Regulation of the immune response requires cross-talkbetween T cells and APC

Immune regulation clearly operates at a number of differentlevels. At its simplest, the removal of antigen can be seen asthe arbiter of continuation versus cessation of a particularresponse. But antigen load can have complex and oftenparadoxical effects on the quality as well as the quantity of aresponse,1 so we argue, based on first principles, that regu-lated antigen expression is a necessary feature of the cogni-tive elements (i.e. the cells) of the immune system. A keyfeature of an immune response is antigen processing by thespecialized APC and presentation of the products to T cells.These T cells can be effectors or regulators of effectors, buttheir function is to act back on the pathogen to limit itsgrowth. Information exchange is required in both directionsfor the APC and T cell compartments to synergize inpathogen removal, and the most obvious example of this is arequirement to control autolysis of APC by CTL. Of courseit is well known that APC can be directly activated by T cellsto up-regulate costimulatory molecules, produce interleukinsand become competent to generate CTL.2–5 Beyond this, weargue that if the APC has some means of recognizing themagnitude and specificity of the T cell response, then theactivation of that set of cells can be appropriately terminated,allowing recognition of other epitopes. The requirement forthis type of regulation, as distinct from conventional APCturnover, is temporal; our hypothesis is about the events thatimmediately follow antigen recognition. The remainder of thepresent article discusses how we think information transfer tothe APC is accomplished, and how this model explains thephenomenon of determinant spreading.

Turning on the T cell requires dynamic TCR clustering

The TCR transduces a graded response according to howmuch and what sort of ligand it finds, and these signals aremoderated by costimulatory signals acquired contextually.It has been recognized for some time that in order to fully

activate a T cell, crosslinking of the TCR must occur ratherthan simple ligation.6 Indeed, T cells stimulated by some anti-CD3 antibodies that cannot crosslink the TCR are renderedanergic, whereas crosslinking of the antibody with a secondantibody or co-crosslinking the TCR with CD4 restores theproliferative signal.7 In fact, it seems likely that ordered TCRoligomerization rather than simple co-localization is requiredat the immunological synapse for recruitment of CD4.8 Thisis borne out by recent mathematical modelling of TCR inter-actions that suggest that dimerization is an essential pre-requisite to T cell activation.9 Oligomerization and furthermultimerization occur in planar membranes, and the TCRcoordinates the ligand complexes as crosslinked patches. Thecore interaction between TCR and ligand is of low affinity,and is therefore likely to promote a dynamic clustering acrossopposed cell membranes.10 The clusters are metastable, butprobably need to be sustained for a time of the order of atleast 1 h, but perhaps as long as 24 h, for full T cell activa-tion to occur.11 Given that MHC–peptide complexes sequen-tially trigger many TCR, resulting in their removal from the T cell, the tertiary complexes are necessarily dynamic innature (more on this point later).

The nature of the APC ligand that binds TCR

Major histocompatibility complex class II molecules areheterodimeric and have a natural propensity to oligomerize.They can form dimers of the heterodimers, called‘superdimers’, in a number of physiological and non-physiological settings. Class II superdimers have been foundin several crystal structures and have been detected bywestern blotting and by immunoprecipitation from B cells.12

The presence of the superdimer at the cell surface has been

Immunology and Cell Biology (1999) 77, 99–104

Theoretical Article

MHC multimerization, antigen expression and the induction of APC amnesia in the developing immune response

RICHARD A LAKE, BRUCE WS ROBINSON and JOHN D HAYBALL

University Department of Medicine, Queen Elizabeth II Medical School, Nedlands, Perth, Western Australia,Australia

Summary Class II multimer formation is an important event in immune recognition. Not only is multimeriza-tion a prerequisite for T cell activation, but it is a signal to APC. In the present article, we propose that multi-merization can result in the specific removal of ligand complexes from the cell surface of the APC, an event whichmay influence the overall pattern of T cell reactivity.

Key words: APC amnesia, determinant spreading, immune regulation, receptor multimerization, T cell activation.

