T Cell Activation

ANRV371-IY27-21 ARI 16 February 2009 13:31

T Cell ActivationJennifer E. Smith-Garvin,1 Gary A. Koretzky,1,2,3

and Martha S. Jordan1,21Abramson Family Cancer Research Institute, 2Department of Pathology and LaboratoryMedicine, 3Department of Medicine, University of Pennsylvania, Philadelphia,Pennsylvania 19104; email: [email protected], [email protected],[email protected]

Annu. Rev. Immunol. 2009. 27:591619

First published online as a Review in Advance onJanuary 8, 2009

The Annual Review of Immunology is online atimmunol.annualreviews.org

This articles doi:10.1146/annurev.immunol.021908.132706

Copyright c 2009 by Annual Reviews.All rights reserved

0732-0582/09/0423-0591$20.00

Key Words

signal transduction, immunoreceptor, integrin

AbstractThis year marks the 25th anniversary of the rst Annual Review ofImmunology article to describe features of the T cell antigen receptor(TCR). In celebration of this anniversary, we begin with a brief intro-duction outlining the chronology of the earliest studies that establishedthe basic paradigm for how the engaged TCR transduces its signals.This review continues with a description of the current state of our un-derstanding of TCR signaling, as well as a summary of recent ndingsexamining other key aspects of T cell activation, including cross talkbetween the TCR and integrins, the role of costimulatory molecules,and how signals may negatively regulate T cell function.

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TCR: T cell antigenreceptor

Signal transduction:biochemical eventslinking surfacereceptor engagementto cellular responses

INTRODUCTION

Twenty-ve years ago, Annual Review of Im-munology published its rst review describingfeatures 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 observationsmade in the decade following the initial descrip-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 understandingof the molecular and biochemical events criti-cal for T cell activation that have emerged fromthe 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 identication of the genesresponsible for the antigen-binding proteins(35). An even earlier approach relied on im-

Figure 1TCR proximal signaling then and now: lling in the gaps. Then (a): TCR signaling mid-1990s (adapted from Reference 2). Ligation ofthe 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 theTCR couples to downstream pathways (b), the molecular basis for Ca2+ inux (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 andits 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 effectorsto 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 Ca2+ from theER into the cytoplasm. STIM contains paired EF hands within the ER lumen, which bind a single Ca2+ molecule. Upon depletion ofER stores, Ca2+-free STIM molecules oligomerize and move to areas of ER/plasma membrane proximity, where they colocalize withand induce dimerization of Orai1 dimers, resulting in a functional CRAC channel and subsequent inux of Ca2+. (d ) Activation of Rasinvolves 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 GRB2and 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 de-ned specicity, hybridomas, or clonal T celltumors in the hope that antibodies would begenerated that would react with the receptorresponsible for binding to antigen (68). Suchantibodies were produced and were then usedrst to demonstrate interference with antigenicresponses and later to perform the initial bio-chemical characterization of the receptor it-self. These studies revealed a complicated cellsurface structure that included proteins reac-tive with antibodies against the nonpolymor-phic CD3 proteins (initially thought of as threepolypeptides, , , and ) (9), as well as variableproteins designated and .

The antigen-binding function of wasobvious early on, by inference owing to theirhighly polymorphic nature and similarity toimmunoglobulin and experimentally owing toearly gene transfer experiments that demon-strated that antigen/major histocompatibilitycomplex (MHC) reactivity tracked with expres-sion of these receptor components (10, 11).However, it was less obvious what role the CD3proteins played in TCR function. Several linesof evidence suggested that the CD3 moleculeswere critical for signal transduction: Unlike ,the CD3 molecules had long cytoplasmic tails,and anti-CD3 antibodies resulted in T cell ac-tivation, although it was difcult to demon-strate signaling function conclusively. One early

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www.annualreviews.org T Cell Activation 593

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PKC: protein kinaseC

PLC: phospholipaseC

PTK: proteintyrosine kinase

Immunoreceptortyrosine-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 linesexpressing without CD3 or CD3 without, thereby creating reagents to investigate theindependent role of the receptor components.These experiments were not successful, as itbecame clear that there is an obligatory coex-pression of with CD3 (12). These studiesdid result, however, in the creation of the rstin a long line of genetically altered Jurkat Tcells that have been instrumental in our under-standing of how the TCR complex couples to itssignaling machinery (13).

EARLY SIGNALING STUDIES

Initial investigations into how the TCR trans-duces its signals began with the observation thatTCR-decient 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 sourceof the Ca2+ increase was shown to be a com-bination of Ca2+ released from an intracellularpool in response to inositol trisphosphate (IP3)production and inux of Ca2+ from outside ofthe cell (15). It was presumed that phorbol es-ters were important because of their ability toactivate protein kinase C (PKC, now known tobe 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 TCRmay function to regulate PLC activity.

Testing this notion proved somewhatdifcult at rst, as PLC regulation was initiallydescribed downstream of heterotrimericguanosine triphosphate (GTP)-binding pro-teins associated with seven-transmembrane

domain receptors. An exhaustive but unsuc-cessful search ensued for the GTP-bindingprotein critical for coupling the TCR toPLC activation. Insight into how the TCRmay initiate PLC activation emerged from aconuence of discoveries in immune cells andother lineages. For example, stimulation of theTCR results in changes in protein phospho-rylation, including inducible phosphorylationof the newly described chain of the CD3complex (16). Furthermore, growth factorreceptors with intrinsic protein tyrosine kinase(PTK) activity also stimulate PLC function.In contrast to seven-transmembrane domainreceptors, however, receptor PTKs stimulateanother PLC isoform, PLC. Although noneof the TCR components possessed enzymaticactivity themselves, cytosolic PTKs of theSrc family (in particular Lck and Fyn) werebeing described in T cells. These PTKs wereassociated either with the TCR (17) or withthe CD4 and CD8 coreceptors (18, 19),both important for TCR signaling. Theseobservations, coupled with the new knowledgethat T cell activation requires PTK function(20), led to a new TCR signaling paradigmin which the TCR recruits cytosolic PTKs toactivate key second messengers.

HOW CD3 TRANSDUCESITS SIGNALS

Testing this model led to a search for addi-tional substrates of the TCR-stimulated PTKs.Among the most attractive candidates were ty-rosines within the CD3 molecules themselvesthat fell within a motif present once withinCD3 , , and and in triplicate within each chain and also in key immunoreceptors on otherimmune cell lineages. The signature ofthese motifs (21), eventually designated im-munoreceptor tyrosine-based activation motifs(ITAMs), is two tyrosines anking a series ofamino acids, including key leucine/isoleucineswith stereotypic spacing. Numerous labora-tories demonstrated that the ITAM tyrosinesare in fact phosphorylated upon TCR lig-ation, but dening the importance of this


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posttranslational modication 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 CD3chains to extracellular and transmembrane do-mains from other proteins (2225). ThesecDNAs 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 cellactivation.

