doi 101098rstb20000644 1071-1076355 2000 Phil Trans R Soc Lond B

Matthew Krummel Christoph Wuumllfing Cenk Sumen and Mark M Davis

cell recognitionminussix views of TminusThirty

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Thirty-six views of T-cell recognition

Matthew Krummel Christoph WIumllcentng Cenk Sumen and Mark M Davis

The Department of Microbiology and Immunology and The Howard Hughes Medical InstituteStanford University School of Medicine Stanford CA 94305 USA

While much is known about the signalling pathways within lymphocytes that are triggered during activa-tion much less is known about how the various cell surface molecules on Tcells initiate these events Toaddress this we have focused on the primary interaction that drives T-cell activation namely the bindingof a particular T-cell receptor (TCR) to peptide^MHC ligands and centnd a close correlation betweenbiological activity and oiexcl-rate that is the most stimulatory TCR ligands have the slowest dissociationrates In general TCRs from multiple histocompatibility complex (MHC) class-II-restricted Tcells havehalf-lives of 1^11s at 25 8C a much narrower range than found with antibodies and suggesting a strongselection for an optimum dissociation rate TCR ligands with even faster dissociation rates tend to beantagonists To observe the eiexclects of these diiexclerent ligands in their physiological setting we made genefusions of various molecules with green pounduorescent protein (GFP) transfected them into the relevantlymphocytes and observed their movements during T-cell recognition using multicolour video micro-scopy We centnd that clustering of CD3plusmn^GFP and CD4^GFP on the Tcell occurs concomitantly or slightlybefore the centrst rise in calcium by the T cell and that various GFP-labelled molecules on the B-cell sidecluster shortly thereafter (ICAM-1 class II MHC CD48) apparently driven byT-cell molecules Most ofthis movement towards the interface is mediated by signals through the co-stimulatory receptors CD28and LFA-1 and involves myosin motors and the cortical actin cytoskeleton Thus we have proposed thatthe principal mechanism by which co-stimulation enhances T-cell responsiveness is by increasing thelocal density of T-cell activation molecules their ligands and their attendant signalling apparatus Incollaboration with Michael Dustin and colleagues we have also found that the formation and stability ofthe TCR-peptide^MHC cluster at the centre of the interaction cap between Tand B cells is highly depen-dent on the dissociation rate of the TCR and its ligand Thus we are able to link this kinetic parameter tothe formation of a cell surface structure that is linked to and probably causal with respect to T-cellactivation

Keywords T-cell receptor Tcell cell recognition multiple histocompatibility complex CD4green pounduorescent protein

1 INTRODUCTION

In 1830 the Japanese artist Hokusai began publishing hisseries of wood block prints entitled Thirty-six views of MtFuji While the concept seems tedious and obsessive atcentrst (and indeed it probably would have been at thehands of say Andy Warhol) the execution was not andthese images remain well known and even iconic to thisday (see wwwcssemonasheduaujwbukiyoehokusaihtml) Similarly in modern biology it has often beenproductive to examine complex natural phenomena froma variety of experimental perspectives

One such phenomenon that we have focused on is thatof transient cell^cell recognition as exemplicented by Tlymphocytes recognizing specicentc peptide^multiple histo-compatibility complexes (MHC) on the surfaces of othercells This process occurs continuously throughout the lifeof an individual and is vital for health No evidence insupport of this statement is more sobering than the fact

that HIV infection the subject of many of the papers inthis issue specicentcally devastates CD4 Tcells and therebyleaves the victim open to many diseases that would ordi-narily never be noticed T cells as a distinct subset oflymphocytes have been a central object of study inimmunology since their discovery four decades ago(Miller et al 1961) and as a consequence all or most ofthe key surface molecules that they rely on for recognitionand auxiliary activities are known Thus Tcells representan interesting (and clinically relevant) model system forstudying how specicentc cell surface molecules mediate cell^cell recognition

