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

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1 Molecular mechanisms in allergy and clinical immunology (Supported by a grant from Merck & Co, Inc, West Point, Pa) Series editor: Lanny J. Rosenwasser, MD T-cell activation through the antigen receptor. Part 1: Signaling components, signaling pathways, and signal integration at the T-cell antigen receptor synapse Andre E. Nel, MD Los Angeles, Calif Part 1 of this review will highlight the basic components and signaling pathways by which the T-cell antigen receptor (TCR) activates mature extrathymic T cells. TCR signaling commences with an early wave of protein tyrosine kinase activation, which is mediated by the Src kinases Lck and Fyn, the 70-kd ζ-associated protein kinase, and members of the Tec kinase family. This early wave of protein tyrosine phosphorylation leads to the activation of downstream signaling pathways, including an increase in intracellular free calcium, protein kinase C, nuclear factor κb and Ras mitogen-activated protein kinase activation. These pathways activate transcription factors, such as activator protein 1, nuclear factor of activated T cells, and Rel proteins, which ultimately lead to the expression of genes that control cellular proliferation, differentiation, anergy, or apoptosis. This review also describes how costimulatory receptors assist in signal transduction and assembly of macromolecular complexes at the TCR contact site with the antigen-presenting cell, also known as the immune synapse. These basic concepts of TCR signal transduction will be used in part 2 to explain how T-cell function can be altered by therapeutic targeting of TCR signaling components, as well as to explain modification of TCR signaling during T H 1/T H 2 differentiation, tolerance, and immune senescence. (J Allergy Clin Immunol 2002;109: ) Key words: T-cell activation, TCR, signal transduction The coordinated activation of T cells by foreign antigen leads to clonal expansion, differentiation, cytotoxic killing, or induction of programmed cell death. T-cell activation is initiated by the T-cell antigen receptor (TCR), which is comprised of a ligand-binding subunit, From the Division of Clinical Immunology/Allergy, Department of Medicine, UCLA School of Medicine, University of California, Los Angeles. Supported by a United States Public Health Service grant (RO-1 AG14992) and a grant from the UCLA Asthma, Allergy, and Immunologic Disease Center (PO-1 AI50495). Received for publication February 13, 2002; revised February 14, 2002; accepted for publication February 18, Reprint requests: Andre Nel, MD, Division of Clinical Immunology/Allergy, UCLA School of Medicine, Le Conte Ave., Los Angeles, CA Mosby, Inc. All right reserved /2002 $ /10/ doi: /mai Abbreviations used AP-1: Activator protein 1 APC: Antigen-presenting cell [Ca 2+ ] i : Intracellular free ionized calcium GEF: Guanine nucleotide exchange factor Grb2: Growth factor receptor bound protein Gads: Grb2 related adapter downstream of Shc Itk: Inducible T-cell kinase IκB: Inhibitory κb protein IκK: IκB kinase IP: Inositolphospholipid ITAM: Immunoreceptor tyrosine-based activation motif JNK: N-terminal c-jun kinase ERK: Extracellular signal regulated kinase LAT: Linker for activated T cells LFA-1: Lymphocyte function associated antigen 1 MAPK: Mitogen-activated protein kinase NF-κB: Nuclear factor κb NFAT: Nuclear factor of activated T cells PKC: Protein kinase C PLC: Phospholipase C PTK: Protein tyrosine kinase PH: Plextrin homology domain PI-3 kinase: Phosphatidyl inositol 3 kinase PI-4,5-P 2 : Phosphatidyl inositol 4,5 biphosphate PTB: Phosphotyrosine-binding domain SLP-76: SH2 domain containing leukocyte protein of 76 kd SH2/SH3: Src homology type 2 or 3 domain SMAC: Supramolecular activation cluster TCR: T-cell antigen receptor Sos: Son of Sevenless WASP: Wiskott-Aldrich syndrome protein ZAP-70: ζ Chain associated protein kinase of 70 kd the α and β chains, and a signaling subunit, namely the CD3ε, γ and δ chains and the TCRζ chain (Fig 1). 1,2 The physiologic ligand for the TCR is foreign peptide bound to the MHC expressed on antigen-presenting cells (APCs), including dendritic cells, macrophages, and B cells. 3,4 Although signals generated by the TCR determine the specificity of the T-cell response to antigen, costimulatory receptors, such as CD2, CD28, CD4, CD8,

2 J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER 5 Nel 759 and integrin molecules, contribute to signal transduction by modulating the response threshold. 5,6 This review will highlight the major aspects of TCR signaling in the peripheral or mature T-cell compartment but will not address TCR signaling in the thymocyte compartment, which has been addressed elsewhere. 7 TCR signaling commences with an early wave of protein tyrosine kinase (PTK) activity, which is mediated by the Src kinases Lck and Fyn, the 70-kd ζ chain associated protein kinase (ZAP-70), and members of the Tec kinase family, inducible T-cell kinase (Itk), Tec, and Txk/Rlk. 8,10 This early wave of protein tyrosine phosphorylation leads to the activation of downstream signaling pathways, including increases in intracellular calcium flux, protein kinase C (PKC), nuclear factor (NF) κb, and Ras mitogen-activated protein kinase (MAPK) activation. 