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Part 1 of this review will highlight the basic components and

signaling pathways by which the T-cell antigen receptor (TCR)

activates mature extrathymic T cells. TCR signaling com-

mences with an early wave of protein tyrosine kinase activa-

tion, which is mediated by the Src kinases Lck and Fyn, the70-kd -associated protein kinase, and members of the Teckinase family. This early wave of protein tyrosine phosphoryla-

tion leads to the activation of downstream signaling pathways,

including an increase in intracellular free calcium, protein

kinase C, nuclear factor B and Rasmitogen-activated pro-

tein kinase activation. These pathways activate transcription

factors, such as activator protein 1, nuclear factor of activated

T cells, and Rel proteins, which ultimately lead to the expres-

sion of genes that control cellular proliferation, differentiation,

anergy, or apoptosis. This review also describes how costimula-

tory receptors assist in signal transduction and assembly of

macromolecular complexes at the TCR contact site with the

antigen-presenting cell, also known as the immune synapse.

These basic concepts of TCR signal transduction will be used

in part 2 to explain how T-cell function can be altered by ther-apeutic targeting of TCR signaling components, as well as to

explain modification of TCR signaling during TH1/TH2 differ-

entiation, tolerance, and immune senescence. (J Allergy Clin

Immunol 2002;109:758-70.)

Key words:T-cell activation, TCR, signal transduction

The coordinated activation of T cells by foreign anti-gen leads to clonal expansion, differentiation, cytotoxickilling, or induction of programmed cell death. T-cellactivation is initiated by the T-cell antigen receptor(TCR), which is comprised of a ligand-binding subunit,

the and chains, and a signaling subunit, namely theCD3,and chains and the TCR chain (Fig 1).1,2 Thephysiologic ligand for the TCR is foreign peptide boundto the MHC expressed on antigen-presenting cells(APCs), including dendritic cells, macrophages, and Bcells.3,4 Although signals generated by the TCR deter-mine the specificity of the T-cell response to antigen,costimulatory receptors, such as CD2, CD28, CD4, CD8,

Molecular mechanisms in allergy and clinical immunology(Supported by a grant from Merck & Co, Inc,West Point, Pa)

Series editor: Lanny J. Rosenwasser, MD

T-cell activation through the antigen

receptor. Part 1: Signaling components,signaling pathways, and signal

integration at the T-cell antigen receptor

synapse

Andre E. Nel, MDLos Angeles, Calif

From the Division of Clinical Immunology/Allergy, Department of Medicine,UCLA School of Medicine, University of California, Los Angeles.

Supported by a United States Public Health Service grant (RO-1 AG14992)

and a grant from the UCLA Asthma, Allergy, and Immunologic Disease

Center (PO-1 AI50495).

Received for publication February 13, 2002; revised February 14, 2002;

accepted for publication February 18, 2002.

Reprint requests: Andre Nel, MD, Division of Clinical Immunology/Allergy,

UCLA School of Medicine, 10833 Le Conte Ave., Los Angeles, CA

90095-1680.

2002 Mosby, Inc. All right reserved.

0091-6749/2002 $35.00 + 0 1/10/124259

doi:10.1067/mai.2002.124259

Abbreviations used

AP-1: Activator protein 1

APC: Antigen-presenting cell

[Ca2+]i: Intracellular free ionized calciumGEF: Guanine nucleotide exchange factor

Grb2: Growth factor receptorbound protein

Gads: Grb2related adapter downstream of Shc

Itk: Inducible T-cell kinase

IB: Inhibitory B protein

IK: IB kinase

IP: Inositolphospholipid

ITAM: Immunoreceptor tyrosine-based activation motif

JNK: N-terminal c-Jun kinase

ERK: Extracellular signalregulated kinase

LAT: Linker for activated T cells

LFA-1: Lymphocyte functionassociated antigen 1

MAPK: Mitogen-activated protein kinase

NF-B: Nuclear factor B

NFAT: Nuclear factor of activated T cellsPKC: Protein kinase C

PLC: Phospholipase C

PTK: Protein tyrosine kinase

PH: Plextrin homology domain

PI-3 kinase: Phosphatidyl inositol 3 kinase

PI-4,5-P2: Phosphatidyl inositol 4,5 biphosphate

PTB: Phosphotyrosine-binding domain

SLP-76: SH2 domaincontaining leukocyte protein

of 76 kd

SH2/SH3: Src homology type 2 or 3 domain

SMAC: Supramolecular activation cluster

TCR: T-cell antigen receptor

Sos: Son of Sevenless

WASP: Wiskott-Aldrich syndrome protein

ZAP-70: Chainassociated protein kinase of 70 kd


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and integrin molecules, contribute to signal transductionby modulating the response threshold.5,6

This review will highlight the major aspects of TCRsignaling in the peripheral or mature T-cell compartmentbut will not address TCR signaling in the thymocyte com-partment, which has been addressed elsewhere.7 TCR sig-naling commences with an early wave of protein tyrosinekinase (PTK) activity, which is mediated by the Src kinas-es Lck and Fyn, the 70-kd chainassociated proteinkinase (ZAP-70), and members of the Tec kinase family,inducible T-cell kinase (Itk), Tec, and Txk/Rlk.8,10 Thisearly wave of protein tyrosine phosphorylation leads tothe activation of downstream signaling pathways, includ-ing increases in intracellular calcium flux, protein kinaseC (PKC), nuclear factor (NF) B, and Rasmitogen-acti-vated protein kinase (MAPK) activation.9,11 These path-ways activate transcription factors that ultimately lead tothe expression of genes that control specific cellularresponses.12,13 This review will also describe how cos-timulatory receptors modify TCR signaling through

assembly of signaling components at the TCRsynapse.5,14,15 In the second part of this review, we willdiscuss how TCR signaling is modified under differentbiologic circumstances and how pharmacologic interven-tion in TCR activation pathways can be used to modulatethe function of the immune system.

