa) + (a), and (rpa) < (r) + (rp). These conditionswill be satisfied if only a small amount of the total associative antibody isinvolved in forming the complex (rpa), and if only a few of the total numberof receptors are receiving an inductive signal. This approximation will bediscussed later. We can then rewrite (4) as:

(A) — (a) + (pa), and substituting for (pa) from (2), we have

Similarly ( D ^ T

From (3), (rpa) = —- -—, and substituting for (rp) using (1),

(rpa) = z r- ^ . Substituting for (a) and (r) using (6) and (7), we have

^ ^^^^- .(V) ^^^-^^ (R)(P)-(A)[K, + (p)l ^^' [K, + (p)] - [K, + (p)] + [K,4 (p)]

(8)


This equation relates the concentration of inductive complexes, (rpa), tothe concentration of receptors, the concentration of antigen, and the bindingconstants of antigen for the receptor and associative antibody. If a cell hasNjt receptors, then there will be nr,,;, complexes formed where, from equa-tion (8),

n,,,.-, (rpa) (p) • (A)NR " (R) - [K,, + (p)] • [K, + (p)]

The number of paralytic signals is given by n ,,, where

-^Kp— — .jy. — I. ,„-. , and substituting for (r) using (7),

^P^ (10)Nn - [K,, + (p)]

If there are 1000 receptors on the precursor cell, and if it is sufficient forone receptor to receive an inductive signal for this cell to be induced, wecan. in principle set

nrpa 1 (P) • (A)

Nn - 1000 - [ K .

to find the concentration of antigen and associative antibody required toinduce the cell. However, such a calculation overlooks the inadequacies onwhich the calculation is based, as I discuss later (see The Inadequacies ofthe Minimal Approach).

Certain observations, however, which do not have to be modified toodrastically when a more realistic picture of induction is considered, can bemade from considering equations (9) and (10).

1) For concentrations of antigen above K and K , K + (p) ^ (p) andK.T + (p) ^ (p), and so equation (9) becomes

n ~ N ( E > ^ NK • (A)n N(P) • (P) (P)

and equation (11) becomes n^, = NR

Thus as the concentration of antigen, (p), rises above K,, and K , the

number of inducing complexes goes down as ^ ^ . However the number(P)

of paralytic signals, which is equal to n,^, increases slowly as (p) increases,and so paralysis is favoured as the concentration of antigen is raised. Thisconsideration provides an explanation of high-zone paralysis.

238 PETER BRETSCHER

2) For concentrations of antigen well below K, and K^Kr, and Ka + (p) = K , and so equation (9) becomes

u • (p) • (A), and equation (10) becomes

+ (p) ^

It can be seen that the number of inductive signals and the number of para-yltic signals both increase linearly with the antigen concentration. Hence theratio of the number of paralytic signals to inductive signals does not change,for (p) < K,,, (p) < Ka, as the antigen concentration is raised. These con-siderations show that this minimal approach does not provide an obviousexplanation for low-zone paralysis.

3) As discussed in 2) above, for antigen concentrations below K^ and K. ,

N • (p) • (A)" •"' K,K,

It can be seen frmn this equation that, if the concentration of associativeantibody, (A), is increased, the concentration of antigen required to givethe cell a given number of inductive signals decreases. For example, if theamount of associative antibody increases ten-fold, the amount of antigenneeded to give the same number of inductive signals to the precursor cellwill decrease ten-fold. Furthermore, the number of paralytic signals theprecursor cell receives will be less at the lower antigen concentration. Forthese two reasons an increase in (A) will decrease- the concentration ofantigen required to induce a response.

lP|=ANTIGeN CONCENTRATION

Figure 10.


4) As shown in 2) above, the number of inductive signals a cell receiveswhen (p) < Kp, and (p) < Ka, is proportional to (p). As shown in 1) above,for (p) > Kr, and (p) > Kj,, the number of inductive signals decreases with

increasing concentration of antigen a s — — . A plot of n,,,, versus (p) will

therefore have the rough shape shown in Figure 10.

From equation (9), n, ^ = ^ - - ? , J£^ ! . ^ ^[Kp + (p)] • [K, + (p)]

The concentration of antigen which gives a maximum number of inductive

signals, i.e. a maximum value of nr,,. , will be given by the conditiondp

(n^pa) = 0, and this condition gives a value of (p) =The actual value of the maximum number of inductive signals is given

by substituting (p) = V(KaKr) into equation (9).

Hence(K,,

N R • (A) • V(K,K,)

HenceHence

Equation (11) shows that the maximum number of inductive signalsdepends on the values of K^ and K,. If the binding between the receptorantibody and associative antibody is very tight, K^ and K, will be very low,and the maximum number of inductive signals, in the presence of a givenamount of associative antibody, (A), will be considerably higher than if K;,and Kr were larger. This is just a quantitative formulation of the intuitivelyreasonable statement that the tighter the binding of the receptor and associa-tive antibody is for antigen, the easier it is to form the (rpa) complexrequired for induction. This shows that the tighter the binding of the receptorand associative antibody is for the antigen, the smaller is the amount ofassociative antibody required to give the precursor cell a given number ofinductive signals.

The Inadequacies of the Minimal Approach

There are four simplifications in the quantitative approach used above which

240 PETER BRETSCHER

limit its validity. I will try to discuss how serious these inadequacies are andto see whether they can be corrected for.

a) If the associative antibody is bound to a cell, there will be interactionsbetween the precursor cell and the cell to which the associative antibody isattached. This means that it is not possible to calculate such parametersas the concentration of associative antibody required to give one inductivesignal to a cell with 1000 receptors in the presence of a given concentrationof antigen. However, as the arguments used above were based on comparingthe likelihood of induction in two different situations, the errors will becommon to both and the conclusion that one situation favours induction overanother situation should still hold.

b) Suppose an antigen A has 20 foreign sites, aj-aoo. In order to induceanti-ai antibody another site on A must be bound by associative antibody.Suppose the antigen A is roughly spherical and that the determinant a . isclose to the determinant ai on the surface of A. It may not be possible forassociative antibody to hind to a2 when the receptor binds to ai, or suchbinding may not allow signal (2) to be dehvered to the precursor cell. Forexample, if associative antibody must be on the surface of a cell for whichit is cytophilic in order for the precursor cell to receive signal ©, there willbe geometrical constraints on the relative positiom of the determinant boundto the receptor and the determinant bound by associative antibody on thesurface of the antigen, see Figure 11. This means that the effective concen-tration of specific associative antibody will be lower by a small factor thanits actual concentration. This is not a serious inadequacy of the minimalapproach.

c) The minimal approach used above assumed that the receptors on theprecursor cell and the associative antibody were monovalent. It will beargued later (see below under Stable 19s Humoral Antibody Synthesis) thatpolymeric antigens, such as polysaccharides, must bind to the receptors onthe B cell at more than one site, and that the receptors on a precursor cellare effectively polyvalent. This in turn suggests that the associative antibody,whatever its location during the inducing event, is effectively polyvalent.For if the effective affinity of the receptor for a polymeric antigen, due tomultivalent binding, were orders of magnitude above that of associative anti-body, paralysis would be greatly favored over induction for a polymericantigen compared to a monomeric antigen. These considerations suggestthat a truer picture of induction might be as shown in Figure 11, as opposedto the picture shown in Figure 9.

