Structure and function of the T cell antigen receptor

Structure and function of the T cell antigen

receptor.

A Weiss

J Clin Invest.

1990;

86(4)

:1015-1022.

https://doi.org/10.1172/JCI114803

.

Research Article

Find the latest version:

http://jci.me/114803/pdf

Perspectives

Structure and Function of the T Cell

Antigen

Receptor

Arthur Weiss

HowardHughesMedicalInstitute,DivisionofRheumatologyand ClinicalImmunology, Departments ofMedicineandofMicrobiology

andImmunology, University ofCalifornia, SanFrancisco,SanFrancisco, California94143

Introduction

Themechanisms bywhich T lymphocytes recognize and

re-spond to antigen are of great importance in understanding

human disease and in thedevelopment of

therapeutic

inter-ventions for immune-mediated diseases. The Tcellresponse to

microorganisms

is

fundamentally important

forsurvival of thehost. Theimportanceof this response isexemplified bythe

impact ofthe current

epidemic

of theacquiredimmune

defi-ciency syndromein whichasubset of T cells is the target ofthe humanimmunodeficiency virus.TheTcellresponseto poly-morphic determinants ofthe

major

histocompatibility

com-plex

(MHC)'

molecules represents the

major

barrier tothe

successful

transplantation

of solidorgans and bone marrow.

InappropriateresponsesbyTcellsto

self-components

canlead

toautoimmune disease.

During the pastfewyears considerableprogress has been made in ourunderstanding ofthe molecular eventsinvolved

in T cellrecognitionand intheprocessesinvolvedininitiating theTcell responseto

foreign antigens.

Alarge number of cell surface molecules on the T cell have beenimplicated inthe

recognition events thatresult in theinitiation ofa Tcell re-sponse. However, of

primary importance

is theTcell

antigen

receptor(TCR)becauseitmustbeinvolved intheregulation ofall

antigen-specific

Tcell responses.

Tcell antigenreceptorrecognition: the uniquenature

of

the

ligand

UnlikeBlymphocytes which utilizemembrane

immunoglob-ulintorecognize antigenic determinants of native proteins, T cells use astructurally distinct

clonally

distributedreceptor to

recognize a different form ofantigen on the surface of an

antigen

presenting

cell(APC) suchasamacrophage,epithelial cell,or Bcell. T cells

simultaneously

recognizeantigenaswell aspolymorphic determinantson aself-MHC molecule,a

phe-nomenon termed "MHC restriction." Understanding MHC

restrictionrequires explanationofthe nature of theantigen as well the T cell receptor that not only recognizes antigen but alsoself-MHCmolecules.

The molecularcharacterization ofthe antigenperceived by

Receivedforpublication16May1990andinrevisedform3 July 1990. 1. Abbreviations used in this paper: APC, antigen presenting cell; MHC,major histocompatibility complex; TCR, T cellantigen recep-tor.

the T cell revealed that,in contrasttoB cells,T cells do not recognize native protein antigens. APC's process exogenous antigens through an acidic choroquin-sensitive endosomal compartment(reviewed in reference 1). Proteolytically treated

antigensorshortpeptidescansubstitute for nativeantigenin

stimulating T cells. This is consistent with the current view that T cellsrecognizeshortpeptidesderived from more

com-plex proteinantigensas aresult ofproteolyticprocessing. It is the source of thepeptide that determines whether it is presented in association with class I (HLA-A,B,C) or class II (HLA-DR, DP, DQ)MHC molecules. Peptidesderived from

proteinssynthesized endogenouslywithin theAPC,including

proteins derivedfrom viruses which have infected thecell,are

presented in association with class IMHC molecules(2). A

similar situation may applyforalloantigens (3). In contrast,

peptides derived from exogenous sources or from antigens which aretakenupthroughendocytic pathways,and

proteo-lytically processed, are presentedin association with class II

MHC molecules (4).