2 RECEPTORS AND LIGANDS INVOLVED IN

aaaaaaaaaaaabbbbbbbbbbb T-CELL RECOGNITION

A surprisingly large number of cell surface moleculesappear to play important roles in T-cell recognitionThese include the ab form of T-cell receptors an anti-body-like heterodimer that is responsible for recognizingparticular peptide^MHC complexes (on either class I orclass II MHC molecules) on another cell This is the keyevent in the decision to recognize one cell versus another

Phil Trans R Soc Lond B (2000) 355 1071^1076 1071 copy 2000 The Royal Society

doi 101098rstb20000644

Author for correspondence (mdaviscmgmstanfordedu)Present address Center for Immunology UT Southwestern MedicalCenter DallasTX 75390-9093 USA

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These heterodimers are always expressed together withthe CD3 polypeptides which are responsible for thesignal transduction (Irving amp Weiss 1991) as the TCRhas no cytoplasmic domain with that capability itself Asfew as ten to 100 of the correct peptide^MHC complexesare sucurrencient for recognition on the antigen-presentingcell (APC) As with other membrane molecules thatrecognize ligands on other cells the acurrennity of a TCR forpeptide^MHC is quite low in the 107 4 to 107 6 m range(table 1) The oiexcl-rates range between 025 and 001s71

and in most systems an increasing oiexcl-rate correlates withloss of T-cell reactivity even to the point where this canresult in interference with an otherwise stimulatory signal(antagonism) Association rates are generally quite low at500^10 000 M71s71 and recent thermodynamic evidencesuggests that this may be due to a mechanism of bindingin which somewhat disordered TCR-binding sites became

more ordered upon ligand binding (Willcox et al 1999Boniface et al 1999) This may correspond to the `inducedcenttrsquo mode of binding employed by DNA-recognitionproteins which also have to discriminate between a largenumber of chemically similar molecules (Boniface et al1999)

Other molecules on the T-cell surface act to facilitaterecognition in ways that are not yet clear These includeLFA-1 which recognizes ICAM-1 on other cells but appar-ently only after it has been converted into a `high acurrennitystatersquo (still in the 1 m m range) by TCR engagement Therole of the LFA-1^ICAM-1 interaction has long beenknown to be important in T-cell adhesion but it is alsoincreasingly clear that it has a signalling role as well

CD4 and CD8 are molecules specicentc to the helper orcytotoxic subsets of T cells respectively and bind class IIor class I MHCs The acurrennity of CD4 for class II MHC

1072 M Krummel and others Thirty- six views ofT-cell recognition

PhilTrans R Soc Lond B (2000)

time (min)1 2 3 4 5 10 60

10 m m

IRM

MHC

ICAM

overlay

Figure 1 T-cell accumulation of MHC and ICAM and the formation of an immunological synapse A representative primaryT cell is shown interacting with a supported lipid bilayer containing two types of GPI-linked molecules MHC (I-Ek) labelledwith Oregon green dye and loaded with moth cytochrome c (88^103) peptide and ICAM-1 labelled with Cy5 (red) The toppanel uses interference repoundection microscopy (IRM) to delineate regions of close T-cell membraneoumlbilayer apposition whichappear progressively darker as demonstrated by the outer ring in the T-cell contact at 1 min The remaining panels show theredistribution of the labelled proteins in the contact area by pounduorescence video microscopy over the course of 1 h MHC is initiallyengaged in the tight outer edge of the contact then actively accumulates into the centre of the synapse where it forms a stablecluster ICAM molecules exhibit a broader distribution throughout the interface in some cases being excluded from the centralMHC region and serve as an adhesive fulcrum for T-cell attachment From Grakoui et al 1999

250

200

150

100

50

00 50 100

(a) (b) (c)

KA (mMndash1)

0 5 15

1koff (s)

0 10 000 20 000

kon (Mndash1 sndash1)

accu

mul

ated

MH

C (

mol

ecul

es m

mndash2

)

