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 2: Role of signaling cascades in T-cell differentiation, anergy, immune senescence, and development of immunotherapy Andre E. Nel, MD, and Ndaisha Slaughter, BS Los Angeles, Calif Part 2 of this review on cellular activation by the T-cell antigen receptor (TCR) will highlight how TCR signaling pathways are adapted to achieve specific biologic outcomes, including different states of T-cell differentiation and the induction of T-cell tolerance. We will also explore how treatment with altered peptide ligands affects TCR signaling to change T-cell differentiation or to induce an anergy state. These changes are accomplished through alteration of protein tyrosine kinase activity, the stoichiometry of phosphorylation of immunoreceptor tyrosinebased activation motifs, intracellular free ionized calcium flux, mitogen-activated protein kinase activity, and transcriptional activation of key cytokine promoters. The CTLA-4 plays an important role in the induction and maintenance of anergy. The second theme will highlight how altered TCR signal transduction, including changes in the compartmentalization of signaling components at the TCR synapse, contributes to decreased T-cell activation during immune senescence. Finally, we will illustrate how the molecular details of TCR activation can be used to modify the function of the immune system. This includes a description of the mechanism of action of altered peptide ligands, CTLA-4Ig, and pharmacologic inhibitors of mitogen-activated protein kinases, nuclear factor κb, and protein kinase C cascades. (J Allergy Clin Immunol 2002;109: ) Key words: T-cell activation, TCR, T H 1/T H 2 differentiation, anergy, therapy, senescence 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, as well as UCLA Asthma, Allergy, and Immune Disease Center grant PO-1 AI Received for publication March 13, 2002; revised March 18, 2002; accepted for publication March 21, Reprint requests: Andre E. Nel, MD, Division of Clinical Immunology/Allergy, UCLA School of Medicine, Le Conte Ave, Los Angeles, CA Mosby, Inc. All rights reserved /2002 $ /10/ doi: /mai Abbreviations used AP-1: Activator protein 1 APC: Antigen-presenting cell APL: Altered peptide ligand [Ca 2+ ] i : Intracellular free ionized calcium camp: Cyclic adenosine monophosphate CsA: Cyclosporin A CREB: camp response element binding factor CREM: camp response element modulator CTLA-4: Cytotoxic T-lymphocyte antigen CTLA-4Ig: Fusion of CTLA-4 with CH domain of IgG ERK: Extracellular signal regulated kinase GATA-3: Zinc-finger transcription factor ITAM: Immunoreceptor tyrosine-based activation motif IκB: Inhibitory κb protein IκBK: IκB kinase JNK: N-terminal c-jun kinase LAT: Linker for activated T cells MAP: Mitogen-activated protein NF-κB: Nuclear factor κb NFAT: Nuclear factor of activated T cells NIP45: NFAT-interacting protein 45 p27 kip1 : 27-kd cyclin-dependent kinase inhibitor PKC: Protein kinase C PLC: Phospholipase C PTK: Protein tyrosine kinase Src: Ross sarcoma oncogene STAT: Signal transducer and activator of transcription TCR: T-cell antigen receptor ZAP-70: ζ-chain associated protein kinase of 70 kd In the first part of this review, we outlined the major signaling components and pathways that play a role in cellular activation by the T-cell antigen receptor (TCR). 1 We have highlighted the role of lipid rafts and supramolecular activation clusters in integrating these pathways into an effective mechanism for signal delivery. 2,3 Because of differences in antigen presentation, variation in TCR affinity for its ligand, and the existence of developmentally different T-cell subsets, the basic scheme of signal transduction needs to be adapted to allow for an appropriate response to the antigen. Part 2 will highlight the changes in TCR signal transduction in the setting of cellular activation by altered peptide ligands (APLs), T- 901

2 902 Nel and Slaughter J ALLERGY CLIN IMMUNOL JUNE 2002 cell tolerance, T H 1/T H 2 differentiation, and immune senescence. We will also show how the molecular details of TCR activation can be used for therapeutic modulation of the immune system. CHANGES IN SIGNAL TRANSDUCTION BY APLs With some exceptions, the spectrum of biologic activities elicited by peptide-mhc ligands correlates with the binding affinity of the TCR. 4-6 Generally speaking, the higher the affinity or slower the off-rate, the better the ability of the ligand to induce a T-cell response. The specificity and avidity of the TCR interaction with its ligand is determined by the primary sequence of the antigenic peptide, which affects its binding to the complementarity-determining regions of the TCR, as well as the peptide-binding groove of the HLA molecule. 7,8 The peptide-binding groove of the HLA molecule consists of a β- pleated sheet, which makes up the floor, and sides composed of α-helixes. 7,8 Although the MHC I groove accommodates shorter (7-15 amino acids) peptides due to convergence of the α-helixes, the MHC II groove binds longer peptides because of an open groove conformation. 8 When bound to the groove, the N-terminal end of the antigenic peptide always points to one side of the groove, whereas the C-terminus is located at the opposite end. This positions the peptide so that some amino acid side chains project upward toward the TCR complementarity-determining region loops, whereas most of the remaining amino acid side chains point into the floor, where they are anchored in specialized pockets. 8,9 Typically, an HLA class I molecule has 6 binding pockets distributed along the length of the groove, 2 to 3 of which are critical as anchors. 8 A small variation in primary peptide sequence can exert a major effect on its ability to interact with either the MHC or the TCR, thereby converting an agonist peptide into a partial agonist or an antagonist. 10 Agonist peptides engage in high-affinity interactions with the TCR and induce a robust T-cell response, whereas partial agonists or antagonists engage in lower-affinity interactions that lead to altered or inhibitory responses. 4-6,10,11 This principle can be used to develop APLs that interfere or modulate the immune response (see Principles of TCR signal transduction can be used therapeutically to modify immune function ). How does treatment with an APL lead to changes in TCR signal transduction? It is useful to recall that the affinity of the TCR for the peptide-mhc complex determines the level of immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation and hence the outcome of the T-cell response to antigen (Fig 1) According to the kinetic proofreading hypothesis, a certain amount of time is required to complete the stepwise phosphorylation and recruitment of downstream signaling molecules by the CD3- and ζ-chain ITAMs. 