9,11 These pathways activate transcription factors that ultimately lead to the expression of genes that control specific cellular responses. 12,13 This review will also describe how costimulatory receptors modify TCR signaling through assembly of signaling components at the TCR synapse. 5,14,15 In the second part of this review, we will discuss how TCR signaling is modified under different biologic circumstances and how pharmacologic intervention in TCR activation pathways can be used to modulate the function of the immune system. INITIAL PHASE OF TYROSINE PROTEIN KINASE ACTIVATION, INCLUDING PHOSPHORYLATION OF IMMUNORECEPTOR TYROSINE-BASED ACTIVATION MOTIFS The earliest recognizable event after TCR engagement by antigen is the induction of tyrosine protein phosphorylation by the Src kinases Lck and Fyn (Fig 1). 2,8,9 How exactly these Src kinases are activated is unclear, but it involves maintenance of an activation-competent state by the removal of inhibitory C-terminal tyrosine phosphate residues by members of the CD45 tyrosine phosphatase family (Fig 1, A). 16 To do so, CD45 participates in the formation of multimeric complexes with the CD4 receptor, which is physically associated with Lck The removal of the C-terminal phosphate residue leads to Src kinase unfolding and activation. 17,18 This conformational change also frees up the autologous Src homology 2 (SH2 domain), which allows Lck to interact with new partners, including ZAP-70 (Fig 1, C). 19 Another contribution to Src kinase activation comes from the sorting of receptor-associated PTKs and tyrosine phosphatases at the TCR contact site with the APC. 5 According to the kinetic segregation or topological constraint theory of T-cell activation, the limited space between the T cell and its participating APC leads to exclusion of large receptors, including the tyrosine phosphatases CD45 and CD148, but allows smaller receptors that regulate PTK activity to remain in the contact zone. 5,20-24 This receptor sorting is critical to the forming of the TCR synapse, which is further explained in Box 1. Once activated, the Src kinases regulate the activation of ZAP-70 and the Tec kinases. 2,8,10,25 This hierarchy is maintained by recruiting the tandem SH2 domains of ZAP-70 to a recognition motif, the immunoreceptor tyrosine-based activation motif (ITAM). 2,26 These ITAMs contain the consensus sequence (D/E)XXYXXL(X) 6-8 YXXL, in which both tyrosines (underlined) serve as substrates for the Src kinases (Fig 1, B and C). 2 These ITAMs appear as a single copy on the CD3γ, δ, and ε chains and as a triplicate repeat on the ζ chain, thereby contributing 10 motifs to each TCR complex (Fig 1). ITAM phosphorylation by a pool of active Src kinases is continuously opposed by tyrosine phosphatases. 20 The repressive effect of the tyrosine phosphatases is removed during the formation of the TCR synapse (Fig 2), allowing the Src kinase to dominate the phosphorylation status of the ITAMs. 5 After it is attached to the ITAM motifs, ZAP-70 is phosphorylated by Lck, leading to activation of the latter kinase (Fig 1, C). 27 In addition, Lck interacts directly with ZAP- 70 through the binding of the Lck SH2 domain to the ZAP-70 py 319 residue. 28 This interaction is critical for sustained ITAM phosphorylation, leading to the recruitment of additional ZAP-70 molecules and Lck substrates, such as the Tec kinase, Itk. 20 It is important to point out that not all the ITAMs are phosphorylated on every occasion the TCR makes contact with an antigen. Instead, the stoichiometry of ITAM phosphorylation and the number of ZAP-70 molecules recruited are dictated by the affinity of the TCR for its peptide ligand. 29,30 This number is limited in case of low-affinity interactions but increases as the affinity increases. The stoichiometry of ITAM phosphorylation therefore serves as a signaling amplification mechanism. 31 In this regard the TCRζ chain migrates as either 21- or 18-kd peptides during SDS-PAGE, depending on the stoichiometry of ITAM phosphorylation. 29,30 The accompanying change in the signaling competency of the ζ chain is further discussed in part 2 of this review. COSTIMULATORY RECEPTORS AFFECT EARLY PTK ACTIVATION In addition to their role in stabilizing TCR interactions with the MHC, CD8 and CD4 play an active role in initiating PTK activity. 16 Although it was originally thought that these Lck-binding coreceptors chaperone the kinase to the ITAMs, more recent data suggest that CD4 and CD8 may actually interact with the MHC after TCR binding to the peptide-mhc complex (Fig 1). 17,32 One explanation is that interaction of the Lck-SH2 motif with the ZAP-70 py 319 residue redirects CD4 or CD8 receptors to the peptide-mhc complex (Fig 1, C). 28 Once anchored at this site, CD4 or CD8 amplifies protein tyrosine phosphorylation by stabilizing the interaction between Lck and ZAP-70. CD28 is another costimulatory receptor that promotes a generalized increase in protein tyrosine phosphorylation. 33 One explanation is that the intracellular tail of CD28 associates with PTKs, including Lck, Tec, and Itk (Fig 3). 34,35 Although CD28 ligation by B7-1 (CD80) and B7-2 (CD86) ligands induces weak protein tyrosine phosphorylation, a major effect of this receptor is to enhance