Studies of the CD3 chimeras led naturally tothe 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-kDaphosphoprotein, 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 andrecruitment of ZAP-70. This converted theTCR with no intrinsic enzymatic function to anactive 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 thetest of intensive investigation. In the 15 yearssince ZAP-70 was cloned, investigators havelled in many of the gaps between the TCRand 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 oflower organisms, including the observation that PTKs could linkto the phosphatidylinositol pathway and the paradigm describingadapter 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 recruitmentof nonreceptor PTKs to enzymatically inactive surface receptorsand the notion that protein tyrosine phosphatases may be positiveas 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 ofother cell surface receptors (e.g., integrins) and other hematopoi-etic lineages. It is clear that, although basic paradigms may besimilar, each cell type and receptor utilizes unique ways to regu-late signal transduction cascades. Additionally, recent studies havedemonstrated that pathways thought to be distinct (e.g., integrinsand immunoreceptors) instead intersect at multiple levels. Fu-ture insights into how diverse signaling pathways are integratedto result in the appropriate biologic response will undoubtedlycontinue to benet from comparing and contrasting activationevents downstream of multiple receptors in different immuneand nonimmune cell lineages.

Adapter protein:cellular protein thatfunctions to bridgemolecular interactionsvia 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 howthese molecules participate in T cell activation,about how signaling complexes are organizedby adapter proteins to bring effector proteinstogether, and about the unexpected intersec-tion of particular signaling pathways (see alsothe side bar, Arguing by Analogy: Comparingand 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 alinear event starting at the receptor and end-ing in the nucleus. Instead, there appears to becomplex feedback and feedforward regulationat each step. Ironically, one of the most central


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pMHC: peptidemajorhistocompatibilitycomplex (MHC)complex

signaling questions that remains is how receptorbinding translates most proximally into an ac-tivating signal. Many models have been pro-posed, but none has yet withstood the rigorof subsequent investigation. This review sum-marizes our current understanding of many ofthese issues and poses some of the intriguingquestions that remain.

INITIATING TCR SIGNALTRANSDUCTION

Since identication of the TCR as a complexconsisting of the variable chains nonco-valently associated with the nonpolymorphicCD3 proteins, considerable work has gone intodening the stoichiometry of these interactingmolecules. We now know that the CD3 proteinsexist as a series of dimers including , , and associated with a single heterodimer. Al-though it has been clear for more than 15 yearsthat CD3 transduces signals from the engagedreceptor via its ITAMs, exactly how ligationof the TCR is translated into the rst signalremains controversial. Current models suggestthat both TCR aggregation and conformationalchanges may play roles in signal initiation.

Two separate but not mutually exclusiveconformational changes within the CD3 cy-toplasmic tails have been proposed as mech-anisms for TCR-inducible ITAM phosphory-lation. The rst mechanism is based on theobservation that, in the presence of acidiclipid designed to mimic the inner leaet ofthe plasma membrane, the cytoplasmic tails ofCD3, and to a lesser extent of CD3, fold orinteract with the lipid in such a way as to pre-vent phosphorylation. Conversely, in aqueoussolution or in the presence of zwitterionic lipid,the CD3 tails are more readily able to be phos-phorylated by Lck (28, 29). These ndings haveled to the hypothesis that in resting T cells theCD3 and/or CD3 tails are tightly associatedwith the lipid-rich inner leaet of the plasmamembrane, rendering them inaccessible to Lckphosphorylation, but following TCR ligation,they are released from the membrane and phos-phorylated. Investigations have begun to test

this hypothesis in T cells, and recently the in-teraction of the CD3 cytoplasmic domain withthe plasma membrane has been conrmed inresting Jurkat T cells by uorescence resonanceenergy transfer (FRET) (29). A second possibleconformational change in CD3 has garneredmuch attention following the observation thatupon TCR ligation a proline-rich region (PRR)in CD3 is exposed and available to recruit anSH3 domain of the adapter protein noncat-alytic tyrosine kinase (Nck). Importantly, thisoccurs prior to CD3 and CD3 ITAM phos-phorylation (30). Investigators speculated thatNck could then recruit and activate effectormolecules required for subsequent ITAM phos-phorylation. However, recent data using micewith retrogenic or knockin mutations of theCD3 PRR have failed to show a requirementfor the CD3/Nck interaction in the activationof peripheral T cells (31, 32). This observationmust be considered in light of the T cell devel-opmental abnormalities in the knockin mice.Resolving the importance of the PRR for ma-ture T cell function requires temporal controlof expression of the mutant.

Although the seminal nding that there isa non-ITAM region in CD3 that is critical forTCR signaling appears rm, several controver-sies remain regarding the CD3 PRR. Thereis disagreement regarding whether the inter-action between Nck and CD3 is constitutiverather than inducible (31, 3335). The role ofthe CD3/Nck interaction in mature T cells isalso controversial, as it has been suggested thatthe interaction (a) is not required for mature Tcell activation (32), (b) is required for signal am-plication and ITAM phosphorylation follow-ing weak TCR ligation (34), or (c) is required forregulation of TCR activity by inhibiting ITAMphosphorylation and promoting TCR degrada-tion (35). Ongoing work will provide insightsinto these controversies.

Although CD3 conformational changes mayexplain the initiation of the intracellular ki-nase cascade, we do not know how pep-tide/MHC (pMHC)/TCR interactions resultin conformational changes in the intracellu-lar regions of the CD3 complex. Models based


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Table 1 Recent models for initial TCR triggering

Model DescriptionPiston-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 regionsof CD3 chains

Receptor deformation The force of the motility of the T cell as it moves over an APC induces TCR triggering only whenpMHC/TCR interactions are of a high enough afnity to withstand the force long enough to undergoa conformational change

Permissive geometry pMHC dimers bind TCR/CD3 dimers inducing a rotational scissor-like conformational change in CD3chain(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 betweenthe T cell and the APC, thereby shifting the enzymatic steady state toward an activating state

Diffusion trapping Expanded kinetic segregation model in which the afnity/diffusion coefcient of the pMHC/TCRdictates the valency of TCRs required to trap the complex in the tight contact zone and thereforeinitiate a productive signal

Pseudodimer Agonist pMHC/TCR recruits a second TCR via interaction with its associated CD4, the second TCRthen binds a coagonist endogenous pMHC forming a stable pseudodimer that triggers signaling viaproximity of ITAMs to CD4-associated Lck and/or by conformational change

on ligand-induced conformational changes inthe TCR and/or TCR aggregation have beenproposed (Table 1). The crystal structuresof several pMHC/TCR complexes have re-vealed little conformational changes in theTCR heterodimer outside of the binding do-main (reviewed in 36). However, owing to thecurrent technical limitations of crystallogra-phy and protein nuclear magnetic resonancespectroscopy, only pMHC/TCR structures inthe absence of transmembrane domains andCD3 components have been elucidated. There-fore, changes between the components of theTCR/CD3 complex in relation to one anotheror to the plasma membrane cannot be ruled out.The rigid, rod-like transmembrane domain ofthe CD3 could mediate a piston-like move-ment of the TCR complex perpendicular tothe 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 bycell-to-cell contact, but this possibility is dif-cult 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 whichthe movement of the T cell as it travels acrossthe antigen-presenting cell (APC) exerts a force

on transient pMHC/TCR interactions. Onlywhen the force required to induce conforma-tional changes in the TCR is less than the forcerequired to break the pMHC/TCR interactionwill TCR triggering occur (38). Alternatively,the permissive geometry model for TCR trig-gering predicts that dimeric pMHCs bind twoTCR/CD3 complexes (39). This interaction re-sults in a rotation of the TCR heterodimersaround one another, displacing the extracellulardomains of the associated CD3 molecules. Thetransmembrane interactions between the CD3and TCR dimers serve as pivot points, and theCD3 movement is scissor-like, exposing previ-ously shielded ITAMs and/or other functionallyimportant domains. This model is consistentwith studies that suggest that TCR aggregationis required for TCR triggering.