4,5,15 Interruption of the TCR interaction with its ligand due to a rapid off-rate might lead to incomplete phosphorylation of the ζ-chain, thereby reducing recruitment and activation of the ζ-chain associated protein kinase of 70 kd (ZAP-70). This might occur during occupancy by a lowaffinity ligand or treatment with an APL, with consequent changes in signal transduction. 4 A synopsis of the key signaling differences in agonist versus APL treatment appears in Table I Briefly, stimulation of naive CD4 + T cells with an agonist induces sufficient assembly of signaling complexes to allow activation of the IL-2 promoter. In this setting there is a sustained and high stoichiometry of phosphorylation of the ζ-chain (p21), linker for activated T cells (LAT) phosphorylation, ZAP- 70 activation, sustained increase of intracellular free ionized calcium [Ca 2+ ] i, extracellular signal regulated kinase (ERK)/N-terminal c-jun kinase (JNK) activation, and nuclear factor of activated T cells (NFAT) translocation to the nucleus (Table I) In contrast, the early biochemical signals induced by APLs include substoichiometric phosphorylation of the ζ-chain (p18), failure to activate ZAP-70, weak and transient [Ca 2+ ] i mobilization, and low levels of ERK and JNK activation (Table I). These changes in TCR signaling are generally insufficient to sustain IL-2 production and might induce an anergic state or skewing of T H 1/T H 2 development (see Changes in TCR signal transduction during T-cell differentiation ). This suggests that in addition to a quantitative reduction in TCR signaling, APLs induce qualitatively different TCR signals. Although it is still unclear whether this includes novel signals, it is possible that monophosphorylated ITAMs might recruit signaling complexes and adapters other than ZAP-70 (Fig 1). 14 This includes negative regulators of TCR signal transduction (Fig 1). Alternatively, biphosphorylated ITAMs could recruit novel signal-transducing molecules (designated B in Fig 1). Therefore, it is clear that the TCR complex is not simply an on-and-off switch but can induce qualitatively different T-cell responses, depending on the avidity of the receptor for its ligand. Further elucidation of the principles of APL signaling might help to improve peptide design for purposes of immunotherapy. ALTERED SIGNAL TRANSDUCTION IN TOLERIZED T CELLS The maintenance of peripheral tolerance is essential for homeostasis in the immune system. Although tolerance has been defined in a number of different ways, the classical definition is that tolerance is an actively maintained state of nonresponsiveness to a specific antigen in a human or an animal previously exposed to the same antigen. 19 The in vitro definition, for which the term anergy is used, is defined as the inability of individual T cells or T-cell clones to produce IL-2 and proliferate on restimulation with an appropriate antigen and an antigenpresenting cell (APC) Anergy can be induced in naive helper T cells during antigen occupancy in the absence of CD28 costimulation and is preventable by means of TCR/CD28 costimulation. 20 This dual signaling requirement to prevent anergy is the cornerstone of the 2-signal hypothesis of T-cell activation, which posits

3 J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER 6 Nel and Slaughter 903 FIG 1. Schematic to explain possible mechanisms by which multiple ITAMs regulate TCR signaling specificity. The TCR signaling apparatus is depicted as a ζ-chain homodimer. A, This model proposes that the stoichiometry of ITAM phosphorylation and TCR signaling potency are directly related. TCR signaling potency refers to both the number and quality of signals, which are induced as explained in Table I. These outcomes are directly related to the avidity of the TCR for its peptide/mhc ligand. B, In this model individual ITAMs recruit distinct activation complexes that couple the TCR to different downstream signaling pathways. The stoichiometry of ITAM phosphorylation, as determined by the avidity of the TCR-ligand interactions, specifies the type of activation complexes that are recruited to the TCR. This panel depicts 2 putative activation complexes, A and B, that associate with monophosphorylated and biphosphorylated ITAMs, respectively. C, This model proposes that ITAMs might play a negative regulatory role on the basis of the ability of monophosphorylated ITAMs to bind inhibitory complexes (I) that interfere with TCR signal transduction. This inhibition might be overcome in the presence of a ligand that interacts with sufficient avidity with the TCR. Adapted from Love and Shores. 14 that optimal T-cell activation is dependent on both TCR (signal 1) and CD28 (signal 2) occupancy (Fig 2). 20,21 The 2-signal concept is also applicable to in vivo immune modulation (Fig 2), as demonstrated by the fact that blockade of CD28 costimulation by a competitive ligand, CTLA-4Ig, can induce a tolerant state to bone marrow and solid organ grafts (see Principles of TCR signal transduction can be used therapeutically to modify immune function ). 22,23 It is important to point out, however, that the true mechanism and correlation of in vitro anergy to in vivo immune tolerance is not clear as yet. Clonal deletion, T-cell apoptosis, and induction of immunoregulatory T-cell subsets are some of the in vivo mechanisms that might substitute for anergy. Given the clinical importance of tolerance to the treatment of transplant rejection, autoimmunity, and allergy, there has been an intense focus on the biochemical events leading to or maintaining the anergic state. Although a lot remains to be learned about changes in TCR signaling in tolerant T cells, a number of studies show that anergic T

