UNIFYING CONCEPTS IN CD28, ICOS AND CTLA4 CO-RECEPTOR SIGNALLING

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1 UNIFYING CONCEPTS IN CD28, ICOS AND CTLA4 CO-RECEPTOR SIGNALLING Christopher E. Rudd and Helga Schneider Many studies have shown the central importance of the co-receptors CD28, inducible costimulatory molecule (ICOS) and cytotoxic T lymphocyte antigen 4 (CTLA4) in the regulation of many aspects of T-cell function. CD28 and ICOS have both overlapping and distinct functions in the positive regulation of T-cell responses, whereas CTLA4 negatively regulates the response. The signalling pathways that underlie the function of each of the co-receptors indicate their shared and unique properties and provide compelling hints of functions that are as yet uncovered. Here, we outline the shared and distinct signalling events that are associated with each of the co-receptors and provide unifying concepts that are related to signalling functions of these co-receptors. IMMUNOLOGICAL SYNAPSE A structure that is formed at the cell surface between a T cell and an antigen-presenting cell; also known as the supramolecular activation cluster (SMAC). The T-cell receptor, numerous signal-transduction molecules and adaptors accumulate at this site. Mobilization of the actin cytoskeleton is required for immunological-synapse formation. Molecular Immunology Section, Department of Immunology, Division of Investigative Science, Faculty of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK. Correspondence to C.E.R. c.rudd@imperial. ac.uk doi: /nri1131 The antigen-receptor complex expressed by T cells that is the T-cell receptor (TCR) and CD3 provides insufficient signals for optimal T-cell function. Instead, a second set of signals is needed to amplify and fine-tune the response, as first proposed in the two-signal hypothesis 1. Present studies point to a situation that is becoming increasingly complex with the identification of a growing number of co-receptors CD28, inducible co-stimulatory molecule (ICOS), cytotoxic T lymphocyte antigen 4 (CTLA4), programmed cell death protein 1 (PD1), CD7 and T-cell immunoglobulin mucin 1 (TIM1) that can be expressed by different subsets of T cells and that can be engaged by overlapping or distinct ligands. Identification of the common and unique signals that are mediated by these coreceptors will be crucial to understand the molecular events that underlie T-cell immunity. This knowledge will also be required for the development of therapeutics that are designed to modulate the immune response. Activation of T cells is initiated by the formation of conjugates between T cells and antigen-presenting cells (APCs) in the context of peptide antigen. Conjugation involves formation of the IMMUNOLOGICAL SYNAPSE, involving a rearrangement of receptors into a central supramolecular activation cluster (csmac) that is enriched with TCRs, CD2 and CD28 molecules surrounded by a peripheral SMAC (psmac) that is enriched with leukocyte function-associated antigen 1 (LFA1) 2,3. Signalling by the TCR CD3 complex is initiated by the recognition of peptide antigen in the context of MHC molecules on APCs a process that involves corecognition of non-polymorphic regions of MHC by the co-receptors CD4 and CD8. Both co-receptors associate with the protein tyrosine kinase (PTK) LCK 4,5 and synapse formation brings CD4 LCK and CD8 LCK complexes into proximity of the TCR complex. This then leads to the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the TCR ζ-chain and the subsequent recruitment of the related PTKs ZAP70 (ζ-chain-associated protein of 70 kda) and SYK (spleen tyrosine kinase) 6. Ultimately, these events lead to the phosphorylation of adaptors, such as the linker for activation of T cells (LAT), which function as scaffolds in the expansion and integration of signalling pathways. By means of recruitment to LAT and SLP76 (SRC homology 2 (SH2)-domain-containing leukocyte protein of 76 kda), phospholipase Cγ1 becomes phosphorylated and activated by members of the TEC family of kinases, thereby increasing intracellular levels of Ca 2+ and activating transcription factors, such as nuclear factor of activated T cells (NFAT) JULY 2003 VOLUME 3