3 760 Nel J ALLERGY CLIN IMMUNOL MAY 2002 FIG 1. The role of the TCR/CD3 complex and the CD4 receptor in the initiation of early protein tyrosine phosphorylation. A, On binding to the peptide/mhc complex, the earliest recognizable event is activation of the Src-kinases, Lck and Fyn. This requires removal of a C-terminal phosphate (red dot) by the tyrosine phosphatase, CD45. This allows the kinase to unfold and to phosphorylate ITAM motifs (blue rectangles in the intracellular domains of CD3δ, ε, γ, and ζ). Tandem ITAM phosphorylations are required for the recruitment of ZAP-70, which attaches by a pair of SH2 domains (yellow half circles). B, Immobilized ZAP-70 is phosphorylated and activated by Lck, which interacts directly with ZAP-70. CD4 interacts with nonpolymorphic MHC domains, serving to stabilize TCR/ligand interactions and promoting further tyrosine phosphorylation. Once activated, ZAP-70 phosphorylates substrates such as LAT, SLP-76, and Vav. Lateral displacement of CD45 and other tyrosine phosphatases from the TCR/APC contact site promotes unopposed PTK activity. induction of protein tyrosine phosphorylation by the TCR. 36,37 An important advance in understanding this synergy has been the discovery that CD28 regulates the assembly of post-tcr components through its effects on lipid rafts. 33 This concept is discussed later on. Suffice to mention here that rafts are membrane domains that are enriched for Src kinases and other signaling components, suggesting that CD28 costimulation leads to the recruitment of PTKs to the TCR synapse, thereby enhancing the ability of the latter receptor to phosphorylate Vav, c-cbl, p62 dok, phospholipase C (PLC) γ1, Lck, and Itk. 36,37 ACTIVATION OF SIGNALING CASCADES DOWNSTREAM OF THE PTK CASCADE The early wave of PTK activity leads to the recruitment, rearrangement, and activation of additional signaling molecules at the TCR contact site with antigen. 11 The ITAMs,

4 J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER 5 Box 1. Rearrangement of TCR-associated signaling molecules, leading to the formation of the immunologic synapse and SMACs 5,15,16 The extracellular portions of the TCR and peptide- MHC molecules are small (approximately 7 nm) compared with several highly abundant cell-surface molecules, such as CD45 (approximately nm) and CD43 (approximately 45 nm), and adhesion molecules, such as LFA-1 (approximately 21 nm, Fig 2). 5,20 Before cell-cell contact, the TCR-CD3 complex is subject to constitutive tyrosine phosphorylation and dephosphorylation. Dephosphorylation dominates, leading to low-level ITAM phosphorylation. Initial LFA-1 intracellular adhesion molecule 1 interactions overcome the barrier to cell-cell contact by means of negatively charged glycoproteins (eg, CD43), which dispel each other at 50- to 100-nm separation of the cell membranes. 21 Integrin binding stops the T cell from migrating and generates a central integrinenriched area surrounded by a close-contact region that includes the bulk of the engaged TCRs. 21,23 Subsequently, the TCR and some of its accessory receptors are transported to the center of the contact area, and the integrins are forced into a surrounding ring (Fig 2). 15,22,23 This molecular arrangement is known as the immunologic synapse (Fig 2) and is responsible for concentrating the TCR, CD3, CD4, and CD28 in the central contact zone at the expense of larger cell-surface receptors, such as CD43 and the family of CD45 tyrosine phosphatases (Figs 2 and 3). 5,20,21 According to the kinetic segregation theory of T- cell activation, this process of receptor sorting leads to the domination of tyrosine phosphorylation at the central contact zone. Accordingly, signaling molecules, such as PTKs (Lck and Fyn), PKCθ, adapter proteins, and GTP-binding proteins (eg, Rac) can be seen to concentrate in the central zone (Fig 3). This assembly of receptors and signaling molecules is also known as SMACs, which are divided into central and peripheral zones (Fig 3). 15 High-affinity TCR engagements within these stabilized zones result in sustained tyrosine phosphorylation of the TCR-associated ITAMs, leading to receptor triggering. 21 However, in case of low-affinity TCR engagement by the peptide-mhc complex, the immunologic synapse is unstable and results in dephosphorylation of the ITAMs on leaving the close-contact zone. This leads to a modification of TCR signaling, as will be discussed in the second part of this review. as well as the adapter proteins, play a key role in the assembly of post-tcr signaling complexes. (For a discussion of adapter molecules, see Box 2 and Fig 4.) Downstream signaling events include the activation of Ras and Rho-family GTPases, MAPK cascades, phosphatidylinositol 3 kinase (PI-3 kinase), PKCθ, and the NF-κB pathway. In addition, Nel 761 FIG 2. The kinetic segregation model of TCR triggering and formation of the immunologic synapse. The presence of large molecules (eg, CD45, CD43, and other glycoproteins) prevents close membrane contact between the T cell and APC, thereby acting to constrain engagement of the peptide-mhc complex (A). By comparison, the TCR-CD3 complex and accessory receptors (eg, CD4 and CD28) are much