4 904 Nel and Slaughter J ALLERGY CLIN IMMUNOL JUNE 2002 TABLE I. Agonist versus APL signaling in naive CD4 + T cells* Agonist Protein tyrosine phosphorylation ζ p21/p (sustained) + (transient) ZAP PLC-γ1 ++ ± LAT +++ [Ca 2+ ] i mobilization +++ (sustained) + (transient) Kinase activity JNK ERK NFAT translocation ++ + IL-2 production ++ ± Macromolecular assembly CD45 association with TCR/Lck + SHP-1 recruitment to TCR synapse Lipid raft integrity Required for Ca 2+ signal? Not required? T-cell differentiation T H 1 T H 2 (± T H 1) APL *The outline and some of the data were taken from Leitenberg and Bottomly. 16 FIG 2. Schematic to explain the role of the CD28 receptor in delivering signal 2. Interference in CD28 binding to CD80 or CD86 by the blocking ligand CTLA-4Ig results in delivery of signal 1 (TCR) only. This leads to interference in IL-2 production in naive T cells, which might result in anergy or alternative biologic outcomes. cells exhibit poor phosphorylation of the TCR-ζ and TCRε chains and are incapable of activating lymphocyte-specific protein tyrosine kinase, ZAP-70, Ras, JNK, and ERK (Fig 3) The TCR also fails to activate activator protein 1 (AP-1) and NFAT binding sites in the IL-2 promoter, but anergic T cells maintain their capacity to induce phospholipase C (PLC) γ1 phosphorylation and [Ca 2+ ] i flux, along with elevated inositol trisphosphate (IP 3 ) levels and

5 J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER 6 Nel and Slaughter 905 FIG 3. Schematic to explain changes in TCR signal transduction in anergic T cells, including the role of the CTLA-4 receptor in inducing the anergic state. These changes include both a subtraction and reduction of signaling required for AP-1 dependent activation of the IL-2 promoter, as well as increased signaling by Fyn kinase and the small GTP-binding protein Rap-1. In addition, anergic T cells maintain PLC-γ1 phosphorylation and ability to generate [Ca 2+ ] i flux. There is also an increase in intracellular camp levels, which might be related to increased Rap-1 activity. This increase in camp might result in increased expression of the cellcycle inhibitor p27 kip1, a purported anergy factor that is responsible for maintenance of a nonresponsive state during TCR occupancy. Hypothetically, p27 kip1 and other anergy factors might be diluted or suppressed by exogenous IL-2 in the culture medium, explaining why this cytokine might induce a recovery from the anergic state. Because T cells must transgress through the cell cycle (in addition to IL-2 production) to avert anergy, it has been proposed that there are 2 distinct states of nonresponsiveness (Table II). 41 Additional anergy factors, which might be involved in negative regulation of the IL-2 promoter, are pcrem and Nil-2a (not shown). The binding sites for CREM and Nil-2a in the IL-2 promoter are shown in Fig 6 in part 1 of this review. 1 a, Change in activity rather than protein abundance. increased Fyn kinase activity (Fig 3). 27,28,30 In addition, anergic T cells exhibit increased intracellular cyclic adenosine monophosphate (camp) levels and increased activity of the small GTP-binding protein Rap-1. 31,32 These findings suggest that anergy induction is dependent on both subtractive, as well as novel, signaling delivery (Fig 3). Because the anergic state can be overcome by IL-2 treatment, it has been proposed that TCR ligation induces the expression of one or more anergy factors, which are responsible for maintenance of the anergic state. 33,34 According to this theory, induction of cellular proliferation by IL-2 might dilute these anergy factors to allow the cell to regain antigen responsiveness. 34 The finding that the cyclin-dependent kinase inhibitor 27-kd cyclin-dependent kinase inhibitor (p27 kip1 ) blocks cell-cycle progression and IL-2 production in anergic T cells in vivo and in vitro suggests that p27 kip1 acts as an anergy factor (Fig 3). 32 p27 kip1 associates with the c-jun coactivator JAB1, thereby acting to disrupt AP-1 binding sites in the IL-2 promoter. 32 Interestingly, p27 kip1 levels are increased by the rise of camp levels in anergic T cells, whereas IL-2 treatment leads to downregulation of p27 kip1 expression. 32 This suggests that the increase in cytosolic camp levels in anergic T cells might be involved in maintaining the anergic state. In addition to regulating p27 kip1 activity, camp activates EPAC (exchange protein directly activated by camp), a guanidine exchanger that catalyzes GTP exchange on Rap A third possible role for camp might be downregulation of the IL-2 promoter by camp response element binding (CREB)/cAMP response element modulator (CREM) complexes. 35 To this end, it was reported that anergic T-cell clones display increased pcreb/pcrem binding to a negative regulatory element, which is situated 180 bp upstream of the start site (Fig 6 from part 1 of this review). 1,35 It is still controversial, however, whether the camp-dependent protein kinase A (PKA) is involved in CREB phosphorylation and binding since PKA activators and inhibitors only have a modest effect on pcreb binding. 36 In addition to PKA, protein kinase Cθ (PKCθ) and ribosomal S6

6 906 Nel and Slaughter J ALLERGY CLIN IMMUNOL TABLE II. Functional comparison of 2 distinct modes of anergy occurring in primary CD4 + T cells in vitro* JUNE 2002 Event Clonal anergy Division arrest anergy Induced by lack of CD28 costimulation Yes No Induced by CTLA-4 signals No Yes Dependence on cell division No Yes Reversed by IL-2 Yes No Associated with p27 kip1 Yes Yes *Data from Wells et al. 40 kinase induce the phosphorylation and activation of CREB, suggesting that other kinases might be involved in CREB/CREM regulators in anergic T cells. 37 Along similar lines, the zinc-finger transcription factor Nil-2a, also known as ZEB, has been proposed to act as an anergy factor. 38,39 As for CREB/CREM, this putative anergy factor interacts with a negative regulatory element situated at 105 in the IL-2 promoter (Fig 6 from part 1 of this review). 1,38 CD28 costimulation has been shown to interfere in Nil-2a mrna expression, suggesting that the transcriptional activation of the gene encoding Nil-2a is regulated by TCR. 38 In addition to the CD28-dependent nonresponsive mode (also known as clonal anergy), tolerance can be induced in some peripheral T-cell populations in the presence of CD28 costimulation (Table II). 40 Although this type of tolerance, also known as division-arrest anergy, is associated with a failure of T cells to proliferate on restimulation with antigen, TCR responsiveness cannot be restored by culturing the cells in IL-2 (Table II). 40 Moreover, although division-arrest anergy is absolutely dependent on the role of a negative regulatory receptor, CTLA-4, this is not an absolute requirement for clonal anergy (Table II). Thus CD28 costimulation is necessary but insufficient to prevent anergy in primary CD4 + T cells. 