Correspondence: Dr RA Lake, University Department ofMedicine, Queen Elizabeth II Medical School, 4th Floor, G Block,Nedlands, Perth, WA 6009, Australia. Email: <rlake@cyllene.uwa.edu.au>

Received 17 August 1998; accepted 6 October 1998.

inferred using antibodies and digitized photomicrography.13

Because the binding groove of class II allows antigenicpeptide to extrude from both ends, these peptides can bechained together so that they can bind simultaneously toadjacent class II molecules. Tandemly repeated epitopes havean increased affinity for class II, and they are more potent atinducing proliferation of T cells than monomeric epitopes.14

Indeed, epitopes that are reiterated up to 32 times are betterstimulators of T cells than the equivalent monomers.15

Crucially, soluble forms of MHC class II multimerize intohigher order structures in the presence of soluble TCR. Thesize of these aggregates is consistent with the formation ofsuperdimers and then oligomers of superdimers.16 The aggre-gates are likely to be low energy states of the molecules insolution and indicate how oligomerization of the minimalTCR/MHC–peptide complexes could occur in membranes.Importantly, the data indicate that the superdimer is a con-stituent in the process of ordered multimerization in planarmembranes and is the first step in the accretion of crosslinkedpatches at the interface of a T cell and APC. Interestingly, thefootprint of different TCR on the same ligand is essentiallythe same, even though there is no conservation of the residuesthat constitute the binding face. The finding that the mode ofrecognition is conserved, has been interpreted to support acommon mechanism of T cell activation that depends upon alogical oligomerization process that may include the co-recognition of other molecules.17 Collectively, theseexperiments suggest that multimerization occurs by TCR-mediated ordering of superdimers.

It is important to recognize that, while the receptor iseffectively monospecific, ligand is presented as a few high-affinity hits in a sea of perhaps 105-non-cognate or low-affinity restriction elements. Because the relative abundanceof any particular ligand is low, the chance of a superdimercontaining the same pair of ligands is orders of magnitudelower.18 Therefore, it is clearly going to be important to deter-mine what is in the contact zone when a T cell interacts withan APC. It cannot all be specific ligand, which suggests thatclass II must be able to oligomerize when the TCR coordin-ates a mixture of ligands with varying affinity. This conjec-ture is consistent with the finding that there is a non-uniformdistribution of the proteins involved in the interactionbetween a T cell and APC, in that non-specific adhesionmolecules such as LFA-1 form a torus or peripheralsupramolecular activation cluster (p-SMAC), around acentral SMAC which contains TCR and ligand. This nidus,the jam in the doughnut, recruits MHC molecules of the sameallele and excludes molecules which are not of the restrictingelement.19 Overall, the evidence suggests that during T cellrecognition of antigen, a proportion of MHC–peptidecomplexes are in the form of multimerized superdimers andthat the arrangement of these complexes is not fixed, but in adynamic clustered equilibrium with TCR.

The affinity of MHC–peptide complexes for TCRregulates clustering

A given T cell can recognize a variety of different ligands,and reciprocally a given ligand can bind to a variety of TCR.The ability to recognize a ligand is only very roughly relatedto the primary amino acid sequence of the interacting

surfaces. Instead, recognition is dependent on shape asdictated by the surface electron density of the receptor andligand. Experimentally, variant ligands have been engineeredby swapping residues that form the secondary amino acidcontacts for the TCR. These variants or altered peptideligands (APL) are recognized as such by the T cell.20 Theremay be a number of different ways that an individual T cellcan respond to APL, each of which is characterized by a dis-tinct set of intracellular signalling events.21 For example,effector responses may be separated from proliferativeresponses, so that cytokines may be secreted by the T cellwithout subsequent cellular proliferation.22 Alternatively,antagonism may be observed, where partial activation of theT cell with an APL renders it incapable of responding to chal-lenge with the native antigen.23,24 It has been demonstratedthat APL signalling occurs because the TCR has a loweraffinity and a faster dissociation rate for these alteredligands.25

CD4 augments the response of a T cell to a partial agonist,but not an antagonist peptide. This can be explained if CD4binds to MHC–peptide complexes at a much slower rate thanthe TCR, which leads to a model of sequential engagementwhere the TCR/MHC–peptide complexes undergo oligomer-ization prior to generating the high affinity CD4 bindingsite.26 This is consistent with the finding that oligomerizationof the TCR/MHC-peptide complexes can occur in theabsence of CD4.16 This model is summarized in Fig. 1.