This modification in our picture of induction has four main consequences,which can be interpreted in a semi-quantitative fashion, but which it isnot easy to express in a rigorous quantitative form.


1) More than one molecule of an antigen, unless the antigen is verylarge, is likely to be involved in induction (see Figure 11). For concentrationsof antigen below the effective binding constants of the receptor and associa-tive antibody for antigen, the number of inductive signals will depend on ahigher power of the concentration of antigen than unity. The number ofparalytic signals will be linearly proportional to the concentration of antigen,and so the ratio of inductive signals to paralytic signals will increase, andhence induction will be favoured over paralysis, as the antigen concentrationis increased. This argument provides an explanation of low-zone paralysis(Bretscher & Cohn 1968).

2) The absolute concentration of a monomeric antigen required to forman inductive complex will be lower in this case compared to the minimalcase considered earlier, as there is cooperative binding of antigen between

PRECURSORCELL

CELL TO WHICHASSOCIATIVE

ANTIBODYMAY BEATTACHED

Figure II.

Transplant. Rev. (1972) 11

242 PETER BRETSCHER

the precursor cell and associative antibody. An inductive signal will also bemore likely to occur.

3) The effective binding constants to the receptor and associative antibodyof a polymeric antigen, which can bind to the receptor and associative anti-body at more than one site, will be much greater than those of a monomericantigen which can only bind at one site to receptor and associative antibody.It is known that the divalent binding of antibody to antigen can increaseits effective binding constant for the antigen many orders of 10 comparedto its binding constant when it binds to the antigen monovalently (Karush1970). This means that the amount of associative antibody required to induceprecursor cells by a polymeric antigen will be much less (by many ordersof 10) than that required to induce a precursor cell by a monomeric antigen.It also means that the concentration of a polymeric antigen required to givethe maximum stimulation to precursor cells will be much lower than thatrequired for a monomeric antigen.

If the effective valency for a polymeric antigen of the receptors on a 19sB cell is greater than the effective valency on a 7s B precursor cell, as I shallargue later (see Stable 19s Humoral Antibody Synthesis), then the amountof associative antibody required to induce the 19s B cell will be less thanthat required to induce the 7s B cell. This follows from the argument thatthe tighter tbe binding of the receptor for the antigen is, the smaller is theamount of associative antibody required to form an inductive complex. Insummary, the response for which one expects the least amount of associativeantibody to be required is a 19s response to a polymeric antigen.

4) The theory demands the formation of a complex between the receptor,antigen and associative antibody in order for the precursor cell to receivean inductive signal. It is not clear, however, whether it is obligatory for theinductive complex to contain more than one antigen molecule if the antigenis monomeric. It is unlikely that a paralytic signal requires a multivalentreceptor to bind as many antigen molecules as are usually involved in theformation of an inductive signal. This would mean that, for (p) < K^ and(p) < Ky, the number of inductive signals would not increase faster withincreasing antigen concentration than the number of paralytic signals, andthe simple explanation of low-zone paralysis given above would not hold.

d) In the simplified approach used above, the number of complexes, nrpa,was assumed to be smaller than the total number of receptors and the totalnumber of associative antibody molecules. It was assumed that (rpa) <(pa) + (a), and that (rpa) < (r) + (rp). Although this assumption is probablyusually valid, there are certain conditions under which this is not so. Forexample, if (A) ^ Ka ^ Kr = c, then at a concentration of free antigenof (p) = c, it can be shown by employing equations (l)-(5) that these


conditions are not satisfied. However if (A) < K,., or " < I, then the(A)

condition that (rpa) < (pa) + (a) is satisfied. To see that this is so, we canuse equation (2).

(pa) = ^ ^ ~ ^ , and as (a) < (A), and - ^ < ' ' ~ k ^ ^ ^'

Hence (pa) < (p).

As the receptors bind to (p) and (pa) with the same binding constant, thecondition that (pa) < (p) means that (rpa) < (rp). More formally,

, , (pa) • (r) . , , (p) • (r)(rpa) = —^-— , and (rp) - —

and SO ^ ^ ^ U ^ <1(rp) K, (p) - (r) (p)

This shows that if (A) < K^, (rpa) < (rp). It can be shown in a similarmanner that if (R) < K , (rpa) < (pa). In order to illustrate what happenswhen this approximation breaks down one can consider an extreme situationwhich may occur for some polymeric antigens. For a polymeric antigenwhich can bind to associative antibody and the receptors on a precursorcell at 5 sites, K;, and K may be as low as 10 --'M. 10 --'M is a concentrationof antigen of about 6 molecules per litre. If there are several molecules of

(A)associative antibody specific for this antigen in a mouse, -^^—> 1. If the

binding constants K,, and K^ are of this low order, one molecule of an anti-gen, given to a mouse, will bind to a precursor cell and associative antibody,and it will thereby induce the precursor cell if one inductive signal is suf-ficient to induce the precursor cell. An equilibrium calculation will not beapplicable in this case, and the sites for the antigen on the precursor cellsand associative antibody will in effect be titrated as the number of antigenmolecules is increased. As the number of antigen molecules given rises abovethe total number of receptor sites and associative antibody sites, the paralysisof precursor cells will be favoured. In this extreme case, induction of pre-cursor cells can occur at very low levels of associative antibody. High-zoneparalysis will occur at very low amounts of antigen.

OTHER VIEWS OF T CELL FUNCTION

It has been widely argued that the function of T cells is to concentrateantigen, or present it in a matrix, to the B cell. The T ceils are usually re-garded as being impartial to whether their presentation of antigen to the B

16"

244 PETER BRETSCHER

cell results in the paralysis or induction of B cells. This view regards T cells(or an antigen-specific product of T cells) as not being obligatory for in-duction of B cells, but as providing a helper function (Edelman 1970, Feld-mann & Basten 1971, Miller 1970, Mitchison 1969, in press, Nossal 1969).

There is no evidence, however, for the associative recognition of antigenin paralysis, as we have discussed earlier (Bretscher & Cohn 1968, 1970),and the only good evidence for associative recognition of antigen comesfrom studying the induction of precursor cells. I shall briefly examine theevidence on which the hypothesis that the function of T cells is to concen-trate or present the antigen to B cells is based.

1) The sensitization of an animal to an antigen P enables a much lowerand subsequent dose of the antigen hP to induce anti-h antibodies than ifthe animal had not been sensitized with P (Mitchison 1967, Rajewsky &Rottlander 1967). The interpretation sometimes given of this result is thatt cells can concentrate or present a matrix of the antigen, at lower dosesof antigen, to the anti-h B cells. In terms of the theory I favour, the in-creased concentration of associative antibody, and its ability to be increasedby the generation of memory t cells, means that the antigen dose needed toform an inducing complex will be lower (see above under QuantitativeAspects of the Theory).