INITIAL PHASE OF TYROSINE PROTEINKINASE ACTIVATION, INCLUDING

PHOSPHORYLATION OF IMMUNORECEPTORTYROSINE-BASED ACTIVATION MOTIFS

The earliest recognizable event after TCR engagementby antigen is the induction of tyrosine protein phosphory-

lation by the Src kinases Lck and Fyn (Fig 1).2,8,9

Howexactly these Src kinases are activated is unclear, but itinvolves maintenance of an activation-competent state bythe removal of inhibitory C-terminal tyrosine phosphateresidues by members of the CD45 tyrosine phosphatasefamily (Fig 1,A).16 To do so, CD45 participates in the for-mation of multimeric complexes with the CD4 receptor,which is physically associated with Lck.16-18 The removalof the C-terminal phosphate residue leads to Src kinaseunfolding and activation.17,18 This conformational changealso frees up the autologous Src homology 2 (SH2domain), which allows Lck to interact with new partners,including ZAP-70 (Fig 1,C).19 Another contribution to Srckinase activation comes from the sorting of receptor-asso-

ciated PTKs and tyrosine phosphatases at the TCR contactsite with the APC.5 According to the kinetic segregation ortopological constraint theory of T-cell activation, the limit-ed space between the T cell and its participating APC leadsto exclusion of large receptors, including the tyrosine phos-phatases CD45 and CD148, but allows smaller receptorsthat regulate PTK activity to remain in the contact zone.5,20-24

This receptor sorting is critical to the forming of the TCRsynapse, which is further explained in Box 1.

Once activated, the Src kinases regulate the activationof ZAP-70 and the Tec kinases.2,8,10,25 This hierarchy is

maintained by recruiting the tandem SH2 domains ofZAP-70 to a recognition motif, the immunoreceptor tyro-sine-based activation motif (ITAM).2,26 These ITAMs con-tain the consensus sequence (D/E)XXYXXL(X)6-8YXXL,in which both tyrosines (underlined) serve as substrates forthe Src kinases (Fig 1,B and C).2 These ITAMs appear asa single copy on the CD3,, and chains and as a tripli-cate repeat on the chain, thereby contributing 10 motifsto each TCR complex (Fig 1). ITAM phosphorylation by apool of active Src kinases is continuously opposed by tyro-sine phosphatases.20 The repressive effect of the tyrosinephosphatases is removed during the formation of the TCRsynapse (Fig 2), allowing the Src kinase to dominate thephosphorylation status of the ITAMs.5

After it is attached to the ITAM motifs, ZAP-70 is phos-phorylated by Lck, leading to activation of the latter kinase(Fig 1,C).27 In addition, Lck interacts directly with ZAP-70 through the binding of the Lck SH2 domain to theZAP-70 pY319 residue.28 This interaction is critical forsustained ITAM phosphorylation, leading to the recruit-

ment of additional ZAP-70 molecules and Lck substrates,such as the Tec kinase, Itk.20 It is important to point outthat not all the ITAMs are phosphorylated on every occa-sion the TCR makes contact with an antigen. Instead, thestoichiometry of ITAM phosphorylation and the numberof ZAP-70 molecules recruited are dictated by the affinityof the TCR for its peptide ligand.29,30 This number is lim-ited in case of low-affinity interactions but increases as theaffinity increases. The stoichiometry of ITAM phosphory-lation therefore serves as a signaling amplification mecha-nism.31 In this regard the TCR chain migrates as either21- or 18-kd peptides during SDS-PAGE, depending onthe stoichiometry of ITAM phosphorylation.29,30 Theaccompanying change in the signaling competency of the

chain is further discussed in part 2 of this review.

COSTIMULATORY RECEPTORS AFFECTEARLY PTK ACTIVATION

In addition to their role in stabilizing TCR interactionswith the MHC, CD8 and CD4 play an active role in ini-tiating PTK activity.16 Although it was originally thoughtthat these Lck-binding coreceptors chaperone the kinaseto the ITAMs, more recent data suggest that CD4 andCD8 may actually interact with the MHC after TCRbinding to the peptide-MHC complex (Fig 1).17,32 Oneexplanation is that interaction of the Lck-SH2 motif withthe ZAP-70 pY319 residue redirects CD4 or CD8 recep-

tors to the peptide-MHC complex (Fig 1, C).28

Onceanchored at this site, CD4 or CD8 amplifies protein tyro-sine phosphorylation by stabilizing the interactionbetween Lck and ZAP-70.