Theprecise mechanisms bywhich MHC molecules

asso-ciate with peptide antigensare notknown. With a few

excep-tionsinvolvingclass IMHC molecules(5),directpeptide

bind-ing has been demonstrated primarily with purified class II

MHC molecules and

peptides

(6). These

peptides

canassociate with class II MHC molecules on the surface oftheAPC, an eventwhichoccurs mostoften experimentallyinvitro, but in

vivo ismoreoftenthought tooccur when peptides generated

withinacompartmentlinkedtotheendocyticpathway

inter-act with classII MHC molecules (4). Peptides derived from endogenously

synthesized

moleculesappear tobindto

imma-ture forms oftheheavy chain ofclass I moleculesin a

pre-Golgicompartment(7). The mechanismresponsible for

degra-dation of endogenously

synthesized

proteins

topeptides isnot known(discussed inreference 2). Variability intheabilityof distinct

peptides

tobindto polymorphic classIIMHC mole-cules has beenwelldocumented (8).Asimilardegreeof

speci-ficity

exists for the interaction of peptides withclass I MHC molecules. Thespecificity of this interactionhelps toexplain

someofthe

previous

observationsonimmuneresponse genes. Theinteractionbetween the peptide and the MHC mole-cule has been mostclearly delineatedfrom the crystal structure

of classIMHCmolecules (9).Thestructure, as might be

per-ceived bythe TCR, consists oftwo a helices, which contain many ofthe class I MHC polymorphic residues, lying on a

floor of eight antiparallel, sheets. Together, theahelicesand , sheetsformagroove inwhichpeptide antigen lies. A similar structuralmotif isthought to holdfor class II MHC molecules. A model has been proposed in which distinct regions of the

TCR interactseparatelywith peptide antigen or with portions

ofthesurrounding MHCmolecule(10).

J.Clin.Invest.

©TheAmerican Society forClinical Investigation, Inc.

0021-9738/90/10/1015/08 $2.00

Structure ofthe T cell antigen receptor and its function

in

antigen recognition

TheTCRisaseven-chainmolecular complex with both vari-able and invariant components(Fig. 1) (1 1). It consists of an antigen/MHC(ligand)binding subunit, a disulfide-linked

het-erodimer (Ti), that is noncovalently associated with a

five-chain complex(CD3 6, E, y,and

{2)

that is thought to play a role in signal transduction (seebelow). The association of Ti with CD3 is intimate and obligatory. Chemical cross-linking of Ti f3 and CD3 y chains on unstimulated T cells has been

accomplished (12).Absenceofthe Ti or chainsin mutant cell lines prevents cell surface expression of the remaining chains

of the TCRcomplex (13, 14). The defective assembly ofthe TCRhas beenassociated withaselectiveTcell immunodefi-ciency(15).

Thestructuraldomains ofTi and CD3 responsible for their

physicalandfunctional association is of considerableinterest.

AfeatureofTi and CD3 that may be important in their

associ-ation is the unusual presence ofoppositely charged amino acids within the transmembranedomains ofall Ti and CD3

chains (1 1). Mutational analysis has provided evidence that the specific conserved basic residues ofTi are required for

structuralassembly ofthecomplex (16). More recentstudies with chimeric molecules demonstrate that the structural and

functional basis oftheassociationbetween Ti and CD3chains is contained within regions ofthe Tichains containingthese transmembrane domains (Tan, L., J. Turner, andA. Weiss, submitted forpublication). Thesedomains also appearto be

responsible for targetingunassembledchainsofthecomplexto a degradative pathway linked to the endoplasmic

reticu-lum(17).

On most human T cellswhich express CD4 or CD8, Ti

consists ofa disulfide-linked 40-44 kD ,B chain and a more

acidicachain of47-54 kD. Eachofthesechainshasconstant

and variabledomainsand eachis derivedfrom immunoglobu-linlikegenes (seebelow). Theaand pchainscontainall of the

information necessary for antigen and MHC

specificity.

cDNAs or rearranged

genomic

DNA

encoding

the a _{and ,B}

chains, derived fromcellswith well-characterized

antigen

and MHCspecificities, can transferboth antigenand MHC

reac-tivitytocell linesor toTcellsof

transgenic mice, respectively

(18, 19).

T

a

Extracellular

Membrane K|

A.

