activación de cels t

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TCR: T cell antigenreceptor

Signal transduction:biochemical eventslinking surface

receptor engagementto cellular responses

INTRODUCTION

Twenty-five years ago, Annual Review of Im-munology published its first review describing

features of the structure we now know as theT cell antigen receptor (TCR) (1). In recog-

nition of this anniversary, this article begins byhighlighting a sampling of seminal observations

made in thedecadefollowing theinitialdescrip-tion of the TCR. The discoveries made dur-

ing this period established the basic paradigmfor how TCR engagement initiates the earli-

est biochemical events leading to cellular ac-tivation, described nicely in an Annual Reviewof Immunology article in 1996 (2) (Figure 1a).

This historical perspective sets the stage for adiscussion of our current state of understanding

of the molecular and biochemical events criti-cal for T cell activation that have emerged from

the work of multiple laboratories.

DESCRIBING THETCR COMPLEX

In the early 1980s, several groups began ex-periments to identify and characterize the anti-

gen receptor on T cells. One approach usednewly developed molecular techniques, ulti-

mately leading to the identification of the genesresponsible for the antigen-binding proteins

(35). An even earlier approach relied on im-

Figure 1

TCR proximal signaling then and now: filling in the gaps. Then (a): TCR signaling mid-1990s (adapted from Reference 2). Ligationthe TCR/CD3 results in activation of Src and Syk family PTKs associated with the intracellular CD3 domains that then activatePLC1 and Ras-dependent pathways. PLC1 hydrolizes PIP2 to form IP3 and DAG. Now (bd): Current understanding of how thTCR couples to downstream pathways (b), the molecular basis for Ca2+ influx (c), and the positive feedback loop responsible for Rasactivation (d). (b) The link between PTKs and downstream pathways is a multimolecular signaling complex nucleated by the adapterproteins LAT, Gads, and SLP-76. Lck activates ZAP-70 to phosphorylate (p) tyrosine residues on LAT, which then recruits Gads anits constitutive binding partner SLP-76. ZAP-70-mediated phosphorylation of SLP-76 results in the recruitment of multiple SH2domaincontaining effector molecules (circles) and adapter proteins (octogons). SH3 domains (shaded overlapping areas) also link effecto

to adapters and contribute to stabilization of the complex. (c) The link between depletion of ER Ca2+

stores and CRAC channelactivation is STIM. TCR-induced IP3 production (see b) results in the activation of IP3 receptors (IP3R), which release Ca

2+ from tER into the cytoplasm. STIM contains paired EF hands within the ER lumen, which bind a single Ca 2+ molecule. Upon depletion ER stores, Ca2+-free STIM molecules oligomerize and move to areas of ER/plasma membrane proximity, where they colocalize witand induce dimerization of Orai1 dimers, resulting in a functional CRAC channel and subsequent influx of Ca 2+. (d) Activation of Rinvolves two Ras GEFs (SOS and RasGRP) in a positive feedback loop. TCR-induced production of DAG (see b) results in themembrane recruitment of RasGRP, where it is phosphorylated and activated by PKC. RasGRP then facilitates the removal of GDPfrom Ras, which can then bind GTP and become activated. GTP-bound Ras then binds SOS, which is bound constitutively to GRBand is inducibly recruited to LAT, increasing its GEF activity, resulting in a positive feedback loop and robust Ras activity.

munization of mice with T cell clones of a

fined specificity, hybridomas, or clonal T ctumors in the hope that antibodies would

generated that would react with the recepresponsible for binding to antigen (68). Su

antibodies were produced and were then ufirst to demonstrate interference with antige

responses and later to perform the initial bchemical characterization of the receptorself. These studies revealed a complicated c

surface structure that included proteins retive with antibodies against the nonpolym

phic CD3 proteins (initially thought of as thpolypeptides, , , and ) (9), as well as varia

proteins designated and .The antigen-binding function of w

obvious early on, by inference owing to thhighly polymorphic nature and similarity

immunoglobulin and experimentally owingearly gene transfer experiments that demo

strated that antigen/major histocompatibi

complex (MHC) reactivity tracked with exprsion of these receptor components (10, 1

However, it was less obvious what role the Cproteins played in TCR function. Several li

of evidence suggested that the CD3 molecuwere critical for signal transduction: Unlikethe CD3 molecules had long cytoplasmic taand anti-CD3 antibodies resulted in T cell

tivation, although it was difficult to demostratesignaling function conclusively. One ea

592 Smith-Garvin Koretzky Jordan


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www.annualreviews.org T Cell Activation 593


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PKC: protein kinaseC

PLC: phospholipaseC

PTK: protein

tyrosine kinaseImmunoreceptortyrosine-basedactivation motif(ITAM): a shortpeptide sequence inthe cytoplasmic tails ofkey surface receptorson hematopoietic cellsthat is characterized bytyrosine residues thatare phosphorylated bySrc family PTKs,

enabling the ITAM torecruit activated Sykfamily kinases

effort involved an attempt to create cell lines

expressing without CD3 or CD3 without

, thereby creating reagents to investigate the

independent role of the receptor components.These experiments were not successful, as it

became clear that there is an obligatory coex-pression of with CD3 (12). These studies

did result, however, in the creation of the firstin a long line of genetically altered Jurkat Tcells that have been instrumental in our under-

standingof how the TCR complex couplesto itssignaling machinery (13).

EARLY SIGNALING STUDIES

Initial investigations into how the TCR trans-

duces its signals began with the observation thatTCR-deficient Jurkat T cells could be stimu-

lated pharmacologically with the combinationof phorbol esters and Ca2+ ionophores (14).This observation led to speculation that en-

gagement of the TCR might stimulate the samesignals that these reagents induced. This no-

tion was tested directly in experiments demon-strating increases in intracellular free Ca2+

following CD3 or TCR stimulation in bothJurkat cells and primary T cells. The source

of the Ca2+ increase was shown to be a com-bination of Ca2+ released from an intracellular

pool in response to inositol trisphosphate (IP3)production and influx of Ca2+ from outside of

the cell (15). It was presumed that phorbol es-

ters were important because of their ability toactivate protein kinase C (PKC, now known to

be a family of enzymes), whose activity is regu-lated physiologically by diacylglycerol (DAG).