10 20

Figure 2 Correlation of MHC cluster density in T-cell contacts with the solution kinetic parameters of the TCR^MHC^peptideinteraction Pooling data from two diiexclerent model antigen systems Hb(64^76) (centlled squares) and moth cytochrome c (88^103)(open squares) demonstrates that when the kinetic factors of acurrennity (a) association rate (b) and dissociation rate (c) aregraphed versus MHC molecules per micrometre squared only dissociation rate (plotted as the inverse) shows a close correlationto accumulated MHC Thus the half-life (t12 ˆ 7 ln2koiexcl) of the molecular interaction between TCRs and cognate MHC^peptide ligands determines the centnal MHC density in T-cell contacts From Grakoui et al 1999

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appears very weak and no direct binding measurementshave been possible For CD8 there are two conpoundictingdata sets In any event the presence of CD4 or CD8 hasthe eiexclect of amplifying the dose response of a given Tcellby ten to 100 times Other key molecules on the T-cellsurface are CD28 and CTLA-4 both of which bind B71(CD80) and B72 (CD86) on other cells and serve torespectively enhance or suppress T-cell activation

CD28 ligation has a major co-stimulatoryrsquo eiexclect onT-cell recognition greatly amplifying activated T-cell

responses and often being required for naive T-cellresponses A number of other T-cell molecules have alsobeen implicated in assisting recognition about whichmuch less is known

3 DISSECTING THE CHOREOGRAPHY OF T-CELL

RECOGNITION USE OF ARTIFICIAL MEMBRANES

How is it that these various molecules collaborate toallow T-cell recognition For many years it has beenknown that clustering TCRs on T cells or Igs on B cellscan articentcially trigger activation Subsequently it hasbeen shown that CD3plusmn chimeras alone when cross-linked could induce T cells to become fully activatedThis is analogous to other systems where receptor dimer-ization or multimerization initiates a signalling cascade(EGF) The fact that this was not the whole story wasshown by the work of Monks et al (1998) who used highresolution antibody staining of centxed T^B couples to showthat TCR LFA-1 and CD28 were not randomly distri-buted in the capsrsquo at the interface but rather had a precise`targetrsquo pattern withTCR and CD28 occupying the `bull-seyersquo LFA-1 enriched in a ring around it on the T celland a minor image of ligands on the B-cell side (egMHC and B7 in the middle ICAM-1 outside) Thisstriking pattern has been dubbed an `immunologicalsynapsersquo and is associated with strong T-cell stimulation

In parallel with this work have been the eiexclorts ofMichael Dustin and his colleagues to develop a dynamicsystem to study the movements of membrane moleculesduring T-cell recognition (Dustin et al 1998) Heresurface molecules that have been engineered to havelipid tails (so that they are laterally mobile) are labelledwith a particular pounduorophore and then placed in a lipidbilayer on a glass slide T cells are then deposited on thisslide and the movement of the diiexclerent molecules isobserved by time-lapse video microscopy The poundatsurface of the membrane on the slide ensures a poundat inter-face and high resolution As shown in centgure 1 when anMHC^peptide complex is labelled with pounduorescein(green) and ICAM-1 is labelled with rhodamine (red) aT cell recognizing these molecules quickly clusters themunderneath itself in a characteristic `synapsersquo patternwith the MHC in the interior and the ICAM-1 in a(largely) concentric ring around it This exactly parallelsthe centndings of Monks et al (1998) but has the addedadvantage of showing the dynamics of the system as wellas the fate of individual T cells A particularly strikingcentnding of this work (Grakoui et al 1999) is that thedensity of MHC clustering is proportional to the mono-meric half-life of the particular peptide^MHC complexwith the TCR (as shown in centgure 2c)oumlthus establishinga link between the kinetic measurements summarized intable 1 with movements of populations of molecules oncell surfaces The data in centgure 2 also centrmly establishthe importance of dissociation rate versus acurrennity orassociation rate in determining the density of MHCclustering Thus the stability of TCR binding to peptide^MHC is critical to synapse formation and this in turn islinked to a robust T-cell response presumably as anincreased density of ligand promotes a higher concen-tration of TCR^CD3 which in turn potentiates internalsignalling