40,41 Apparently, T cells are also required to transgress through the cell cycle to prevent anergy, implying that failure of IL-2 production or cell-cycle progression induces distinct states of nonresponsiveness (Table II). 41 An interesting biochemical hallmark of both forms of anergy is elevated p27 kip1 levels (Table II). 40 In contrast to the stimulatory effects of CD28, crosslinking of CTLA-4 inhibits IL-2 production, cell-cycle progression, and activation of TCR-induced cyclins. 22,42,43 The fact that CD28 and CTLA-4 are homologous receptors that bind to the same APC ligands (B7-1/CD80 and B7-2/CD86) yet induce opposite effects suggests a delicate balance between their regulatory activities. 44 A shift of this balance in one direction or the other might influence to what extent an individual T cell will respond. It might be relevant that although CD28 is constitutively expressed on the cell surface, CTLA-4 is absent from naive T cells and requires prior cellular activation for its expression. 44,45 Interestingly, the majority of newly expressed CTLA-4 molecules do not localize directly at the cell surface but accumulate in an endosomal compartment, where they are sequestered by an adapter protein, AP50 (Fig 3). 46,47 AP50 interacts with the Y 201 motif in the CTLA-4 tail, but phosphorylation of that site leads to receptor translocation to the cell surface (Fig 3). 47 TCRassociated Src and Tec protein tyrosine kinases (PTKs) are likely involved in Y 201 phosphorylation. When expressed on the cell surface, CTLA-4 is able to modulate several aspects of TCR signaling, including inhibition of protein tyrosine phosphorylation; JNK, ERK, and nuclear factor (NF) κb activation; and transcriptional activation of NF-κB, NFAT, and AP-1 regulatory elements ,47-50 This could explain several of the TCR signaling changes depicted in Fig 3. It has been proposed that CTLA-4 regulates or recruits tyrosine phosphatases to the TCR to accomplish these subtractive signaling effects (Fig 3). 47,51 It has been demonstrated that the SH2-containing protein tyrosine phosphatase SHP-2 can associate with the py 201 motif. However, this finding is controversial because mutational deletion of the CTLA- 4 cytoplasmic tail failed to demonstrate a need for that domain in signal transduction. 51,52 It has therefore been suggested that a major function of CTLA-4 is to act as a competitive inhibitor for B7 binding to CD28 (Fig 3). 44 In this regard CTLA-4 functions only when its ligand is present on the same APC that participates in TCR ligation. In contrast, CD28 ligation can occur in trans ie, ligation by a B7 ligand that is expressed on a bystander cell. 44,47 A third explanation for the biologic effects of CTLA-4 is disruption of signaling arrangements in the immunologic synapse (Fig 3). 44 This could occur by sequestrating key signaling proteins from the synapse. In this regard it is noteworthy that both CTLA-4 and B7-1 form homodimers and that a single CTLA-4 homodimer can bind 2 B7 molecules. 44 This allows the formation of multimeric lattices, which might disrupt the organized assembly of key signaling components in the immunologic synapse. Finally, CTLA-4 might deliver an unidentified signal that affects cell-cycle transition and therefore assists in anergy induction (Fig 3). 43 It is interesting that the phenotypically distinct CD4 + /CD25 + T-cell subset, which is responsible for the maintenance of in vivo immune tolerance, exhibits constitutive CTLA-4 expression. 53 Cross-linking of CTLA-4 induces secretion of TGF-β, which provides a possible explanation for the immunoregulatory capabilities of this cellular subset. 54 Much remains to be learned, however, about the role of CTLA-4 and the mechanism of action of the CD4 + /CD25 + T-cell subset.

7 J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER 6 Nel and Slaughter 907 FIG 4. Schematic to explain how differences in TCR signal transduction on the basis of the potency of the stimulus or the type of dendritic cell (DC) might determine T H 1 and T H 2 differentiation. A, A strong stimulus will lead to a high stoichiometry of ITAM phosphorylation, with subsequent generation of sustained [Ca 2+ ] i flux, JNK, and NF-κB activation (A). Under these conditions, a range of transcription factors is introduced in the nucleus, which, in the presence of T-bet and STAT4, will favor the activation of T H 1 genes. (Fig 4 continued on next page.) CHANGES IN TCR SIGNAL TRANSDUCTION DURING T-CELL DIFFERENTIATION Differences in the organization of TCR-associated signaling complexes might shed light on the differentiation of naive T cells into effector subsets with unique cytokine and immune regulatory profiles. T H 1 cells produce IFN-γ and TNF-α and promote cell-mediated immunity, whereas T H 2 cells are characterized by the production of IL-4, IL-5, IL-10, and IL-13 and promote humoral immunity. Cytokines play a key role in the development of T H 1 and T H 2 subsets (Table III). 16,55,56,64,65,71-73 For instance, the presence of IL-4 during T-cell priming promotes T H 2 differentiation and inhibits T H 1 development, and the presence of IFN-γ inhibits T H 2 generation and promotes T H 1 differentiation. 55 How this cytokine milieu is established is the subject of intense research interest but includes the possibility that TCR signaling plays a role in T H 1/T H 2 skewing (Table III). 16,55,56 In this regard it is known that a change in TCR signaling potency can determine the type of cytokine that will be induced (Fig 4). 16,56-61 TCR signaling potency refers to both the number and quality of signals that are induced (Table I). Thus in the absence of a dominant cytokine, low-potency TCR signals preferentially generate IL-4 production and T H 2 differentiation. 16,56-61 The same outcome can also be achieved with very low doses of an antigen or an APL with a low avidity for the TCR complex In contrast, priming of naive T cells with optimal doses of an agonist peptide tends to favor IL-2 production and T H 1 differentiation in vivo and in vitro (Fig 4). 62 There are several possible explanations for why potent TCR signals might promote T H 1 differentiation but suppress T H 2 development. One possibility is that stimulation of naive T cells with optimal doses of an agonist peptide provides a good stimulus for IL-2 production, which promotes T H 1 differentiation or interferes in T H 2 differentiation. 16,56,62 Therefore it is relevant that those strong agonists generate sustained [Ca 2+ ] i flux, which is required for activation of the IL-2 promoter (Table I). 16 In contrast, developing T H 2 cells exhibit diminished and transient [Ca 2+ ] i flux, which fails to support sustained IL-2 production on TCR ligation (Fig 4). 16,63 Although the potency of the TCR stimulus is a determining factor in T H 1/T H 2 differentiation, this does not