I CD3/T3 1

Cytoplasm

Figure1.Schematicrepresentationof thestructureofthe T cell anti-gen receptor. Thesingle-letter abbreviationsfor basic andacidic aminoacids contained withinthemembrane isshown.

Phosphoryla-tion sitesontheA, y, and 6chainsare notmeant todepict stoichi-ometryorpositionsof these modifications.

The

reactivity

ofTcellsfor

antigenic peptides

associated withclassI orII MHC moleculesis dependentontheselection of cells

expressing

TCRswith

specificity

forclassI orclass II MHC

during thymic

ontogeny

through

aprocess termed

thy-mic education. Immaturethymocytes which coexpress CD4

andCD8 cellsurface moleculesand express low levelsofTi

afi

are

"positively

selected" andallowed tocontinue their

devel-opmental

maturation

(20).

This maturational process

ulti-mately

results in CD8orCD4

single positive

cellsthat express

high levels of

a/fl

TCR which

recognize

class I or II MHC

molecules,

respectively.

However,

during

the maturational

process, elimination ofTcells whichexpress a/,8 TCR that

reacttoostrongly with self

antigens

occursthroughanactive suicideprocess

(21, 22).

Aberranciesin thesemechanismsmay

permit

the emergence ofautoreactiveTcells.

Cyclosporin

A can prevent clonal deletion in the

thymus resulting

in the emergence ofT cellswhich would otherwise be deleted

(23).

This may

explain

why some transplant

patients

or animals

treated with

cyclosporin

manifest autoimmune

phenomena

(24, 25).

Peripheral T cellsarecommonly divided into subpopula-tions based on their

expression

ofCD4 and CD8. The strong

biasofCD4+ andCD8+ Tcells

expressing

a/fl

TCRfor

ex-tracellularandintracellular infectious

organisms, respectively,

is determined

during thymic

selection and is also better

un-derstoodin thecontextoftheinteractionof the CD4 and CD 8 molecules with MHC molecules and the functions ofMHC molecules. CD8 binds to class I MHC molecules

(26).

This

helpsto

explain

why CD8 Tcells tend to be involved in the responseto

endogenously synthesized antigens

thatareclassI

MHC restricted. Most ofthese CD8 T cells have

cytolytic

function,

and this function contributestothe elimination of virally infectedcells.Incontrast, CD4 bindstoclass II MHC molecules

(27).

This is consistent withthe strongbias ofCD4 T cells, which usually function as

helper

cells, for

antigenic

peptides

derivedfrom exogenoussources orfrom the

endocy-tic

pathway

whichassociate withclass II MHCrestricted

anti-gens. These CD4 T cells

respond

to

_organisms

which do not

use hostcell

biosynthetic machinery.

OnadistinctsubsetofTcells which

generally

do not

ex-press CD4 or CD8,

0.5-10%

of

peripheral

T

cells,

another

heterodimertermed the Ti y6 isassociated withtheCD3

com-plex

(28).

Liketheaand /3

chains,

theyand a chainsarealso

derived from

immunoglobulinlike

genes

(reviewed

in

refer-ence

28).

However, unlike the cells that express Ti

a13,

the

antigen reactivity

ofthe cells

bearing

these receptors has beena matterof considerablecontroversy.

Cells

expressing

Ti

_yb

arethefirst

antigen

receptor-bearing

cellstoappear

during

thymic

ontogeny

(29, 30)

butthese

re-ceptors have very limited structural

diversity

and represent the precursors ofTcells that

populate

the murine

epidermis

(31,

32).

Later

during

ontogeny, the Ti 6y chains on

developing

thymocytes

exhibit greater

diversity (33).

In some

species

(chicken

and

mouse),

it has been shown thatTcells

expressing

the Ti

'y

referentially

localizewithin

epithelial layers

of tissues

(34,

35).

Thesecells may havea

unique

role incertainimmune responses to

antigens

that could be

presented

by epithelial

cells.