Thirty-six views ofT-cell recognition M Krummel and others 1073

Phil Trans R Soc Lond B (2000)

long-lived TCRndashpMHCcomplexes allowconcerted CD4ndashMHCbinding and thispermits signalling

APC

APC

APC

APCT cell

T cell

pre-contact configuration

TCR

MHC II

CD4

peptide

initial scanning

recognition

TCRs and CD4are enriched inthe trailing edge

TCRs scan MHCcomplexes

Ca2+

Ca2+

cellular activity inducedby signalling andorco-stimulation inducecoalesence ofTCRndashpMHC clustersleading to sustainedsignalling

effector functions

synapse formation

Figure 3 A model for checkpoints in T-cell activation It issuggested that the progression to eiexclector functions involvesthe ability of the TCR to (i) scan MHC complexes then(ii) form long-lived TCR^pMHC complexes therebyrecruiting CD4 which then (iii) initiates a cellularreorientation event stabilizing existing TCR complexeswhile recruiting more TCRs to prolong the response Thecentnal concentguration with TCRs highly localized into themiddle of the interface is thus a result of traversing a numberof checkpoints for TCR binding and pMHC recognition

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4 LABELLING T CELLS DIRECTLY BEADS AND

GREEN FLUORESCENT PROTEINS

While extremely useful in decentning and following themolecules on APCs that are the ligands forT-cell recogni-tion this type of system is not as well suited for studyingT-cell molecules This is because it is the Tcell that is theactive partner here with the movements of its moleculesdriving the action of molecules on the APCs The T-cellmolecules are also linked to the cytoskeleton in ways thatare not well decentned and so it is not possible to reproducethis on a glass slide Therefore we have labelled intact Tcells in two diiexclerent ways (i) by attaching large beads(4^5 m m) either via antibodies to cell surface moleculesthat are neutral with respect to activation or throughbiotinylated lipids and streptavidin-coated beads(WIumllcentng amp Davis 1998) or (ii) using green pounduorescentprotein (GFP) fusions to the cytoplasmic regions ofvarious cell surface proteins (WIumllcentng et al 1998Krummel et al 2000) Using the centrst procedure we havebeen able to show that there is a distinct cell surfacetransport mechanism on T cells that is linked to thecytoskeleton and is triggered through the co-stimulatoryrsquoreceptors CD28 and LFA-1 This eiexclect results in thebeads bound to the anterior of the T cell translocating tothe interface with an APC beginning at about 4 minafter the centrst calcium poundux and probably continuingthroughout the centrst 30^60min of recognition We believethat this mechanism serves to deliver T-cell surface mol-ecules and their subcellular components to the synapseregion and is likely to be the chief reason that moleculesaccumulate there much more quickly than one wouldexpect based on passive diiexclusion It may also explainwhy cytoskeletal inhibitors such as cytocholasin D inter-fere so profoundly with T-cell recognition (and yet haveno eiexclect on APC WIumllcentng et al 1998) Similar obser-vations regarding a co-stimulation-driven transportmechanism have been reported by Viola et al (1999) whohave linked it to lipid `raftrsquo movement using a cholera-toxin-staining reagent Thus it may be that there is acytoskeletal linkage to membrane rafts that transports

them (and their cargo) to the interface In any event thetransport of key recognition molecules to the T-cell^APCinterface would elevate the concentration of such mole-cules and thereby potentiate T-cell activation As notedelsewhere (Cornall et al 1998) even modest increases inthe number of signalling molecules can have a signicentcanteiexclect on the sensitivity of lymphocyte activation In lightof this data we have proposed that the entire enhance-ment eiexclect seen with co-stimulation inTcells may be dueto this type of eiexclect (WIumllcentng amp Davis 1998)