8 908 Nel and Slaughter J ALLERGY CLIN IMMUNOL JUNE 2002 FIG 4. B, Weaker stimuli or activation by type II DCs induces a limited stoichiometry of ITAM phosphorylation, which results in transient [Ca 2+ ] i flux and a modified panel of transcription factors that favor activation of the IL-4 promoter. In addition to the role of GATA-3, c-maf, STAT6, and NIP45 in this event, the relative ratio of nuclear NFATc versus NFATp/NFAT4 might be an important determining factor. The JNK cascade, as well as the pattern of oscillatory Ca 2+ waves, might help to determine the NFAT ratio. TABLE III. Determining factors in T H 1/T H 2 differentiation Factor T H 1 differentiation T H 2 differentiation Cytokine milieu 55 IL-4, IFN-γ, IL-12 + IL-18 IL-4, IFN-γ Transcription factors 55,71 T-bet, STAT4 by GATA-3, c-maf, STAT6, NIP45, junb, NFATc by NFATp/NFAT4 Chromatin remodeling 72 Required for activation of the gene encoding IFN-γ Required for activation of the gene encoding IL-4 TCR complexing to CD4 and CD45 16,56 Required Not required Role of lipid rafts 56 TCR association with rafts No association CD28/B7 costimulation 73 IFN-γ production minimally affected Absolutely required for IL-4, IL-5, and IL-10 production Influence of JNK isoforms 64,65 JNK2 enhances JNK1 interferes TCR signaling potency 16,56 Potent agonists/strong stimuli enhance Low-potency signals/apls/low antigen dose enhance Type of APC 55 IL-12 producing type I dendritic cells Type II dendritic cells T-bet, T-box expressed in T cells. imply that weak signaling is a sine qua non for T H 2 differentiation. In this regard it is important to keep in mind that a host of transcription factors are involved in the activation of the IL-4 promoter and in T H 2 development (Fig 4). 55,68,69 These include signal transducer and activator of transcription 6 (STAT6); GATA-3 (a zinc-finger transcription factor), c-maf (a basic leucine zipper transcription factor), and NFAT-interacting protein 45 (NIP45), 2 of which (STAT6 and GATA-3) are directly related to occupancy of the IL-4 receptor by its ligand (Fig 4). 55,64 There are a number of ways in which TCR signaling might negatively affect the ability of these transcription factors to induce IL-4 production. One possibility is differential activation of the Jun kinase cascade (Fig 4). 65 In this regard it

9 J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER 6 Nel and Slaughter 909 is known that delayed JNK activation occurs in precursor CD4 + T cells during antigen stimulation. 65 However, after differentiation of these cells into effector T H 2 cells, antigen stimulation fails to induce JNK activation. 65 In contrast, TCR occupancy leads to JNK activation in T H 1 cells (Fig 4), which express similar levels of JNK protein as T H 2 cells. 66 Therefore, it is interesting that JNK1 negatively regulates T H 2 development as a result of its ability to induce nuclear export of NFATc (Fig 4). 67 Although JNK can phosphorylate NFAT4, there is no evidence for NFATc phosphorylation, and the mechanism for the nuclear export of NFATc remains unknown. 68 NFATc is required for the activation of the IL-4 promoter, whereas 2 additional family members, NFATp and NFAT4, exert negative regulatory effects on this gene. 16,64,69-71 By decreasing the nuclear NFATc abundance, JNK indirectly favors T H 1 development. This effect is strengthened by direct involvement of JNK2 in T H 1 differentiation, probably as a result of its ability to induce the expression of the β 2 subunit of the IL-12 receptor. 16,55 IL-12 receptor occupancy leads to increased IFN-γ production, which actively promotes T H 1 differentiation (Fig 4). 66 It is likely that in the setting of T H 1 differentiation, JNK2 acts in synergy with other transcription factors, such as T-box expressed in T cells (T-bet) and STAT4, which positively regulates IFNγ production (Fig 4). 55,64 NFAT and NF-κB transcription factors contribute to the expression of the gene encoding IFN-γ, suggesting that sustained [Ca 2+ ] i flux and activation of the IκB protein kinases are involved (Fig 4). The relative abundance of the NFATc versus the NFATp and NFAT4 isoforms in the nucleus could determine whether TCR engagement results in T H 1 or T H 2 differentiation. 16,56 Thus a relatively high NFATc to NFATp plus NFAT4 ratio should favor IL-4 expression and T H 2 differentiation, whereas a reversal of that ratio could interfere in the IL-4 production, thereby favoring T H 1 differentiation (Fig 4). 16,56 Whether these differences in the nuclear abundance of NFAT isoforms, as well as JNK activation, are related to TCR signaling potency or different modes of [Ca 2+ ] i oscillation is unclear. One theory has been that sustained [Ca 2+ ] i flux promotes the nuclear relocation of NFAT proteins, as well as JNK1 activation (Fig 4). 16 Because of the effect of JNK1 on NFATc nuclear export, only NFATp and NFAT4 will accumulate in the nucleus, thereby favoring T H 1 differentiation (Fig 4). 62 Thus, in the setting of a potent agonist or APCs that promote strong TCR signaling, sustained [Ca 2+ ] i release and a high stoichiometry of ITAM phosphorylation could promote T H 1 differentiation (Fig 4). An accompanying increase in c- Jun, NFAT, and NF-κB transcription factors will ensure that IFN-γ is induced in these cells. 55 In contrast, lowpotency signals or antigen presentation by an APC that favors T H 2 differentiation might induce a short but still sufficient burst of [Ca 2+ ] i flux to allow NFAT translocation to the nucleus (Fig 4). 16 In the absence of JNK activation, NFATc will dominate, thereby favoring IL-4 production and T H 2 differentiation (Fig 4). 16 The increase in nuclear AP-1 activity that is required for activation of the IL-4 promoter might reflect ERK activation by the TCR (Fig 4). 64 In addition to the role of the TCR, a number of costimulatory receptors exert an effect on the polarity of T- cell differentiation (Table III). This includes contributions by CD4, CD45, and CD Although CD28 costimulation has been shown to be an absolute requirement for IL-4, IL-5, and IL-10 production during in vitro stimulation, this finding is not compatible with the fact that CD28 is required for JNK and NF-κB activation. 