2) Some antigens can still induce a normal response in animals depletedof both T and t cells. The best studied of these antigens are polymeric. Thosefavouring the hypothesis that the role of T cells is to present the antigen tothe B cells argue that the antigen, being a polymer, has the correct form ofrepeated identical detenninants to induce the B cell without the requirementof a T cell or an antigen specific product derived from a T cell (Andersson& Blomgren 1971, Feldmann & Basten 1971). There is evidence in certaincases, however, that specific T cells or associative antibody molecules derivedfrom T cells are still present in these animals. On the theory I favourit is expected that the 19s response to polymeric antigens would bethe last to be effected by depleting an animal of t and T cells (see aboveunder Quantitative Aspects of the Theory, and later under 'Thymus-Inde-pendent Antigens').

3) Neonatal thymectomy impairs the humoral response to certain antigensby removing the source of new t cells (Miller 1961). In such an animal theresponse to a standard dose of an antigen, such as sheep red blood cells(SRBC), is feeble compared to a control, sham-thymectomised mouse. Higherdoses of antigen can result in an almost normal response (Sinclair & Elliot1968, Taylor & Wortis 1968). This result has been interpreted as showingthat T cells concentrate the antigen (Taylor 1969). This result is under-standable on the theory I favour in the same manner as point 1) above.


'THYMUS-INDEPENDENT ANTIGENS'

According to the theory described above the specific induction of B cells byantigen requires the presence of specific T cells, or associative antibodyderived from a T cell. As shown above, considerably less specific associativeantibody, by several orders of 10, is expected to be required to induce a!9s humoral response to polymeric antigens than to monomeric antigens.I will examine here the evidence for the hypothesis that some antigens are'thymus-independent.' in the sense that such antigens, by virtue of possessingrepeating identical determinants, can induce their corresponding specific Bcells by binding directly to the receptors on the B cell. On this view, theinduction of B cells by these antigens occurs in the absence of associativerecognition.

All the experiments to be described are based on the following kind ofargument. If T and t cells are not required for the induction of B cells by a'thymus-independent' antigen, then either decreasing or increasing the num-ber of specific T and t cells in the animal should not affect the inductionof specific B cells by the antigen. For example, neonatal thymectomy affectsthe response of mice to certain antigens, such as SRBC (sheep red bloodcells) and BSA (bovine serum albumin), by removing the source of newlygenerated t cells, but does not affect the response to keyhole limpet hemo-cyanin (Humphrey et al. 1964). There are four antigens for which theevidence that they are 'thymus-independent' has been claimed to be particu-larly strong. They are polyvinylpyrrolidone (PVP), E-Coli lipopolysaccharide(LPS) (Andersson & Blomgren 1971), type III pneumococcal polysaccharide(SIII) (Howard et al. 1971b), and a polymer of the flagellin derived fromSalmonella Adelaide (POL) (Feldmann & Basten 1971). Each of theseantigens expresses many identical antigenic units.

Polyvinylpyrrolidone or PVP

Adult mice were (partially) depleted of both t and T cells by thymectomy,irradiation two to three weeks later, and were reconstituted with 10' syn-geneic bone marrow cells. Their response to various antigens was examinedtwo or three weeks later. I shall refer to these mice as T cell-depleted mice.

Normal mice, T cell-depleted mice, and T cell-depleted mice given 4.5 x10" syngeneic thymus cells, were challenged with PVP. There were nosignificant differences in the response to PVP in these three groups of miceeight days after immunization. The immunization of T cell-depleted micewith SRBC produced no detectable anti-SRBC antibody, but T cell-depletedmice given 4.5 x 10' thymus eells produced a good anti-SRBC response.

246 PETER BRETSCHER

These observations have been interpreted as showing that PVP is a 'thymus-independent' antigen, and that SRBC is a 'thymus-dependent' antigen.

T cell-depleted mice were injected with both SRBC and PVP, and noantibody titre against SRBC could be detected. When the conjugate SRBC-PVP was injected, however, the anti-SRBC titre was almost as high asthat of a T cell-depleted animal reconstituted with 4.5 x 10' thymus cellsand challenged with SRBC. This result shows that in these T cell-depletedmice, an anti-SRBC B cell can be induced by SRBC-PVP, but not by SRBC.These results are quite understandable on the theory described above ifquantitative considerations are taken into account. As PVP is a polymericantigen, its effective binding to both the receptor on the precursor cell andassociative antibody will be relatively tight and it will therefore appear tobe relatively 'thymus-independent'. SRBC-PVP can bind more tightly toassociative antibody via the polymeric PVP molecule than can SRBC, andtherefore SRBC-PVP will require less specific associative antibody to inducean anti-SRBC response than will SRBC.

We can analyze more formally what the result of coupling PVP to SRBCshould be on the induction of an anti-SRBC precursor cell as follows. Themaximum number of inductive signals is given by equation (11).

. , NK (A)

Suppose that the anti-SRBC binding constants of the receptor and associa-tive antibody are of the order 10"^M, and the effective anti-PVP bindingconstants are of the order of 10" -'M. The amount of associative antibodyspecific for SRBC and PVP is assumed to be identical. For the inductionof an anti-SRBC precursor by SRBC,

and for the induction of an anti-SRBC precursor by SRBC-PVP,

(nrpoVax, - jQ-s ^ ]0''^^"+ 2.10"^^ ^ 10"8

Thus the maximum number of inductive signals that an anti-SRBC pre-cursor will receive will be about four fold greater when SRBC-PVP isinjected into an animal than when SRBC are injected. This is not a largedifference. However, the concentration of antigen at which the number ofinductive signals is a maximum is given by p = \/(KaK,,), and for SRBCthis will be about lO^M and for SRBC-PVP it will be about 10 ^^M. Thenumber of paralytic signals the anti-SRBC precursor receives will be very


different at an effective concentration of the antigen of 10 ' M and lThese considerations suggest that coupling PVP to SRBC enables an anti-SRBC precursor cell to be induced by allowing the cell to receive themaximum number of inductive signals without receiving many paralyticsignals, rather than by increasing by a large factor the total number of in-ductive signals it can receive.

TTie alternative view that antigens with repeating identical determinantsare 'thymus-independent' does not explain these results. On this alternativeview. SRBC cannot express the required repeated identical determinants toinduce a B cell directly as it is a 'thymus-dependent' antigen. Hence theanti-SRBC response raised in T cell-depleted mice by injecting SRBC-PVPis paradoxical, as it implies that there are T cells or associative antibodyspecific for PVP in these T cell-depleted animals.

The hypothesis that there are either some associative antibody moleculesor T cells specific for PVP in these T cell-depleted mice is further supportedby another experiment. A T cell-depleted animal, challenged with SRBC-PVP and given spleen cells immune to PVP, produces a larger anti-SRBCresponse than an animal given the same number of spleen cells immune toE. coli lipopolysaccharide. This shows that immunity to the carrier enhancesthe anti-SRBC response, and is consistent with associative recognition beingrequired for the induction of anti-SRBC antibody on challenge with SRBC-PVP in these T cell-depleted mice.

E-coli lipolysaccharide or LPS

This antigen was studied in an identical manner to PVP with similar results.However LPS may stick to the surface of lymphoid cells and thus affect theresponse in a manner described later, (see Reconstitution of the Responseof T Cell-Depleted Spleen Cells by Various Substances).