Concurrent studies in other systems haddemonstrated that a single enzymatic reaction,

hydrolysis of the membrane PI(4,5)P2 (phos-pholipid phosphatidylinositol 4,5 bisphos-

phate) by phospholipase C (PLC), generatesboth IP3 and DAG, suggesting that the TCR

may function to regulate PLC activity.Testing this notion proved somewhat

difficult at first, as PLC regulation was initially

described downstream of heterotrimericguanosine triphosphate (GTP)-binding pro-

teins associated with seven-transmembrane

domain receptors. An exhaustive but uns

cessful search ensued for the GTP-bindprotein critical for coupling the TCR

PLC activation. Insight into how the TCmay initiate PLC activation emerged from

confluence of discoveries in immune cells aother lineages. For example, stimulation of

TCR results in changes in protein phosphrylation, including inducible phosphorylatof the newly described chain of the C

complex (16). Furthermore, growth facreceptors with intrinsic protein tyrosine kin

(PTK) activity also stimulate PLC functiIn contrast to seven-transmembrane dom

receptors, however, receptor PTKs stimulanother PLC isoform, PLC. Although no

of the TCR components possessed enzymaactivity themselves, cytosolic PTKs of

Src family (in particular Lck and Fyn) wbeing described in T cells. These PTKs w

associated either with the TCR (17) or w

the CD4 and CD8 coreceptors (18, 1both important for TCR signaling. Th

observations, coupled with the new knowledthat T cell activation requires PTK funct

(20), led to a new TCR signaling paradigin which the TCR recruits cytosolic PTKs

activate key second messengers.

HOW CD3 TRANSDUCESITS SIGNALS

Testing this model led to a search for adtional substrates of the TCR-stimulated PTK

Among the most attractive candidates were rosines within the CD3 molecules themsel

that fell within a motif present once withCD3 , , and and in triplicate within eac

chainandalsoinkeyimmunoreceptorsonothimmune cell lineages. The signature

these motifs (21), eventually designated imunoreceptor tyrosine-based activation mo

(ITAMs), is two tyrosines flanking a seriesamino acids, including key leucine/isoleucin

with stereotypic spacing. Numerous labo

tories demonstrated that the ITAM tyrosinare in fact phosphorylated upon TCR l

ation, but defining the importance of t



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posttranslational modification for TCR func-

tion was not trivial. Because the CD3 moleculescould not be expressed without the chains,

several groups created chimeric molecules fus-ing the cytoplasmic domains of individual CD3

chains to extracellular and transmembrane do-mains from other proteins (2225). These

cDNAs in chimeric proteins were then trans-fected into T cell lines selected for TCR loss.Antibody crosslinking of the extracellular do-

main of the chimeras recapitulated all theknown TCR-mediated signaling events lead-

ing to cellular activation. Mutation of the keyITAM tyrosines (or altering their spacing) abro-

gated the ability of the chimeras to activate thecells, thus demonstrating that tyrosine phos-

phorylation of the CD3 ITAMs is an earlyand requisite step for TCR-mediated T cell

activation.Studies of the CD3 chimeras led naturally to

the question of the purpose for ITAM phospho-

rylation. Unlike growth factor receptors whoseintrinsic enzymatic activity is enhanced by tyro-

sine phosphorylation, the CD3 molecules haveno such effector function on their own. Investi-

gators speculated, therefore, that tyrosine phos-phorylation of the ITAMs might serve as dock-

ing sites for interactions with other proteins.Indeed, it was soon shown that phosphory-

lated CD3

(and later other ITAM-containingproteins) is a recruitment site of a 70-kDa

phosphoprotein, the Syk kinase family memberZAP-70 (-associated protein of 70 kDa) PTK

(26). A model therefore emerged that engage-

ment of the TCR leads to Src family PTK ac-tivity resulting in ITAM phosphorylation and

recruitment of ZAP-70. This converted theTCR with no intrinsic enzymatic function to an

active PTK able to phosphorylate a spectrumof substrates leading to a myriad of down-

stream signals that, when integrated appropri-ately (along with signals from other corecep-

tors), lead to T cell activation (27).The basic tenets of this model have stood the

test of intensive investigation. In the 15 yearssince ZAP-70 was cloned, investigators have

filled in many of the gaps between the TCR

and initiation of effector functions. Much has

ARGUING BY ANALOGY: COMPARINGAND CONTRASTING SIGNALING PATHWAYSBY MULTIPLE RECEPTORS AND LINEAGES

Key insights important for understanding T cell activation havecome from studies in nonimmune mammalian cells and cells of

lower organisms, including the observation that PTKs could linkto the phosphatidylinositol pathway and the paradigm describing

adapter proteins as integrators of signal transduction. T cell biol-ogists have also provided unique insights to their nonimmunol-

ogist colleagues. Examples include a mechanism for recruitment

of nonreceptor PTKs to enzymatically inactive surface receptorsand the notion that protein tyrosine phosphatases may be positive

as well as negative regulators of PTK pathways.Within the immune system, biologists studying TCR signal-

ing have been the donors and recipients of information that hasbeen useful for investigators examining the signaling pathways of

other cell surface receptors (e.g., integrins) and other hematopoi-etic lineages. It is clear that, although basic paradigms may be

similar, each cell type and receptor utilizes unique ways to regu-late signal transduction cascades. Additionally, recent studies have

demonstrated that pathways thought to be distinct (e.g., integrinsand immunoreceptors) instead intersect at multiple levels. Fu-

ture insights into how diverse signaling pathways are integrated

to result in the appropriate biologic response will undoubtedlycontinue to benefit from comparing and contrasting activation

events downstream of multiple receptors in different immuneand nonimmune cell lineages.

Adapter protein:cellular protein that

functions to bridgemolecular interactiovia characteristicdomains able tomediateprotein/protein orprotein/lipidinteractions

been learned about substrates of the PTKs (in-

cluding Src, Syk, and more recently Tec fam-ily members) activated by the TCR and how

these molecules participate in T cell activation,about how signaling complexes are organized

by adapter proteins to bring effector proteinstogether, and about the unexpected intersec-

tion of particular signaling pathways (see alsothe side bar, Arguing by Analogy: Comparing

and Contrasting Signaling Pathways by Mul-tiple Receptors and Lineages). With the ac-

cumulation of data, it has also become clearthat signaling via the TCR complex is not a

linear event starting at the receptor and end-

ing in the nucleus. Instead, there appears to becomplex feedback and feedforward regulation

at each step. Ironically, one of the most central



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Table 1 Recent models for initial TCR triggering

Model Description

Piston-like movement An external force from the APC/T cell interaction pushes the TCR/CD3 complex in a piston-like

fashion perpendicular to the plasma membrane, exposing activating motifs on the intracellular region

of CD3 chains

Receptor deformation The force of the motility of the T cell as it moves over an APC induces TCR triggering only when

pMHC/TCR interactions are of a high enough affinity to withstand the force long enough to underg

a conformational changePermissive geometry pMHC dimers bind TCR/CD3 dimers inducing a rotational scissor-like conformational change in CD

chain(s) that reveal previously hidden intracellular activation motif(s)

Kinetic segregation Signal initiation occurs because of exclusion of inhibitory molecules in the tight contact zone between

the T cell and the APC, thereby shifting the enzymatic steady state toward an activating state