The second and more powerful approach to the issue ofhow to follow the dynamics of molecules onTcells involvesthe use of GFP fusions This method has proven widelyuseful in a great number of biological applicationspreviously (Tsien 1998) but it can be argued that its mostsignicentcant impact could be in the understanding of cellsurface molecules The chemistry of soluble proteins can bereadily studied in solution (at least in theory) whereas thedynamics of cell surface proteins is not easily studied or canonly be studied to the sort of centrst approximation illustratedby the data in table 1These measurements are necessary inorder to understand the monomeric interaction dynamicbut are inherently unable to provide information regardingthe eiexclects of movement in a bilayer^raft the eiexclects ofpolyvalency or the cooperativity between diiexclerent types ofmolecules (either directly or through their respectivesignalling pathways) or the consequences of spatial segre-gation at the interface (synapse formation) GFP fusionsallow us to study individual molecules at 37 8C in theirnative context Our early eiexclorts in this regard showed thatICAM-1^GFP on B cell APCs clustered rapidly in thesynapse with a T cell beginning about 20 s after the centrstrise in intracellular free calcium (which we can monitorsimultaneously) This clustering of ICAM-1 did not occurin cells that lacked co-stimulatory ligands and served topotentiate calcium signalling and by inference the T-cellresponse (WIumllcentng et al 1998)

Our most recent GFP work has focused on T-cell mol-ecules where we have labelled CD3plusmn (the most importantof the CD3 chains with respect to signalling) TCRa andCD4 In none of these cases did the GFP fusion (to the

1074 M Krummel and others Thirty-six views ofT-cell recognition

PhilTrans R Soc Lond B (2000)

Table 1 Kinetic parameters of T-cell surface molecules ligand binding measured by surface plasmon resonance

T-cell molecule ligand species KD ( m M) kon (M71s71) koiexcl (s71) references

TCR MHC peptidea mouse 90^2 11 000^850 025^001 Davis et al 1998Savage et al 1999

TCR MHC SAg (SEA) mouse 23 4800 001 Redpath et al1999

CD8aa MHCI human 200^30 100 000^1200 18^005 Wyer et al 1999Garcia et al 1996

CD28 CD80 (B7-1) human 4 660 000 16 Van der Merweet al 1997

CTLA-4 CD80 (B7-1) human 04 940 000 043 Van der Merweet al 1997

CD2 CD48 rat 90^60 100 000 6 Van der Merweet al 1993

CD2 CD58 (LFA-3) human 22^9 400 000 4 Van der Merweet al 1994

CD62L GlyCAM human 105 100 000 10 Nicholson et al1998

a Range includes agonist peptides only for both MHC class I and II

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cytoplasmic domain) have any noticeable eiexclect on T-cellactivation or signal transduction To overcome theproblem of the non-planar nature of the T-cell^APCcouple we have also constructed a video pounduorescencemicroscopy apparatus which uses a moveable objective tocollect optical sections (20^30 at 05^10 m m thick)through the cell couples allowing us to view the interfacein three dimensions (Krummel et al 2000) Using thissystem we have been able to show that TCR^CD3 clus-ters rapidly to the synapse coincident with the centrstcalcium poundux and either remains there if activation isstable or dissipates and re-forms if it is not FurthermoreCD4 GFP initially co-localizes to the centre of the inter-face but then is dispersed to the outer edges soon after-wards This runs contrary to the notion of CD4 as a co-receptor that serves to stabilize TCR^peptide^MHCinteractions (Janeway 1992) and instead suggests that itmay be acting to potentiate only the early phases ofrecognition CD4 GFP lacking its cytoplasmic domainalso behaves in the same way so this pattern of movementcould not be orchestrated by a cytoskeletal linkage toCD4 but instead might be the result of some type ofphysical exclusion occurring at the centre of the synapse(Krummel et al 2000)