Pneumococcal Polysaccharide type lU, or SIII

i) Adult mice were (partially) deprived of both t and T cells by thymect-omy, irradiation with 85OR, and then reconstituted with 5 x 10^ syngeneicbone marrow cells. Two months later their response to SIII was assayed,and compared with control mice which had only been irradiated and recon-stituted with bone marrow cells. The response of test mice and control micewere comparable, and only slightly lower than that of normal mice. Thetheory explains these observations on the ground that a 19s response to apolymeric antigen is expected to generally require very little specific asso-ciative antibody derived from T cells.

248 PETER BRETSCHER

ii) Rabbits sensitized with SIII and BGG (bovine ;• globulin) show ahigher response to SIII on challenge with the conjugate SlII-BGG thanrabbits sensitized with SIU (Paul et al. 1971). This observation will bediscussed at greater length later, see under Stable 19s Humoral AntibodySynthesis. The observation shows that sensitizing against the carrier to SIIIcan increase the response to SIII, and is consistent with associative recogni-tion being r«|uired for the induction of anti-SIII B cells by SIII, but isdifficult to reconcile with the hypothesis that SIII has the correct arrange-ment of repeated, identical determinants such that it induces anti-SIII B cellsdirectly.

Polymeric form of Flagellin from Salmonella Adelaide, or POL

This antigen is a polymer of about 300 units of flagellin. Flagellin itself isa reasonably strong immunogen, as neonatally thymectomized mice respondto flagellin as well as normal mice (Martin & Miller 1969). Other criteria,however, show that the monomer is a 'thymus-dependent' antigen (Feldmann& Basten 1971). The fact that flagellin is a strong immunogen suggests thatit has many foreign sites. It will be particularly difficult to show that the19s humoral response to POL is 'thymus-dependent' as POL is a polymerof flagellin which itself is a strong immunogen.

Spleen cells from adult thymectomized, lethally irradiated, and bone mar-row reconstituted mice show the same response to POL as control mice,which have been sham-thymectomized, lethally irradiated and bone marrowreconstituted. Such T cell-depleted mice were drained by the thoracic duct,and their spleens were treated with anti-f^ antiserum and complement, in aneffort to further remove T and t cells. (The antigen S occurs on t and Tcells but not on B cells.) These spleen cells supported a normal 19s responseagainst POL compared to spleen cells from control mice.

These observations on the immunogenicity of POL in T cell-depletedspleen cultures are similar to those described above which showed thatPVP, LPS, and SIII could induce a good response in T cell-depleted animals.For these antigens, however, the response to a conjugate of the 'thymus-independent' antigen and a 'thymus-dependent' antigen was studied, andevidence against the view that these antigens can induce B cells directly wasobtained. Comparable experiments have not, to my knowledge, been per-formed with POL. I shall later suggest an experiment which, according tothe theory, will show that the response to POL is 'thymus-dependent', (seeunder 7n Vitro Paralysis').

These observations on the ability of spleen eells from T cell-depleted miceto be induced by POL have one unique feature over other experiments on

CONTROL OE ANTIBODY SYNTHESIS 249

'thymus-independent' antigens. The number of spleen cells incubated withPOL, and which result in a humoral response against POL, is of the orderof 10" cells. If a response against POL required the presence of a unispecificT cell, the frequency of this cell would have to be at least one per 10^ spleencells in T cell-depleted mice. As the number of t and T cells in T cell-depleted mice is probably at least 10^ fold lower than in normal mice, thefrequency of this unispecific T cell would have to be of the order of oneper 10* spleen cells in a normal mouse. This calculation is a conservativeone, and the high figure of 10"* for the frequency of an anti-POL T cellstrongly suggests that the induction of B cells by POL does not require aspecific T cell. According to the theory this requires that specific associativeantibody, secreted by a T cell, can be involved in the induction of precursorcells. A rough calculation follows which shows that it is not unreasonablethat there should be enough specific associative antibody in a mouse whichhas been completely depleted of t and T cells for a response to take placeagainst POL.

If it is necessary for associative antibody to be secreted by a T cell inorder for precursor cells to be induced, there must always be some secretedspecific associative antibody present in order to obtain a response to anyantigen. The amount of free associative antibody specific for flagellin maybe insufficient to induce anti-flagellin B cells when flagellin is initially givento a mouse. The flagellin may have to induce specific t cells in the presenceof secreted associative antibody before anti-flagellin B cells can be efficientlyinduced. Let the concentration of secreted associative antibody specific forflagellin, before flagellin is administered to the mouse, be («•). The flagellinwill induce t cells such that the concentration of secreted associative anti-body is, say, 1000 (a), and at this concentration of associative antibodyspecific B cells can be induced by flagellin. The quantitative formulation ofthe theory shows that the amount of associative antibody required to inducean anti-flagellin B cell by POL is much less than that required to inducethe same cell by flagellin. If POL binds trivalently to both the precursor celland to associative antibody, then the effective binding constants of thesemolecules for POL will be roughly 10'" times lower than the binding con-stants of these molecules for the monomer. The amount of associative anti-body required to induce an anti-flagellin B cell by POL will be corre-spondingly lower. There are many more sites on POL to which associativeantibody can bind to than there are on the monomer. As POL is made upof about 300 flagellin units, the amount of associative antibody required byPOL to induce a B cell will be less by a factor of about 10- on this accountcompared to the amount of associative antibody required by the monomerto induce a B cell. Thus if a concentration of 1000 (o) is required to induce

250 PETER BRETSCHER

an anti-flagellin B cell by flagellin, a concentration of about -—-^—r-^-

^ 10" (a) will be required to induce the same B cell by POL. If the halflife of associative antibody is, say, five days, it will take about 150 days forthe original concentration of anti-flagellin associative antibody of (a) to decayto 10"''(a). Although the figures used in this calculation are somewhatarbitrary, they do show that if t and T cells are completely removed from ananimal it is not surprising, in terms of the theory, that the subsequentresponse to an antigen such as POL is not affected for a very long time.These considerations strongly favour the view that T cells secrete specificassociative antibody which can then be involved in inducing precursor cells.

High-Zone Paralysis by 'Thymus-independent Antigens'

Most of the antigens discussed here paralyse animals when given at highdoses (Andersson 1969, Howard et al. 1971a, Sjoberg 1972). This is under-standable on the theory that associative recognition is required for the in-duction but not for the paralysis of antibody synthesis; this expectationdepends on induction requiring associative recognition. The phenomenon ofhigh-zone paralysis is not readily accounted for on the hypothesis that theseantigens induce their corresponding B cells by binding to them directly inthe absence of associative recognition.

STABLE 19S HUMORAL ANTIBODY SYNTHESIS

The humoral response to certain antigens appears to be predominantly a19s response. Such antigens as SIII (Howard et al. 1971a), c-carbohydrate(Sher & Cohn, 1972), and dextran (Carson et al., pers. comm.) induce pro-longed 19s specific antibody synthesis, and the usual switch to 7s antibodysynthesis does not occur.

For the sake of simplicity, I shall assume that there are separate B pre-cursor eells for 19s and 7s antibody producing cells, though tho argumentcan be modified if there is a common precursor cell. 19s and 7s moleculeshave a common light chain, and in the rabbit the heavy chain variable regiongenetic marker is present on the heavy chain of both 19s and 7s molecules.It therefore does not seem plausible to attempt to account for the inductionof continued 19s antibody synthesis by these polymeric antigens on theassumption that 19s and 7s B cells have receptors with different specificities.