CD28 is another costimulatory receptor that promotesa generalized increase in protein tyrosine phosphoryla-tion.33 One explanation is that the intracellular tail ofCD28 associates with PTKs, including Lck, Tec, and Itk(Fig 3).34,35 Although CD28 ligation by B7-1 (CD80) andB7-2 (CD86) ligands induces weak protein tyrosine phos-phorylation, a major effect of this receptor is to enhance


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induction of protein tyrosine phosphorylation by theTCR.36,37 An important advance in understanding thissynergy has been the discovery that CD28 regulates theassembly of post-TCR components through its effects onlipid rafts.33 This concept is discussed later on. Suffice tomention here that rafts are membrane domains that areenriched for Src kinases and other signaling components,suggesting that CD28 costimulation leads to the recruit-ment of PTKs to the TCR synapse, thereby enhancing the

ability of the latter receptor to phosphorylate Vav, c-Cbl,p62dok, phospholipase C (PLC) 1, Lck, and Itk.36,37

ACTIVATION OF SIGNALING CASCADESDOWNSTREAM OF THE PTK CASCADE

The early wave of PTK activity leads to the recruitment,rearrangement, and activation of additional signaling mol-ecules at the TCR contact site with antigen.11 The ITAMs,

FIG 1. The role of the TCR/CD3 complex and the CD4 receptor in the initiation of early protein tyrosine phos-

phorylation. A, On binding to the peptide/MHC complex, the earliest recognizable event is activation of the

Src-kinases, Lck and Fyn. This requires removal of a C-terminal phosphate (red dot)by the tyrosine phos-

phatase, CD45. This allows the kinase to unfold and to phosphorylate ITAM motifs (blue rectanglesin the

intracellular domains of CD3, , , and ). Tandem ITAM phosphorylations are required for the recruitment

of ZAP-70, which attaches by a pair of SH2 domains (yellow half circles). B, Immobilized ZAP-70 is phos-

phorylated and activated by Lck, which interacts directly with ZAP-70. CD4 interacts with nonpolymorphic

MHC domains, serving to stabilize TCR/ligand interactions and promoting further tyrosine phosphorylation.

Once activated, ZAP-70 phosphorylates substrates such as LAT, SLP-76, and Vav. Lateral displacement of

CD45 and other tyrosine phosphatases from the TCR/APC contact site promotes unopposed PTK activity.


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as well as the adapter proteins, play a key role in the assem-bly of post-TCR signaling complexes. (For a discussion ofadapter molecules, see Box 2 and Fig 4.) Downstream sig-naling events include the activation of Ras and Rho-familyGTPases, MAPK cascades, phosphatidylinositol 3 kinase(PI-3 kinase), PKC, and the NF-B pathway. In addition,

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TCR ligation leads to the phosphorylation and activation of

PLC-1, which initiates inositolphospholipid (IP) turnoverand intracellular free ionized calcium ([Ca2+]i) flux. Asummation of these effects leads to transcriptional activa-tion of biologically important genes.

Contribution of IP turnover, [Ca2+]i flux, andPKC activation to TCR signaling

PLC-1 tyrosine phosphorylation is one of the majordeterminants for Ca2+ signaling. Although it was initiallyproposed that Lck regulates PLC-1 phosphorylation, it isclear that this event involves 3 different types of PTK

FIG 2. The kinetic segregation model of TCR triggering and for-

mation of the immunologic synapse. The presence of large mole-

cules (eg, CD45, CD43, and other glycoproteins) prevents close

membrane contact between the T cell and APC, thereby acting to

constrain engagement of the peptide-MHC complex (A). By com-

parison, the TCR-CD3 complex and accessory receptors (eg, CD4

and CD28) are much smaller, which requires large molecules to

migrate laterally to allow the smaller receptors to interact. This

small-scale segregation of receptors leads to the formation of a

specialized contact zone known as the immunologic synapse (B).

Because large-sized tyrosine phosphatases (eg, CD45) are exclud-

ed from the synapse, tyrosine kinases dominate, preparing the

receptor for triggering. TCR triggering, with the assistance of co-

stimulatory receptors, initiates active large-scale segregation of

receptors, signaling molecules, and cytoskeletal elements, lead-

ing to the formation of SMACs (Fig 3).

Box 1. Rearrangement of TCR-associatedsignaling molecules, leading to the

formation of the immunologic synapseand SMACs5,15,16

The extracellular portions of the TCR and peptide-MHC molecules are small (approximately 7 nm) com-pared with several highly abundant cell-surface mole-cules, such as CD45 (approximately 28-50 nm) andCD43 (approximately 45 nm), and adhesion mole-cules, such as LFA-1 (approximately 21 nm, Fig 2).5,20

Before cell-cell contact, the TCR-CD3 complex is sub-ject to constitutive tyrosine phosphorylation anddephosphorylation. Dephosphorylation dominates,leading to low-level ITAM phosphorylation. InitialLFA-1intracellular adhesion molecule 1 interactionsovercome the barrier to cell-cell contact by means ofnegatively charged glycoproteins (eg, CD43), whichdispel each other at 50- to 100-nm separation of thecell membranes.21 Integrin binding stops the T cell

from migrating and generates a central integrin-enriched area surrounded by a close-contact regionthat includes the bulk of the engaged TCRs.21,23 Sub-sequently, the TCR and some of its accessory receptorsare transported to the center of the contact area, and theintegrins are forced into a surrounding ring (Fig2).15,22,23 This molecular arrangement is known as theimmunologic synapse (Fig 2) and is responsible forconcentrating the TCR, CD3, CD4, and CD28 in thecentral contact zone at the expense of larger cell-sur-face receptors, such as CD43 and the family of CD45tyrosine phosphatases (Figs 2 and 3).5,20,21