smaller, which requires large molecules to migrate laterally to allow the smaller receptors to interact. This small-scale segregation of receptors leads to the formation of a specialized contact zone known as the immunologic synapse (B). Because large-sized tyrosine phosphatases (eg, CD45) are excluded from the synapse, tyrosine kinases dominate, preparing the receptor for triggering. TCR triggering, with the assistance of costimulatory receptors, initiates active large-scale segregation of receptors, signaling molecules, and cytoskeletal elements, leading to the formation of SMACs (Fig 3). TCR ligation leads to the phosphorylation and activation of PLC-γ1, which initiates inositolphospholipid (IP) turnover and intracellular free ionized calcium ([Ca 2+ ] i ) flux. A summation of these effects leads to transcriptional activation of biologically important genes. Contribution of IP turnover, [Ca 2+ ] i flux, and PKC activation to TCR signaling PLC-γ1 tyrosine phosphorylation is one of the major determinants for Ca 2+ signaling. Although it was initially proposed that Lck regulates PLC-γ1 phosphorylation, it is clear that this event involves 3 different types of PTK

5 762 Nel J ALLERGY CLIN IMMUNOL MAY 2002 FIG 3. Cross-section of the mature immunologic synapse showing the arrangement of receptors, signaling molecules, and cytoskeletal proteins in the SMACs. In the mature synapse TCR/CD3, CD28, and several signaling molecules congregate in the center of the SMACs, also known as the csmac. A second group of molecules, including the adhesion receptors LFA-1 and intracellular adhesion molecule 1 and the cytoskeletal protein talin form a ring around the csmac, to form the outer ring of the peripheral or psmac. The inner ring of the psmac includes the CD2 receptor, which interacts with LFA-3 or CD48 on the opposing APC. Box 2. Adapter proteins and their interactive domains Adapter proteins are defined as molecules that lack intrinsic enzymatic activity yet are able to contribute to signaling by mediating intermolecular interactions. 38 Key adapter proteins involved in T-cell signaling include LAT, SLP-76, Grb-2, and Gads. 38 LAT is a type III transmembrane protein with a long cytoplasmic tail that includes 9 tyrosine-based motifs. 39,40 When phosphorylated by ZAP-70, these motifs serve as docking sites for specific SH2 proteins, including PLC-γ 1, Grb2, PI-3 kinase, and Gads (Fig 4). 40 As a result, LAT controls [Ca 2+ ] I flux and Ras/ERK, NFAT/AP-1, and PI-3 kinase activation. 40 Importantly, LAT contains a palmitoyl tail, 41 which allows it to concentrate in lipid rafts. SLP-76 is an adapter protein with SH2- and proline-rich motifs that associate with Vav, Gads, Itk, LAT, and Grb2 (Fig 4). 38 The roles of Gads and Grb2 are discussed above. Other adapter molecules that play a role in T- cell activation include the Cbl family, the Crk family, the Dok family, Lnk, Nck, Shc, Src-like adapter protein, Srclike adapter protein 130/Fyb, and TCR-interacting molecule; these are reviewed elsewhere. 38 In addition to SH2 domains, intermolecular interactions are mediated by SH3, phosphotyrosine binding (PTB), and PH domains (Fig 4). 42,43 Generally speaking, these are modular structures comprised of 40 to 150 amino acids. The SH2, SH3, and PTB domains are shaped into ligand-binding pockets that recognize 3 to 6 amino acid motifs in adjacent proteins. SH2 domains are phosphotyrosine-binding modules that recognize the consensus sequence pyxxυ (where py represents phosphotyrosine and υ represents any hydrophobic amino acid, Figs 1 and 3). SH3 domains interact with proline-containing peptides that conform to the sequences R/KxxPxxP (class 1) or PxxPxR/K (class 2). 44 An example is Grb2 binding to Sos (Fig 4, B). PTB domains recognize NPxY sequences in which the tyrosine residue may or may not be phosphorylated. PH domains share a similar fold to the PTB domain but interact with D3-phosphorylated IPs. 43 Examples include PLC-γ 1 and Vav interactions with the plasma membrane (Fig 4). activity, namely Lck, ZAP-70, and Tec kinases (Fig 4, A). 45 Two adapter molecules, linker for activated T cells (LAT) and SH2 domain containing leukocyte protein of 76 kd (SLP-76), contribute to the formation of that signaling module (Fig 4, A). 39,45 Consensus phosphotyrosine residues (Box 2) are responsible for the recruitment of the PLC-γ1 SH2 domains to LAT and the vicinity of the surface membrane. 40 The role of SLP-76 in this setting is the recruitment of a Tec kinase, which ultimately phosphorylates PLC-γ1 (Fig 4, A). 45 LAT stabilizes SLP-76 binding to this macromolecular complex through the interposition of an adapter protein, Grb2 related adapter downstream of Shc (Gads; Fig 4, A). 45 PLC-γ1 tyrosine phosphorylation leads to its catalytic activation and cleavage of phosphatidyl inositol-4,5 biphosphate (PI-4,5-P 2 ), which is located in the inner leaflet of the plasma membrane (Fig 5). This generates inositol-1,4,5-trisphosphate, which releases Ca 2+ from endoplasmic reticulum storage sites (Fig 5). 46 Once these stores are depleted, store-operated Ca 2+ channels in the