TCR aggregation has long been impli-cated as a mechanism for TCR triggering. Inthe classic aggregation model, crosslinking ofTCR/CD3 complexes with multimeric pMHCenables close contact and transphosphorylationbetween the CD3 tails and associated PTKs.Clustering of several TCR complexes also mayresult 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 byobservations that soluble multimeric but notmonomeric pMHC can trigger TCR activa-tion. However, recent biochemical and micro-scopic studies suggest that preformed TCRaggregates are present on nonactivated T cells(reviewed in 40). In the kinetic segregationmodel, adhesion and other accessory moleculeswith short extracellular domains such as CD2initiate close contact zones between an APCand a T cell (41). Inhibitory phosphatases withlong extracellular domains such as CD45 areexcluded because of their size. TCR complexesthat engage pMHC on the APC surface remainin 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 theclose 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 afn-ity and diffusion coefcient of the pMHC/TCRinteraction. Another model has been proposedthat suggests that endogenous pMHCs amplifysignals produced by the rare agonist pMHCsby promoting TCR aggregation and a subse-quent phosphorylation cascade (40). Similarly,the pseudodimer model proposes that an ago-nist pMHC/TCR can recruit a second TCR in aCD4-dependent manner, and this second TCRbinds a coagonist endogenous pMHC, forminga stable pseudodimer that could trigger signal-ing through ITAM/Lck proximity or throughCD3 conformational changes (43). Many ofthese models are not mutually exclusive, and itis likely that TCR aggregation, conformationalchanges within the TCR complex, and exclu-sion of inhibitory molecules are all required

for TCR triggering, perhaps in a stepwisefashion.

PROXIMAL SIGNALINGCOMPLEX

The earliest step in intracellular signaling fol-lowing TCR ligation is the activation of Src(Lck and Fyn) PTKs, leading to phosphoryla-tion of the CD3 ITAMs. Recruitment of ZAP-70 follows, leading to a cascade of phospho-rylation events. The past decade has seen thedescription of a subcellular assembly and acti-vation of an adapter protein nucleated multi-molecular signaling complex (Figure 1b). Thiscomplex is responsible for propagating theTCR/PTK signal into multiple and diverse dis-tal signaling pathways.

Among the most important of the ZAP-70targets are the transmembrane adapter proteinlinker for the activation of T cells (LAT) andthe cytosolic adapter protein Src homology 2(SH2) domaincontaining leukocyte phospho-protein of 76 kDa (SLP-76) (44, 45). Thesetwo adapters form the backbone of the com-plex that organizes effector molecules in thecorrect spatiotemporal manner to allow for theactivation of multiple signaling pathways. Theimportance of these adapters is underscored bystudies showing that the loss of either LAT orSLP-76 results in a near complete loss of TCRsignal transduction reminiscent of Syk/ZAP-70or Lck/Fyn double-decient T cells (4648).

LAT contains nine tyrosines that are phos-phorylated upon TCR engagement, which bindthe C-terminal SH2 domain of PLC1, the p85subunit of phosphoinositide 3-kinase (PI3K),and the adapters growth factor receptor-boundprotein 2 (GRB2) and GRB2-related adapterdownstream of Shc (Gads) (reviewed in 47).SLP-76 is then recruited to phosphorylatedLAT via their mutual binding partner Gads(49). SLP-76 itself contains three modulardomains: an N-terminal acidic domain withthree phosphorylatable tyrosines that interactwith the SH2 domains of Vav1, Nck, and IL-2-induced tyrosine kinase (Itk); a PRR thatbinds constitutively Gads and PLC1; and a


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C-terminal SH2 region that can bind adhesionand degranulationpromoting adapter protein(ADAP) and hematopoietic progenitor kinase1 (HPK1) (reviewed in 46). Although LAT andSLP-76 serve to nucleate this large signalingcomplex, the effector molecules themselves alsoare important for stabilizing the complex. Forexample, the Tec family kinase Itk is requiredin a kinase-independent manner for the re-cruitment of the guanine nucleotide exchangefactor (GEF) Vav1 to the APC contact site,whereas Vav1 is required for optimal SLP-76phosphorylation and recruitment to LAT aswell as for Itk activation (5052). These andother data suggest that the formation of thecomplex is more complicated than the linearmodel most often invoked for simplicity. For ex-ample, PLC1 directly binds to SLP-76, LAT,and Vav1 as well as to its activating kinaseItk (reviewed in 53). It is thought that theseinteractions 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 preciseallosteric and perhaps stoichiometric changeswithin the multimolecular complex that allowfor signal transduction.

To investigate more precisely the impor-tance of these complex interactions in primaryT cells, several laboratories have generatedmice expressing transgenic or knockin muta-tions in specic binding regions in variousmolecules involved in proximal signaling (5557). 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, aswould 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 doesnot result in a loss of interaction with their pro-posed binding partners, although it does resultin 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, thesestudies suggest that SH2/phosphotyrosine in-teractions may play important regulatory rolesfor 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 conformationalswitch allowing kinase activity (58). Consistentwith 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 specicallythe 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 byspecic domain/domain interactions within thecomplex.

The proximal signaling complex resultsin the activation of PLC1-dependent path-ways including Ca2+- and DAG-inducedresponses, cytoskeletal rearrangements, andintegrin activation pathways. Ligation of co-stimulatory receptors such as CD28 augmentsthese pathways. Below, we discuss the mecha-nisms by which these pathways are activated andregulated.

PLC1 ACTIVATIONAND SIGNAL TRANSDUCTION

Following TCR ligation, PLC1 is found inthe proximal signaling complex bound to SLP-76, Vav1, and LAT, where it is phosphorylatedand activated by Itk. Activated PLC1 then hy-drolyzes the membrane lipid PI(4,5)P2, produc-ing the second messengers IP3 and DAG. Thesetwo messengers are essential 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 andthe 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 themembrane through PH domain interactionswith PIP3, which has been locally generatedby Lck-induced PI3K activity (reviewed in 61).At the membrane, Lck phosphorylates Itk, andthe SH2 and SH3 domains of Itk interact withphosphorylated tyrosine 145 and the PRR ofSLP-76, respectively (6264). The role of Vav1and ZAP-70 in Itk activation is not understoodbut may relate to their involvement in the phos-phorylation of SLP-76 or PI3K activation (45,51, 52). A second Tec family kinase, Rlk, can alsophosphorylate PLC1, resulting in a relativelymild defect in Itk-decient mice and requiringthe study of Rlk/Itk double-decient mice tobetter understand the role of Tec kinases in Tcell activation (reviewed in 61).