According to the kinetic segregation theory of T-cell activation, this process of receptor sorting leads

to the domination of tyrosine phosphorylation at thecentral contact zone. Accordingly, signaling mole-cules, such as PTKs (Lck and Fyn), PKC, adapterproteins, and GTP-binding proteins (eg, Rac) can beseen to concentrate in the central zone (Fig 3). Thisassembly of receptors and signaling molecules is alsoknown as SMACs, which are divided into central andperipheral zones (Fig 3).15 High-affinity TCRengagements within these stabilized zones result insustained tyrosine phosphorylation of the TCR-asso-ciated ITAMs, leading to receptor triggering.21 How-ever, in case of low-affinity TCR engagement by thepeptide-MHC complex, the immunologic synapse isunstable and results in dephosphorylation of the

ITAMs on leaving the close-contact zone. This leadsto a modification of TCR signaling, as will be dis-cussed in the second part of this review.


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activity, namely Lck, ZAP-70, and Tec kinases (Fig 4,A).45 Two adapter molecules, linker for activated T cells(LAT) and SH2 domaincontaining leukocyte protein of76 kd (SLP-76), contribute to the formation of that sig-naling module (Fig 4,A).39,45 Consensus phosphotyrosineresidues (Box 2) are responsible for the recruitment of thePLC-1 SH2 domains to LAT and the vicinity of the sur-face membrane.40 The role of SLP-76 in this setting is therecruitment of a Tec kinase, which ultimately phosphory-lates PLC-1 (Fig 4,A).45 LAT stabilizes SLP-76 binding

to this macromolecular complex through the interpositionof an adapter protein, Grb2related adapter downstreamof Shc (Gads; Fig 4,A).45

PLC-1 tyrosine phosphorylation leads to its catalyticactivation and cleavage of phosphatidyl inositol-4,5biphosphate (PI-4,5-P2), which is located in the innerleaflet of the plasma membrane (Fig 5). This generatesinositol-1,4,5-trisphosphate, which releases Ca2+ fromendoplasmic reticulum storage sites (Fig 5).46 Once thesestores are depleted, store-operated Ca2+ channels in the

FIG 3. Cross-section of the mature immunologic synapse showing the arrangement of receptors, signaling

molecules, and cytoskeletal proteins in the SMACs. In the mature synapse TCR/CD3, CD28, and several sig-

naling molecules congregate in the center of the SMACs, also known as the cSMAC. A second group of mol-

ecules, including the adhesion receptors LFA-1 and intracellular adhesion molecule 1 and the cytoskeletalprotein talin form a ring around the cSMAC, to form the outer ring of the peripheral or pSMAC. The inner

ring of the pSMAC includes the CD2 receptor, which interacts with LFA-3 or CD48 on the opposing APC.

Box 2. Adapter proteins and theirinteractive domains

Adapter proteins are defined as molecules that lack intrinsic enzymatic activity yet are able to contribute to sig-naling by mediating intermolecular interactions.38 Key adapter proteins involved in T-cell signaling include LAT,SLP-76, Grb-2, and Gads.38 LAT is a type III transmembrane protein with a long cytoplasmic tail that includes 9 tyro-sine-based motifs.39,40 When phosphorylated by ZAP-70, these motifs serve as docking sites for specific SH2 pro-teins, including PLC-1, Grb2, PI-3 kinase, and Gads (Fig 4).

40 As a result, LAT controls [Ca2+]I flux and Ras/ERK,NFAT/AP-1, and PI-3 kinase activation.40 Importantly, LAT contains a palmitoyl tail,41 which allows it to concentrate

in lipid rafts. SLP-76 is an adapter protein with SH2- and proline-rich motifs that associate with Vav, Gads, Itk, LAT,and Grb2 (Fig 4).38 The roles of Gads and Grb2 are discussed above. Other adapter molecules that play a role in T-cell activation include the Cbl family, the Crk family, the Dok family, Lnk, Nck, Shc, Src-like adapter protein, Src-like adapter protein 130/Fyb, and TCR-interacting molecule; these are reviewed elsewhere.38

In addition to SH2 domains, intermolecular interactions are mediated by SH3, phosphotyrosinebinding (PTB),and PH domains (Fig 4).42,43 Generally speaking, these are modular structures comprised of 40 to 150 amino acids.The SH2, SH3, and PTB domains are shaped into ligand-binding pockets that recognize 3 to 6 amino acid motifs inadjacent proteins. SH2 domains are phosphotyrosine-binding modules that recognize the consensus sequence pYxx(where pY represents phosphotyrosine and represents any hydrophobic amino acid, Figs 1 and 3). SH3 domainsinteract with proline-containing peptides that conform to the sequences R/KxxPxxP (class 1) or PxxPxR/K (class 2).44

An example is Grb2 binding to Sos (Fig 4,B). PTB domains recognize NPxY sequences in which the tyrosine residuemay or may not be phosphorylated. PH domains share a similar fold to the PTB domain but interact with D3-phos-phorylated IPs.43 Examples include PLC-1 and Vav interactions with the plasma membrane (Fig 4).