A logical loss of TCR

After it has received an activation signal, a T cell is renderedincapable of further TCR-mediated stimulation because theTCR is rapidly internalized and targeted for lysosomal deg-radation.27 This uncouples the TCR from its membrane-anchored signalling molecules and therefore reduces thecell’s sensitivity to subsequent stimulation with antigen. Thismakes sense when intracellular signalling pathways areshared. For example, the growth stimulus imparted by IL-2receptor ligation, which is a second and independent signalfor T cell growth, could be confused or re-emphasizedinappropriately by further TCR ligation if the TCR remainedfunctional at the cell surface. Both of these pathways involvethe intracellular recruitment and activation of protein tyro-sine kinases such as p56lck.28,29 Loss of TCR allows for theamplification of a low-abundance signal and means that asingle ligand has the capacity to serially trigger some 200 TCR.30 But the loss of TCR may also be a way of main-taining the dynamic nature of the complex, because eachTCR can be replaced by one of identical specificity. Thesechanges may help the lattice come to an energy-minimizedstate in which ligand is coordinated in a favourable pattern.We hypothesize that the composition of the complexaccreting at the contact sites between the opposed cellsdetermines the fate of the T cell and the APC. The rules that determine the fate of the T cell may well be differentfrom those that regulate the APC, but may depend, in part,on the ratio of superdimers containing the same epitope tothose containing different epitopes.

This loss of surface TCR has been relatively easy tofollow because, only very unusually, have more than tworeceptor specificities been observed in a single T cell.31 In

RA Lake et al.100

contrast, specific MHC–peptide complexes constitute only asmall fraction of surface MHC, and their fate after interactionwith TCR is uncertain. We believe they can be internalizedafter turning on the T cell (Fig. 1). This is not an outlandishhypothesis because MHC molecules are known to constitu-tively enter endocytic pathways within APC. For class Imolecules the process is spontaneous and selective andbroadly similar to the internalization and recycling of otherreceptors, such as the transferrin receptor.32 Class II mole-cules are also constitutively endocytosed and recycled.33

Importantly, truncation of either one of the alpha or betacytoplasmic tails blocks the internalization pathway, whereasit has no effect on the invariant chain-dependent presentationof native antigen. Thus the cytoplasmic tails are not requiredfor the conventional presentation pathway, but jointlycontribute a signal for internalization of class II molecules.34

APC amnesia: where have all the peptides gone?

We have observed that if peptide-pulsed APC are exposed tocognate T cells, then these APC are incapable of stimulatinga second set of T cells of the same specificity (Fig. 2d). Thisinability to stimulate is not due to the production of solublefactors or to anergy induction because superaddition ofpeptide allows proliferation of the second set of cells toproceed (Fig. 2e). The effect is antigen-specific because afirst set of T cells with different peptide specificity has onlya limited effect on the ability of the second set of T cells toproliferate (Fig. 2c). We have termed this effect ‘APCamnesia’, because it appears as though the APC have lost allmemory of the antigenic peptide. It is important to note thatAPC amnesia does not occur when the first set of T cells isactivated by a different ligand on the same APC. This iseasily accomplished by pulsing the APC with both peptides,a finding which effectively rules out non-specific explana-tions of the phenomenon based on cell clustering. In fact,competition between proliferating T cells is evident(compare Fig. 2a,c), and this is probably because the T cellsform conjugates with APC. In a similar fashion, anergic T cells have been shown to inhibit proliferation of otherpotentially reactive T cells by competing for both the surfaceof the APC as well as any locally produced IL-2.35