DAG-MEDIATED SIGNALINGPATHWAYS

TCR-induced production of DAG results inthe activation of two major pathways involvingRas 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 proteinkinase (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 extracellularsignal-regulated kinase 1 (Erk1) and Erk2. Erkkinase activity results in the activation of thetranscription factor Elk1, which contributes tothe activation of the activator protein-1 (AP-1)( Jun/Fos) transcription complex via regulationof Fos expression. Additionally, Erk activity canresult 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 GEFsand is suppressed by GTPase-activating pro-teins (GAPs). Two Ras GEFs are presentin T cells, son of sevenless (SOS) and Rasguanyl nucleotide-releasing protein (RasGRP).

RasGRP appears more dominant for early ac-tivation of Ras, as SOS cannot compensatefor RasGRP deciency (6668). RasGRP is in-ducibly recruited to the membrane through aDAG-binding domain (69), where it is phos-phorylated by PKC (70). SOS is constitu-tively bound to the adapter protein GRB2,and upon TCR stimulation, the GRB2 SH2domain is recruited to and binds phospho-rylated tyrosines on LAT, thereby bringingSOS into the proximal signaling complex,where it can facilitate the localized activa-tion of Ras (71). The signicance of thesetwo modes of Ras activation was unclear un-til recently, when it was shown that RasGRP-dependent RasGTP production catalyzes SOSactivity, resulting in a positive feedback loopand robust TCR-induced Ras activation (72)(Figure 1c). This unied model explains theRasGRP dominance, as RasGRP would be re-quired to initiate the production of RasGTP,which upon reaching threshold levels wouldcatalyze SOS activity and further amplify thesignal.

The second major signaling pathway regu-lated by DAG is mediated by PKC, a PKCfamily member that contains a lipid-bindingdomain specic for DAG, which is importantfor recruiting PKC to the plasma membranefollowing T cell activation. However, phospho-rylation of PKC by Lck (reviewed in 73) maybe required to induce a conformational changethat enables binding to the lipid phosphatidylserine (PS), which in turn enhances binding toDAG, resulting in PKC activation (74). Otherproximal signaling molecules, including Vav1,PI3K, and 3-phosphoinositide-dependent ki-nase 1 (PDK1), also play roles in PKC local-ization, but details of their contributions are notcompletely dened (73).

One critical pathway that PKC regulatesis NF-B activation. Because both the canoni-cal and noncanonical activation of this pathwayin T cells is described in detail in this volumeof Annual Review of Immunology [see review byKarin and colleagues (75)], we highlight onlythe major steps in the classical activation ofNF-B downstream of the TCR. The NF-B


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family of transcription factors consists of vemembers. 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 sometime, the specics of how PKC activation leadsto nuclear import of NF-B in T cells are stillbeing elucidated.

Over the past several years, the identi-cation and characterization of a lymphocyte-specic activation complex have provided someinsight 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 ofCARMA1, which is required for CARMA1oligomerization and association with Bcl10 (77,78). MALT1 binds to Bcl10 and contributesto the degradation of the regulatory subunitof the IKK complex (IKK) by facilitating itspolyubiquitination, possibly via activation ofthe E3 ubiquitin ligase tumor necrosis factorreceptor-associated factor 6 (TRAF6) (79, 80).Degradation of this regulatory subunit allowsfor 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 ofNF-B activation (81, 82).

One additional component of the complexthat was recently identied as a CARMA1-

associating protein is ADAP (83). ADAP,originally dened as a SLP-76-binding part-ner, associates with the MAGUK domain ofCARMA1 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/orstabilize 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 theendoplasmic reticulum (ER) membrane, lead-ing to the release of ER Ca2+ stores intothe cytoplasm. Depletion of ER Ca2+ trig-gers a sustained inux of extracellular Ca2+

through the activation of plasma membraneCa2+ 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 beenidentied by their biophysical properties, andit has just been in the past few years thatthe pore-forming subunit of the channels wasidentied as the four-transmembrane domaincontaining molecule Orai1 (8587). Addition-ally, studies in the past few years have revealedthe sensor for depleted ER Ca2+ stores andthe activator of CRAC channels as stromal in-teraction molecule (STIM) (88, 89). STIM isan 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, whereone hand binds a single Ca2+ ion with lowafnity (88, 89). Two STIM proteins, STIM1and STIM2, are found in mammals, and re-cent work has shown that STIM1 is importantfor 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 clustersof 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. Recent work has shown thatSTIM1 oligomerization is sufcient to inducepunctae formation and CRAC channel activa-tion independent of Ca2+ store depletion (95).Further studies showed that the C-terminalcoiled-coil domain of STIM1 alone can inducedimerization of Orai1 dimers, which is suf-cient for CRAC channel activation independentof punctae formation and store depletion (96).How STIM1 oligomers translocate to areas ofER plasma membrane apposition and how, oncethere, they induce the Orai1 tetramerization re-main unknown. Interestingly, the WASp familyverprolin 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 tobe the dominant mode of Ca2+ entry in T cells,but other Ca2+ channels exist. Their relevanceremains unclear (reviewed in 84).

TCR-induced increases in intracellularCa2+ levels result in the activation of Ca2+

and calmodulin-dependent transcription fac-tors, including myocyte-enhancing factor 2(MEF2) and downstream regulatory elementantagonist modulator (DREAM), as well as sig-naling proteins, including the phosphatase cal-cineurin and the Ca2+-calmodulin-dependentkinase (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, therebyintegrating signaling pathways, resulting in dif-ferential gene expression patterns and func-tional outcomes, depending on the context ofthe 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 includ-ing IL-2. In contrast, NFAT activity in the ab-sence of AP-1 activation induces a pattern ofgene expression that ultimately results in T cellanergy and a characteristic lack of IL-2 pro-duction (100). It is still unclear whether NFATisoforms are cooperating with other transcrip-tion factors or are functioning as dimers toinduce the anergic transcriptional pattern (re-viewed in 101). The regulatory T cell lineagespecic transcription factor forkhead boxprotein 3 (FOXP3) also cooperates with NFATand antagonizes NFAT/AP-1 gene transcrip-tion, resulting in Treg functional gene expres-sion and a lack of IL-2 production (102). Finally,NFAT family members can also cooperate withSTAT proteins to induce either Th1 or Th2differentiation through expression of T-bet orGATA3, respectively (99).

ACTIN AND CYTOSKELETALRESPONSES

When a T cell is presented with cognate anti-gen by an APC, signals from the TCR initiatea program of actin cytoskeletal rearrangementsthat results in polarization and activation of theT cell (reviewed in 104). Actin reorganization isessential for T cell function, as actin polymer-ization inhibitors impede T cell/APC interac-tions (105) and abolish proximal TCR signals(106).