Diffusion trapping Expanded kinetic segregation model in which the affinity/diffusion coefficient of the pMHC/TCR

dictates the valency of TCRs required to trap the complex in the tight contact zone and therefore

initiate a productive signal

Pseudodimer Agonist pMHC/TCR recruits a second TCR via interaction with its associated CD4, the second TCR

then binds a coagonist endogenous pMHC forming a stable pseudodimer that triggers signaling via

proximity of ITAMs to CD4-associated Lck and/or by conformational change

on ligand-induced conformational changes in

the TCR and/or TCR aggregation have beenproposed (Table 1). The crystal structures

of several pMHC/TCR complexes have re-vealed little conformational changes in the

TCR heterodimer outside of the binding do-main (reviewed in 36). However, owing to the

current technical limitations of crystallogra-phy and protein nuclear magnetic resonance

spectroscopy, only pMHC/TCR structures in

the absence of transmembrane domains andCD3 components have beenelucidated. There-

fore, changes between the components of theTCR/CD3 complex in relation to one another

or to the plasma membrane cannot be ruled out.The rigid, rod-like transmembrane domain of

the CD3 could mediate a piston-like move-ment of the TCR complex perpendicular to

the plasma membrane after ligand binding, ina manner similar to that reported for the as-

partate receptor (37). Such a model would re-quire external forces that could be provided by

cell-to-cell contact, but this possibility is dif-

ficult to reconcile with the ability of solubleligands to activate the TCR. Similarly, an ex-

ternal force is required for the recently pro-posed receptor deformation model, in which

the movement of the T cell as it travels acrossthe antigen-presenting cell (APC) exerts a force

on transient pMHC/TCR interactions. Only

when the force required to induce conforma-tional changes in the TCR is less than the force

required to break the pMHC/TCR interactionwill TCR triggering occur (38). Alternatively,

the permissive geometry model for TCR trig-gering predicts that dimeric pMHCs bind two

TCR/CD3 complexes (39). This interaction re-sults in a rotation of the TCR heterodimers

around one another, displacing the extracellulardomains of the associated CD3 molecules. The

transmembrane interactions between the CD3

and TCR dimers serve as pivot points, and theCD3 movement is scissor-like, exposing previ-

ouslyshielded ITAMs and/or other functionallyimportant domains. This model is consistent

with studies that suggest that TCR aggregationis required for TCR triggering.

TCR aggregation has long been impli-cated as a mechanism for TCR triggering. In

the classic aggregation model, crosslinking ofTCR/CD3 complexes with multimeric pMHC

enables close contact and transphosphorylationbetween the CD3 tails and associated PTKs.

Clustering of several TCR complexes also may

result in competition for membrane lipid be-tween the CD3 chains, resulting in dissocia-

tion of the cytoplasmic domains from the mem-brane and subsequent ITAM phosphorylation



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PI3K:phosphoinositide3-kinase

(28). Aggregation is supported strongly by

observations that soluble multimeric but notmonomeric pMHC can trigger TCR activa-

tion. However, recent biochemical and micro-scopic studies suggest that preformed TCR

aggregates are present on nonactivated T cells(reviewed in 40). In the kinetic segregation

model, adhesion and other accessory moleculeswith short extracellular domains such as CD2initiate close contact zones between an APC

and a T cell (41). Inhibitory phosphatases withlong extracellular domains such as CD45 are

excluded because of their size. TCR complexesthat engage pMHC on the APC surface remain

in the close contact zone, where they are seg-regated from phosphatases and are able to ini-

tiate signaling. TCR complexes that do not en-gage pMHC are free to diffuse outside of the

close contact zone. Shortening the extracellulardomains of inhibitory molecules or lengthen-

ing the extracellular domains of adhesion or ac-

cessory molecules can abrogate TCR signaling,suggesting that kinetic segregation is an impor-

tant aspect of TCR signal initiation (reviewedin 41). This notion was expanded with the dif-

fusion trapping model in which immobilizationor trapping of pMHC/TCRs in the close con-

tact zones is responsible for TCR signal initi-ation (42). The valency, or degree of aggrega-

tion, of the pMHC/TCR required for trappingand therefore triggering depends on the affin-

ity anddiffusion coefficient of thepMHC/TCRinteraction. Another model has been proposed

that suggests that endogenous pMHCs amplify

signals produced by the rare agonist pMHCsby promoting TCR aggregation and a subse-

quent phosphorylation cascade (40). Similarly,the pseudodimer model proposes that an ago-

nistpMHC/TCRcanrecruitasecondTCRinaCD4-dependent manner, and this second TCR

binds a coagonist endogenous pMHC, forminga stable pseudodimer that could trigger signal-

ing through ITAM/Lck proximity or throughCD3 conformational changes (43). Many of

these models are not mutually exclusive, and itis likely that TCR aggregation, conformational

changes within the TCR complex, and exclu-

sion of inhibitory molecules are all required

for TCR triggering, perhaps in a stepw

fashion.

PROXIMAL SIGNALINGCOMPLEX

The earliest step in intracellular signaling f

lowing TCR ligation is the activation of S(Lck and Fyn) PTKs, leading to phosphorytion of the CD3 ITAMs. Recruitment of ZA

70 follows, leading to a cascade of phosphrylation events. The past decade has seen t

description of a subcellular assembly and acvation of an adapter protein nucleated mu

molecular signaling complex (Figure 1b). Tcomplex is responsible for propagating

TCR/PTK signal into multiple and diverse dtal signaling pathways.

Among the most important of the ZAPtargets are the transmembrane adapter protlinker for the activation of T cells (LAT) a

the cytosolic adapter protein Src homolog(SH2) domaincontaining leukocyte phosph

protein of 76 kDa (SLP-76) (44, 45). Thtwo adapters form the backbone of the co

plex that organizes effector molecules in tcorrect spatiotemporal manner to allow for

activation of multiple signaling pathways. Timportance of these adapters is underscored

studies showing that the loss of either LATSLP-76 results in a near complete loss of TC

signal transduction reminiscent of Syk/ZAPor Lck/Fyn double-deficient T cells (4648)

LAT contains nine tyrosines that are ph

phorylatedupon TCRengagement, which bthe C-terminal SH2domainof PLC1,thep

subunit of phosphoinositide 3-kinase (PI3and the adapters growth factor receptor-bou

protein 2 (GRB2) and GRB2-related adapdownstream of Shc (Gads) (reviewed in 4

SLP-76 is then recruited to phosphorylaLAT via their mutual binding partner Ga

(49). SLP-76 itself contains three modudomains: an N-terminal acidic domain w

three phosphorylatable tyrosines that interwith the SH2 domains of Vav1, Nck, and I

2-induced tyrosine kinase (Itk); a PRR t

binds constitutively Gads and PLC1; an



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C-terminal SH2 region that can bind adhesion

and degranulationpromoting adapter protein(ADAP) and hematopoietic progenitor kinase