55,73 We have already discussed the involvement of these pathways in activation of a T H 1 (IFN-γ) promoter. 55 Although we lack a suitable explanation for this paradox, this illustrates the complexity of the signaling events involved in T H 1/T H 2 differentiation and the necessity to learn more about the specific molecular events involved in these processes. The schemes depicted in Fig 4 should therefore be seen as preliminary. CHANGES IN TCR SIGNAL TRANSDUCTION DURING IMMUNE SENESCENCE Aging leads to a decline in the ability to mount rigorous T-cell responses to newly encountered antigens, as well as previously encountered (recall) antigens. 74 This decline manifests as a decrease in delayed-type hypersensitivity responses, diminished ability to respond to vaccination, and increased susceptibility to virulent viral and mycobacterial infections. 74 This decline in cellular function occurs in parallel with changes in the composition of T-cell subsets, as well as altered ability of T cells to respond to TCR libation. 75,76 Phenotypic changes include an expansion of the memory CD4 + subset, whereas changes in TCR signal transudation are characterized by early changes in tyrosine and serine/threonine-specific kinase activity, decreased ζ-chain and LAT phosphorylation, decreased Raf-1/MEK/ERK activation, decreased CD28-dependent JNK activation, decreased [Ca 2+ ] i flux, impaired NFATc translocation to the nucleus, and decreased IL-2 production (Fig 5) Changes in ZAP- 70 activity are still controversial, with some workers reporting a decrease in ZAP-70 activation, whereas others observed no change. 76 These changes in TCR signaling are not due to decreased expression of TCR/CD3 components, which remain normal. 79 However, there is a decline in CD28 abundance in the CD8 subset. 80,81 This broad decline in TCR signal transduction during immune senescence raises the question as to whether a large number of individual signaling components are simultaneously targeted by the aging process or whether these changes occur as a result of disrupted function of a common upstream component. With regard to the latter possibility, it is known that aging affects the compartmentalization of signaling components at the TCR synapse. 76,82-84 Confocal microscopy has revealed that the majority of CD4 + T cells from aged mice fail to efficiently relocate PKCθ,Vav oncogene, and LAT to the immune synapse (Fig 5) These changes occur in parallel with reduced NFATc relocation to the nucleus. 84 Although these findings might reflect expansion of the memory T- cell pool during aging, it has been shown that similar

10 910 Nel and Slaughter J ALLERGY CLIN IMMUNOL JUNE 2002 FIG 5. Schematic to explain altered TCR signal transduction during immune senescence. These changes include decreased activity of several afferent TCR signaling components, notably a decrease in the stoichiometry of ITAM phosphorylation and PTK activity and decreased activation of MAP kinase cascades. This decrease might be due to a broad decline in TCR signaling components or might be the result of altered compartmentalization of signaling components at the TCR synapse. Possible mechanisms to explain these changes in signaling assembly at the TCR synapse include decreased expression and function of the CD28 receptor, altered lipid raft trafficking, change in lipid raft composition, and cytoskeletal assembly. Ultimately, these changes lead to decreased NFAT and AP-1 transcription activity in the nucleus, with a failure in IL- 2 production. changes occur in naive T cells during immune senescence. 83,84 This suggests that some of the TCR signaling defects that occur during aging could be due to defective immune synapse formation (Fig 5). Although the molecular basis for defective synapse formation is unknown, it has been demonstrated that aging leads to changes in the F-actin polymerization at the site of the TCR synapse (Fig 5). 82 These cytoskeletal changes might contribute to decreased movement of kinase substrates and coupling factors to the TCR synapse. 83 An age-dependent decline in CD28 surface density in some CD8 + T-cell subsets could contribute to these cytoskeletal changes because CD28 plays an important role in regulation of the cytoskeleton, as discussed in part 1 of this review. 1,80,81 Moreover, a functional decline in the quality of CD28 signaling during aging has been documented. 85 A possible contribution to altered compartmentalization of signaling components during immune senescence is a change in lipid raft composition (Fig 5). In this regard it could be relevant that the surface membrane of senescent T cells exhibits a higher cholesterol-phospholipid content than cells of younger individuals. 86,87 It is also well known that changes in membrane microviscosity with aging are associated with lower proliferative responses to lectin stimulation. 86,87 A study of the role of lipid rafts in TCR signaling decline during senescence is therefore indicated. This information can be helpful in finding new ways to boost the function of the immune system during aging. PRINCIPLES OF TCR SIGNAL TRANSDUCTION CAN BE USED THERAPEUTICALLY TO MODIFY IMMUNE FUNCTION Advances in the understanding of TCR signaling pathways have led to the development of novel immunomodulators and promise to deliver new forms of immunotherapy for allergic diseases, autoimmunity, malignancy, and transplant rejection. A classification of the currently available drugs and emerging new therapies appears in Table IV. 22,23,89-91,94-105, This classification is based on whether these agents target the TCR/CD3 complex, costimulatory receptors, or individual signaling components (Table IV). With regard to targeting signaling pathways, it is important to consider that all signaling components do not contribute equally to TCR signaling and that a considerable degree of redundancy exists in these pathways. The relative contribution of individual TCR pathways to global signal transduction has been addressed by calculating the amount of energy that is consumed by each pathway during mitogenic stimulation. 88 With concanavalin A as the stimulus, it was calculated that 84% of