2 APC CD80/ CD86 CD80/ CD86 B7H CD28 CTLA4 ICOS T cell CD28 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CTLA4 KMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN ICOS PI3K (SH2) GRB2/GADS (SH2) PI3K (SH2)/PP2A AP1/AP2 ITK (SH3) SHP2 PI3K (SH2) LCK (SH3) GRB2/GADS (SH3) TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL Figure 1 Structure of the cytoplasmic domains of human CD28, ICOS and CTLA4. The cytoplasmic domains of CD28, inducible co-stimulatory molecule (ICOS) and cytotoxic T lymphocyte antigen 4 (CTLA4) have a common Tyr-Xaa-Xaa-Met motif that binds to the SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase (PI3K). Each co-receptor also has unique features. Within this motif CD28 has an asparagine (N) that is, ptyr-xaa-asn-met for binding of the SH2 domain of growth-factor receptor-bound protein 2 (GRB2), and possibly GRB2-related adaptor protein (GADS), with tandem GRB-SH3-domain binding to the carboxyterminal proline residues. Similarly, CTLA4 has a unique Gly-Val-Tyr-Val-Lys-Met motif, which in a non-phosphorylated form binds to tetrameric adaptor complexes AP1 (AP47) and AP2 (AP50). CTLA4 also has a classic Lys-Xaa-Xaa-Pro-Xaa-Xaa-Pro SH3-domain binding motif, whereas CD28 has a Pro-Arg-Arg-Pro motif that might bind to IL-2 inducible T-cell kinase (ITK), and a carboxy-terminal Pro-Tyr-Ala-Pro-Pro-Arg that binds to the SH3 domain of LCK and GRB2 or GADS. ICOS is noteworthy with the absence of proline-rich motifs. With such an effective machinery for TCR signalling, why is there the need for additional signals from different co-receptors? This article reviews our present understanding of signalling by the co-receptors CD28, ICOS and CTLA4, and the role of their common and specific pathways in the regulation of T-cell function. Co-receptors CD28, ICOS and CTLA4 CD28, ICOS and CTLA4 are members of the immunoglobulin supergene family, each with a variable immunoglobulin-like domain that binds to cognate ligands expressed by APCs. Although CD28 and CTLA4 bind CD80 (B7-1) and CD86 (B7-2), ICOS binds to the related molecule B7H (also known as B7RP1, LICOS or GL50). A crucial Met-Tyr-Pro-Pro-Pro-Tyr loop in the CD28 and CTLA4 immunoglobulin-like domain provides structural specificity for the interaction with CD80 and CD86 (REF. 8). Although the crystal structure of CD28 has not been solved, the structure of CTLA4 in a complex with CD80 and CD86 has been determined. Whereas CD28 interacts with a single binding domain 9, each CTLA4 dimer binds two independent bivalent B7 molecules in the formation of a zipper-like matrix 10, which might explain why CTLA4 has a fold higher binding avidity. In this arrangement, CTLA4 might also preferentially bind CD80 (REF. 11). CD28 is expressed by naive and activated T cells and is required for optimal production of cytokines and proliferation In general, naive or primary T-cell responses are more dependent on CD28 than are secondary responses. This is especially true for T helper 2 (T H 2) cells, the secondary responses of which can operate without the need for CD28 ligation. In this case, other co-receptors, such as ICOS, provide signals that substitute for the co-receptor. In keeping with the importance of CD28, CD28-deficient mice have markedly reduced responses to antigen 15. CD28 can reduce the number of TCRs that must be ligated for a response, lower the activation threshold and allow a response to low-affinity peptides 16. CD28 can also alter the balance between T H 1 and T H 2-cell differentiation 17 and prevent the induction of non-responsiveness (anergy) and cell death (apoptosis) 18. ICOS