1 (HPK1) (reviewed in 46). Although LAT andSLP-76 serve to nucleate this large signaling

complex, the effector molecules themselvesalsoare important for stabilizing the complex. For

example, the Tec family kinase Itk is requiredin a kinase-independent manner for the re-cruitment of the guanine nucleotide exchange

factor (GEF) Vav1 to the APC contact site,whereas Vav1 is required for optimal SLP-76

phosphorylation and recruitment to LAT aswell as for Itk activation (5052). These and

other data suggest that the formation of thecomplex is more complicated than the linear

modelmostofteninvokedforsimplicity.Forex-ample, PLC1 directly binds to SLP-76, LAT,

and Vav1 as well as to its activating kinaseItk (reviewed in 53). It is thought that these

interactions collectively are required to stabi-

lize PLC1 in the correct conformation withinthe complex to allow for its optimal activity

(54). Advances in biochemical and structuraltechniques are needed to elucidate the precise

allosteric and perhaps stoichiometric changeswithin the multimolecular complex that allow

for signal transduction.To investigate more precisely the impor-

tance of these complex interactions in primaryT cells, several laboratories have generated

mice expressing transgenic or knockin muta-tions in specific binding regions in various

molecules involved in proximal signaling (55

57). Tyrosine to phenylalanine mutations inSLP-76 at residues 112 and 128 or 145 in pri-

mary thymocytes and T cells do not result ina loss of SLP-76/Vav1/Nck/Itk interactions, as

would be expected from earlier phosphopeptidemapping studies and studies in cell lines (57).

However, these tyrosine mutations still resultin severe defects in downstream signaling path-

ways consistent with defective Vav1 or Itk activ-ity. Similarly, mutation of tyrosines of Vav1 does

not result in a loss of interaction with their pro-posed binding partners, although it does result

in dysregulated Vav1-dependent signaling (55).

Although the continued interactions of these

proteins seen by immunoprecipitation exper-

iments are likely due to tertiary interactionswith other domains or other molecules, these

studies suggest that SH2/phosphotyrosine in-teractions may play important regulatory roles

for the activation of effector molecules. Indeed,structural studies have suggested that the in-

teraction between the SH2 domain of Itk anda phosphotyrosine results in a conformational

switch allowing kinase activity (58). Consistent

with these data, investigators recently showedin Jurkat T cell lines that an Itk/SLP-76 in-

teraction is required for Itk kinase activity, al-though we do not yet know if it is specifically

the SH2/phosphotyrosine interaction that me-diates this kinase activity (59). Further stud-

ies are required to determine how the activ-ities of molecules beyond Itk are affected by

specific domain/domain interactions within thecomplex.

The proximal signaling complex resultsin the activation of PLC1-dependent path-

ways including Ca2+- and DAG-induced

responses, cytoskeletal rearrangements, andintegrin activation pathways. Ligation of co-

stimulatory receptors such as CD28 augmentsthese pathways. Below, we discuss the mecha-

nismsbywhichthesepathwaysareactivatedandregulated.

PLC1 ACTIVATIONAND SIGNAL TRANSDUCTION

Following TCR ligation, PLC1 is found in

the proximal signaling complex bound to SLP-76, Vav1, and LAT, where it is phosphorylated

and activated by Itk. Activated PLC1 then hy-drolyzes the membrane lipidPI(4,5)P2, produc-

ingthesecondmessengersIP3 andDAG. Thesetwomessengers areessential for T cell function,

and therefore the regulation of PLC1 acti-vation has been the subject of intensive stud-

ies. Localization of PLC1 to the proximalsignaling complex is dependent on LAT and

the Gads-binding region of SLP-76 (54). Ac-

tivation of PLC1 is dependent on Itk kinaseactivity that, in turn, is dependent on Vav1,

Lck, ZAP-70, LAT, and SLP-76 (52, 60, 61).



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Following TCR ligation, Itk is recruited to the

membrane through PH domain interactionswith PIP3, which has been locally generated

by Lck-induced PI3K activity (reviewed in 61).At the membrane, Lck phosphorylates Itk, and

the SH2 and SH3 domains of Itk interact withphosphorylated tyrosine 145 and the PRR of

SLP-76, respectively (6264). The role of Vav1and ZAP-70 in Itk activation is not understoodbut may relate to theirinvolvement in the phos-

phorylation of SLP-76 or PI3K activation (45,51,52). A secondTec family kinase, Rlk, canalso

phosphorylate PLC1, resulting in a relativelymild defect in Itk-deficient mice and requiring

the study of Rlk/Itk double-deficient mice tobetter understand the role of Tec kinases in T

cell activation (reviewed in 61).

DAG-MEDIATED SIGNALINGPATHWAYS

TCR-induced production of DAG results inthe activation of two major pathways involving

Ras and PKC. Ras is a guanine nucleotidebinding protein and is required for the acti-

vation of the serine-threonine kinase Raf-1,which initiates a mitogen-associated protein

kinase (MAPK) phosphorylation and activa-tion cascade. Raf-1 is a MAPK kinase kinase

(MAPKKK) that phosphorylates and activatesMAPK kinases (MAPKKs), which in turn phos-

phorylate and activate the MAPKs extracellular

signal-regulated kinase 1 (Erk1) and Erk2. Erkkinase activity results in the activation of the

transcription factor Elk1, which contributes tothe activation of the activator protein-1 (AP-1)

( Jun/Fos) transcription complex via regulationof Fos expression. Additionally, Erk activity can

result in the transcriptional activation of sig-nal transducer and activator of transcription 3

(STAT3) and in the serine phosphorylation ofLck (reviewed in 65).

Ras is only active in the GTP-boundstate, and its activation is facilitated by GEFs

and is suppressed by GTPase-activating pro-

teins (GAPs). Two Ras GEFs are presentin T cells, son of sevenless (SOS) and Ras

guanyl nucleotide-releasing protein (RasGRP).

RasGRP appears more dominant for early

tivation of Ras, as SOS cannot compensfor RasGRP deficiency (6668). RasGRP is

ducibly recruited to the membrane througDAG-binding domain (69), where it is ph

phorylated by PKC (70). SOS is constitively bound to the adapter protein GR

and upon TCR stimulation, the GRB2 SHdomain is recruited to and binds phosph

rylated tyrosines on LAT, thereby bring

SOS into the proximal signaling compwhere it can facilitate the localized acti

tion of Ras (71). The significance of thtwo modes of Ras activation was unclear u

til recently, when it was shown that RasGRdependent RasGTP production catalyzes S

activity, resulting in a positive feedback loand robust TCR-induced Ras activation (

(Figure 1c). This unified model explains tRasGRP dominance, as RasGRP would be

quired to initiate the production of RasGTwhich upon reaching threshold levels wo

catalyze SOS activity and further amplify

signal.The second major signaling pathway re

lated by DAG is mediated by PKC, a PKfamily member that contains a lipid-bind

domain specific for DAG, which is importfor recruiting PKC to the plasma membra

following T cell activation. However, phosphrylation of PKC by Lck (reviewed in 73) m

be required to induce a conformational chanthat enables binding to the lipid phosphati

serine (PS), which in turn enhances bindingDAG, resulting in PKC activation (74). Ot

proximal signaling molecules, including Va

PI3K, and 3-phosphoinositide-dependent nase 1 (PDK1), also play roles in PKC loc

ization, butdetails of their contributions arencompletely defined (73).