6 J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER 5 Nel 763 FIG 4. Examples of key adapter proteins that play a role in TCR signaling. A shows the formation of a signaling complex that regulates IP turnover and [Ca 2+ ] i flux. The importance of modular binding domains in facilitating interactions between LAT, SLP-76, Gads, and Itk is demonstrated: SH2 () ), SH3 (υυυυ), PH (vvvv), and PR (filled circles). B shows the use of modular domains in the formation of signaling complexes that lead to Vav and Ras activation, respectively. The former complex includes LAT/Gads/SLP-76 and PI-3 kinase, and the latter involves LAT/Grb2 and Sos. surface membrane allow extracellular Ca 2+ influx. 46 Sequential Ca 2+ release from both internal and external stores leads to a sustained elevation of [Ca 2+ ] i,which acts as an important trigger for transcriptional activation in the nucleus (Fig 5). 12,47 The spatiotemporal characteristics of [Ca 2+ ] i signaling (transient, sustained, or oscillatory) are important in determining which genes are activated. For instance, sustained [Ca 2+ ] i elevation is critical for the activation of the IL-2 promoter by the calcineurin pathway (Fig 5). 12,47 Ca 2+ activates calcineurin, a calcium-calmodulin dependent serine phosphatase that dephosphorylates the nuclear factor of activated T cells (NFAT), which includes 4 isoforms (Fig 5). 47,48 This leads to the intranuclear translocation of NFAT proteins, which occupy proximal and distal NFAT binding sites in the IL-2 promoter (Fig 5). 12 Interference in calcineurin activation by cyclosporin A and FK506 form the basis for the immunosuppressive activities of these drugs (Fig 5). 48 Another important function of IP turnover is PKC activation T cells express multiple functionally distinct PKC isoforms that can be classified into the classical PKCs (α, β 1, β 2, and γ), which are regulated by calcium, diacylglycerol, and phospholipids; novel PKCs (δ, ε, ν, and θ), which are regulated by diacylglycerol and phospholipids; and atypical PKCs (ζ and λ), which lack Ca 2+ - or diacylglycerol-binding domains. 52 A major recent finding is that among the available PKCs, only the θ isoform is recruited to the site of TCR engagement (Fig 6). 51 In that location, PKCθ is involved in the activation of the NF-κB pathway and possibly also the N-terminal c-jun kinase (JNK) cascade (see below). 53 Regulation of p21ras and MAPK cascades TCR ligation leads to a rapid accumulation of the active GTP-bound form of p21ras in the vicinity of the T-cell membrane (Fig 4, B). 54,55 Ras plays a critical role in

7 764 Nel J ALLERGY CLIN IMMUNOL MAY 2002 cytokine gene expression, particularly activation of the IL- 2 promoter and T-cell proliferation (Fig 5). 56 The Ras guanine nucleotide binding cycle is controlled by the counterregulatory effects of guanine nucleotide exchange factors (GEFs), which promote the activation of Ras, and GTPase-activating proteins, which stimulate the intrinsic GTPase activity of Ras, thereby resulting in GTP hydrolysis and inactivation of Ras. 54,55 Although there are several possible avenues for Ras activation in T cells, a well-characterized pathway is the involvement of the adapter protein growth factor receptor bound protein (Grb2) and Son of Sevenless (Sos), a GEF (Fig 4, B). 57 In this assembly the Grb2-SH2 domain is recruited to a consensus phosphotyrosine motif on LAT, 40 whereas Grb2-SH3 domains bind to proline-rich regions in Sos (Fig 4, B). Although it has been suggested that Ras activation by phorbol esters involves a PKC member, there is no evidence for a direct interaction between PKC and Ras, arguing that either PKC or the phorbol ester may activate a Ras GEF. Once activated, Ras couples to multiple effector pathways, including activation of the extracellular signal-regulated kinase (ERK) cascade, as well as linkage to Rho GTPases. 56,58 The ERKs belong to the MAPK family, which are activated by a cascade involving a MAP3K (Raf-1) and a MAP2K (MEK-1 or MEK-2; Fig 7). 58,59 p21ras interacts directly with the serine-threonine kinase Raf-1, which is activated in a complex fashion at the surface membrane. Once activated, the ERKs play an essential role in the expression of the activator protein 1 (AP- 1) transcription factor c-fos, as well as c-myc. 59 c-fos is involved in transcriptional regulation of AP-1 response elements in the IL-2 promoter (Fig 5). 60 In addition to the ERK cascade, the p38 MAPK and JNK cascades play a role in TCR signaling (Fig 7). 59,61,62 In contrast to abundant ERK expression, the JNK1 and JNK2 isoforms are present in low quantities in primary T cells and require prior TCR engagement for their expression. 63 Subsequent activation of JNK is CD28 dependent (Fig 7) and is required for activation of the IL-2 promoter, particularly the CD28 response element (CD28RE, Fig 5). 61,62 In murine studies it has been shown that the major target of the JNK2 isoform is the IFN-γ gene and that knockout of JNK2 impairs T H 1 development. 64 In contrast, the major role of JNK1 is interference in T H 2 development, as exemplified by increased IL-4 and IL-5 production in JNK1 knockout mice. 65 This effect may be explained by the ability of JNK1 to enhance the nuclear export of NFATc, which is required for the activation of the IL-4 promoter. 65 Similarly, the p38 MAPK cascade regulates IFN-γ gene expression in T H 1 cells but apparently does not affect T H 2 cytokines. 