and CTLA4 are restricted in their expression to activated and memory T cells. ICOS regulates the production of interleukin-4 (IL-4), immunoglobulinisotype class switching and the formation of germinal centres By contrast, CTLA4 functions as a potent negative regulator of the T-cell response. Co-ligation of CTLA4 with TCR, or with TCR and CD28 or TCR and ICOS inhibits proliferation 23,24. CTLA4-deficient mice develop massive lymphoadenopathy, autoimmunity and early death 25,26. On the basis of this evidence, it has been proposed that CTLA4 raises the threshold for TCR signalling, although there is some debate as to whether CTLA4-deficient T cells are hyperresponsive to antigen 14,27. In either case, CTLA4 signalling seems to terminate T-cell responses, which is of potential relevance in situations of inappropriate activation. Co-receptor cytoplasmic domains The first clue as to the nature of co-receptor signalling derives from the composition of the cytoplasmic domains and their interaction with intracellular proteins. CD28, ICOS and CTLA4 have surprisingly small cytoplasmic domains of 41, 35 and 37 amino-acid residues, respectively (FIG. 1). Common to each is a Tyr- Xaa-Xaa-Met consensus motif: a Tyr-Met-Asn-Met motif in CD28, a Tyr-Met-Phe-Met motif in ICOS and a Tyr-Val-Lys-Met motif in CTLA4. This motif functions as a binding site for the SH2 domains of p85 an adaptor subunit of the lipid kinase phosphatidylinositol 3-kinase (PI3K) 19,28 33 (BOX 1). Phosphorylation of the tyrosine residue in this motif allows p85 to bind, whereas specificity is determined by the adjacent residues, in particular the methionine in the plus three position. Of the two SH2 domains of p85, the aminoterminal version, which has a 10-fold higher avidity for the Tyr-Xaa-Xaa-Met motif, predominates 28.The binding affinities of p85 for CD28 and CTLA4 are almost identical to that for motifs in growth-factor receptors, such as the platelet-derived growth-factor receptor (PDGFR), indicating a similar mechanism in the recruitment and generation of D3 lipids 28,33. Besides common residues, CD28 has an Asn residue in the plus two position that is absent in ICOS or CTLA4. This is a signature residue for SH2-domain binding of the adaptor growth-factor receptor-bound protein 2 (GRB2) 34. GRB2 is comprised of one SH2 domain flanked by two SH3 domains that bind to the guaninenucleotide exchange factor Son of sevenless (SOS) an activator of the GTPase RAS. Co-precipitation NATURE REVIEWS IMMUNOLOGY VOLUME 3 JULY

3 D3 LIPIDS Phosphatidylinositol (4,5)- bisphosphate (PtdIns(4,5)P 2 ) is phosphorylated on the D3 position of the inositol ring to yield phosphatidylinositol (3,4,5)-trisphosphate (PtdInsP 3 ). Conversion of PtdInsP 3 to PtdIns(3,4)P 2 by SH2-domain-containing inositol phosphatase (SHIP2) can also occur. Different pleckstrin-homology domains bind to PtdInsP 3 and/or PtdIns(3,4)P 2. Box 1 Phosphatidylinositol 3-kinase (PI3K) experiments have shown binding of CD28 to GRB2, but at low levels compared with PI3K This might be due to competition for the same site by PI3K, or possibly by GRB2-related adaptor protein (GADS) 38. Additional studies are required to elucidate the exact nature of competitive binding to this site. The CTLA4 Tyr-Val-Lys-Met site is unique in that it binds the clathrin-adaptor protein complexes AP1 (µ1 AP47) and AP2 (µ2 AP50) Only the non-phosphorylated motif binds the complex, with specificity that is determined by adjacent upstream residues. Another difference relates to the proline residues in CD28 and CTLA4 that are not present in ICOS. Intriguingly, CTLA4 has a classic (Arg/Lys)-Xaa-Xaa- Pro-Xaa-Xaa-Pro motif for SH3-domain binding (FIG. 1) that is, Lys-Met-Pro-Pro-Thr-Glu-Pro although a putative ligand has not been identified. This might be due to the overlap of this motif with the Tyr-Val-Lys- Met motif. Best-fit computational analysis identifies the motif as