One critical pathway that PKC regulais NF-B activation. Because both the cano

cal and noncanonical activation of this pathwin T cells is described in detail in this volu

ofAnnual Review of Immunology [see reviewKarin and colleagues (75)], we highlight o

the major steps in the classical activationNF-B downstream of the TCR. The NF-



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family of transcription factors consists of five

members. In resting cells, NF-B is found inthe cytosol associated with inhibitor of NF-B

(IB) family members that keep NF-B frommoving into the nucleus. Upon T cell activa-

tion, IB is phosphorylated by the IB kinase(IKK) complex, ubiquitinylated, and degraded,

allowing NF-B to translocate into the nucleus,where it activates genes involved in the func-tion, survival, and homeostasis of T cells (re-

viewed in 76). Although we have known thegeneral pathway of NF-B activation for some

time, the specifics of how PKC activation leadsto nuclear import of NF-B in T cells are still

being elucidated.Over the past several years, the identifi-

cation and characterization of a lymphocyte-specific activation complex have provided some

insight into this question. Following TCRstimulation, a trimolecular complex forms be-

tween CARMA1 [caspase recruitment domain

(CARD) and membrane-associated guanylatekinase (MAGUK)-containing scaffold pro-

tein], the CARD-containing adapter proteinBcl10, and mucosa-associated lymphoid tis-

sue lymphoma translocation gene 1 (MALT1)(73, 76). The assembly of this CBM com-

plex (CARMA1/Bcl10/MALT1) is regulatedby PKC through its phosphorylation of

CARMA1, which is required for CARMA1oligomerization and association with Bcl10 (77,

78). MALT1 binds to Bcl10 and contributes

to the degradation of the regulatory subunitof the IKK complex (IKK) by facilitating its

polyubiquitination, possibly via activation ofthe E3 ubiquitin ligase tumor necrosis factor

receptor-associated factor 6 (TRAF6) (79, 80).Degradation of this regulatory subunit allows

for phosphorylation of IB by the IKK cat-alytic subunits, subsequent IB degradation,

and release of NF-B, resulting in NF-B nu-clear localization and gene activation. Recently,

MALT1 was shown to enhance NF-B signal-ing through its ability to degrade the deubiq-

uitinating enzyme A20, a negative regulator of

NF-B activation (81, 82).One additional component of the complex

that was recently identified as a CARMA1-

associating protein is ADAP (83). ADAP,

originally defined as a SLP-76-binding part-ner, associates with the MAGUK domain of

CARMA1 following T cell stimulation. Inves-tigators proposed that this interaction might al-

ter the conformation of CARMA1 and enhanceits association with Bcl10 and MALT1 and/or

stabilize the CBM complex.

Ca2+-MEDIATED SIGNALINGPATHWAYS

Ca2+ ions are universal second messengers ineukaryotic cells. The IP3 generated by TCR-

stimulated PLC1 activity stimulates Ca2+-permeable ion channel receptors (IP3R) on the

endoplasmic reticulum (ER) membrane, lead-ing to the release of ER Ca2+ stores into

the cytoplasm. Depletion of ER Ca

2+

trig-gers a sustained influx of extracellular Ca2+

through the activation of plasma membrane

Ca2+ release-activated Ca2+ (CRAC) channelsin a process known as store-operated Ca2+ en-

try (SOCE) (reviewed in 84) (Figure 1d). Fordecades the CRAC channels had only been

identified by their biophysical properties, andit has just been in the past few years that

the pore-forming subunit of the channels wasidentified as the four-transmembrane domain

containing molecule Orai1 (8587). Addition-ally, studies in the past few years have revealed

the sensor for depleted ER Ca2+ stores and

the activator of CRAC channels as stromal in-teraction molecule (STIM) (88, 89). STIM is

an ER-resident transmembrane protein with aC-terminal cytoplasmic coiled-coil motif and,

within the ER lumen, an N-terminal sterile

motif (SAM) and paired EF hands, where

one hand binds a single Ca2+ ion with lowaffinity (88, 89). Two STIM proteins, STIM1

and STIM2, are found in mammals, and re-cent work has shown that STIM1 is important

for the initial robust phase of SOCE, whereasSTIM2 is important for maintaining basal Ca2+

levels and sustaining the late phase of SOCE

(90, 91). Following ER Ca2+ depletion, STIM1molecules aggregate in clusters that preferen-

tially localize to sites of ER plasma membrane



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NFAT: nuclear factorof activated T cells

apposition, where they colocalize with clusters

of Orai1, forming punctae (9294). Thebiochemical mechanism by which STIM1 cou-

ples Ca2+ depletion to CRAC activation isnot yet fully understood and is an area of in-

tense investigation. Recentwork hasshown that

STIM1 oligomerization is sufficient to induce

punctae formation and CRAC channel activa-tion independent of Ca2+ store depletion (95).Further studies showed that the C-terminal

coiled-coil domain of STIM1 alone can inducedimerization of Orai1 dimers, which is suffi-

cientfor CRACchannel activation independentof punctae formation and store depletion (96).

How STIM1 oligomers translocate to areas ofER plasmamembraneappositionand how, once

there, they induce theOrai1 tetramerization re-main unknown. Interestingly, the WASp family

verprolin homologous protein (WAVE2) com-plex is also required for SOCE activity, througha mechanism that remains to be fully elucidated,

although independent of its function in actinremodeling (97, 98). CRAC channels appear to

be the dominant mode of Ca2+ entry in T cells,but other Ca2+ channels exist. Their relevance

remains unclear (reviewed in 84).TCR-induced increases in intracellular

Ca2+ levels result in the activation of Ca2+

and calmodulin-dependent transcription fac-

tors, including myocyte-enhancing factor 2(MEF2) and downstream regulatory element

antagonist modulator (DREAM), as well as sig-

naling proteins, including the phosphatase cal-cineurin and the Ca2+-calmodulin-dependent

kinase (CaMK), that in turn activate a vari-ety of transcription programs (reviewed in 99).

Activated calcineurin dephosphorylates mem-bers of the nuclear factor of activated T cells

(NFAT) family, leading to their translocationto the nucleus. In the nucleus, NFAT iso-

forms can form cooperative complexes with avariety of other transcription factors, thereby

integrating signaling pathways, resulting in dif-ferential gene expression patterns and func-

tional outcomes, depending on the context of

the TCR signal. The most well-studied inter-action is NFAT/AP-1, which integrates Ca2+

and Ras signals and results in the expression of

genes important for T cell activation inclu

ing IL-2. In contrast, NFAT activity in the sence of AP-1 activation induces a pattern

gene expression that ultimately results in T canergy and a characteristic lack of IL-2 p

duction (100). It is still unclear whether NFAisoforms are cooperating with other transcr

tion factors or are functioning as dimersinduce the anergic transcriptional pattern (viewed in 101). The regulatory T cell lineag

specific transcription factor forkhead bprotein 3 (FOXP3) also cooperates with NF

and antagonizes NFAT/AP-1 gene transcrtion, resulting in Treg functional gene expr

sionandalackofIL-2production(102).FinaNFAT family members can also cooperate w

STAT proteins to induce either Th1 or Tdifferentiation through expression of T-bet

GATA3, respectively (99).