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surface membrane allow extracellular Ca2+ influx.46

Sequential Ca2+ release from both internal and externalstores leads to a sustained elevation of [Ca2+]i, which actsas an important trigger for transcriptional activation in thenucleus (Fig 5).12,47 The spatiotemporal characteristics of[Ca2+]i signaling (transient, sustained, or oscillatory) areimportant in determining which genes are activated. For

instance, sustained [Ca2+

]i elevation is critical for the acti-vation of the IL-2 promoter by the calcineurin pathway(Fig 5).12,47 Ca2+ activates calcineurin, a calcium-calmod-ulindependent serine phosphatase that dephosphorylatesthe nuclear factor of activated T cells (NFAT), whichincludes 4 isoforms (Fig 5).47,48 This leads to the intranu-clear translocation of NFAT proteins, which occupy prox-imal and distal NFAT binding sites in the IL-2 promoter(Fig 5).12 Interference in calcineurin activation bycyclosporin A and FK506 form the basis for the immuno-suppressive activities of these drugs (Fig 5).48

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Another important function of IP turnover is PKCactivation.49-51 T cells express multiple functionally dis-tinct PKC isoforms that can be classified into the classi-cal PKCs (,1,2, and ), which are regulated by cal-cium, diacylglycerol, and phospholipids; novel PKCs (,, , and ), which are regulated by diacylglycerol andphospholipids; and atypical PKCs ( and ), which lack

Ca2+

- or diacylglycerol-binding domains.52

A majorrecent finding is that among the available PKCs, only the isoform is recruited to the site of TCR engagement (Fig6).51 In that location, PKC is involved in the activationof the NF-B pathway and possibly also the N-terminalc-Jun kinase (JNK) cascade (see below).53

Regulation of p21ras and MAPK cascades

TCR ligation leads to a rapid accumulation of the activeGTP-bound form of p21ras in the vicinity of the T-cellmembrane (Fig 4, B).54,55 Ras plays a critical role in

FIG 4. Examples of key adapter proteins that play a role in TCR signaling. A shows the formation of a sig-naling complex that regulates IP turnover and [Ca2+]i flux. The importance of modular binding domains in

facilitating interactions between LAT, SLP-76, Gads, and Itk is demonstrated: SH2 ()), SH3 (), PH (vvvv),

and PR (filled circles). B shows the use of modular domains in the formation of signaling complexes that

lead to Vav and Ras activation, respectively. The former complex includes LAT/Gads/SLP-76 and PI-3 kinase,

and the latter involves LAT/Grb2 and Sos.


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cytokine gene expression, particularly activation of the IL-2 promoter and T-cell proliferation (Fig 5).56 The Ras gua-nine nucleotide binding cycle is controlled by the counter-regulatory effects of guanine nucleotide exchange factors(GEFs), which promote the activation of Ras, andGTPase-activating proteins, which stimulate the intrinsicGTPase activity of Ras, thereby resulting in GTP hydroly-sis and inactivation of Ras.54,55 Although there are severalpossible avenues for Ras activation in T cells, a well-char-acterized pathway is the involvement of the adapter pro-tein growth factor receptorbound protein (Grb2) and Sonof Sevenless (Sos), a GEF (Fig 4,B).57 In this assemblythe Grb2-SH2 domain is recruited to a consensus phos-photyrosine motif on LAT,40 whereas Grb2-SH3 domainsbind to proline-rich regions in Sos (Fig 4,B). Although ithas been suggested that Ras activation by phorbol estersinvolves a PKC member, there is no evidence for a directinteraction between PKC and Ras,arguing that either PKCor the phorbol ester may activate a Ras GEF.

Once activated, Ras couples to multiple effector path-

ways, including activation of the extracellular signal-reg-ulated kinase (ERK) cascade, as well as linkage to RhoGTPases.56,58 The ERKs belong to the MAPK family,which are activated by a cascade involving a MAP3K(Raf-1) and a MAP2K (MEK-1 or MEK-2; Fig 7).58,59

p21ras interacts directly with the serine-threonine kinaseRaf-1, which is activated in a complex fashion at the sur-face membrane. Once activated, the ERKs play an essen-tial role in the expression of the activator protein 1 (AP-1) transcription factor c-Fos, as well as c-myc.59 c-Fos isinvolved in transcriptional regulation of AP-1 responseelements in the IL-2 promoter (Fig 5).60

In addition to the ERK cascade, the p38 MAPK andJNK cascades play a role in TCR signaling (Fig 7).59,61,62

In contrast to abundant ERK expression, the JNK1 andJNK2 isoforms are present in low quantities in primary Tcells and require prior TCR engagement for their expres-sion.63 Subsequent activation of JNK is CD28 dependent(Fig 7) and is required for activation of the IL-2 promot-er, particularly the CD28 response element (CD28RE,Fig 5).61,62 In murine studies it has been shown that themajor target of the JNK2 isoform is the IFN-gene andthat knockout of JNK2 impairs TH1 development.

64 Incontrast, the major role of JNK1 is interference in TH2development, as exemplified by increased IL-4 and IL-5production in JNK1 knockout mice.65 This effect may beexplained by the ability of JNK1 to enhance the nuclearexport of NFATc, which is required for the activation of

the IL-4 promoter.65

Similarly, the p38 MAPK cascaderegulates IFN-gene expression in TH1 cells but appar-ently does not affect TH2 cytokines.