Alternatively,

these cells may represent an

_important

surveillance

mechanism, recognizing

altered

epithelial

tissue

that may be

injured by

environmentalexposure. The limited

diversity

in the Ti 6yin themurinedentritic

epidermal

Tcells has

suggested

that these receptorsreactwithawell conserved

antigen, i.e., heat shockproteins expressed bysuchdamaged

tissues (31). Reactivity to heat shock proteins by Tcells

ex-pressing Tiy6recentlyhas been reported (36, 37). Increases in Ti

y/6-bearing

cells have also been noted in granulomatous lesions of leprosy and cutaneousleishmaniasis(38). Moreover, mycobacterial antigen reactivity of y/6 Ti-bearing cells iso-latedfrom rheumatoid synovial fluid or fromsites of

myco-bacterial inoculationhas beendescribed (39,40).Hovever,the

preciseroleofTcellsexpressingTi

y6

in host immunityawaits thedevelopmentof better invitrosystemsandthe

demonstra-tionof theirpolyclonalresponses towelldefined antigens.The role of MHC molecules or class I MHC-related CDl gene products insuchantigen responsesremainstobedetermined. Both Ti af3 and

y6

areassociated withtheCD3 complex. CD3is comprised ofthe noncovalentlyassociatedhomologous 6, E, and y chains and associated disulfide-linked homo- or

heterodimers ofv-vor

_t-q

chains (11, 41, 42, 43). The v

chain has little homologytothe CD3 chainsbutishomologous

tothe ychainofthe IgE Fc receptorwhich isalso expressed as

adisulfide-linked dimer(44). Theqchainrepresents an

alter-natively spliced proteinproductof the chain gene (45). It has been suggested thatthe r-D or v-xndimers aredistinct sub-units of the TCR, separate from the CD3 complex, perhaps withdistinct functionsinsignaltransduction (see below) (43,

46). Recently, ther- s chain homodimerhas also been shown to be expressed on natural killercells in the absence of the TCR or other CD3 components (47). On these cells, the v

chain is associated with CD16(an Fc receptor) (48, 49) or as yetunidentified proteins(47).

Itiswidelyassumed that CD3 playsarole in

signal

trans-duction. This notion has been supported

by

the observation thatanti-CD3mAbsmimicthefunction of

antigen

in

activat-ingT cells and from studies with somaticcell mutantswith defective TCR-mediated

signal

transductionfunction

(50, 51).

Moreover, the structural complexity ofthe cytoplasmic do-mainsoftheCD3 chains (40-115 amino acids) (11,43)

com-paredto thescant information contained in the

cytoplasmic

tails oftheTi chains (5-12 amino

acids)

is consistent with this notion. CD3 ispresumedtoreceiveasignal fromthe ligand-occupied Tiandtransmit thisstateofreceptor occupancyby activating intracellular

signal

transduction mechanisms. The role ofconformational changes,

cross-linking,

oraggregation

in theactivation ofCD3isnotknown.

Genes

encoding

the

Ti

chains and their

functions

The genesencodingthe Ti a,

#,

-y, and 6chainsareorganized

ina mannersimilarto

immunoglobulin

(Ig)genes(reviewed in reference 52 and Fig. 2 A). During thymic ontogeny, these genes undergo recombination which involves thejoining of individual variable (V), diversity (D), andjoining (J) region

gene segments

(Fig.

2 B). As a result, a large repertoire of distinctTCRaregenerated anddistributedinaclonalfashion.

Rearrangement of these gene segments proceeds inanordered andregulatedprocess.

The mechanism ofTCR gene segment recombination is similar to the one for Ig genes in pre-B cells (53). During recombination, thejoining ofV/D

(fi

and

6),

V/J(a and y), D/D

(6),

orD/J(fiand6) regionsegmentsresultsin thelooping

outand deletion oftheinterveningDNA. The deleted

prod-uctshavebeenrecoveredfrommousethymusascircularDNA segments (54). In the severe combined

immunodeficiency

(SCID) mouse model the recombination processisdefective

A

V.-_IDC)

|_

k / A- fe oN rICXNCC HiC

aipnaodeI[a

beta

gamma

B

3erm',ne

Early rrymocyte

late-hyrnocyte

-FPer'ieva' T -el

delta alpha

D J C V J.-.SO C

-.,%nMRIII Mil MO I I *IMO

on-,4

V --4 J C1 J C2

x---~~~

Hw

1Xillo

4|-11-~~

_il

A

n

__0

d

il

_Hm

Ml

Figure 2.(A)Organization of theTcellreceptor genes.(B)Example of the orderedstochasticrearrangementofthe#chainof the Tcell

receptorduringTcell ontogeny.

and results in the imprecise joining of the gene segments

(55).