ACTIN AND CYTOSKELETALRESPONSES

When a T cell is presented with cognate angen by an APC, signals from the TCR initi

a program of actin cytoskeletal rearrangemethat results in polarization and activation of

T cell (reviewed in 104). Actin reorganizatioessential for T cell function, as actin polym

ization inhibitors impede T cell/APC intertions (105) and abolish proximal TCR sign

(106).

T cell/APC conjugation results in morphlogical changes, as the stimulated T cell roun

up and accumulates filamentous actin (F-actat the stimulatory interface. These changes

thought to depend on a TCR-induced increin plasma membrane fluidity and a decre

in cellular motility. Cessation of motilityassociated with TCR-induced Ca2+-depend

phosphorylation and deactivation of the myomotor protein MyH9; however, the signal

pathway linking the TCR to this event has nyet been defined fully (107). Plasma membra

fluidity is increased, in part, by the TCR- a

Vav1-dependent transient dephosphorylatof ERM (ezrin, radixin, and moesin) prote

resulting in the loss of their ability to link t



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plasma membrane to the actin cytoskeleton

(108). Ca2+ signaling and integrin activationdownstream of the TCR result in additional

modifications of actin-associated proteins thatmay play roles in altering plasma membrane

rigidity (104).Accumulation of F-actin at the T cell/APC

interface is the result of TCR-induced lo-calized activation of multiple actin regulatoryand polymerizing pathways, the best stud-

ied of which involves the actin-related pro-teins 2/3 (Arp2/3) complex, although Arp2/3-

independent pathways also contribute to thisprocess (109). Activation of Arp2/3 requires its

interaction with nucleation-promoting factors(NPF) including Wiskott-Aldrich syndrome

protein (WASp), WAVE2, and hematopoieticcell lineagespecific protein 1 (HS1). WASp is

recruited to the site of TCR activation throughits interaction with the SLP-76-associated

adapter protein Nck, where it is activated via

Vav1-dependent stimulation of the Rho fam-ily GTPase Cdc42 (110). Vav1-mediated acti-

vation of a second Rho family GTPase, Rac1,results in the activation of WAVE2 (104). Given

the proposed reliance of WASp and WAVE2on Vav1-mediated GTPase activation, it is in-

teresting that actin-dependent processes thatare defective in Vav1-deficient T cells can

be rescued with the expression of a GEF-inactive Vav1 mutant, suggesting that other

Rac and Cdc42 GEFs may be able to sup-port TCR-induced actin changes (55). This

result also suggests that other Vav1 functions

are important for TCR-induced actin changes.Consistent with this is the observation that,

through its protein interaction domains, Vav1may contribute to WAVE2 and WASp activa-

tion through the recruitment of Dynamin2, aGTPase that is important for TCR-induced

actin dynamics (111).Activation of the T cell in response to an

APC also results in the polarization of the Tcell, whereby the microtubular organizing cen-

ter (MTOC) moves toward the T cell/APCcontact site (112). Although polarization of the

MTOC has long been observed as a hallmark of

productive T cell/APC conjugation, the signal-

cSMAC: centralsupramolecularactivation cluster

pSMAC: peripherasupramolecular

activation cluster

ing mechanisms responsible for this movement

remain undefined. Recent data suggest, how-ever, that the adapter protein ADAP (a com-

ponent of the SLP-76-nucleated complex) mayplay a role through its interaction with the mi-

crotubule motor protein dynein (113). Move-ment of the MTOC appears essential for the

formation of the immunological synapse (IS).The IS is an organized structure that devel-opsatthecontactsitebetweentheTcellandthe

APC. It is composed of two concentric regionsbased on molecular composition: the TCR-

rich central supramolecular activation cluster(cSMAC), surrounded by the integrin-rich pe-

ripheral SMAC (pSMAC) (114, 115). Althoughthe IS was described approximately 10 years

ago, its precise role in T cell activation re-mains unclear. Initially, the concentration of

receptors in the cSMAC led to the proposalthat the cSMAC is the site of enhanced recep-

tor engagement and prolonged signaling (115).

However, later studiesshowed that TCRsignalspeak prior to cSMAC formation and suggested

that the cSMAC is primarily the site of TCRdegradation (116, 121, 122). More recently, it

has been proposed that the cSMAC is the siteof both TCR signal enhancement and TCR

degradation, andthe balance between these twoprocesses is determined by the quality of the

antigen such that the cSMAC can serve to am-plify weak agonist signals (117). Most studies of

the cSMAC have focused on the TCR and itsassociated molecules. However, a newly defined

subregion of the cSMAC that is rich in CD28, a

costimulatory molecule (see below), and PKCbut relatively devoid of the TCR points to the

potential importance of the cSMAC for costim-ulatory molecule signal transduction (118).

Although the precise roles of the cSMACremain controversial, it is now well accepted

that the initiation of TCR signals occurs in pe-ripheral microclusters that begin to form prior

to IS formation. pMHC/TCR ligation resultsin the formation of intracellular microclusters

that contain the TCR complex and associatedsignaling molecules, including LAT and SLP-

76 (120). These clusters initiate and can sus-

tain Ca2+ signals (121). The clusters persist



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Inside-out signaling:signals initiated byengagement ofimmunoreceptors thatlead to conformationalchanges and clustering

of integrins, therebyincreasing the affinityand avidity of theintegrins for theirligands

for a short time, after which they converge

toward the cSMAC (121, 122). Formation andtranslocation of the clusters are dependent on

F-actin dynamics, and new clusters continue toform even after the mature IS is established

(121123). Integrins play a key role in sus-taining microclusters, emphasizing their im-

portance for T cell activation (124).Opposite the MTOC and IS, another or-dered structure forms, known as the distal pole

complex (DPC). Although the role of the DPCis not known for certain, investigators specu-

late that the DPC is critical for sequesteringnegative regulators away from the TCR acti-

vation complex (104). Additionally, the DPCmay contribute to the polarization of key

signaling molecules that may be required fordistinguishing memory versus effector fate de-

cisions in recently divided cells (125). Forma-tion of this complex is dependent on F-actin

and the rephosphorylation of ERM proteins

that migrate to the distal pole linking signal-ing molecules to the cytoskeleton (126).

TCR signaling cascades and pathwaysdownstream of actin reorganization are inter-

twined and difficult to tease apart, as manyof the effector molecules involved have mul-

tiple enzymatic and adapter functions. WASp,WAVE2, and Vav1 signals play roles in TCR-

induced signalingthat appearto be independentof their roles in actin responses (52, 97, 127).