A minimal assumption to explain this phenomenon appears to be that thepolymeric nature of these antigens is essential to the continued 19s humoralantibody synthesis. The easiest way of recognizing the polymeric nature of


an antigen is by binding to it, during the inducing event, polyvalently. Ipropose that these polymeric antigens can bind to a 19s B cell at more sitesthan they can on a 7s B cell. The effective binding constant by the receptorson the 19s B cell for the antigen will be tighter than that of the receptors onthe 7s B cell.

It can be seen from equation (11)

that the maximum number of inductive signals is dominated by the higherof the two binding constants K, and K,. If the effective binding for a poly-meric antigen by associative antibody and the receptors on a 19s B cell istighter than the binding of the receptors on a 7s B cell considerably lessassociative antibody will be needed to give the 19s B cell a given numberof inductive signals that will be needed to give a 7s B cell the same numberof inductive signals. In this ease less associative antibody would be requiredto induce I9s B cells than 7s B cells at an optimal antigen concentration.

It is known that SIII is not rapidly metabolized by mice, and that it con-tinuously aids the removal of specific antibody. This continuous removalof antibody is known as the treadmilling phenomenon (Howard et al. 1970).When SIII is injected into an animal, both t and B cells will be induced.19s antibody will be preferentially induced over 7s antibody if the concen-tration of associative antibody is low. Consider the situation in which no 7sB cells are present. The induced 19s antibody will feedback on the inductionof both 19s B and t cells, by removal of SIII and thus lowering its concen-tration. After a time a semi-steady state is reached when the number of19s antibody forming cells, and presumably the antigen concentration andnumber of T cells, do not change much with time. This steady state levelof T cells must be higher than that required to induce 19s cells at the steadystate concentration of antigen, and I suggest that it is lower than thatrequired for inducing 7s precursor cells at this antigen concentration. Hencethe 7s B cells present are not induced.

These considerations are also relevant to the observation that a responseto antigens, other than those described above, is usually characterized bya switch from 19s humoral antibody production to 7s humoral antibodyproduction. When the antigen is initially injected the concentration of specificassociative antibody will be low. If some of the antigen can bind to thereceptors on a 19s B cell with a higher valency than it can to the receptorson a 7s B ceil, the induction of 19s B cells will be favoured over the induc-tion of 7s B cells. As associative antibody is induced, and consequently itsconcentration raised, the induction of precursor cells will not be so limited

252 PETER BRETSCHER

by the concentration of associative antibody. These considerations providea partial explanation for the switch from 19s to 7s humoral antibody pro-duction.

This explanation for steady 19s humoral antibody synthesis predicts that7s B cells will be induced if the number of specific T cells is artificiallyraised. As described above, rabbits sensitized with SIII and BGG show ahigher response on challenge with the conjugate SIII-BGG than do rabbitssensitized with SIII (Paul et al. 1971). It would be particularly interesting todetermine whether this increased titre to SIII is due in part to 7s antibody.

ABNORMAL INDUCTION

Recent evidence shows that an abnormal form of induction can be experi-mentally produced (Katz et al. 1971a,b, Kreuth & Williams 1971, McCullagh1970). In one of these experiments (McCullagh 1970), a rat was renderedunresponsive to SRBC. This implies that there were insufficient B and/orT and t cells for a response to occur. When such a rat was given, besidesSRBC, spleen cells from a rat of an histoincompatible strain, which had alsobeen rendered unresponsive to SRBC, a large number of anti-SRBC antibodyforming cells were induced 48 hours after cell transfer. TTiese antibodyforming cells were shown to be of host origin, demonstrating that the hosthad anti-SRBC B cells. Furthermore, an anti-SRBC response was not ob-served if the spleen cells came from a rat made tolerant of recipient skingrafts. This evidence shows that it is by virtue of their ability to recognizehost antigens that the donor cells enable host B cells to be induced. As theunresponsive recipient had a sufficient number of anti-SRBC B cells togenerate a large response when given histoincompatible spleen cells, hisunresponsive state must have been due to an insufficiency of specific t andT cells. The proposed interpretation of these results is shown in Figure 12,in which the normal and abnormal induction of a B cell are depicted so thatthey can be compared.

ASSOCIATIVEAHTIBODISPECIFIC

FOBAMII SEN

tMTIBODTSPECIFIC

ron a CELL

Figure 12.


The binding of antigen to the receptor on the B cell leads to the cellreceiving signal ®, and the associative antibody, on or derived from a donorT cell, binding to a foreign histoincompatible antigen on the B cell, leadsto the cell receiving signal (D. The B cell has no way of distinguishingthis case, in which signal @ is delivered to it as a result of associative anti-body binding to the B cell itself, from the usual one in which associativeantibody binds to the ant:igen.

Further experiments support the crucial feature of this interpretation thatthe donor cells provide an antigen-specific molecule which has specificityfor host cells (Katz et al. 1971b). Guinea pigs, primed with a hapten proteinconjugate hR, were injected with allogeneic spleen cells, which could reactagainst the host, and with the hapten conjugated onto another protein Swhich did not cross-react with R. A sharp increase in the anti-h titre wasobserved, and to obtain the highest anti-h titre both the conjugate hS andthe allogeneic spleen cells were required. These results can be interpretedin terms of abnormal induction. The anti-h memory cells of the host receivesignal © as a result of hS binding to their receptors. Associative antibody,derived from the donor cells, binds to the surface of the anti-h memory cells,thereby delivering signal ©. If the spleen cells come from a donor whichhas ail the antigens of the recipient as well as having some antigens whichare foreign to the recipient, an increased anti-h response is not observed.This result is consistent with the requirement that the donor cells musthave the immunolo^cal capability of reacting against the host B eells. It isnot consistent with the hypothesis that any strong immunological reaction,caused by injecting foreign spleen cells, gives rise to a long-range factor,which is not antigenic-specific and which enhances the response. Neither

ASSOCIATIVEANTIBODYSPECIFIC

FOR t CELL

Figure 13.

254 PETER BRETSCHER

are the phenomena described above, and interpreted in terms of abnormalinduction, readily explicable on the hypothesis that the function of T cells,or the associative antibody they may secrete, is to concentrate or present theantigen in a matrix to B cells.

The similarity of the proposed mechanisms for the normal induction ofB and t cells strongly suggests that t cells can also be abnormally inducedas shown in Figure 13.

The existence of abnormal induction may be relevant to an understandingof the development of generalized autoimmunity. A theory for the develop-ment of generalized autoimmunity, as displayed by NZB mice, has beenproposed and is described elsewhere (Bretseher, submitted for publication).

THE ASYMMETRY IN THE REQUIREMENT FOR

SPECIFIC B AND t CELLS FOR THE INDUCTION OF

HUMORAL ANTIBODY SYNTHESIS

If T ceils must secrete associative antibody in order for associative antibodyand antigen to induce precursor cells, B cells can in principle be inducedin the absence of specific t and T cells. For most antigens, however, therewill be insufficient specific associative antibody to induce B cells in theabsence of T cells. In an animal does not possess a sufficient number of Tcells to induce B cells efficiently, there will be a further requirement forspecific t cells, which can, be induced in the presence of T cells, to providemore specific T cells.