5 CONCLUSION

A summary of what we have learned thus far isschematized in centgure 3 Here we see a T-cell blast (thatis one that had been in contact with ligand a week or sobeforehand) encountering an APC of the `professionalrsquovariety (B cells dendritic cells or macrophages) whichpossesses co-stimulatory ligands If an initial survey ofthe APCrsquos membrane turns up a sucurrencient number (ca tento 100) of peptide^MHC complexes that can stably bindto its TCRs small clusters of TCR^CD3 CD4 can formperhaps purely by chemical attraction as seen in solutionby Reich et al (1997) and this may allow the centrst releaseof calcium from intracellular stores It also leads tosynapse formation in which much larger clusters of TCR^CD3 form probably driven by the co-stimulatory trans-port mechanism described above which also serves tocluster MHC molecules on the APC side by an eiexclectrsquo Bymeans and for reasons that are unknown synapse forma-tion is also accompanied by the segregation of LFA-1 andother molecules to the outer ring area where they arejoined by CD4 The establishment and maintenance ofsuch a synapse for minutes or more correlates with arobust (and irreversible) commitment to T-cell activationand the release of cytokines in the case of T helper cells(parallel studies on cytotoxic T cells have not yet beendone but will presumably yield similar results)

Why is synapse formation important We think thatT-cell recognition is an inherently `noisyrsquo process with alarge number of false starts While this noisiness is prob-ably a consequence of the extreme sensitivity of T cells tominute quantities of a particular antigen this wouldmake the danger of autoimmunity much more probableThus synapse formation may serve as a proofreadingprocess in which a larger area of the APC membrane issurveyed for ligand and only if additional examplesrsquo arefound does the Tcell commit to the eiexclectorrsquo phrase char-acteristic of full activation

Clearly we are just at the beginning of a new apprecia-tion of how cell surface molecules on T cells scan cells inthe body and arrive at a decision as to whether or not toachieve full activation This decision depends on cytoske-leton and signalling pathways inside the cell as well as theligands available to the T-cell surface molecules It is alsodependent on synapse formation although the purpose ofthis particular concentguration of cell surface molecules isnot clear beyond the clustering of TCR^CD3 complexesand the likely cascade of tyrosine kinase activity thatwould ensue Another mystery concerns the eventsleading up to the initial activation eventsoumlor ratherwhat distinguishes these from the random `noisersquo that a Tcell encounters from the large number of peptide^MHCson various cell surfaces that it would encounter everyworking day It is also mysterious how co-stimulatorysignals trigger the movement of cell surface moleculesand what diiexclerent signals through LFA-1 and CD28 aredoing (as they are synergistic they must be diiexclerent insome way) Thus we have many phenomena operating inthe same time-frame that are linked to the various cellsurface molecules and are somehow integrated to contri-bute to T-cell recognition Clearly more `viewsrsquo of greatermolecular clarity will be needed to properly understandhow this works but much progress has been made in thelast few years and it may not be long before we can graspthe main elements and their relationships

We thank the National Institutes of Health and the HowardHughes Medical Institute for research support and StephanieWheaton for expert secretarial assistance

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Cornall R J Cyster J G Hibbs M L Dunn A ROtipoby K L Clark E A amp Goodnow C C 1998Polygenic autoimmune traits Lyn CD22 and SHP-1 arelimiting elements of a biochemical pathway regulating BCRsignaling and selection Immunity 8 497^508

Davis M M Boniface J J Reich Z Lyons D Hampl JArden B amp Chien Y 1998 Ligand recognition by ab T cellreceptors A Rev Immunol 16 523^544

Dustin M L (and 10 others) 1998 A novel adaptor proteinorchestrates receptor pattering and cytoskeletal polarity in Tcell contacts Cell 94 667

Garcia K C Scott C A Brunmark A Carbone F RPeterson P A Wilson I A amp Teyton L 1996 CD8enhances formation of stable T-cell receptorMHC class Imolecule complexes Nature 384 577^581