Therefore, loss of different actin regulators mayresult in complex TCR signaling defects (128).

Future studies are required to understand fully

the feedforward and feedback mechanisms thatdefine the interdependence between cytoskele-

tal dynamics and T cell activation.

TCR INSIDE-OUT SIGNALINGTO INTEGRINS

Integrins are heterodimeric receptors re-

sponsible for mediating cell/cell or cell/matrixadhesions. Key T cell integrins include leuko-

cyte functionassociated antigen-1 (LFA-1)

and very late antigen-4 (VLA-4), which bindtheir respective ligands intercellular adhe-

sion molecule (ICAM) and vascular cell ad-

hesion molecule (VCAM) and fibronectin

other immune cells, endothelial cells, fibrolasts, and extracellular matrix proteins. Acti

tion of integrins (increasing their affinity aavidity for ligand) is critically dependent

biochemical events initiated by the TCRprocess designated inside-out signaling (12

Although the pathway from the TCR to ingrin activation has not been completely eludated, TCR-mediated activation of several k

signaling molecules as well as TCR-inducactin/cytoskeletalchanges havebeen implica

in this process.A central regulator of inside-out signal

is the small GTPase Ras-proximity-1 (RapRap1 enhances T cell activation by mediati

TCR-induced adhesion to ICAM-1. This coclusion is based on studies utilizing overexpr

sion of dominant-negative and constitutivactive forms of Rap1 (130, 131) and more

cently by analysis of mice deficient in Rap

in which TCR-induced adhesion to ICAMis markedly reduced (132). The importance

understanding therole of Rap1 is clear, as mutions in Rap1-mediatedintegrin activation h

been linked to leukocyte adhesiondeficiensyndrome, a disease that can lead to severe b

terial infections (reviewed in 129).Many proximal signaling molecules t

comprise the early TCR signalosome, incling LAT, SLP-76, and PLC1, are required

integrin and Rap1 activation (reviewed in 1(Figure 2). However, there are downstream

fectors that have a more selective role in

tegrin activation, including the adapter ptein ADAP. T cells from ADAP-deficient m

are defective in TCR-induced LFA-1 clusting and adhesion to ICAM (133). The asso

ation of ADAP with SLP-76 appears to be quiredfor integrin activation, as overexpress

of ADAP but not a mutant of ADAP that canot bind to SLP-76 enhances integrin funct

following TCR ligation (134, 135). Althoumice expressing the reciprocal mutation in

SH2 domain of SLP-76 have been generaand share several characteristics with ADA

deficient mice, TCR-induced integrin acti

tion in these mice has not yet been reported



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16/31


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how Akt mediates T cell growth and survival

downstream of CD28.The CD28-mediated generation of PIP3

also serves as a docking site for the PHdomain of Itk.Although Itk inducibly associates

with the LAT/SLP-76/Gads/PLC1 complex

that forms following TCR ligation, its local-

ization and activation also depends on PI3K-generated PIP3 (61). Itk can also associate di-rectly with CD28 via the CD28 proximal PxxP

motif (61). It is possible that this interactionkeeps Itk close to Lck (which binds to the dis-

tal PxxP motif of CD28), allowing for Lck-mediated phosphorylation and activation of Itk

(149) and enhanced Ca2+ flux, another charac-teristic of CD28-mediated signaling. Despite

the fact that Itk and CD28 associate with oneanother, studies in Itk-deficient T cells demon-

strate that some CD28 signaling is still intact inthe absence of Itk, suggesting that this interac-tion may not be that critical for CD28 signal-

ing (150). Although the proline motifs in thetail of CD28 are required for CD28-mediated

proliferation and IL-2 production, these motifsare dispensable for Bcl-xl upregulation (151).

This function appears to be more reliant on theproximal YMNM p85 binding site (151). Thus,

CD28 can differentially regulate proliferationand survival in activated T cells.

Another more recently described functionof CD28 is induction of arginine methyla-

tion. Following CD28 ligation, protein argi-

nine methyltransferase activity increases, andarginine methylation of multiple proteins, in-

cluding Vav1, is induced. Vav1 arginine methy-lation appears to occur in thenucleusandcorre-

lates with IL-2 production (152). Although theprecise biologic significance of this posttrans-

lational modification is unknown, this pathwaymay provide yet another mechanism by which

CD28 regulates TCR signaling.Many of the pathways described above are

activated by TCR ligation alone; however, themagnitude of the response is considerably aug-

mented with CD28 coligation. This observa-

tion has led to speculation that CD28 engage-ment results primarily in a quantitative rather

than a qualitative change in T cell activation

parameters (141). Although this appears to be

true, it is also true that quantitative differencesin signaling can result in qualitatively distinct

functional outcomes.CD28-deficient mice exhibit dampened im-

mune responses to a variety of infectious agents(reviewed in 141). Although these studies

demonstrate the importance of costimulationby CD28, notall immuneresponses areseverely

impacted by its loss (153). Such observations in-

dicate that molecules other than CD28 can pro-vide costimulation for T cells. Indeed, multiple

surface receptors have been described as havingcostimulatory functions. Included among these

areCD2, CD5, CD30, 4-1BB, OX40, induciblecostimulator (ICOS), and LFA-1. For this re-

view, we focus on the CD28-related proteinICOS and two members of the tumor necro-

sis factor receptor (TNFR) family to provideexamples of how costimulatory molecules can

link to downstream effectors, either directly orthrough adapter proteins.

Unlike CD28, which is expressed at con-

stantlevelsonbothrestingandactivatedTcells,ICOS is induciblyexpressed on activatedT cells

(154). ICOS deficiency results in impaired im-mune responses, similar to yet not as severe

as those observed in CD28 knockout models,suggesting that these two molecules may func-

tion in similar pathways (155). Indeed, ICOSshares several structural features with CD28,

including a YMXM motif in its cytoplasmictail that associates with p85 (155). This site

is presumed to be responsible for PI3K-drivenAkt and/or Itk activation observed downstream

of CD3/ICOS stimulation, which likely con-

tributes to the similarities seen in CD3/CD28and CD3/ICOS-stimulated cells (156). ICOS

does not induce IL-2 gene transcription asCD28 does. Failure to induce IL-2 has been

attributed, at least in part, to the inability of theYxxM motif of ICOS to associate with Grb2,

an association that is present via this motif inCD28 (157). Therefore, although ICOS acti-

vates genes similar to those induced by CD28,there are differences in the degree to which par-

ticular genes are expressed. These differenceshave in vivo relevance, as mice doubly deficient



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Dok-1 and -2, are expressed in T cells. Co-

ordinated deletion or knockdown of theseproteins results in increased TCR cytokine

production and proliferation; prolonged phos-phorylation of ZAP-70, LAT, SLP-76, and