a fit for the SH3 domain of IL-2-inducible T-cell kinase (ITK). By contrast, CD28 has two Pro-Xaa- Xaa-Pro motifs, the first of which (Pro-Arg-Arg-Pro) might bind to ITK 42. The second carboxy-terminal Pro- Tyr-Ala-Pro-Pro-Arg motif binds to the SH3 domain of LCK, resulting in activation of the kinase 43, and the SH3 domain of GRB2 (REF. 36). Constitutive GRB2 binding to the same motif has been identified, as has tandem SH2 SH3-domain binding to phosphorylated Tyr-Met-Asn-Met and Pro-Tyr-Ala-Pro motifs Class 1A forms of PI3K are formed by non-discriminate binding between catalytic p110α, p110β or p110δ subunits and various p85α, p50, p85β or p55 adaptors. Whereas p110α and p110β forms are widely expressed, p110δ is preferentially expressed by immune cells. Each isoform has an animo-terminal p85-binding domain, a RAS-binding domain, a C2 domain and a lipid/serine kinase domain. The lipid kinase phosphorylates the 3 hydroxyl position of phosphoinositides (PtdIns) phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ) seems to function as the main in vivo substrate. p110δ-deficient mice show defects in the differentiation and function of T cells 163. Conversely, the loss of phosphatase and tensin homologue (PTEN), a phosphatase that removes the 3 hydroxyl phosphate, leads to lymph-node hyperplasia and autoimmune disease 164. Importantly, D3 lipids anchor to the inner face of the plasma membrane where they bind and recruit various proteins with pleckstrin-homology domains. Certain pleckstrin-homology domains, such as in PtdIns-dependent kinase 1 (PDK1), bind only PtdInsP 3, whereas others, such as in protein kinase B (PKB), bind both PtdIns(3,4)P 2 and PtdInsP 3. The importance of pleckstrin-homology domains in PI3K signalling is most clearly illustrated by mutations in the pleckstrin-homology domain of Bruton s tyrosine kinase (BTK), in which impaired binding of PtdInsP 3 results in the development of X-linked agammaglobulinaemia 165. The adaptor/regulatory p85 subunit has two SRC-homology 2 (SH2) domains, which mediate binding to phosphotyrosine motifs, an SH3 domain and a BCRhomology domain (BH) that is related to a GTP-activating (GAP) domain. This module binds to GTP-bound forms of RAC and CDC42 (REF. 166). In this way, the subunit might generate signals independently of p110. Consistent with this, a CD28 p85 chimeric protein that is unable to bind to p110 can cooperate with RAC1 to activate transcription of the gene encoding IL-2 (REF. 167). Deletion of the BCRhomology domain abrogated this function. Whereas T-cell development is unaffected in p85α-knockout mice (probably due to subunit redundancy), p85αβ deficiency is embryonic lethal 168,169. With these different modes of recruitment, a single co-receptor has the potential to engage several signalling proteins. The presence of a shared Tyr-Xaa-Xaa-Met motif amongst the co-receptors indicates an overlapping function, although differences in other residues might elicit roles that are unique to each receptor. Common co-receptor pathways: PI3K One crucial property that is shared by CD28, ICOS and CTLA4 is the recruitment of class 1A forms of PI3K 19,28 33 (BOX 1). They function as the main receptor-associated reservoirs for the kinase on the surface of T cells. This contrasts with the TCR CD3 complex, which associates with low levels of kinase, possibly mediated by binding of PI3K to the SH3 domain of the SRC-family PTK member FYN, or to the adaptors LAT or TCR-interacting molecule (TRIM) 44,45. Each of these co-receptors lacks an intrinsic kinase for autophosphorylation, and so require an interaction with adjacent PTKs. Candidate kinases include LCK, FYN, TEC and RLK 43, LCK is a promising candidate as it binds to CD4, CD8 and CD28, which places it at the interface of signals 1 and 2. The binding of CD28 to LCK leads to kinase activation and sustained signalling at the immunological synapse 49. Both TCR and