11 J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER 6 Nel and Slaughter 911 TABLE IV. Drugs or treatment modalities that target the TCR signaling cascade Target drug/agent Mechanism of action Clinical or research use TCR/CD3 1. Anti-CD3 mab 1. T-cell depletion and tolerization 1. Organ transplantation complex (OKT3, humanized anti-cd3 mab) Peptide therapy 2. Blocking peptides interfere in 2. Blocking peptides/apls are used successand APLs antigen-specific responses and fully to treat allergic and autoimmune induce tolerance or skewed responses in mice Fel d 1 peptides T H 1/T H 2 differentiation. APLs suppress allergic responses and asthma in induce T-cell tolerance or skewed human subjects. 103 PLA 2 peptides induce T H 1/T H 2 differentiation tolerance to bee venom in human subjects. 123 APLs and copolymer 1 have proven beneficial for multiple sclerosis trials. 104,105 Costimulatory 1. CTLA-4Ig 22, Blocks CD28 engagement by 1. Extensive use in animals to prevent receptors CD80/CD86. Induces anergy and allograft (kidney, heart, pancreatic-islet) long-term tolerance caused by rejection. 23,94 Interferes in autoimmune interference in delivery of signal. disease (eg, murine lupus models) Anti-CD4 mab T-cell depletion Successful treatment of psoriasis in human (keliximab) subjects. 99 Ex vivo tolerization of T cells to prevent graft-versus-host disease during human bone marrow transplantation. 98 Ca 2+ /calcineurin 1. CsA 89 Both drugs inhibit NFAT CsA and FK506 are widely used as first-line pathway 2. Tacrolimus translocation to the nucleus immunosuppressants in organ and bone (FK506) 89 and interfere in IL-2 production marrow transplantation in human and cellular proliferation. 90 subjects Occational use in systemic lupus erythematosus and rheumatoid arthritis in human subjects. 125,126 Tacrolimus is also useful as a topical agent for treatment of severe atopic dermatitis. 127 MAP kinase 1. ERK: PD98059 Relatively specific inhibition of the Experimental drugs used extensively to cascades : U0126 ERK, JNK, and p38 MAP kinase interfere in T-cell activation in vitro. 2. JNK: SP cascades during TCR ligation Although MEK 1 inhibitors (PD98059, 3. p38: SB in vitro ,118 U0126) exert potent inhibitory effects on : RWJ67657 CD3/CD28-induced IL-2 production, the p38 MAP kinase inhibitors do not affect IL-2 production but interfere in IL-5 production in allergen-reactive T H cells. 113,115,118 RWJ67657 has been used in experimental studies in human subjects and animals to suppress endotoxin-induced cytokine production and clinical symptoms. 116,117 NF-κB cascade 1. IκBα superrepressor 1. Abrogates p65 nuclear Agents 1, 4, and 7 are experimental and have translocation 111 not been used to suppress antigen-specific 2. Glucocorticoids 2. Increase IκBa expression 109 immune responses. The IκBα superrepressor 3. Aspirin 3. Inhibitor of IκB kinase β 110 and cyclopentenone PGs induce apoptosis 4. Lactacystin 4. Proteosome inhibitor: prevents in primary human T cells activated through release of Rel proteins 121 the TCR CsA 5. Proteosome inhibitor FK Abrogates c-rel nuclear translocation Cyclopentenone PGs 7. IκB kinase β inhibitor 111 PL-dependent/ Rottlerin 120 Interferes in PKCθ in T cells and No animal or human studies to date show an Ca 2+ -independent interferes in NF-κB activation and effect on antigen-induced responsiveness. PKC inhibitors IL-2 production. Also inhibits PKCδ. Src kinase activity Pyrinidine derivatives Interfere in induction of IL-2 Potential clinical use of Src kinase inhibitors (PP1 and PP2) 128 synthesis and T-cell proliferation demonstrated by success of Gleevac but not in cell survival 128 (STI571) in treatment of chronic myeloid leukemia 119

12 912 Nel and Slaughter J ALLERGY CLIN IMMUNOL JUNE 2002 the energy was spent on the PTK/PLC-γ1/PKC pathway. 88 This could be further subdivided into energy expenditures of 30% and 54%, respectively, toward the PLC-γ1 [Ca 2+ ] i /calcineurin and PKC pathways. 88 Activation of mitogen-activated protein (MAP) kinase cascades were included in the PKC pathway and required 40% of the total energy expenditure. 88 The remaining 16% of the energy was required for processes that could not be measured by the energetic approach, including assembly of macromolecular complexes. 88 Although there is not enough space to discuss the individual actions or use of every agent listed in Table IV, the reader is referred to part 1 of this review, in which the mechanism of action of cyclosporin A (CsA) and tacrolimus (FK506) is illustrated in Fig 6 of that article. 1 Although CsA is a well-established drug for suppression of cellular immune function in organ transplantation or autoimmune disease, an even more potent calcineurin inhibitor, tacrolimus, has been introduced for the treatment of transplant rejection and atopic dermatitis. 89 Like CsA, tacrolimus complexes with a cytosolic receptor protein, FKBP-12, which functions as an immunophilin that inhibits the function of the calcineurin complex. 90 The choice between CsA and tacrolimus for immunosuppression requires awareness of their side-effect profiles, which have been reviewed elsewhere. 91 Sirolimus (rapamycin), like CsA and tacrolimus, is a prodrug that complexes with an immunophilin to exert its immunesuppressive effects. 92 Although sirolimus binds to FKBP- 12, this drug-immunophilin complex interacts with the target of rapamycin (TOR) instead of calcineurin. 93 TOR is a kinase-like protein that regulates enzymatic processes involved in G1-to-S phase cell-cycle transition. Therefore the major effect of sirolimus is to interfere in IL-2 and IL-4 induced T- and B-cell proliferation, rather than directly targeting TCR activation events. 92 This agent is used as a maintenance immunosuppressant or as adjuvant therapy to calcineurin inhibitors. Because sirolimus has a relatively low index of toxicity, its combination with CsA lowers the immunosuppressive dose of the latter drug, and this leads to a decrease in CsA toxicity. 92 The effectiveness of CD28 costimulatory blockade was first demonstrated in animal experiments with CTLA-4Ig, an engineered fusion protein that binds with high affinity to the CD28 ligands B7-1 and B ,23,94 Subsequently, this antagonist has been used successfully to suppress allograft rejection and to induce long-lasting graft acceptance in animals. 22 In addition, preclinical studies in primates have confirmed a key role for CD28/B7 in allograft rejection, which can be prevented with CTLA-4Ig treatment. 95,96 CTLA-4Ig has also been used to treat autoimmune disease in animals. 