59 The p38 MAPK cascade also play a role in the induction of apoptosis in CD8 + T cells. 59 Regulation of PI-3 kinase activity In addition to PLC-γ1 activation, PTKs affect IP turnover through the involvement of PI-3 kinase. 66 This kinase is comprised of regulatory (p85) and catalytic (p110) subunits and is recruited through the modular SH2 domain on the p85 subunit to LAT or TCR-interacting molecule adapters (Fig 4, B). 41,67 Activated PI-3 kinase phosphorylates the D-3 position of the IP ring, thereby converting PI-4,5-P 2 and PI-4-P to PI-3,4,5-P 3 and PI-3,4-P 2,respectively. 63 These D-3 phosphorylated IPs interact with the plextrin homology (PH) domains of PLC-γ 1,Tec, and Vav and serve to anchor these proteins at the plasma membrane (Fig 4, B). 43 PI-3 kinase is also responsible for the recruitment and activation of the serine-threonine kinase, protein kinase B (Akt), which plays a role in cellular survival. 68 Regulation of NF-κB activation NF-κB activation is dependent on a multisubunit 700- to 900-kd cytosolic signaling complex. 69,71 This complex includes the catalytically active IκB kinases (IκK) IKKα and IKKβ, which are arranged into IKKα-IKKβ heterodimers by a noncatalytic IKK subunit, IKKγ (Fig 8) The activated IKK complex initiates the phosphorylation and proteolytic degradation of the inhibitory IκBα and IκBβ proteins. This leads to the release of NF-κB transcription factors (p65, c-rel, RelA, and p50), which are sequestered in the cytosol by IκBα and IκBβ, allowing these factors to enter the nucleus and initiate transcriptional activation of genes involved in cellular proliferation and survival A key gene target is the CD28RE in the IL-2 promoter (Fig 5). 72 This is a combinatorial response element that requires c-rel, as well as AP-1, transcription factors for full activity. Therefore it is interesting that, as for JNK activation, NF-κB activation is dependent on CD28 costimulation (Fig 8). 72,73 In fact, these costimulatory requirements are in excellent agreement with the key role of CD28 in IL-2 production in unprimed T cells. Another important target for the NF-κB pathway and the CD28 receptor is the bcl-x L promoter, which expresses an NF-κB response element 860 bp upstream of the start site. 74 Increased Bcl-x L expression plays a critical role in cellular survival during CD28 costimulation. 75 The exact mechanism by which the NF-κB cascade is initiated by TCR ligation still needs to be clarified but involves PKCθ recruitment to the TCR synapse (Fig 8). 53,76 This recruitment is dependent on the involvement of Vav and assembly of the cytoskeleton, suggesting that one or more IKK components bind to a cytoskeletal scaffold (Fig 8). 77 PKCθ contributes to the activation of the IKK complex through its ability to phosphorylate IKKβ (Fig 8). 53,77 Interestingly, pharmacologic interference in PKCθ activity abrogates NF-κB activation and IL-2 production. 53 Role of CD28 in the activation of downstream NF-κB and JNK signaling cascades Activation of the IL-2 promoter by CD28 costimulation is dependent on the synergistic activation of the JNK and NF-κB cascades. 61,62,73,74 Although the precise mechanisms of engagement of these cascades are unclear, it is relevant that the CD28 tail includes several binding motifs that may be involved in signal transduction, including 4 tyrosine residues (Fig 5). 34,35 The py 170 motif serves as a docking site for PI-3 kinase and Grb2,

8 J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER 5 Nel 765 FIG 5. Signaling domains of the CD28 receptor (numbering according to the murine protein sequence). The 41 aa cytoplasmic tail contains 4 tyrosine residues, 2 of which (Y 170 and Y 188 ) are phosphorylated during CD28 ligation by B7 ligands. py 170 recruits PI-3 kinase, which may play an indirect role in Vav activation by allowing this protein to dock to D3-phosphorylated IP in the surface membrane. CD28 further assists in the activation of Vav by enhancing its tyrosine phosphorylation by either a TCR- or CD28-associated PTK. Vav is involved in cytoskeletal assembly through the activation of Rac-1 and also plays a role in the recruitment of PKCθ and possibly JNK activation. Notice that through its effects on the cytoskeleton, CD28 may also be responsible for the polarization of lipid rafts at the TCR synapse. The constitutive association of PTKs, such as Lck, with these rafts may lead to a generalized increase in protein tyrosine phosphorylation on TCR engagement. Two proline-rich domains (PR1 and PR2) are functionally involved in the recruitment and the activation of the Tec kinase, Itk. In addition, these PR domains are recognized by the SH3 domain of Lck and may play a role in the activation of that kinase. Although the exact role of these PTKs in CD28 signaling is unclear, they could contribute to phosphorylation of the Y 170 and Y 188 residues, as well as to the overall increase in PTK activity during CD28 costimulation. FIG 6. Components of the various MAPK families and their relationship to TCR and CD28 costimulation. The MAPKs are phosphorylated and activated by MAP2Ks, which, in turn, are activated by several MAP3Ks. A variety of transcription factors are phosphorylated and activated by these cascades, as demonstrated.