T cell/APC conjugation results in morpho-logical changes, as the stimulated T cell roundsup and accumulates lamentous actin (F-actin)at the stimulatory interface. These changes arethought to depend on a TCR-induced increasein plasma membrane uidity and a decreasein cellular motility. Cessation of motility isassociated with TCR-induced Ca2+-dependentphosphorylation and deactivation of the myosinmotor protein MyH9; however, the signalingpathway linking the TCR to this event has notyet been dened fully (107). Plasma membraneuidity is increased, in part, by the TCR- andVav1-dependent transient dephosphorylationof ERM (ezrin, radixin, and moesin) proteins,resulting in the loss of their ability to link the


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plasma membrane to the actin cytoskeleton(108). Ca2+ signaling and integrin activationdownstream of the TCR result in additionalmodications of actin-associated proteins thatmay play roles in altering plasma membranerigidity (104).

Accumulation of F-actin at the T cell/APCinterface 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 itsinteraction with nucleation-promoting factors(NPF) including Wiskott-Aldrich syndromeprotein (WASp), WAVE2, and hematopoieticcell lineagespecic protein 1 (HS1). WASp isrecruited to the site of TCR activation throughits interaction with the SLP-76-associatedadapter protein Nck, where it is activated viaVav1-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). Giventhe proposed reliance of WASp and WAVE2on Vav1-mediated GTPase activation, it is in-teresting that actin-dependent processes thatare defective in Vav1-decient T cells canbe rescued with the expression of a GEF-inactive Vav1 mutant, suggesting that otherRac and Cdc42 GEFs may be able to sup-port TCR-induced actin changes (55). Thisresult also suggests that other Vav1 functionsare 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-inducedactin dynamics (111).

Activation of the T cell in response to anAPC 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 theMTOC has long been observed as a hallmark ofproductive T cell/APC conjugation, the signal-

cSMAC: centralsupramolecularactivation cluster

pSMAC: peripheralsupramolecularactivation cluster

ing mechanisms responsible for this movementremain undened. 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 theformation of the immunological synapse (IS).

The IS is an organized structure that devel-ops at the contact site between the T cell and theAPC. 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 yearsago, its precise role in T cell activation re-mains unclear. Initially, the concentration ofreceptors in the cSMAC led to the proposalthat the cSMAC is the site of enhanced recep-tor engagement and prolonged signaling (115).However, later studies showed that TCR signalspeak prior to cSMAC formation and suggestedthat the cSMAC is primarily the site of TCRdegradation (116, 121, 122). More recently, ithas been proposed that the cSMAC is the siteof both TCR signal enhancement and TCRdegradation, and the balance between these twoprocesses is determined by the quality of theantigen such that the cSMAC can serve to am-plify weak agonist signals (117). Most studies ofthe cSMAC have focused on the TCR and itsassociated molecules. However, a newly denedsubregion of the cSMAC that is rich in CD28, acostimulatory molecule (see below), and PKCbut relatively devoid of the TCR points to thepotential importance of the cSMAC for costim-ulatory molecule signal transduction (118).

Although the precise roles of the cSMACremain controversial, it is now well acceptedthat the initiation of TCR signals occurs in pe-ripheral microclusters that begin to form priorto IS formation. pMHC/TCR ligation resultsin the formation of intracellular microclustersthat 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 clusteringof integrins, therebyincreasing the afnityand avidity of theintegrins for theirligands

for a short time, after which they convergetoward the cSMAC (121, 122). Formation andtranslocation of the clusters are dependent onF-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 polecomplex (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 keysignaling 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-actinand the rephosphorylation of ERM proteinsthat 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 difcult 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 signaling that appear to 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 fullythe feedforward and feedback mechanisms thatdene 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 bronectin onother immune cells, endothelial cells, brob-lasts, and extracellular matrix proteins. Activa-tion of integrins (increasing their afnity andavidity for ligand) is critically dependent onbiochemical events initiated by the TCR, aprocess designated inside-out signaling (129).Although the pathway from the TCR to inte-grin activation has not been completely eluci-dated, TCR-mediated activation of several keysignaling molecules as well as TCR-inducedactin/cytoskeletal changes have been implicatedin this process.

A central regulator of inside-out signalingis the small GTPase Ras-proximity-1 (Rap1).Rap1 enhances T cell activation by mediatingTCR-induced adhesion to ICAM-1. This con-clusion is based on studies utilizing overexpres-sion of dominant-negative and constitutivelyactive forms of Rap1 (130, 131) and more re-cently by analysis of mice decient in Rap1A,in which TCR-induced adhesion to ICAM-1is markedly reduced (132). The importance ofunderstanding the role of Rap1 is clear, as muta-tions in Rap1-mediated integrin activation havebeen linked to leukocyte adhesiondeciencysyndrome, a disease that can lead to severe bac-terial infections (reviewed in 129).

Many proximal signaling molecules thatcomprise the early TCR signalosome, includ-ing LAT, SLP-76, and PLC1, are required forintegrin and Rap1 activation (reviewed in 129)(Figure 2). However, there are downstream ef-fectors that have a more selective role in in-tegrin activation, including the adapter pro-tein ADAP. T cells from ADAP-decient miceare defective in TCR-induced LFA-1 cluster-ing and adhesion to ICAM (133). The associ-ation of ADAP with SLP-76 appears to be re-quired for integrin activation, as overexpressionof ADAP but not a mutant of ADAP that can-not bind to SLP-76 enhances integrin functionfollowing TCR ligation (134, 135). Althoughmice expressing the reciprocal mutation in theSH2 domain of SLP-76 have been generatedand share several characteristics with ADAP-decient mice, TCR-induced integrin activa-tion in these mice has not yet been reported.


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Pathway A

Pathway B

Pathway C

Actin reorganization

TCR/CD3Integrins

Inside-out signaling

DAG

PTK

SLP-76

LAT

ADAP RIAMSKAP55

ADAP

ADAP

RIAM

SKAP55

VAV1

WAVE2

TALIN

ARP 2/3

ITK

PLC1 PKC

PKD1 RAPL

RAP1GADS

Figure 2A model of integrin activation. Upon TCR ligation, the LAT/SLP-76-nucleated signalosome assembles.This complex allows for the activation of three pathways necessary for inside-out activation of integrins.Vav1 and Itk contribute to the actin reorganization required for integrin mobility ( pathway A). Mobilizationof the ADAP/SKAP55/RIAM complex is necessary for activated Rap1 plasma membrane localization( pathway B). An active Rap1/RIAM complex induces the association of talin with integrin tails, perhapsresulting in altered integrin afnity. Thirdly, PLC1-mediated generation of DAG leads to PKD1 activationand association with Rap1 ( pathway C). This interaction is required for Rap1 activation and contributes toRap1 recruitment. Rap1 in turn recruits RAPL, and subsequent RAPL binding to L subunits results inintegrin clustering and afnity changes.