Akt; and the development of a lupus-like re-nal diseasewith highantidouble-stranded DNA

antibody titers (167, 168). Dok can asso-ciate with several negative regulators includ-ing SHIP-1 (SH2 domaincontaining inositol

phosphatase), Csk, and Ras GTPase-activatingprotein (RasGAP); however, how these associ-

ations mediate the negative regulatory role ofDok proteins remains to fully elucidated. Re-

cent structure/function studies in T cell linesshowed that the phosphotyrosine-binding do-

main(PTB)ofDok-1and-2canbindtheITAMmotif in CD3, making it tempting to specu-

late that Dok may compete with ZAP-70 forITAM binding (167). Whether this interaction

is relevant in vivo remains to be shown. Dok

proteins can also be recruited inducibly to aLAT-, Grb2-, and SHIP-1-containing molecu-

lar complex (168). This finding is intriguing andbegs the question as to how LAT participates in

both positive and negative signaling pathways.Determining the domains necessary for the as-

sembly of this negative regulatory complex andwhether it is separate from or a part of the stim-

ulatory LAT-containing complex may provideinsight into how early phosphorylation events

are regulated.It has been suggested that SLP-76 can also

be a target of negative regulation. The serine/

threonine kinase HPK1 inducibly binds to theSH2 domain of SLP-76. This kinase has both

positive and negative effects on TCR signaltransduction; however, its role as a promi-

nent negative regulator was confirmed re-cently by the description of HPK1-deficient

mice. HPK1-deficient T cells exhibit en-hanced phosphorylation of several early sig-

naling molecules, and HPK1-deficient miceare more susceptible to experimental autoim-

mune encephalomyelitis (EAE) than are wild-type mice (169). As a possible explanation for

these phenotypes, two groups have shown that

HPK1 can phosphorylate a serine residue

SLP-76 that mediates recruitment of 14-family members (169, 170). 14-3-3 prote

havebeenimplicatedintheregulationofsevesignaling pathways. Thus, although the p

cise mechanism by which HPK1 disrupts TCsignal transduction remains unclear, identifi

tion of a SLP-76/14-3-3 interaction may incate that this mechanism involves a conserv

method of signal transduction regulation.

The role of a novel family of proteins in rulating TCR signaling has become apprecia

more recently. The suppressor of T cell rector signaling (Sts) family of proteins conta

two members, Sts-1 (TULA2) and -2 (TULCombined deficiency in these proteins leads

hyperproliferative T cells and an increased sceptibility to autoimmunity (171). When t

phenotype of Sts-1 and -2 knockout mice wdescribed, the mechanism of negative regu

tion was unclear. Although still not understofully, the C-terminal phosphoglycerate mut

domain of Sts-1 acts as a phosphatase w

specificity for Syk and, to a lesser degree, family members, thus providing a model

how Sts-1 may negatively regulate TCR snal transduction (172). Interestingly, Sts-2 h

very little phosphatase activity and, in cell lmodels, can enhance ZAP-70 activation (17

Recent studies demonstrating that Sts-2 cbind to and induce the degradation of c-C

(a negative regulator of TCR signaling; see blow) have provided a mechanism by which St

may act as a positive regulator of T cell actition (174). Whether net ZAP-70 activity is u

mately increased or decreased in response to

in vivo challenge likely depends on the relatexpression of these two family members.

Cbl proteins play an important role as native regulators of T cell signaling. Two fam

members, c-Cbl and Cbl-b, are immune moulators, as deletion of each gene in vivo leads

hypercellularity and, in the case of Cbl-b, sptaneous mulitorgan infiltration (175, 176). B

c-Cbl and Cbl-b facilitate the ubiquitinationproteins, targeting them for degradation. Th

also mediate the downregulation of the TC



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following stimulation with antigenic peptides

and have been recently shown to play a role inthe dissipation of the early signaling complex

(176178). Thus, Cbl proteins, by way of theirability to regulate protein degradation, provide

anexampleofyetanothermeansbywhichTCRsignaling is terminated.

Similar to the necessity of signals providedby the TCR to be complemented by costim-ulatory molecules, TCR-generated regulatory

signals are also aided by coreceptor signals.Cytotoxic T lymphocyte antigen-4 (CTLA-4)

and programmed death-1 (PD-1) are two ex-amples of such receptors that limit the ex-

pansion and activation of TCR-triggered Tcells. These molecules are found on activated

T cells with peak expression 2448 h afterstimulation. Genetic studies have documented

the importance of both for maintaining self-tolerance. By 56 days after birth, CTLA-4-

deficient mice exhibit activated peripheral T

cells, splenomegaly, and lymphocytic infiltratesinto nonlymphoid organs (179). Although less

striking, PD-1-deficient mice also develop au-toimmune features, including the development

of a lupus-like disease by 6 months of age (180).Like costimulatory receptors, inhibitory recep-

tors utilizemotifs andmolecular pathways simi-lar to those used by the TCR when propagating

a negative signal; they may even provide dock-ing sites for these shared molecules (181, 182).

Another mode by which inhibitory receptorshave been proposed to function is to utilize the

same ligand and/or signaling molecules as cos-

timulatory receptors, thus setting up the poten-tial for competition or sequestration of ligands

or key substrates. This means is best illustratedby CTLA-4 and CD28, which share the lig-

ands CD80 and CD86 and counter each otherin the regulation of cell cycle proteins, cytokine

expression, and Cbl-b expression.

CONCLUSIONS

As in the first 10 years after identification of

the TCR, the past 15 years have seen a dra-

matic expansion in our understanding of thebiochemical pathways triggered downstream ofthe TCR. We now appreciate more fully howPTKs couple to effectors and second messen-

gers via the utilization of adapter proteins, howRas and NF-B pathways are activated, how

Ca2+ ions are sensed, that TCRsignaling can be

required for the activation of other cell surfacereceptors, and that the actin cytoskeleton does

more than just dictate cell shape. Studies inves-tigating signal transduction by costimulatory

molecules have highlighted the importance ofsignal amplification, and discoveries of spon-

taneous or induced models of autoimmunityhave brought negative regulation of these sig-

naling pathways to the forefront. However, asoften noted in this review, links between many

signaling molecules and cellular outcomes re-main poorly defined. Future studies will be

required to fill gaps in our knowledge andlikely will reveal new and unexpected interac-

tions between currently known and unknown

molecules.This review primarily focused on the signal

transduction pathways in naive T cells. Overthe next 15 years, it will be important to ap-

ply current and new knowledge to other T celllineages, including T regulatory cells, natural

killer T cells, and memory T cells, to estab-lish whether these paradigms are universal. In

the coming years, the population-based analy-ses that established the field of signal transduc-

tion will be driven toward single cell analyses.We can anticipate that this technological shift

will allow for better analysis of human T cells

and the application of basic science to humandisease.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings that

might be perceived as affecting the objectivity of this review.



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ACKNOWLEDGMENTS

We thank Christopher P. Garvin for figure design, Dr. Art Weiss for helpful comments, and JustStadanlick for editorial assistance.

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