9 766 Nel J ALLERGY CLIN IMMUNOL MAY 2002 FIG 7. Schematic to explain synergistic activation of the IL-2 promoter by Ca 2+ /calcineurin, Ras/MAP, and NFκB cascades. The role of cyclosporin A and tacrolimus (FK506) in disrupting activation of the IL2 gene is demonstrated. The CD28 response element (CD28RE) is a combinatorial element that requires activation of both the NF-κB and JNK cascades. Both cascades are dependent on CD28 costimulation. The role of the negative regulatory elements CREB/CREM and NRE-A in the induction of T-cell tolerance is discussed in part 2 of this review. whereas py 188 has been shown to be critical for JNK activation and transcriptional activation of the IL-2 promoter. 35 In addition to these phosphotyrosines, the CD28 tail contains 2 proline-rich regions (PR1 and PR2) that may be functionally involved in Lck and Tec kinase recruitment and activation. 34,35,78,79 How exactly the phosphotyrosine and PR motifs relate to PTK activation and distal signaling cascades is still unclear. We do know, however, that CD28 assists in the assembly of the cortical cytoskeleton and recruitment of lipid rafts to the TCR synapse. 33 Regulation of the cytoskeleton involves the sustained phosphorylation and activation of Vav by CD28. Vav is a GEF for Rac-1, which plays a role in cytoskeletal assembly and possibly also the activation of the JNK cascade (Fig 3). It is interesting that the MAP3K in the JNK cascade, JNK kinase kinase, assembles at the TCR synapse and interacts directly with the Lck-associated adapter proteins. 81,82 This suggests that the JNK cascade is activated in the vicinity of the TCR synapse by signaling components that are assembled by means of TCR-CD28 costimulation. Lipid rafts, which are enriched for Lck, may be involved in this event, in addition to contributing to the overall increase in PTK activity during CD28 costimulation. How the NF-κB pathway may fit into the above scheme of CD28 signal transduction is uncertain but could involve the recruitment of PKCθ to the TCR synapse. PKCθ recruitment requires Vav activation and cytoskeletal

10 J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER 5 Nel 767 FIG 8. Schematic to explain the role of CD28, lipid rafts, and the cytoskeleton in the recruitment and activation of PKCθ and the IKK complex. This diagram shows how activation of PI-3 kinase and Vav by the CD28 receptor leads to assembly of the actin cytoskeleton. It also highlights the role of PKCθ in the activation of the IKK complex and the subsequent NF-κB activation. TABLE I. Key costimulatory effects of the CD28 receptor 1. Enhanced protein tyrosine phosphorylation by the TCR 2. Activation of the JNK and NF-κB cascades 3. Cellular proliferation caused by increased expression and stabilization of IL-2 mrna 4. Decreased threshold for the activation of naive T cells through assembly of signaling components at the TCR synapse and SMACs 5. Assembly of the cortical cytoskeleton and polarization of lipid rafts 6. Regulation of T H 1/T H 2 balance by enhancing IL-4, IL-5, and IL-10 production 7. Regulation of cellular migration through effects on chemokine (MIP-1α, MIP-1β) production, as well as chemokine receptor (CXCR4, CCR5) expression 8. Enhanced T-cell survival through increased Bcl-x L expression 9. Essential for the homeostasis of the regulatory CD4 + CD25 + subset 10. Prevents anergy through its effect on IL-2 production and cellcycle progression assembly. 80 Accordingly, PKCθ has been shown to be critical for NF-κB activation in murine knockout studies. 80 Because of its ability to regulate specific signaling cascades, as well as to promote a generalized increase in protein tyrosine phosphorylation, CD28 controls a wide range of responses in naive CD4 + T cells, including a decrease of the TCR signaling threshold (Table I). In contrast, the major effect of CD28 in memory T cells is to enhance the TCR response, whereas its role in CD8 + T cells is less clearly defined. This is in contrast to its TABLE II. Raft-associated signaling components that play a role in TCR activation Constitutive Lck Fyn Rac LAT CD2 CD5 CD9 GPI-linked receptors MEKK2, JNK kinase kinase; GPI, glycophosphatidylinositol. Inducible Vav PKCθ MEKK2 IKKα, β, γ ZAP-70 TCRζ CD3ε essential role in naive T cells. The contribution of CD28 to TCR signaling in naive T cells has therefore been described as signal 2 to highlight the major contribution to the delivery of signal 1 by the TCR. 83 This 2-signal concept has particular relevance in understanding T-cell tolerance and is further discussed in part 2 of this review. DYNAMIC INTEGRATION OF SIGNALS BY THE TCR SYNAPSE: ROLE OF LIPID RAFTS, CYTOSKELETON, AND SUPRAMOLECULAR ACTIVATION CLUSTERS Although a considerable amount of information on TCR signaling has been obtained through the use of antibodies that ligate TCR or CD3, in vivo TCR activation require a contribution by APCs and accessory receptors,