59 The p38 MAPKcascade also play a role in the induction of apoptosis inCD8+ T cells.59

Regulation of PI-3 kinase activity

In addition to PLC-1 activation, PTKs affect IPturnover through the involvement of PI-3 kinase.66 Thiskinase is comprised of regulatory (p85) and catalytic(p110) subunits and is recruited through the modular

SH2 domain on the p85 subunit to LAT or TCR-interact-ing molecule adapters (Fig 4, B).41,67 Activated PI-3kinase phosphorylates the D-3 position of the IP ring,thereby converting PI-4,5-P2 and PI-4-P to PI-3,4,5-P3and PI-3,4-P2, respectively.

63 These D-3 phosphorylatedIPs interact with the plextrin homology (PH) domains ofPLC-

1, Tec, and Vav and serve to anchor these proteins

at the plasma membrane (Fig 4,B).43 PI-3 kinase is alsoresponsible for the recruitment and activation of the ser-ine-threonine kinase, protein kinase B (Akt), which playsa role in cellular survival.68

Regulation of NF-B activation

NF-B activation is dependent on a multisubunit 700-to 900-kd cytosolic signaling complex.69,71 This complexincludes the catalytically active IB kinases (IK) IKKand IKK, which are arranged into IKK-IKK het-erodimers by a noncatalytic IKK subunit, IKK(Fig 8).69-71

The activated IKK complex initiates the phosphorylationand proteolytic degradation of the inhibitory IB and

IB proteins. This leads to the release of NF-B tran-scription factors (p65, c-Rel, RelA, and p50), which aresequestered in the cytosol by IB and IB, allowingthese factors to enter the nucleus and initiate transcription-al activation of genes involved in cellular proliferation andsurvival.69-71 A key gene target is the CD28RE in the IL-2promoter (Fig 5).72 This is a combinatorial response ele-ment that requires c-Rel, as well as AP-1, transcription fac-tors for full activity. Therefore it is interesting that, as forJNK activation, NF-B activation is dependent on CD28costimulation (Fig 8).72,73 In fact, these costimulatoryrequirements are in excellent agreement with the key roleof CD28 in IL-2 production in unprimed T cells. Anotherimportant target for the NF-B pathway and the CD28

receptor is the bcl-xL promoter, which expresses an NF-Bresponse element 860 bp upstream of the start site.74

Increased Bcl-xL expression plays a critical role in cellularsurvival during CD28 costimulation.75

The exact mechanism by which the NF-B cascade isinitiated by TCR ligation still needs to be clarified butinvolves PKC recruitment to the TCR synapse (Fig8).53,76 This recruitment is dependent on the involvement ofVav and assembly of the cytoskeleton, suggesting that oneor more IKK components bind to a cytoskeletal scaffold(Fig 8).77 PKC contributes to the activation of the IKKcomplex through its ability to phosphorylate IKK (Fig8).53,77 Interestingly, pharmacologic interference in PKCactivity abrogates NF-B activation and IL-2 production.53

Role of CD28 in the activation of down-

stream NF-B and JNK signaling cascades

Activation of the IL-2 promoter by CD28 costimula-tion is dependent on the synergistic activation of the JNKand NF-B cascades.61,62,73,74 Although the precisemechanisms of engagement of these cascades areunclear, it is relevant that the CD28 tail includes severalbinding motifs that may be involved in signal transduc-tion, including 4 tyrosine residues (Fig 5).34,35 The pY170

motif serves as a docking site for PI-3 kinase and Grb2,


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FIG 6. Components of the various MAPK families and their relationship to TCR and CD28 costimulation.

The MAPKs are phosphorylated and activated by MAP2Ks, which, in turn, are activated by several MAP3Ks.

A variety of transcription factors are phosphorylated and activated by these cascades, as demonstrated.

FIG 5. Signaling domains of the CD28 receptor (numbering according to the murine protein sequence). The 41

aa cytoplasmic tail contains 4 tyrosine residues, 2 of which (Y170 and Y188) are phosphorylated during CD28 lig-

ation by B7 ligands. pY170 recruits PI-3 kinase, which may play an indirect role in Vav activation by allowing this

protein to dock to D3-phosphorylated IP in the surface membrane. CD28 further assists in the activation of Vav

by enhancing its tyrosine phosphorylation by either a TCR- or CD28-associated PTK. Vav is involved in cytoskele-

tal assembly through the activation of Rac-1 and also plays a role in the recruitment of PKC and possibly JNK

activation. Notice that through its effects on the cytoskeleton, CD28 may also be responsible for the polarization

of lipid rafts at the TCR synapse. The constitutive association of PTKs, such as Lck, with these rafts may lead to

a generalized increase in protein tyrosine phosphorylation on TCR engagement. Two proline-rich domains (PR1

and PR2) are functionally involved in the recruitment and the activation of the Tec kinase, Itk. In addition, these

PR domains are recognized by the SH3 domain of Lck and may play a role in the activation of that kinase.

Although the exact role of these PTKs in CD28 signaling is unclear, they could contribute to phosphorylation of

the Y170 and Y188 residues, as well as to the overall increase in PTK activity during CD28 costimulation.