We are aware of the preliminary nature of our experi-ments and would not wish to overinterpret them. But it isclear from other studies that T cells can exert a selectivedepletion of class II molecules from the surface of APC. Thishas been described independently by two other groups,although the specificity and functional consequences of thisloss have not been detailed.36,37 Vidovic et al. noted globalbut allele-restricted loss of MHC class II,37 whereas Manca,using cloned, irradiated HIV gp120 peptide-specific T cells,found specificity as described here.36 In our experiments,unstimulated, unirradiated naive T cells were used, indicat-ing that amnesia is likely to be occurring in a situation thatphysiologically parallels that which is likely to be present inlymph nodes. This is an important point, because it is likelythat T cells need to acquire an activated phenotype beforethey are capable of signalling backwards to the APC. It isimportant to note here that amnesia is distinct from any con-stitutive global turnover of class II molecules: amnesia ispeptide specific. Antigen-presenting cell amnesia may alsooccur in a class I system and could explain how APC canprime CD8+ T cells with different specificities while failingto interact with a second set of T cells with identical speci-ficity.38

To summarize, we are suggesting that the processes ofMHC multimerisation and amnesia are linked. This is basedon published work, on the evidence described above and onsome recent unpublished experiments using mutated MHCmolecules although we acknowledge that these twoprocesses may be independent.

APC amnesia 101

Figure 1 A model of T cell and APC interaction involving TCR-mediated MHC class II oligomerization. Chronologically, eventsare depicted from top to bottom of the figure. Antigen recognitionby a T cell results in localized accretion of both the TCR andantigenic MHC–peptide complexes at the cellular interface.Oligomerization precedes binding of the coreceptor CD4, thusfull T cell activation, and is the rate limiting step of the interac-tion for the T cell. T cell receptors are triggered and internalized,but are replaced by others of identical specificity, and the struc-ture of antigen in the contact zone becomes more ordered as allof the specific MHC–peptide complexes are recruited to thelattice. At a critical point, the accretion complex is internalizedfrom the surface of the APC. If localized oligomerization at theinterface between the T cell and APC does not occur, the outcomeis partial activation of the T cell, accompanied by TCR internal-ization alone. Antigen-presenting cells with internalizedMHC–peptide complexes cannot stimulate more T cells of thesame specificity.

Significance of APC amnesia

While there is extensive literature describing the processes ofantigen acquisition, processing and presentation by APC,there is little known of the fate of antigenic peptides boundto MHC class II molecules after recognition by reactive

T cells. We know that these complexes can associate intohigher ordered structures in a step-wise process of TCR-mediated oligomerization and that this process is the rate-limiting step of T cell activation, ultimately influencing theeffector arm or quality of the immune response. We proposethat antigenic complexes are internalized by the APC as afunction of coordinated accretion between molecules onopposed cell membranes. This system limits the exposure ofspecific antigenic complexes to the immune system andtherefore has the capacity to limit the extent of the immuneresponse. Such a mechanism is teleologically attractive andwould be expected to have fundamental consequences for ourunderstanding of mechanisms of host defence. We predictthat APC amnesia will depend on co-recognition of othermolecules on the surface of the T cell. Thus, the status of theT cell will determine the extent of APC amnesia.

The significance of APC amnesia lies in the ability of thecells of the immune system to correspond with each other. Anability to control antigen expression enables communicationfrom the effector population back to the APC without therequirement to influence the antigen itself. By limiting theimmune response to each epitope of a complex antigen, intra- and intermolecular determinant spreading would bepromoted. This would occur because dominant epitopeswould be sequentially removed from the individual APCfollowing T cell stimulation, allowing less dominant deter-minants to be presented by that APC without competition.39

This process would be aided by the up-regulation of expres-sion of costimulatory molecules on the APC. Importantly,dominance in the overall T cell response would be preservedbecause other APC, newly arrived in the lymph node fromtheir site of antigen uptake, would still present dominantepitopes, provided of course that the antigen was still presentat that site. This process might explain how determinantspreading, which is crucial for host defence, can be ‘guaran-teed’, particularly because it is clear that the immune systemdoes have the capacity to respond to antigens such as viruseswith a surprising degree of dominance when analysed usingsensitive means such as MHC–peptide tetramer technology.40