The defect in this mouse results in profound T and B cell defects. Whethersome casesof SCID inmanresult from de-fects in the process of recombination awaits further investiga-tion. The recombinase isthoughttoplayarole in the

numer-ouschromosomal translocations and inversions that involve the TCR genes in T celltumors(52).

Each of the human TCR genes hasadifferent organization

withvaryingdegreesofpotential diversitygeneratedby recom-bination of the availableV, D,and Jregionsegments(Fig.2). Becausethe TCR isatwo-chain structure, the various combi-nations of partner chains is quite large.However,nonrandom pairing ofthe protein products ofatleast theyand 6 chains has been welldocumented(31, 56, 57).This nonrandom pairing

doesnotnecessarilyreflectanincompatibilityof the V regions inassemblyofthe yand 6 chainproteins,but may result from

thymicorperipheralselection.

The potential diversity ofthe TCR would appear much smaller than forIgbecause theIgheavy chainlocuscontainsa

muchlargernumberofVregionsegments(inexcessof 1,000).

However, thepotential repertoireof TCRs has been estimated

tobe muchlarger(reviewedinreferences10and52). Diversity

is greatlyincreasedbyflexibilityatthe3'junction ofVandD

segmentjunctionswhen recombinedtoD orJ segments. In

addition, variable numbers of additional nucleotides

fre-quently found atthejunctions ofV/D, V/J, D/D, and D/J segmentsgreatlyincreasediversityof the TCR. These

nucleo-tides,termed Nregions,areaddedthroughtheactionof termi-nal deoxynucleotidyl transferase. Finally, the ability ofthe 6

locus to use a variable number ofD segments also greatly amplifiesthepotential for

diversity

because each segment used hasflexibilityatthemarginof thejunctionandvariable

num-bers ofN-region nucleotides are added at eachjoint. These

various mechanisms contributetothegeneration ofa

poten-I

iI

tiallyenormous repertoire of TCRs, estimated at1017and 1020

distinct

af:

and oyb heterodimers, respectively (10, 52). The unusual organization of the a/6 locus bears special mention. The 6 C, J,and D segments are located between the

J,,-

and

V-region

segments. Theaand 6locicanshare

V-region

segments, although onlyasmallnumber ofVregions actually appear to be utilized in the recombined 6 genes (28). As a consequence ofV-region segment recombination to Ja seg-ments,the 6 region C,J,and D segments are deleted. Specific regions flanking the 6 locus have been identified which appear toplayarole in6genedeletion(58). In

a/l

TCR-bearing cells, both alleles of the6locus are usually deleted.

Polymorphismsin the TCR loci have been described. Use

ofdifferentconstantregionsegments as well as an allelic poly-morphism of the constant region of the ygene account for the observed disulfide- and nondisulfide-linked protein products

of different sizes(28). PolymorphismsinV regionshave also been observed. Atleastoneofthesepolymorphismshas been

associatedwithahumandisease, multiple sclerosis(59). Recent studies suggestimportant insights into the

patho-genesisandtherapyof diseasemay be obtained by the

identifi-cation ofparticularTCRVregion segments thatare preferen-tiallyused in responsestodefinedantigensand in

autoimmu-nity. Progress inidentifyingTCR Vregion segments that are

preferentiallyexpressed in complex mixturesofresponding T cells hascomefrom the isolationofsomeTCRV

region-spe-cific monoclonal antibodies and the application of quantita-tive polymerase chain reactiontechnology.Anexcellent exam-ple ofan oligoclonal response ofpathogenic T cells is the rodent model ofexperimental allergic encephalomyelitis