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whereas pY188 has been shown to be critical for JNKactivation and transcriptional activation of the IL-2 pro-moter.35 In addition to these phosphotyrosines, the CD28

tail contains 2 proline-rich regions (PR1 and PR2) thatmay be functionally involved in Lck and Tec kinaserecruitment and activation.34,35,78,79

How exactly the phosphotyrosine and PR motifs relateto PTK activation and distal signaling cascades is stillunclear. We do know, however, that CD28 assists in theassembly of the cortical cytoskeleton and recruitment oflipid rafts to the TCR synapse.33 Regulation of thecytoskeleton involves the sustained phosphorylation andactivation of Vav by CD28. Vav is a GEF for Rac-1, whichplays a role in cytoskeletal assembly and possibly also the

activation of the JNK cascade (Fig 3). It is interesting thatthe MAP3K in the JNK cascade, JNK kinase kinase,assembles at the TCR synapse and interacts directly with

the Lck-associated adapter proteins.81,82

This suggeststhat the JNK cascade is activated in the vicinity of theTCR synapse by signaling components that are assembledby means of TCR-CD28 costimulation. Lipid rafts, whichare enriched for Lck, may be involved in this event, inaddition to contributing to the overall increase in PTKactivity during CD28 costimulation.

How the NF-B pathway may fit into the above schemeof CD28 signal transduction is uncertain but could involvethe recruitment of PKC to the TCR synapse. PKCrecruitment requires Vav activation and cytoskeletal

FIG 7. Schematic to explain synergistic activation of the IL-2 promoter by Ca2+/calcineurin, Ras/MAP, and NF-

B cascades. The role of cyclosporin A and tacrolimus (FK506) in disrupting activation of the IL2gene is

demonstrated. The CD28 response element (CD28RE) is a combinatorial element that requires activation of

both the NF-B and JNK cascades. Both cascades are dependent on CD28 costimulation. The role of the

negative regulatory elements CREB/CREM and NRE-A in the induction of T-cell tolerance is discussed in part

2 of this review.


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assembly.80 Accordingly, PKC has been shown to be crit-ical for NF-B activation in murine knockout studies.80

Because of its ability to regulate specific signalingcascades, as well as to promote a generalized increase inprotein tyrosine phosphorylation, CD28 controls a widerange of responses in naive CD4+ T cells, including adecrease of the TCR signaling threshold (Table I). Incontrast, the major effect of CD28 in memory T cells isto enhance the TCR response, whereas its role in CD8+ Tcells is less clearly defined. This is in contrast to its

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essential role in naive T cells. The contribution of CD28to TCR signaling in naive T cells has therefore beendescribed as signal 2 to highlight the major contributionto the delivery of signal 1 by the TCR.83 This 2-signalconcept has particular relevance in understanding T-cell

tolerance and is further discussed in part 2 of this review.

DYNAMIC INTEGRATION OF SIGNALS BY

THE TCR SYNAPSE: ROLE OF LIPID RAFTS,CYTOSKELETON, AND SUPRAMOLECULAR

ACTIVATION CLUSTERS

Although a considerable amount of information onTCR signaling has been obtained through the use of anti-bodies that ligate TCR or CD3, in vivo TCR activationrequire a contribution by APCs and accessory receptors,

TABLE II. Raft-associated signaling components that play

a role in TCR activation

Constitutive Inducible

Lck Vav

Fyn PKC

Rac MEKK2LAT IKK,,CD2 ZAP-70

CD5 TCRCD9 CD3GPI-linked receptors

MEKK2, JNK kinase kinase; GPI, glycophosphatidylinositol.

FIG 8. Schematic to explain the role of CD28, lipid rafts, and the cytoskeleton in the recruitment and acti-

vation of PKC and the IKK complex. This diagram shows how activation of PI-3 kinase and Vav by the

CD28 receptor leads to assembly of the actin cytoskeleton. It also highlights the role of PKC in the activa-

tion of the IKK complex and the subsequent NF-B activation.

TABLE I. Key costimulatory effects of the CD28 receptor

1. Enhanced protein tyrosine phosphorylation by the TCR

2. Activation of the JNK and NF-B cascades3. Cellular proliferation caused by increased expression and sta-

bilization of IL-2 mRNA

4. Decreased threshold for the activation of naive T cells through

assembly of signaling components at the TCR synapse andSMACs

5. Assembly of the cortical cytoskeleton and polarization of lipid

rafts

6. Regulation of TH1/TH2 balance by enhancing IL-4, IL-5, and

IL-10 production

7. Regulation of cellular migration through effects on chemokine

(MIP-1, MIP-1) production, as well as chemokine receptor

(CXCR4, CCR5) expression

8. Enhanced T-cell survival through increased Bcl-xL expression

9. Essential for the homeostasis of the regulatory CD4+CD25+

subset

10. Prevents anergy through its effect on IL-2 production and cell-

cycle progression


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which introduces an additional level of complexity. Thisincludes the requirement that the TCR and accessoryreceptors be assembled in the immunologic synapse (Fig2). This contact site also serves as an assembly site forsignaling molecules on the inner leaflet of the T-cellmembrane, an arrangement known as the supramolecularactivation cluster (SMAC; Box 1).5,15,20,84

Which stimuli regulate the formation of the TCRsynapse and assembly of the SMACs? We have alreadydiscussed the role of topological constraint in determin-ing which receptors participate in TCR synapse (Box1).5,20 This is a passive process that prepares the TCR foractive large-scale segregation of signaling molecules,which also involves several costimulatory receptors, thecortical cytoskeleton, and lipid rafts.20,84,85