Although the proposed mechanisms for the induction and paralysis of Band t cells are identical, the actual number of these two cell types requiredfor the induction of a humoral response to a monomeric antigen with fewforeign sites will be very different. For example, if an animal has 100 B cellsspecific for such an antigen, and a few specific T cells and 5 specific t cells,he may not respond to the antigen at ali. The animal may be unable togenerate enough specific associative antibody before the 100 B and 5 t cellsare paralysed. On the other hand, an animal which has 5 specific B cells,a few T cells and 100 t cells, may respond to such an antigen very well, ashe has the ability to induce a large number of specific t cells giving rise toa large number of specific T cells. These considerations show that there isan asymmetry in the requirement for B and t cells in the induction ofhumoral antibody synthesis.

More specific t cells will be required for the induction of B eells by amonomeric antigen with a few foreign sites than will be required for a strongmonomeric antigen with many foreign sites in the presence of the same


number of specific T cells. A polymeric antigen can induce a few t eellsvery efficiently in the presence of a few T cells, and requires fewer T cellsto induce a B cell. A more rapid humoral response to a strong polymericantigen would be expected to occur in an animal with 100 B cells, 3 T cellsand 5 t cells, than in an animal with 5 B cells, 3 T cells and 100 tcells.

A rabbit paralysed to BSA can be induced to make anti-BSA antibodiesby injecting a cross reacting antigen, HSA (human serum albumin), at atime when the rabbit is still unresponsive to a challenge with BSA. Theseanti-BSA antibodies are specific for determinants which are shared by HSAand BSA (Weigle 1964). This result shows that there are anti-BSA B cellsin the unresponsive animal which can be induced by HSA. These anti-BSAB cells are not induced if the animal is challenged with BSA as there aresufficient anti-BSA t and T cells to induce them. However, there are suf-ficient anti-HSA t and T cells to induce those B cells which have receptorswhich can bind to determinants shared by BSA and HSA when HSA isadministered to the animal. Suppose the time for the generation of a newanti-BSA B cell is the same as that for generating a new anti-BSA t cell.The asymmetry in the requirement for the presence of anti-BSA B cells andanti-BSA t cells, even in the presence of a few specific T cells, to be ableto induce a humoral response against the weak immunogen BSA will resultin the B cell population appearing to recover faster than the t eell population.The slowness with which the t ceil population appears to recover is alsopresumably due to the requirement for the presence of T cells in order toinduce t cells, (see later. Evidence for the Associative Recognition of AntigenDuring the Induction of t cells).

The rate at which the B and t eell population are paralysed, followingthe administration to mice of a paralysing dose of HGG (human y-globulin),has been studied (Weigle et al. 1971). It has been shown that the populationof B cells are paralysed much more slowly than the population of t cells, andthat their recovery from a paralysed state is also much more rapid. Theseresults are understandable even if the time required to paralyse an indi-vidual specific B and an individual specific t cell are the same, and if therate at which new specific B and t cells are generated is also the same. Theasymmetry seen in the paralysability of the B and t cell population must atleast partly reflect the asymmetry in the requirement for B and t cells inorder to induce a humoral response against HGG. TTiese and other similarobservations should not be interpreted as showing that individual B cellsare paralysed more slowly than individual t cells, or that B cells requirehigher doses of antigen than t cells to be paralysed. These observations donot suggest that the mechanism for paralysing B and t cells are fundamentallydifferent (Mitchison 1971b, Weigle et al. 1971).

256 PETER BRETSCHER

In the HGG system referred to above the time required to induce anunresponsive B cell population was comparable to the time required for theB cell population to recover from its unresponsive state. If the frequencyat which new anti-HGG B cells are generated had been higher by a factorof 10 it is possible that the B cell population would have appeared to beunparalysed at all times after the injection of a paralysing dose of HGG.As the rate of generation of new B cells specific for an antigen depends onthe number and nature of the determinants on the antigen, it is possible thatfor some antigens it will be difficult to show B cell paralysis. For example,if the average time between the generation of successive specific B cells, ig,is less than the time required to paralyse a B cell, Tp, under conditions ofexcess antigen, there will always be some specific B cells present in theparalysed animal which can be induced to respond under favorable condi-tions (see below, under Other Views of Paralysis).

OTHER VIEWS OF PARALYSIS

I have been tacitly assuming that the paralysis of precursor cells is irrevers-ible. The best evidence that precursor cells do not recover from their para-lysed state comes from experiments in which thymectomised animals areparalysed. Such animals do not recover, or recover much more slowly, fromtheir unresponsive state than unthymectomised animals. This observationstrongly suggests that recovery from an unresponsive state requires thegeneration of new t cells rather than the recovery of paralysed t cells (Claman& Talmage 1963, Taylor 1969).

Spleen cells from mice specifically paralysed to SIII show a rapid 'escape'from their paralysed state when transferred to 900-R irradiated, syngeneicmice. It is known that some SIII antigen was carried over with the spleencells from the paralysed mice. The response was clearly seen four days aftercell transfer. This result has been interpreted as showing that a paralysedcell could recover from its paralysed state (Howard et al. 1972). However,a less drastic interpretation is possible. The donor, though unresponsive, hadsome recently generated specific B cells present which had not been ir-reversible paralysed (see above under The Asymmetry in the Requirementfor Specific B and t Cells for the Induction of Humoral Antibody Synthesis).These specific B cells could not be induced in the paralysed animal becausethe antigen concentration was too high to allow associative recognition tooccur even if there were sufficient specific associative antibody present. Isuggest that these B cells, on being transferred to an irradiated syngeneicmouse, were induced by the antigen carried over with the donor cells, in the


presence of the host's X-ray resistant T cells or secreted associative antibody.This suggestion is plausible because an anti-SIII B cell requires very littleassociative antibody to be induced (see above under Thymus-independentAntigens'). A variation of this explanation is that new B cells specific forSIII were generated from the donor cells in the X-rayed recipient. A similarrapid 'escape' from tolerance has been observed for another 'thymus-indepen-dent' antigen, LPS (Sjoberg 1972).

Other evidence has been obtained which is interpreted as showing thatspecific antibody is required for some antigens to paralyse their precursorcells (Diener & Feldmann 1970). Some of this evidence will be discussedbelow, see under 'In Vitro Paralysis'. It has also been suggested that a weakinductive event may lead to low-zone paralysis, and that exhaustive stimula-tion may lead to high-zone paralysis (Jerne 1968, Stcrzl 1968). These sug-gestions, besides leading to various other difficulties, do not account for thefact that some antigens, for example deaggregated HGG, are paralytic at alldoses at which they can be seen to have any effect (Golub & Weigle 1969).

'IN VITRO PARALYSIS'

Although claims for having achieved in vitro paralysis have been obtainedwith two different antigens, 1 shall only consider those experiments usinga fragment of flagellin, and the polymer of flagellin, or POL. This systemhas been studied extensively (Diener & Armstrong 1969, Diener & Feldmann1970) and my discussion of this system also applies to the observations madewith the other antigen (Britton 1969).