(EAE) (60). These T cells respond to peptides derived from

myelin basic protein and expressa restricted set of TCR V,

and

_V#

gene segments. Development of diseasehas been pre-vented in mice by usingpeptide analogues ofthepathogenic peptide (61)orby vaccination with

synthetic

peptidesderived

fromtheVa sequenceoftheTCRexpressed bythepathogenic

Tcells (62).Established active EAEin mice has been treated

successfully with monoclonal antibody reactivewith this

_V's

(63). Inspired bythesestudiesinrodents, recentexamination ofpatients with

multiple

sclerosishassuggested the

involve-mentofoligoclonalTcell populations (64).Toxic shock syn-drome may also result from thepolyclonal activationofTcells which expressparticularV: region-derived products reactive

with staphylococcal enterotoxins (65). These observations

suggest

therapeutic

approachestowards

preventing

or

treating

human diseasesinvolving oligoclonalTcell responses.

Somatic mutation ofTCR genes doesnotappeartooccur.

Thisis in markedcontrast toIg genes where somaticmutation

playsamajorrole ingeneratingantibodies withhigher

affini-tiesforantigen. Somaticallymutated residues inIgarefound

throughoutthevariableregion.Incontrast, theoverwhelming

degreeofvariability ofthe TCR iscontainedin the sequences

ofthe VDJ junction, comparable to the CDR3 domain of

immunoglobulinwhichis involved in formation of the pocket

oftheantibody bindingsite. Theseobservationsledtoamodel for antigen recognition by the TCR, which emphasizes why somatic mutation ofTCR genes would notbe desirableand thatthevariabilityin the TCR should be concentrated in the VDJjunction (10). Accordingtothis model, the TCRaand d chainsassumeaconformation similartothat Ig. TheV

regions

makecontactwithMHCmoleculeahelices,whereasthe VDJ

junction interactswith the associated peptide. Somatic

muta-tion would impairMHC recognition, thusexplaining the ab-senceof somatic mutationof the TCR. This model is consis-tent with the limited number of TCR V regions that have

evolved fortherequisiteMHCrecognitionand places a greater importance upon VDJ junctional diversity for peptide antigen

recognition.

Role

of the

T

cell antigen receptor in signal transduction

Theinitiationof an immune response not only requires recog-nition of antigen by the TCR, but this recogrecog-nition event must betranslatedintoatransmembranesignal.This signal, in turn, leads to a cascadeofintracellular eventswhichinfluence cel-lularresponses such as the transcriptional activation of

lym-phokineandlymphokinereceptor genes, cell proliferation, or

activation ofthe cytolytic effector mechanism. Although transmembrane signalling by the TCR may not be the sole

signal transductioneventrequired forTcellactivation, it

cer-tainly mustplay aprimary role inregulating antigen-specific activation ofTcells.Defectivesignal transductionby the TCR has beenassociatedwith acongenital immunodeficiency syn-drome(66).

Two early signal transduction pathways are activated by

stimulation ofthe TCR: theinositolphospholipidsecond

mes-sengerpathwayandatyrosine kinase pathway. Stimulation of

Tcells byantigenoranti-TCRmAbs induces rapid, large, and

sustained increases in cytoplasmic free calcium

([Ca2+[i)

as

well as theactivation ofthe calcium- and

phospholipid-depen-dent serine and threonine kinase, protein kinase C (PKC). Manystudiessuggest that thesebiochemical changesare physi-ologically importanteventsinitiatedas aresultofTCR

stimu-lation(reviewedinreference 67).

The rise in

[Ca2+]i

and theactivation ofPKC have been observed inawidevarietyof cells in responsetostimulation of manydistinct receptorswhich regulate theinositol

phospho-lipid second messengerpathway (68). This involves

receptor-mediated activation of a family of intracellular enzymes, termed

phospholipase

C(PLC) (69).Theisozyme ofPLC that is activated by TCR stimulation isnot known. All of these enzymes cleave

phosphatidylinositol 4,5-bisphosphate

to

yield

twopotent intracellular second messengers: inositol

1,4,5-tris-phosphate

_(UP3)

and diacylglycerol (DG). These two potent second messengers are

responsible

for the mobilization of

[Ca2"]J

and theactivation ofPKC,

respectively.