In addition to its inducible interactionwith SLP-76, ADAP constitutively associateswith Src kinaseassociated phosphoprotein of55 kDa (SKAP55). The ADAP/SKAP55 com-plex is important for proper localization of ac-tivated Rap1 (134). How ADAP and SKAP55recruit Rap1 to the membrane was unclear un-til recently when investigators showed that athird adapter, Rap1-GTP-interacting adaptermolecule (RIAM), which is also constitutivelybound to SKAP55, associates with activatedRap1 upon TCR ligation, resulting in Rap1movement to the membrane (136). How RIAMitself relocalizes to the plasma membrane fol-lowing T cell stimulation is currently unknown,although it is hypothesized that recruitmentmay be through the inducible interaction ofPI(3,4)P2 (a product of T cell activation) andthe pleckstrin homology (PH) domain of RIAM(137).

In addition to mobilization of theADAP/SKAP55/RIAM complex that isdownstream of early TCR signals and is likelySLP-76 dependent, Rap1-mediated integrinactivation is also dependent on other TCR-triggered signals, including those leading toactivation of PKC. One PKC target importantfor Rap1 activation is the serine-threoninekinase protein kinase D1 (PKD1) (also knownas PKC). Following TCR stimulation, Rap1associates with the PH domain of PKD1 (138).Interestingly, it is this interaction and not thekinase activity of PKD1 that is required forRap1 membrane recruitment and activation.Because PKD1 also inducibly recruits theRap1 GEF C3G (Crk SH3 domain GEF) tothe membrane in a TCR-dependent fashion,it is tempting to speculate that this eventcontributes to the dependence of Rap1 onPKD1 for its activation (138).


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Costimulation:signals delivered to Tcells by cell surfacereceptors other thanthe TCR itself thatpotentiate T cellactivation

Activated Rap1 can also associate with theeffector regulator of cell adhesion and polariza-tion enriched in lymphoid tissues (RAPL). Thisinteraction is coincident with RAPL membranelocalization and necessary for binding of RAPLto the L subunit of LFA-1. In cell line models,this association is important for LFA-1 cluster-ing as well as afnity modulation (139).

In addition to regulating the activationof Rap1, signals from the TCR also reg-ulate cytoskeletal attachments to integrins.One cytoskeletal binding protein important forintegrin activation is talin. Recent studies inplatelets have demonstrated that Rap1 ac-tivity enhances the association of talin with-integrin subunits through association withRIAM (140). It is possible that formation ofa talin/RIAM/Rap1 complex may enable talinto bind integrins, which may induce the high-afnity ligand-binding state. Talin is not theonly actin-binding protein implicated in TCR-induced integrin activation. Vinculin, WAVE2(98), and the Arp2/3 complex also play roles inthis process, and dening the precise steps thatlink early TCR signals to activation of thesemolecules is an area of active investigation.

COSTIMULATION

One central tenet of T cell activation is that sig-naling solely through the TCR results in a non-responsive state (anergy) in which T cells arerefractory to restimulation. Coligation of othercell surface receptors provides additional sig-nals required for anergy avoidance and produc-tive T cell activation. Although many cell sur-face receptors can enhance signaling throughthe TCR, CD28 does so more robustly thanother costimulatory molecules.

Numerous studies have shown that CD28promotes T cell proliferation, cytokine produc-tion (via gene transcription and mRNA stabil-ity), cell survival, and cellular metabolism (re-viewed in 141). One key effector downstreamof CD28 is PI3K. Following binding of CD28to its ligands CD80 or CD86 on APCs, thep85 regulatory subunit of PI3K associates witha pYMNM motif located in the cytoplasmic

tail of CD28 (142). This regulatory subunit re-cruits the p110 catalytic subunit of PI3K, whichconverts PIP2 to phosphatidylinositol (3,4,5)trisphosphate (PIP3) at the membrane. Local-ized PIP3 generation serves as a docking site forthe PH domains of PDK1 (3-phosphoinositide-dependent protein kinase 1) and its targetAkt.

Akt phosphorylates multiple proteins, en-abling it to affect numerous cellular responses.Activation of Akt enhances the nuclear translo-cation of NF-B, which has positive effectson the expression of prosurvival genes includ-ing Bcl-xl. Emerging data suggest that Akt ac-complishes this function by associating withCARMA1 and facilitating the assembly of theCBM complex (143, 144), a step critical forNF-B activation (see above). The ability ofAkt to promote prosurvival gene expression,coupled with the ability of Akt to inhibit tran-scription factors that promote cell cycle arrest,results in Akt-driven cell survival and prolifera-tion (141). Akt also affects optimal transcriptionof NFAT-regulated genes, such as IL-2. Onewell-known target of Akt is GSK-3 (glycogen-synthase kinase 3), a serine-threonine kinasethat promotes nuclear export of NFAT (145).Thus, inactivation of GSK-3 by Akt might beone pathway responsible for prolonged NFATnuclear localization and thus IL-2 transcrip-tion following CD28 costimulation. Recently,a GSK-3-independent mechanism by whichAkt may regulate NFAT activity was suggested.This model posits that phosphorylated NFATis bound by the scaffolding protein Homer,thus inhibiting access of calcineurin to NFAT(146). Investigators proposed that CD28 lig-ation induces Akt-mediated phosphorylationof Homer, resulting in its dissociation frompNFAT. Unbound pNFAT would then be sus-ceptible to calcineurin phosphatase activity andnuclear entry. Whether this pathway is Akt de-pendent remains to be rigorously tested. Lastly,TCR/CD28 coligation increases the cell sur-face expression of the insulin transporter Glut1,leading to increased glucose uptake and glycol-ysis (147, 148), which is also mediated by Akt.Together, these data provide a framework for


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how Akt mediates T cell growth and survivaldownstream of CD28.

The CD28-mediated generation of PIP3also serves as a docking site for the PHdomain of Itk. Although Itk inducibly associateswith the LAT/SLP-76/Gads/PLC1 complexthat 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 PxxPmotif (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+ ux, another charac-teristic of CD28-mediated signaling. Despitethe fact that Itk and CD28 associate with oneanother, studies in Itk-decient 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-mediatedproliferation 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 the nucleus and corre-lates with IL-2 production (152). Although theprecise biologic signicance of this posttrans-lational modication is unknown, this pathwaymay provide yet another mechanism by whichCD28 regulates TCR signaling.

Many of the pathways described above areactivated 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 ratherthan a qualitative change in T cell activation

parameters (141). Although this appears to betrue, it is also true that quantitative differencesin signaling can result in qualitatively distinctfunctional outcomes.

CD28-decient mice exhibit dampened im-mune responses to a variety of infectious agents(reviewed in 141). Although these studiesdemonstrate the importance of costimulationby CD28, not all immune responses are severelyimpacted by its loss (153). Such observations in-dicate that molecules other than CD28 can pro-vide costimulation for T cells. Indeed, multiplesurface receptors have been described as havingcostimulatory functions. Included among theseare CD2, 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 canlink to downstream effectors, either directly orthrough adapter proteins.

Unlike CD28, which is expressed at con-stant levels on both resting and activated T cells,ICOS is inducibly expressed on activated T cells(154). ICOS deciency results in impaired im-mune responses, similar to yet not as severeas 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 siteis presumed to be responsible for PI3K-drivenAkt and/or Itk activation observed downstreamof CD3/ICOS stimulation, which likely con-tributes to the similarities seen in CD3/CD28and CD3/ICOS-stimulated cells (156). ICOSdoes not induce IL-2 gene transcription asCD28 does. Failure to induce IL-2 has beenattributed, 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 decient


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in CD28 and ICOS are severely defective ingenerating immune responses (158).