Grakoui A Bromley S K Sumen C Davis M M ShawA S Allen P M amp Dustin M L 1999 The immunologicalsynapse a molecular machine controlling T cell activationScience 285 221^227

Irving B amp Weiss A 1991 The cytoplasmic domain of theT cell receptor plusmn chain is sucurrencient to couple to receptor-associated signal transduction pathways Cell 64 891^901

Janeway C A 1992 The T cell receptor as a multicomponentsignalling machine CD4CD8 coreceptors and CD45 in Tcell activation A Rev Immunol 10 645^674

Krummel M F Sjaastad M WIumllcentng C amp Davis M M2000 Diiexclerential clustering of CD4 and CD3plusmn during T cellrecognition Science (In the press)

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Phil Trans R Soc Lond B (2000)

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Matsui K Boniface J J Reay P A Schild H Fazekas de StGroth B amp Davis M M1991Lowacurrennity interactionof peptide^MHC complexeswithTcell receptor Science2541788^1791

Miller J F A P 1961 Immunological function of the thymusLancet 2 748^749

Monks C R Freiberg B A Kupfer H Sciaky N amp KupferA 1998 Three-dimensional segregation of supramolecularactivation clusters inTcells Nature 395 82^86

Nicholson M W Barclay A N Singer M S Rosen S Damp Van der Merwe P A 1998 Acurrennity and kinetic analysisof L-selectin (CD62L) binding to glycosylation-dependentcell-adhesion molecule-1 J Biol Chem 273 763^770

Redpath S Alam S M Lin C M OrsquoRourke A M ampGascoigneN R J1999 Cutting edge trimolecular interactionof TCR with MHC class II and bacterial superantigen shows asimilar acurrennity to MHCpeptide ligands J Immunol163 6^10

Reich Z Boniface J J Lyons D S Borochov N WachtelE J amp Davis M M 1997 Ligand-specicentc oligomerization ofT-cell receptor molecules Nature 387 617^620

Savage P A Boniface J J amp Davis M M 1999 A kineticbasis for Tcell receptor repertoire selection during an immuneresponse Immunity 10 485^492

Tsien R Y 1998 The green pounduorescent protein A Rev Biochem67 509^544

Van der Merwe P A Brown M H Davis S J amp BarclayA N 1993 Acurrennity and kinetic analysis of the interaction of

the cell adhesion molecules rat CD2 and CD48 EMBO J 124945^4954

Van der Merwe P A Barclay A N Mason DW Davies E AMorgan B P Tone M Krishnam A K Ianelli C ampDavis S J 1994 Human cell-adhesion molecule CD2 bindsCD58 (LFA-3) with a very low acurrennity and an extremely fastdissociation rate but does not bind CD48 or CD59Biochemistry 33 10149^10160

Van der Merwe P A Bodian D L Denke S Linsley P ampDavis S J 1997 CD80 (B7-1) binds both CD28 and CTLA-4with a low acurrennity and very fast kinetics J Exp Med 185393^403

Viola A Schroeder S Sakakibara Y amp Lanzavecchia A1999 T lymphocyte costimulation mediated by reorganizationof membrane microdomains Science 283 680

Willcox B E Gao G F Wyer J R Ladbury J E Bell J IJakobsen B K amp Van der Merwe P A 1999 TCR binding topeptide^MHC stabilizes a poundexible recognition interfaceImmunity 10 357^365

WIumllcentng C amp Davis M M 1998 A receptorcytoskeletal move-ment triggered by co-stimulation during T cell activationScience 282 2266^2269

WIumllcentng C Sjaastad M D amp Davis M M 1998 Visualizingthe dynamics of Tcell activation ICAM-1 migrates rapidly tothe T cellB cell interface and acts to sustain calcium levelsProc Natl Acad Sci USA 95 6302^6307

1076 M Krummel and others Thirty- six views ofT-cell recognition

PhilTrans R Soc Lond B (2000)

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