Spleen cells are incubated at 31°C for six hours with various concentra-tions of POL and the fragment of flagellin, washed, and then challenged invitro with an immunogenic dose of POL. It is found that spleen cells pre-treated in this way with high doses of POL cannot be induced to produceanti-POL humoral antibody secreting cells, but these pretreated spleen cellscan be induced by antigens which do not cross-react with POL. Spleen cellspretreated with lower doses of POL or with the fragment of flagellin areinduced by immunogenic doses of POL. If it is assumed that POL containsan average number of 300 flagellin monomers (Ada et al. 1964), it can becalculated that the approximate concentration of POL required to make thesespleen cells unresponsive is 10"^°M.

Pretreatment of the spleen cells with doses of POL, corresponding to amolarity of POL of 10 '"M, results in the spleens being uninducible by animmunogenic dose of POL. On the other hand, pretreatment of the spleencells with the fragment, up to doses corresponding to a molarity of the

Transplant. Rev. (1972) II "

258 PETER BRETSCHER

fragment of lO'^M, have no effect on the response of the spleen cells to asubsequent challenge with an immunogenic dose of POL (Diener & Feld-mann 1970). According to the theory the fragment of flagellin, given at aconcentration of lO^M which is probably above the binding constant of thereceptors on most of the precursor cells specific for the fragment, shouldbe able to deliver signal ® to a precursor cell just as well as POL. The con-clusion drawn from these results, that POL is paralytic whereas the fragmentis not, is difficult to reconcile with both the theory and the in vivo results.In adult rats the fragment is found to be paralytic whereas the polymer isnot (Ada et al. 1967).

I suggest that POL can bind at least divalently to the precursor cells andassociative antibody and that the immune response is thus blocked. The rateof dissociation for the complex of a molecule bound divalently to an anti-body is of the order of one hour (Karush 1970), and if the molecule is tri-valently bound it will be considerably longer. As the fragment does notparalyse precursor cells, when given at high concentrations, in six hours,I suggest POL has blocked the response in a manner similar to that by whicha hapten can (Brownstone et al. 1966). However, I suggest that POL blocksthe response even after the spleen cells are washed as it is effectively irre-versibly bound to the specific associative antibody and to the anti-POLprecursor cells. This suggestion is favoured by the observation that POLblocks the response at concentrations of about 10"^"M, which implies thatPOL blocks the response by binding at least divalently to anti-POL asso^ciative antibody and precursor cells.

This interpretation of 'in vitro paralysis' makes one easily testable predic-tion. 'Paralysed spleen cells' which have been washed should be rescuabieand induced if T cells specific for POL are added to them. A convenientsource of such cells which would not contain any functional anti-POL B cellsis irradiated spleen cells sensitized to POL. This rescue experiment wouldshow that the 'in vitro paralysis' is reversible, and therefore different fromwhat is known about in vitro paralysis of t cells (see above, under OtherViews of Paralysis). The experiment suggested would also show that POLis a 'thymus-dependent' antigen.

It has also been found that a given concentration of the fragment offlagellin can induce 'in vitro paralysis' if a particular amount of specific anti-flagellin antibody is added (Diener & Feldmann 1970). The requirement forspecific antibody can be interpreted as showing that the fragment cannotbind 'irreversibly' to anti-flagellin precursor cells, but that certain complexesof the fragment and anti-flagellin antibody can do so. The suggestion basedon these observations that antibody may be required for monomeric antigensto induce paralysis of precursor cells in vitro is not likely.


EVIDENCE FOR THE ASSOCIATIVE RECOGNITION OF ANTIGEN

DURING THE INDUCTION OF I CELLS

There is indirect evidence for the associative recognition of antigen beingrequired for the induction of associative antibody as described above (seeThe Regulation of the Formation of Associative Antibody). A number offurther observations can be most plausibly interpreted as showing that theassociative recognition of antigen is required for t cell induction, but fornone of these observations is such an interpretation the only possible one.I shall briefly describe some of these experiments and their interpretationin terms of the theory.

1. Three months after rabbits have been paralysed to BSA, at a time whenthey are still unresponsive to a normally immunogenic dose of BSA, theinjection of HSA, which cross-reacts with BSA, results in the induction ofanti-BSA antibody. All these anti-BSA antibody molecules are absorbablewith HSA. This experiment has been described above, and interpreted asshowing that there are sufficient anti-BSA B cells to be induced by HSA, butthat there are insufficient anti-BSA t and T cells for the anti-BSA B cellsto be induced on the animal being challenged with BSA. A subsequentchallenge with BSA, after the unresponsive state to BSA has been brokenwith HSA, results in the de novo synthesis of anti-BSA antibody. If thoseanimals that have been made unresponsive to BSA are not given HSA beforethe challenging dose of BSA, anti-BSA antibody is not induced (Weigle et al.1971). As these animals that are unresponsive to BSA are known to haveanti-BSA B cells which are inducible by HSA, these results suggest that thechallenge with HSA resulted in the induction of t cells specific for determi-nants common to BSA and HSA. A subsequent challenge with BSA canthen result in the induction of anti-BSA B celts. These results can be mostsimply interpreted as showing that BSA cannot induce the few t cells presentwhich are specific for determinants common to BSA and HSA, but that HSAcan do so. This suggests that the associative recognition of antigen is requiredfor t cell induction.

2. Mice can be rendered unresponsive to SRBC by irradiation, reconsti-tution with bone marrow cells, and the injection of massive doses of SRBC.Such mice must have insufficient B cells, and/or t and T cells, to respondto SRBC. Such mice, which presumably still contain some SRBC, cannotbe reconstituted with thymus cells. If HRBC (horse red blood cells), whichcross-react slightly with SRBC, are given with the thymus cells, a goodresponse of anti-SRBC antibody is induced. The majority of this anti-SRBCantibody cannot be absorbed with HRBC (Gershon & Kondo 1972). Theunresponsive mouse must possess anti-SRBC B cells as anti-SRBC antibody

260 PETER BRETSCHER

is induced when no B cells have been given to the animal. The unresponsivestate must be due to an insufficiency of T and/or t cells. However, thymuscells, when given alone, fail to reconstitute the animal. This observationsuggests that the thymus cells cannot be induced by SRBC, and on thetheory the most straight forward interpretation of this result is that the micehave insufficient anti-SRBC T cells to induce the thymus or t cells. T cellsspecific for HRBC are present in the mouse, however, and so HRBC caninduce those thymus cells which have specificity for determinants shared byHRBC and SRBC. These newly generated anti-SRBC T cells will enablefurther anti-SRBC t cells and B cells to be induced. A simple prediction ofthis interpretation is that a small number of irradiated spleen cells from anormal mouse, as a source of anti-SRBC T cells, will, if given with thethymus cells, enable an anti-SRBC response to occur in the absence ofHRBC. The reconstituting activity of these irradiated spleen cells shouldbe abolished if they are treated with anti-6' serum and complement as Tcells, as opposed to most other cell types, bear the & antigen on theirsurface. Certain observations obtained in in vitro spleen cultures could beinterpreted in a similar fashion (Hartmann 1970), but I do not have thespace to describe them here.