Outside of the CD28 family of costimu-latory molecules, the OX40 (CD134) and 4-1BB (CD137) members of the TNFR familyprovide costimulation upon engagement withtheir ligands OX40L and 4-1BBL. Like CD28,OX40 or 4-1BB ligation induces activation ofPI3K/Akt, NF-B, JNK, and p38 MAPK (159).However, unlike CD28 and ICOS, OX40 and4-1BB do not directly associate with proteinkinases but rather link to downstream sig-naling through the TRAF (TNFR-associatedfactor) family of adapter proteins. The pro-teins involved in linking TRAF signaling toNF-B and the JNK/p38 pathways have notbeen completely elucidated in primary T cells.However, studies in other systems implicateTRAFs themselves in the direct recruitment ofthe IKK complex as well as of serine/threoninekinases that initiate signaling to JNK and p38(reviewed in 160).

Many of the same genes are regulated down-stream of CD28 and TNFR family members.One reason for such overlap may be due tothe timing of receptor expression. CD28 is ex-pressed early and is critical for induction ofan immune response. It promotes expressionof several other costimulatory molecules in-cluding ICOS, OX40, and 4-1BB. Once ex-pressed, these receptors prolong or sustain animmune response and, in the case of OX40and 4-1BB, are important for memory T cellformation (159). The use of alternative meansby which to activate these similar pathways,e.g., direct binding to kinases versus use ofadapter proteins, may also allow for differentialnegative regulation of these pathways. In fact,some TRAF proteins negatively regulate NF-B; thus, association of TNFRs with differentTRAF family members can modulate the im-mune response (160). Lastly, a role for manycostimulatory molecules, including ICOS and4-1BB, in the development and/or function ofregulatory T cells is becoming increasingly ap-parent (161). This layer of complexity will haveto be taken into account when deciphering theroles of costimulatory molecules in vivo.

NEGATIVE REGULATIONOF TCR SIGNALING

As outlined above, signaling through the TCRtriggers an array of signals that activate multipleeffector pathways. Activation of these pathwaysis regulated to ensure that T cells respond toappropriate ligands and for the proper dura-tion. As with positive regulation of T cell sig-naling, negative regulation is mediated throughboth TCR-generated signals and those emanat-ing from other cell surface receptors (Table 2).

Even the most proximal TCR signalingevents are actively regulated. For example, mul-tiple proteins contribute to the regulation ofLck activity. C-terminal Src kinase (Csk) is re-sponsible for phosphorylating Lck on its in-hibitory tyrosine residue (Y505) and maintain-ing Lck in an inactive state (162, reviewed in163). Countering this is the phosphatase CD45that dephosphorylates the inhibitory site allow-ing for Lck autophosphorylation and activa-tion. Interestingly, CD45 can also limit Lckactivity by dephosphorylating its active site(163). Whether CD45 negatively or positivelyimpacts TCR signaling is likely to be con-trolled by its proximity to TCR-stimulated ef-fector molecules during TCR engagement andwhether CD45 itself is in an enzymatically fa-vorable conformation.

An additional layer of Lck regulation initi-ated by TCR signals has been proposed. As ameans to explain how the TCR can distinguishbetween strong and weak ligands, investigatorsshowed that weak or antagonistic TCR ligationresults in rapid Lck-mediated phosphorylationof SHP1 (SH2 domaincontaining protein-tyrosine phosphatase) (164). SHP1 then de-phosphorylates the active site of Lck, resultingin cessation of the TCR signal. Conversely, inthe presence of strong or agonistic TCR lig-ation, Erk is rapidly activated and phosphory-lates Lck on Ser59. This activity is thought toprevent SHP1 binding, thus keeping Lck ac-tive to sustain TCR signals and further amplifyErk activity. The extent to which this regulatoryloop operates in vivo in the context of agoniststimulation awaits the generation and analysis


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Table 2 Sampling of inhibitors of TCR signalinga

Molecule Interactions and/or function Deficiency phenotypeCsk (162, 163) (kinase) Phosphorylates inhibitory Y505 of Lck Csk knockdown in primary T cells: increased

proliferation and IL-2 productionHPK1 (169, 170) (kinase) Phosphorylates SLP-76 providing 14-3-3 binding

siteIncreased SLP-76, PLC1, LAT, ZAP-70, Vav1phosphorylation

Enhanced T cell proliferationEnhanced disease in EAE model

SHP1 (164, 165)(phosphatase)

Dephosphorylates Lck and ZAP-70Association with Lck inhibited by Erk activation

Defective positive selectionSplenomegaly, lymphadonopathyAlopecia, inamed tissues

Sts-1 (171, 172, 174)(phosphatase)

Dephosphorylates ZAP-70Binds c-Cbl and ubiquitinylated proteins

Sts-1/ Sts-2/: hyperproliferative T cells andincreased susceptibility to EAE

Dok-1, Dok-2 (167, 168)(adapters)

Binds Csk, RasGAP, SHIP-1PTB domain can associate with CD3 ITAMs

Dok-1/Dok-2/: increased T cell cytokineproduction, enhanced pZAP-70, pLAT, and pErk

Lupus-like renal diseaseProduction of antidouble-stranded DNAantibodies

c-Cbl, Cbl-b (175177)(E3 ligase)

Catalyzes attachment of ubiquitin to proteinsPromotes TCR downregulation

c-Cbl/: splenomegaly, increased TCRexpression and ZAP-70 phosphorylation

Cbl-b/: multiorgan lymphocytic inltration,increased IL-2 production, autoantibodyproduction

CTLA-4 (179, 182)(inhibitory receptor)

Binds ligands CD80 and CD86Recruits phosphatases SHP1 and PPA2

Multiorgan lymphocytic inltratesSplenomegalyElevated serum immunoglobulin

PD-1 (180, 181)(inhibitory receptor)

Binds ligands PD-L1 and PD-L2Recruits SHP1

Lupus-like diseaseIncreased serum immunoglobulin

aAbbreviations: Csk, C-terminal Src kinase; CTLA-4, cytotoxic T lymphocyte antigen-4; Dok, downstream of kinase; HPK1, hematopoietic progenitorkinase 1; ITAM, immunoreceptor tyrosine-based activation motif; LAT, linker for the activation of T cells; PD-1, programmed death-1; PD-L1/PD-L2,PD-1 ligands; PLC, phospholipase C; PPA2, protein phosphatase A2; PTB, phosphotyrosine-binding domain; RasGAP, Ras GTPase-activating protein;SHIP-1, SH2 domaincontaining inositol phosphatase 1; SHP1, SH2 domaincontaining tyrosine phosphatase 1; SLP-76, SH2 domaincontainingleukocyte phosphoprotein of 76 kDa; Sts-1, suppressor of T cell receptor signaling; ZAP-70, -associated protein of 70 kDa.

of mice harboring a mutation at the indicatedLck serine residue.