Lipid rafts are glycosphingolipid- and cholesterol-enriched membrane microdomains that are biochemical-ly defined as an ordered lipid phase that is insoluble inlow concentrations of nonionic detergents at 4C.86 Asignificant fraction of acylated, myristoylated, and gly-

cophosphatidylinositol-linked proteins are anchored inlipid rafts and therefore packaged to participate in cellu-lar events, such as signal transduction (Table II).87

Although TCR-CD3 ligation exerts an independent effecton lipid raft trafficking, CD28, CD2, CD5, and lympho-cyte functionassociated antigen 1 (LFA-1) play a majorrole in recruiting lipid rafts to the TCR synapse. 33,85,88,89

This process requires from 10 to 60 minutes for comple-tion.23 A list of constitutive and newly recruited raft sig-naling molecules appears in Table II. Scrutiny of this listreveals an overlap with the SMAC components describedin Fig 6; this suggests that rafts are involved in theassembly of the SMACs. Although the exact moleculardetails of raft recruitment to the site of TCR engagement

is still unclear, the effect of costimulatory receptors, suchas CD28, on the assembly of the actin cytoskeleton islikely to play a role (Fig 3).14,22,90

TCR engagement induces a dynamic reorganization ofthe cortical cytoskeleton, with the assistance of costimu-latory receptors, such as CD28, CD2, and LFA-1.91,93

Best characterized is the role of CD28, which is requiredfor prolonged tyrosine phosphorylation and activationof Vav-1, the GEF responsible for Rac-1 andCdc42.34,35,79,92,93 LAT, SLP-76, and PI-3 kinase areinvolved in localizing Vav at the plasma membrane (Fig4,B). Activated Cdc42 interacts with the Wiscott-Aldrichsyndrome protein (WASP), which controls the actin reg-ulatory complex,Arp2/3 (Fig 8).94-96 In its inactive state,

WASP assumes a closed conformation that fails to bindto Arp2/3, but in the presence of activated Cdc42, thisprotein opens up, leading to the recruitment and activa-tion of Arp2/3.94-96 The adapter protein Nck assists inWASP activation (Fig 8). This leads to actin polymeriza-tion at the TCR contact site in addition to the assemblyof cytoskeletal proteins, such as profilin, talin, vinculin,ezrin, radixin, and moesin.97

The cytoskeleton has an important structural role inshaping the contact zone between the T cell and theAPC.93 In addition, the cytoskeleton plays an active role

in signal transduction, as exemplified by the fact thatdrugs that inhibit actin polymerization interfere with T-cell activation.98 One possibility is that actin filamentsmay establish a scaffold for the assembly of signalingcomplexes.93 This may involve novel adapter proteinsthat link signaling components to actin filaments. PKCis recruited to the SMACs in this manner (Figs 6 and 8)and may contribute to cytoskeletal assembly on the basisof its ability to phosphorylate the cytoskeletal proteinmoesin.77,99 An additional role for the actin scaffoldmay be to sustain signaling by preventing the degrada-tion of signaling components.100 Finally, the actin scaf-fold may play a role in lipid raft trafficking to the TCRsynapse. Details of the specific molecular connectionsbetween the cytoskeleton and ordered lipid domainremain to be clarified.

The formation of the TCR synapse and the SMACsprovides a stable arrangement through which the TCRcan establish a threshold for T-cell activation.20,84,99 Thisthreshold is dependent on an optimal number (approxi-

mately 50) and avidity of TCR-MHC peptide interac-tions.21 The cumulative effect of multiple integrated sig-naling events delivers a high-fidelity deterministic signal,which leads to full T-cell activation. When, however,these arrangements are disrupted by low-avidity TCRinteractions with the peptide-MHC complex, an alternateform of signaling and biologic outcome may ensue. Thisaspect is further discussed in part 2 of this review.

SUMMARY

All considered, TCR signaling is a dynamic event thatcan elicit a variety of T-cell responses, depending on thecellular subset and the conditions under which the TCR

is engaged by the specific MHC-peptide ligand. Whetherthe cell responds to delivery of the TCR signal by prolif-eration, differentiation, apoptosis, anergy, developmentof memory, or cytotoxic killing depends on the quality ofthe TCR signal, as well as a host of modifying factors,such as cellular subset, costimulatory receptors, type ofAPC, cytokine milieu, and the ultimate success of T cellsin achieving their immunomodulatory effects. Althoughit is difficult in a review of this size to describe in detailthe contribution of TCR signaling to every biologic out-come, I have attempted, by way of the IL-2 promoter(Fig 5), to demonstrate how a variety of signaling path-ways are integrated into the activation of a gene that isinvolved in T-cell proliferation and apoptosis. To further

demonstrate how TCR signaling is modified duringTH1/TH2 differentiation, treatment with altered peptideligands, tolerance induction, and immune senescence, wewill use the basic concepts outlined in part 1 to elucidatethose aspects in part 2 of this review.

I thank Mr Boyd Jacobson, manager of the Illustration Department,

National Jewish Medical Center, for skillful design of the artwork, and

Mr Photi Christofas for skillful assistance in preparing the manuscript.

Because of space constraints, it was not possible to site the seminal

contributions from a large number of investigators to this field.


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