97 Costimulation blockers are among the most promising future therapies to achieve tolerization of transplanted or autologous antigens. 22 To date, however, relatively few clinical trials have been undertaken with CTLA- 4Ig in human subjects (Table IV). 98,99 We have discussed the ability of altered TCR ligands to exert dominant negative effects on T-cell function. It has now been demonstrated that APLs exert immune regulatory effects in vivo and can be used to reverse experimental autoimmune disease and allergic inflammation in animal models These effects have been ascribed to the induction of in vivo tolerance or skewed T H 1/T H 2 differentiation. An altered myelin basic protein peptide has been used to induce T H 2 skewing in patients with multiple sclerosis, and glatiramer acetate (copolymer 1), a mixture of synthetic polypeptides, has been used in controlled clinical studies to benefit patients with relapsingremitting multiple sclerosis. 104,105 APLs have also been used to induce T H 1 skewing or anergy in human T-cells clones that respond to the major house dust mite allergen Der p Fel d 1 peptides are effective for suppressing cat allergic responses, including asthma, in human subjects. 106 Vaccination with APLs might therefore become an important form of immunotherapy in the future. 102 The use of specific drug inhibitors to interfere in the MAP kinase and NF-κB cascades (Table IV) is still experimental and has not been used for immunomodulation in vivo. Glucocorticoids (eg, prednisone) have an effect on these cascades and are widely used as antiinflammatory and immunosuppressive agents. Although the major anti-inflammatory and immunosuppressive actions of glucocorticoids involve their ability to complex to steroid receptors that inhibit proinflammatory genes, occupied steroid receptors also interfere directly in the function of NF-κB and AP-1 transcription factors. 107,108 In addition, corticosteroids induce the expression of IκBα, which prevents the intranuclear translocation of the NF-κB transcription factor p This leads to decreased activation of NF-κB dependent genes. Similarly, the nonsteroidal anti-inflammatory drugs aspirin and sodium salicylate are agents that exert inhibitory effects on the NF-κB pathway, although this is not the major mechanism of action of these drugs. More specifically, these drugs interfere in the phosphorylation of IκBα through inhibition of the upstream kinase IκB kinase β. 110 More recently, it has also been demonstrated that cyclopentenone prostaglandins (eg, PGA 1 and 15- deoxy 12,14 prostaglandin J2) act as potent inhibitors of IκB kinase β, 121 and these lipid inhibitors, as well as sodium salicylate, can be used to induce apoptosis in antigen-responsive T cells. 111 Apoptosis is likely the result of interference in Bcl-x L expression, which is dependent on the NF-κB pathway. 111 Although ERK and p38 MAP kinase inhibitors have been used to interfere in cellular responses after TCR ligation (Table IV), these agents have not been used to disrupt antigen-specific immune responses in vivo An orally active p38 MAP kinase inhibitor, RWJ 67657, has been used successfully to suppress endotoxininduced clinical effects (eg, fever) and cytokine (TNF-α, IL-8, and IL-6) production in human volunteers. 116 Moreover, p38 MAP kinase inhibitors exert antiinflammatory effects in the murine lung. 117 The ability of p38 MAP kinase inhibitors to interfere in allergeninduced IL-5 synthesis without affecting IL-2 production in human T-cell clones suggests that these agents might be helpful in treating allergen-induced eosinophilic

13 J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER 6 Nel and Slaughter 913 inflammation in human subjects and animals. 115 In contrast, ERK and JNK inhibitors interfere in IL-2 production and might be useful to limit clonal expansion of activated T cells in vivo. 113,114,118 Interestingly, ERK inhibitors do not interfere in anergy induction. 118 Although the Bcr-Abl inhibitor STI571 (Gleevec) has been used with dramatic success in chronic myeloid leukemia, Src kinase inhibitors have not been used as immunosuppressants in vivo. 119 However, given the key role of Lck in TCR activation, it is possible that agents that target Src PTKs (eg, pyrimidine derivatives [PP1 and PP2]) might have clinical benefits in the immune system. 128 Similarly, although a variety of PKC inhibitors have been shown to affect T-cell activation in vitro, these drugs have not been used in clinical studies on the immune system. 120 CONCLUSION Knowledge about the molecular pathways of T-cell activation now allows us to dissect the molecular basis for differential signaling through the TCR, in addition to allowing us to develop new immunotherapies. In this review we have focused on modification of TCR signaling during T H 1/T H 2 differentiation, tolerization, stimulation with APLs, and immune senescence. Equally important is the adaptation of TCR signaling to induction of apoptosis, memory development, and cytotoxic killing. Although it is not possible to cover all of these topics here, it should be clear from the examples chosen that TCR signaling is a dynamic process that can be modified to meet the biological needs of the immune system in defense against foreign antigens. It should also be clear that these signaling modifications are complicated events and that we are just beginning to understand the molecular details of signaling specificity. We predict the rapid pace of research and progress in this area will lead to augmentation and modification of the signaling paradigms depicted in Figs 1 to 5. In addition, the progress made in developing novel agents that interfere or modify TCR signaling holds the promise for delivering new immunotherapies in the future. We 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 of the manuscript. Because of space constraints, it was not possible to cite the seminal contributions from a large number of the investigators to this field. REFERENCES 1. Nel AE. T-cell activation through the antigen receptor (TCR). Part 1. Signaling components, signaling pathways and signal integration at the TCR synapse. J Allergy Clin Immunol 2002; Shaw AS, Dustin ML. Making the T cell receptor go the distance: a topological view of T cell activation. Immunity 1997;6: Dustin ML, Shaw AS. Costimulation: building an immunological synapse. Science 1999;283: Germain RN, Stefanova I. 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