The initial rise in

_[Ca2"]J

isthoughttoresult from the

ac-tionofIP3ona

specific

receptor within the

endoplasmic

retic-ulum which causes the release of calcium from

sequestered

stores(70). The sustainedincrease in

_[Ca2+]i

depends

upona

transmembrane fluxof calcium (71)and may involve the

re-sponseofanill-defined calciumchanneltoinositol

phosphates

(72). However, it ispossiblethatapassivetransmembrane flux is only required to replenish inositol-sensitive intracellular

storesof calcium.

While the precise mechanism by which the rise in

[Ca2+],

leadstosubsequent cellular responses isnotknown,it is

likely

thatcalcium-activatedkinasesplayan

important

role. A

sus-tained rise in the

[Ca2+]i

appearsto be necessary for several later cellular responses(73, 74).The bestcharacterized ofthese cellular responses isthe

transcriptional

activation of the IL-2 gene.Sustainedsecond messenger

generation

may be

required

because theactivation ofthe IL-2 gene isnota

primary

gene

activationevent.Itdependsupon theprior transcriptional

ac-tivation of other immediate early acac-tivation genes(75). Indi-rectevidence would suggest that animportantaction of cyclo-sporin A,apotentimmunosuppressive agent used commonly in modem transplantation, is to inhibit events which result from this increase in

_[Ca2+]j,

but before the transcriptional activation of the IL-2 gene (75, 76).

The other second messenger derived from the inositol phospholipid pathway is 1,2-diacylglycerol. Both diacylgly-ceroland phorbol estersactivate PKC(77). Several different

forms ofPKC have beenidentifiedbut theimportanceof the various isozymes isnotknown.Theseisozymescanbe differ-entially expressedin Tcells. Several T cell responses are not

dependentonPKC,3(78).HowPKC contributestolater cel-lular responses onceactivated is not clear. It is likelythat a

cascade of intracellular substrates may be involved in the

re-sponse,

including

PKC-activated

transcriptional

factors (75).

Inaddition, two PKC substrates are componentsofthe TCR

complex, the CD3 y and a chains (79).

Phosphorylation

of thesechainsmay be involved inregulatingTCR function.

HowtheTCRactivates PLC isnotclear.Evidence froma

varietyofsystemshas suggestedthatguanine nucleotide bind-ing(G)proteinsmayserve tocouplesomereceptors,

particu-larly thosewithseventransmembranedomains, toPLC(80).

The TCR contains seventransmembrane domains, albeit in-volving seven polypeptidescomprisingthe receptor complex.

Indirect evidence suggests that the Tcell antigen receptor is

coupledtoPLCviaaGprotein. However, alternative

mecha-nismsinvolvingthetyrosine kinase pathwayin theregulation ofPLCactivation have recentlygained support(see below).

Indeed, relatively little progresshas been made inidentifying proteins that interact with the receptor uponligand binding.

However,recently, twointegralmembraneglycoproteins of34 and38kDhave beenidentified which interact withthe

recep-toruponligand

binding

(81).Thefunction oftheseproteins is

not known, but they mayplay a role in signal transduction

because they do not interact with the TCR in somatic cell mutants that are defective in TCR-mediated activation ofPLC.

TheTCR alsoactivatesatyrosine kinase activitythatisnot

intrinsic to the TCR (82, 83). Stimulation ofthe TCR with antigen orwith mAbs results inthephosphorylationon

tyro-sine residues ofseveral

proteins

including

the TCR v chain.

Candidates forthekinase includelck and

fyn,

membersofthe

src

family

of tyrosine kinases (84, 85). Ithas been shown that

lck physically interacts withCD4 and CD8(86). Cross-linking ofCD4 results inincreased Ick activityand the phosphoryla-tion ofthe TCR rchain (87). Veryrecent studiessuggest an

interaction between the TCR and fyn(88). How the TCR is

coupledtothetyrosinekinasepathway isnotknown, butit is independent ofthecoupling of the TCR to PLC because

hy-bridoma variants which are defective in TCR-induced PLC

activationstillactivatethetyrosine kinasepathway (83).