RECONSTITUTION OF THE RESPONSE OF T CELL DEPLETEDSPLEEN CELLS BY VARIOUS SUBSTANCES

It has been shown that the response to SRBC by spleen cells, partiallydepleted of t and T cells, can be reconstituted with various factors. Forexample, certain batches of foetal bovine serum contain one or more factors,which, if added to the medium in which the T cell-depleted spleen cells arecultured, will restore the response of these depleted cells to SRBC (Byrd1971). Such spleen cells can also be reconstituted with supernatants fromcultures of syngeneic spleen cells (Gorcynski et al. 1972) and with super-natants from a mixture of spleen cells from two histoincompatible strainsof mice (Dutton et al. 1971). The main interest of such observations, fromthe point of view of the theory, is whether such factors can be regarded aseither providing a B cell with signal (D, or whether such factors enable aB cell to bypass the requirement for signal ®.

Suppose that associative antibody must be bound to a cell for which itis cytophilic in order for signal @ to be delivered to the precursor cell, asseems most plausible. Signal (D could either be mediated by a membrane-membrane interaction between the precursor cell and the cell for whichassociative antibody is cytophilic, or the interaction of associative antibodywith antigen could lead to the release of a molecule of short range which


mediates signal @. Signal © cannot be mediated by a molecule with a longrange as this would lead to the conversion of a paralytic signal for one pre-cursor cell into an inductive signal in the presence of the induction ofanother precursor cell. Although signal @ may be mediated by a diffusiblemolecule, it would be essential that such a molecule can act over only a veryshort distance. It therefore seems unlikely that supernatants from culturesof various cells would contain the molecule that may mediate signal (2). Itis possible, however, that some of these supernatants from various cellscould contain associative antibody specific for either the antigen or the Bcell, which would allow the B cell to be either normally or abnormallyinduced in the presence of antigen.

Non-specific factors, which might be loosely called mitogens. could actin various ways in restoring the response. I list below some of these ways.

1) Any factor, which sticks to B cells, especially if it sticks to these cellsin an aggregated form, could lead to the abnormal induction of these cells.For example, if pokeweed mitogen can stick to a B cell, the B cell couldpresumably be abnormally induced. If the sticking factor is itself polymeric,or if it sticks to the cell in an aggregated form, only a small amount ofassociative antibody specific for the factor would be required to abnormallyinduce the B cell. The abnormal induction of the B cell might well appearto be 'thymus-independent'. This suggestion may be relevant to an inter-pretation of the observation that pokeweed mitogen stimulates T cell-depleted spleen cells to synthesize ;'M antibody molecules (Parkhouse et al.1972).

2) A factor could also stick to the surface of the remaining cells whichcan be induced to secrete, or give rise to cells which can secrete, associativeantibody. These cells could be abnormally induced as described for B cellsin 1) above.

3) The factor could improve the culture conditions in some undefinedway, allowing a residual response to be much increased.

4) The factor could stick to a precursor cell in a particular way leadingto effects on the ceil surface that are equivalent to signal (D. This wouldbo a less likely but interesting possibility.

SUMMARY

Of the main assumptions of the theory there is one for which there is nogood evidence. This assumption is that there is competition between theinduction and paralysis of t cells.

The theory, besides providing an explanation of self-nonself discrimina-tion, accounts for several kinds of observation;

262 PETER BRETSCHER

1) Certain antigens, when given in small doses to an animal, result in theanimal being unresponsive to a subsequent, normally immunogenic dose,of the antigen. The occurence of low-zone paralysis is explained by thetheory in its quantitative formulation.

2) Medium doses of antigen induce the formation of humoral antibodysynthesis.

3) High doses cf antigen paralyse an animal. This high-zone paralysis isexplained by the impossibility of associative recognition, and consequentlythe induction of precursor cells, when the antibody receptors and associativeantibody are saturated with antigen.

4) All doses of antigen, above a certain lower limit, maintain the unre-sponsive state of the animal to the antigen.

5) The fragmentation of an antigen increases the paralytic potency of theantigen, whereas aggregation of the antigen increases its immunogenicity.

6) Non-immunogenic molecules are paralytic.7) Antibodies against a non-immunogenic molecule, or hapten h, are

induced if the hapten is coupled to an antigen P, against which the animalcan. respond, when the animal is challenged with hP.

8) Those antigens which appear to be 'thymus-independent' are allpolymeric antigens, and the theory provides an explanation for this fact.

9) In T cell-depleted mice an immune response to antigens other than'thymus independent' antigens does not occur. The ability of these T cell-depleted mice to respond to a 'thymus-dependent' antigen when they arechallenged with a conjugate of a thymus-independent' and 'thymus-depen-dent' antigen is explained by the theory.

10) Some polymeric antigens induce 19s humoral antibody synthesisalmost exclusively.

11) An animal made unresponsive to an antigen A can, before it hasrecovered from its unresponsive state to A, be induced to synthesize anti-Aantibody on being challenged with an antigen B which cross-reacts with A.All the induced anti-A antibody can be obsorbed with B.

12) The induction of the cross-reacting antibody described in 11) isinhibited if A is given simultaneously with B, when B is a weak immunogen.

13) The sensitization of an animal with a carrier P enhances the animal'sresponse to a hapten h when the animal is challenged with hP.

14) A paralysed animal can be reconstituted with normal spleen cells ifparalyzing amounts of antigen are not present in the animal.

15) Thymectomy delays or prevents the recovery from an unresponsivestate.

16) The phenomenon of abnormal induction finds a natural explanationin terms of the theory.


The interpretation of the theory in cellular terms is consistent with a verywide range of observations in cellular immunology. One open, question iswhether the secretion by T cells of associative antibody is paralysable and/orinducible.

There are three principal predictions of the theory:1) An in vitro response to POL can be induced with 10^ T cell-depleted

spleen cells. In order to explain the occurrence of this response in terms ofthe theory in a reasonable manner it is necessary to postulate that associativeantibody, secreted by a T cell, can be involved in the fonnation of aninductive signal to a precursor cell. The response to POL by these spleencells strongly suggests that T cells secrete active associative antibody.

2) t cell induction requires the presence of antigen and specific associativeantibody.

3) There is some evidence that the paralysis of precursor cells is irre-versible, and the theory predicts that 'in vitro paralysis', as seen in the frag-ment of flagellin and POL systems, is misleading. The theory predicts thatthese 'paralysed' spleen cells can be rescued by providing them with anti-flagellin T cells. This rescue experiment would also show that POL is a'thymus-dependent' antigen.

ADDENDUM

Experiments to be reported shortly* (Feldmann, pers. comm.) show that Tcells secrete an antigen-specific molecule which is cytophilic for a macro-phage type cell. Macrophages bearing a complex of the antigen and thisantigen specific molecule can replace the requirement for T cells in theinduction of B ceils. I consider this to be strong evidence for the correctnessof the first of the three predictions of the theory described above.

* Feldmann, M., May 1972, J. exp. Med.Feldmann, M. & Basten, A., July 1972, / . exp. Med.Feldmann, M., 1972, submitted for publication.

ACKNOWLEDGEMENTS

I thank the California Division of the American Cancer Society for a Dern-ham Fellowship. This work was supported by a National Institutes of HealthGrant (A-I05875) and a Training Grant (CA-05213) to Melvin Cohn. I alsothank B. Coffman, B. Geckeler and J. D. Watson for giving me their com-ments on the manuscript.

264 PETER BRETSCHER

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The Control of Humoral and Associative Antibody Synthesis

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