The importance of SHP1 function is evi-dent, as mice decient in SHP1 develop severeautoimmunity (165). SHP1 can be recruitedquickly to the TCR complex via its associationwith Lck. However, SH2 domaincontainingphosphatases are typically recruited to phos-phorylated ITIMs (immunoreceptor tyrosine-based inhibitory motifs) present in the cytoplas-mic tails of cell surface receptors. Recently, theITIM-bearing inhibitory receptor carcinoem-bryonic antigen-related cell adhesion molecule

1 (CEACAM1) was identied as a potential can-didate for SHP1 recruitment in human T cells(166). CEACAM1 upregulation occurs hoursafter stimulation; thus, SHP1 likely regulatesinitial TCR signaling events as well as late sig-naling termination events. Given the impactSHP1 activity has on immune regulation, itwill be important to understand exactly howSHP1 is recruited into the TCR-ligated sig-naling complex.

Adapter proteins also play a critical rolein negatively regulating TCR signals. Thedownstream of kinase (Dok) adapter proteins,


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Dok-1 and -2, are expressed in T cells. Co-ordinated deletion or knockdown of theseproteins results in increased TCR cytokineproduction and proliferation; prolonged phos-phorylation of ZAP-70, LAT, SLP-76, andAkt; and the development of a lupus-like re-nal disease with high antidouble-stranded DNAantibody titers (167, 168). Dok can asso-ciate with several negative regulators includ-ing SHIP-1 (SH2 domaincontaining inositolphosphatase), 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) of Dok-1 and -2 can bind the ITAMmotif in CD3, making it tempting to specu-late that Dok may compete with ZAP-70 forITAM binding (167). Whether this interactionis relevant in vivo remains to be shown. Dokproteins can also be recruited inducibly to aLAT-, Grb2-, and SHIP-1-containing molecu-lar complex (168). This nding is intriguing andbegs the question as to how LAT participates inboth 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 eventsare regulated.

It has been suggested that SLP-76 can alsobe a target of negative regulation. The serine/threonine kinase HPK1 inducibly binds to theSH2 domain of SLP-76. This kinase has bothpositive and negative effects on TCR signaltransduction; however, its role as a promi-nent negative regulator was conrmed re-cently by the description of HPK1-decientmice. HPK1-decient T cells exhibit en-hanced phosphorylation of several early sig-naling molecules, and HPK1-decient miceare more susceptible to experimental autoim-mune encephalomyelitis (EAE) than are wild-type mice (169). As a possible explanation forthese phenotypes, two groups have shown that

HPK1 can phosphorylate a serine residue inSLP-76 that mediates recruitment of 14-3-3family members (169, 170). 14-3-3 proteinshave been implicated in the regulation of severalsignaling pathways. Thus, although the pre-cise mechanism by which HPK1 disrupts TCRsignal transduction remains unclear, identica-tion of a SLP-76/14-3-3 interaction may indi-cate that this mechanism involves a conservedmethod of signal transduction regulation.

The role of a novel family of proteins in reg-ulating TCR signaling has become appreciatedmore recently. The suppressor of T cell recep-tor signaling (Sts) family of proteins containstwo members, Sts-1 (TULA2) and -2 (TULA).Combined deciency in these proteins leads tohyperproliferative T cells and an increased sus-ceptibility to autoimmunity (171). When thephenotype of Sts-1 and -2 knockout mice wasdescribed, the mechanism of negative regula-tion was unclear. Although still not understoodfully, the C-terminal phosphoglycerate mutasedomain of Sts-1 acts as a phosphatase withspecicity for Syk and, to a lesser degree, Srcfamily members, thus providing a model forhow Sts-1 may negatively regulate TCR sig-nal transduction (172). Interestingly, Sts-2 hasvery little phosphatase activity and, in cell linemodels, can enhance ZAP-70 activation (173).Recent studies demonstrating that Sts-2 canbind to and induce the degradation of c-Cbl(a negative regulator of TCR signaling; see be-low) have provided a mechanism by which Sts-2may act as a positive regulator of T cell activa-tion (174). Whether net ZAP-70 activity is ulti-mately increased or decreased in response to anin vivo challenge likely depends on the relativeexpression of these two family members.

Cbl proteins play an important role as neg-ative regulators of T cell signaling. Two familymembers, c-Cbl and Cbl-b, are immune mod-ulators, as deletion of each gene in vivo leads tohypercellularity and, in the case of Cbl-b, spon-taneous mulitorgan inltration (175, 176). Bothc-Cbl and Cbl-b facilitate the ubiquitination ofproteins, targeting them for degradation. Theyalso mediate the downregulation of the TCR


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following stimulation with antigenic peptidesand 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, providean example of yet another means by which TCRsignaling is terminated.

Similar to the necessity of signals providedby the TCR to be complemented by costim-ulatory molecules, TCR-generated regulatorysignals 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 activatedT cells with peak expression 2448 h afterstimulation. Genetic studies have documentedthe importance of both for maintaining self-tolerance. By 56 days after birth, CTLA-4-decient mice exhibit activated peripheral Tcells, splenomegaly, and lymphocytic inltratesinto nonlymphoid organs (179). Although lessstriking, PD-1-decient mice also develop au-toimmune features, including the developmentof a lupus-like disease by 6 months of age (180).Like costimulatory receptors, inhibitory recep-tors utilize motifs and molecular pathways simi-lar to those used by the TCR when propagatinga 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 thesame ligand and/or signaling molecules as cos-timulatory receptors, thus setting up the poten-tial for competition or sequestration of ligandsor 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, cytokineexpression, and Cbl-b expression.

CONCLUSIONS

As in the rst 10 years after identication ofthe 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, howCa2+ ions are sensed, that TCR signaling can berequired for the activation of other cell surfacereceptors, and that the actin cytoskeleton doesmore than just dictate cell shape. Studies inves-tigating signal transduction by costimulatorymolecules have highlighted the importance ofsignal amplication, 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 manysignaling molecules and cellular outcomes re-main poorly dened. Future studies will berequired to ll gaps in our knowledge andlikely will reveal new and unexpected interac-tions between currently known and unknownmolecules.

This review primarily focused on the signaltransduction 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, naturalkiller T cells, and memory T cells, to estab-lish whether these paradigms are universal. Inthe coming years, the population-based analy-ses that established the eld of signal transduc-tion will be driven toward single cell analyses.We can anticipate that this technological shiftwill allow for better analysis of human T cellsand the application of basic science to humandisease.

DISCLOSURE STATEMENT

The authors are not aware of any afliations, memberships, funding, or nancial holdings thatmight be perceived as affecting the objectivity of this review.


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ACKNOWLEDGMENTS

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

LITERATURE CITED

1. Haskins K, Kappler J, Marrack P. 1984. The major histocompatibility complex-restricted antigen re-ceptor on T cells. Annu. Rev. Immunol. 2:5166

2. Cantrell D. 1996. T cell antigen receptor signal transduction pathways. Annu. Rev. Immunol. 14:2

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T Cell Activation