Insight into the function ofthe tyrosine kinase pathway may comefrom studies ofthe CD45(T200)familyof proteins. Theseproteins, whichareexpressed on all leukocytes

includ-ingTcells, have duplicated tyrosine phosphatase domains in

their cytoplasmicdomains (89). The regulationofatyrosine kinasepathway could be intimatelylinkedto theregulation of

this

tyrosine

phosphatase. In supportof this notion, aTcell clone that fails to express CD45 does not produce IL-2 or

proliferate in response to antigen or anti-TCR mAb (90). Moreover, recentstudieswith a CD45 negative cell line suggest that the function of CD45 is essential for the activation of PLC by the TCR (91). Increased tyrosine phosphorylation of lck has been observed in some CD45 negative mutants (92). These studies suggest that CD45 mayregulate the activity of T cell tyrosine kinases and together they may have a complex rela-tionship with the PLC that is activated by the TCR.

Whereas the relationship of the tyrosine kinase pathway

and the inositol phospholipid pathway has not been estab-lished, the studies performed with CD45 negative mutants

suggest that thetyrosine kinase may regulate the activation of PLC.This is supported by kinetic studies that suggest that the tyrosine kinase is activated before PLC (93). Direct phosphor-ylation of PLC and itsassociation with stimulated epidermal

growthfactor and theplatelet-derivedgrowth factor receptors has been observed (94). Although the enzymatic activity of PLC was not shown to be altered by such phosphorylation, such studies suggest that this event may be important in

re-ceptor-mediated activation of the inositolphospholipid

path-way. Thus, in T cells, activation ofa tyrosine kinase may represent theprimary event which thenserves to activate or

regulatePLCactivity,possiblybyphosphorylation.

Therelative importance ofthetyrosine kinaseand inositol

phospholipid pathways in later cellular responses associated with T cell activation is not known. The requirement and

involvementoftheinositolphospholipidpathway in the

acti-vation of T cells leading to IL-2production was recently chal-lenged bytheisolation ofamurineTcellhybridoma variant

which produced normal levelsofIL-2 uponTCRstimulation

butfailedtomanifestdetectableinositol phospholipid second

messengerproduction (95). Theexplanation forthedefectin

thiscell has beenrelatedto recentstudies which indicatethata

distinct form oftheTCR,containing

r-q

dimers, is coupled

tothe inositolphospholipid pathway (46).

Fromthestudiesreportedtodateitwould appear that the

activation of theinositol phospholipid pathwaycanleadtoT cell activation responses. Thishasbeen furthersupported by more recentexperiments inwhichaheterologous receptorthat

activatestheinositol phospholipidpathway, the human

mus-carinic receptorsubtype 1, whenexpressed in T cellscan in-duce T cell activation responses (Desai, D., and A. Weiss, unpublished data). Nevertheless, in order to understand the

relative contributions ofthe twopartially characterized signal

transduction pathways and any other signal transduction

events regulated bythe TCRaswell as by other cell surface

molecules on the T cell, more progressin understanding how

signal transduction eventsinitiated attheplasma membrane regulate subsequentintracellular responses is required.

Conclusion

The Tcellantigen receptorisanextraordinarily complex cell

surfacereceptor. Its

importance

in

regulating

T cell recogni-tion andactivation makesit a critical component in all host

immuneresponsesfortheclinician.Moreover, the studyofthe Tcell receptor has proved to be an important model not only

fortheimmunologist, but alsoforthe moleculargeneticistto

studyTCR generegulationandrecombination as well as lym-phokine gene regulation, for the developmental biologist to

studythymic ontogeny, for thecell biologistto study the

and for the physiologist to study signal transduction. Thus, the study ofthe TCR is likely to yield important insights into basic biologicalprocessesand human disease.

Acknowledaments

The author would like to thank Dr. Gary Koretzky forhis critical reading of the manuscript. This work wassupported inpart by Na-tional Institutes of Health grantGM39553.

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