The kinetics of removal of MHC–peptide complexes fromAPC surfaces following T cell recognition is yet to be deter-mined. If relatively slow, it may represent a means of ensur-ing that all first responses to complex antigens induce a broadspectrum of cytokines in the responding T cell population, ashas been described.41 This is because the reduced numbers ofMHC–peptide complexes that follow partial removal fromthe surface of the APC after T cell recognition would alter thestrength of the signal given to T cells by APC. This is a majorfactor in determining the type of response generated, that is,whether a Th1 or Th2 pattern is induced.1 Thus, a reductionin the density of specific complexes on the surface would beexpected to alter the type of immune response generated.

The process of APC amnesia is likely to be constitutive,guaranteeing that ongoing responses to self antigens, whichappear to be occurring continuously, are limited in strength,exhibit diversity and generate Th2 cytokines to regulate anypotentially damaging self-reactive T cells. Failure of thisamnesia mechanism may therefore contribute to autoimmunereactions, for example, by permitting powerful, oligoclonalreactions with a Th1 phenotype. It will be interesting toobserve, as the mechanism for APC amnesia is unravelled,

RA Lake et al.102

Figure 2 Antigen-presenting cell amnesia depends upon TCRrecognition of MHC–peptide complexes. Carboxyfluoresceindiacetate succinimidyl ester (CFSE) is a fluorescent dye thatlabels all cells homogeneously and offers the opportunity to studythe replication status of individual cells within a culture. Becausethe mean fluorescence intensity of daughter cells is halved at eachdivision, the number of times a particular cell has transited cellcycle can be estimated in the context of other phenotypic markersin a different fluorescence channel. Antigen-presenting cells werepulsed with either one or both peptides, influenza virus haemag-glutinin (HA) and ovalbumin (OVA) and then incubated with HA-or OVA-specific T cells. After 18 h, HA-specific T cells that werelabelled with CFSE, were added to each of the cultures and theirfluorescence intensity analysed 48 h later. PP is the parent peakand includes all the non-proliferating T cells; DP are the daugh-ter peaks which include all the various stages of proliferating Tcells. (a) Irrelevant peptide (OVA): no proliferation; (b) specificpeptide (HA): proliferation; (c) OVA + HA peptide with APC pre-exposed to OVA-specific cells: proliferation; (d) OVA + HApeptide with APC pre-exposed HA-specific cells: proliferationblocked; (e) OVA + HA peptide with APC pre-exposed to HA-specific cells with HA peptide added: proliferation restored.

whether any of the known MHC associations with autoim-munity correspond to structural changes in the MHC mole-cules that are required to transmit an internalization signal.The corollary to this is that during inflammatory processes,such as viral infection, amnesia may be reduced. This wouldalso serve to stimulate powerful, oligoclonal reactions with aTh1 phenotype, the type that are observed during antiviralresponses.40 It will be crucial to study the effect of APCmodulating factors on this process and to compare it in DC,B cells and macrophages from various anatomical sites.

Because a particular TCR cannot know whether it willultimately be restricted by class I or class II molecules, themechanism of signal transduction is likely to be conservedbetween CD4+ and CD8+ T cells. We anticipate that APCamnesia will be also be conserved in class I systems, and thiscould confer an ability to regulate the potential auto-cytotoxicity of CTL.

Conclusion

We have argued, based on our own observations and a largebody of published work, that during T cell activation theprocess of TCR-directed clustering generates a dynamicframework between two cell membranes with potentiallydiverse signalling properties. We reason that cognateMHC–peptide complexes are not inert, but that orderedmultimerization of these complexes is an essential com-ponent of the process of activation. This produces additionalbackwards signals to the APC and results in the specific lossof the complexes, a process that may affect further T cellactivation, differentiation and determinant spreading.

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