11 768 Nel J ALLERGY CLIN IMMUNOL which introduces an additional level of complexity. This includes the requirement that the TCR and accessory receptors be assembled in the immunologic synapse (Fig 2). This contact site also serves as an assembly site for signaling molecules on the inner leaflet of the T-cell membrane, an arrangement known as the supramolecular activation cluster (SMAC; Box 1). 5,15,20,84 Which stimuli regulate the formation of the TCR synapse and assembly of the SMACs? We have already discussed the role of topological constraint in determining which receptors participate in TCR synapse (Box 1). 5,20 This is a passive process that prepares the TCR for active large-scale segregation of signaling molecules, which also involves several costimulatory receptors, the cortical cytoskeleton, and lipid rafts. 20,84,85 Lipid rafts are glycosphingolipid- and cholesterolenriched membrane microdomains that are biochemically defined as an ordered lipid phase that is insoluble in low concentrations of nonionic detergents at 4 C. 86 A significant fraction of acylated, myristoylated, and glycophosphatidylinositol-linked proteins are anchored in lipid rafts and therefore packaged to participate in cellular events, such as signal transduction (Table II). 87 Although TCR-CD3 ligation exerts an independent effect on lipid raft trafficking, CD28, CD2, CD5, and lymphocyte function associated antigen 1 (LFA-1) play a major role in recruiting lipid rafts to the TCR synapse. 33,85,88,89 This process requires from 10 to 60 minutes for completion. 23 A list of constitutive and newly recruited raft signaling molecules appears in Table II. Scrutiny of this list reveals an overlap with the SMAC components described in Fig 6; this suggests that rafts are involved in the assembly of the SMACs. Although the exact molecular details of raft recruitment to the site of TCR engagement is still unclear, the effect of costimulatory receptors, such as CD28, on the assembly of the actin cytoskeleton is likely to play a role (Fig 3). 14,22,90 TCR engagement induces a dynamic reorganization of the cortical cytoskeleton, with the assistance of costimulatory receptors, such as CD28, CD2, and LFA-1. 91,93 Best characterized is the role of CD28, which is required for prolonged tyrosine phosphorylation and activation of Vav-1, the GEF responsible for Rac-1 and Cdc42. 34,35,79,92,93 LAT, SLP-76, and PI-3 kinase are involved in localizing Vav at the plasma membrane (Fig 4, B). Activated Cdc42 interacts with the Wiscott-Aldrich syndrome protein (WASP), which controls the actin regulatory complex, Arp2/3 (Fig 8) In its inactive state, WASP assumes a closed conformation that fails to bind to Arp2/3, but in the presence of activated Cdc42, this protein opens up, leading to the recruitment and activation of Arp2/ The adapter protein Nck assists in WASP activation (Fig 8). This leads to actin polymerization at the TCR contact site in addition to the assembly of cytoskeletal proteins, such as profilin, talin, vinculin, ezrin, radixin, and moesin. 97 The cytoskeleton has an important structural role in shaping the contact zone between the T cell and the APC. 93 In addition, the cytoskeleton plays an active role MAY 2002 in signal transduction, as exemplified by the fact that drugs that inhibit actin polymerization interfere with T- cell activation. 98 One possibility is that actin filaments may establish a scaffold for the assembly of signaling complexes. 93 This may involve novel adapter proteins that link signaling components to actin filaments. PKCθ is recruited to the SMACs in this manner (Figs 6 and 8) and may contribute to cytoskeletal assembly on the basis of its ability to phosphorylate the cytoskeletal protein moesin. 77,99 An additional role for the actin scaffold may be to sustain signaling by preventing the degradation of signaling components. 100 Finally, the actin scaffold may play a role in lipid raft trafficking to the TCR synapse. Details of the specific molecular connections between the cytoskeleton and ordered lipid domain remain to be clarified. The formation of the TCR synapse and the SMACs provides a stable arrangement through which the TCR can establish a threshold for T-cell activation. 20,84,99 This threshold is dependent on an optimal number (approximately 50) and avidity of TCR-MHC peptide interactions. 21 The cumulative effect of multiple integrated signaling events delivers a high-fidelity deterministic signal, which leads to full T-cell activation. When, however, these arrangements are disrupted by low-avidity TCR interactions with the peptide-mhc complex, an alternate form of signaling and biologic outcome may ensue. This aspect is further discussed in part 2 of this review. SUMMARY All considered, TCR signaling is a dynamic event that can elicit a variety of T-cell responses, depending on the cellular subset and the conditions under which the TCR is engaged by the specific MHC-peptide ligand. Whether the cell responds to delivery of the TCR signal by proliferation, differentiation, apoptosis, anergy, development of memory, or cytotoxic killing depends on the quality of the TCR signal, as well as a host of modifying factors, such as cellular subset, costimulatory receptors, type of APC, cytokine milieu, and the ultimate success of T cells in achieving their immunomodulatory effects. Although it is difficult in a review of this size to describe in detail the contribution of TCR signaling to every biologic outcome, I have attempted, by way of the IL-2 promoter (Fig 5), to demonstrate how a variety of signaling pathways are integrated into the activation of a gene that is involved in T-cell proliferation and apoptosis. To further demonstrate how TCR signaling is modified during T H 1/T H 2 differentiation, treatment with altered peptide ligands, tolerance induction, and immune senescence, we will use the basic concepts outlined in part 1 to elucidate those aspects in part 2 of this review. I thank Mr Boyd Jacobson, manager of the Illustration Department, National Jewish Medical Center, for skillful design of the artwork, and Mr Photi Christofas for skillful assistance in preparing the manuscript. Because of space constraints, it was not possible to site the seminal contributions from a large number of investigators to this field.

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