CD28 can engage PI3K and produce D3 LIPIDS phosphatidylinositol (3,4)-bisphosphate (PtdIns(3,4)P 2 ) and phosphatidylinositol (3,4,5)- trisphosphate (PtdInsP 3 ). TCR activation is almost certainly required for CD28 phosphorylation and PI3K recruitment, although independent engagement of the co-receptor activates PI3K. The main lipid that is produced as a result of the activation process in T cells is PtdInsP 3. Activation of PI3K occurs within seconds of TCR ligation and results in the localization of PtdInsP 3 at the interface between T cells and APCs, and the consequential recruitment of proteins that have a pleckstrin-homology domain 50,51. As we discuss, the importance of the CD28 PI3K complex in the regulation of T-cell responses is a subject of much debate. Studies with the PI3K inhibitors wortmannin and LY indicate that PI3K is required for TCR CD28-induced production of cytokines and proliferation 52. The requirement for the CD28 PI3K complex is likely to depend on the efficacy of the TCR-mediated induction of PtdInsP 3. Low-affinity peptides would be expected to generate lower levels of PtdInsP 3, and as a consequence be more dependent on CD28-associated PI3K. Not surprisingly, peptide affinity determines the relative requirement for CD28 in T-cell responses. Similarly, the requirement for CD28 is also likely to depend on the engagement of other PI3K-associated co-receptors, such as CTLA4 and ICOS. Each of these co-receptors would be expected to alter the overall levels of phospholipids and affect the threshold of activation. This might explain why the role of the CD28 PI3K complex in cytokine production and proliferation has varied in different studies. Initial studies with mouse T-cell hybridomas indicated a requirement for the binding of PI3K to CD28 for the production of IL-2 that is induced by antibody specific for CD3. This was observed by mutation of the 546 JULY 2003 VOLUME 3

4 Tyr (that is, loss of binding of GRB2, GADS and PI3K) and Met residues (that is, loss of binding of PI3K only) of the Tyr-Xaa-Xaa-Met motif of CD28 (REFS 30,36,53,54). However, concurrent studies with Jurkat T cells found the Tyr-Xaa-Xaa-Met motif to be dispensable 31,32. This discrepancy is probably due to loss of the expression of phosphatase and tensin homologue (PTEN) by Jurkat T cells, which obviates the need for additional lipids provided by the co-receptor 55. In fact, in Jurkat T cells, the overproduction of D3 lipids might even inhibit T-cell function 56. We have also found that expression of constitutively active p110 in these cells inhibits the production of IL-2 (J. Kang and C.E.R., unpublished observations), which underscores the abnormal nature of Jurkat T cells and the difficulty in using them to evaluate the role of PI3K. Similarly, transgenic approaches have produced mixed results. T cells from CD28-deficient mice that express a (Tyr Phe)-Met-Asn-Met mutant CD28 transgene have reduced production of IL-2 in the early to mid phases (0 48 hours) of activation induced by CD3-specific antibody 57. However, at later stages of activation, cytokine levels were not reduced, possibly due to the appearance of other co-receptors with associated PI3K. The same cells showed markedly reduced proliferation and IL-2-dependent graft-versus-host responses. Another study showed that the mutant CD28 transgene in CD28-deficient mice had no effect on the proliferation or production of IL-2 (REF. 58). In another system, retroviral expression of the mutant protein in CD28- deficient TCR-transgenic T cells led to reduced proliferation, which varied with the dose of specific peptide 59. The level of production of IL-2 at a single time point was only marginally reduced. Clearly, these studies indicate that the CD28 PI3K interaction is not obligatory for the production of IL-2. However, most transgenic models have been selected for high-affinity peptide TCR interactions and so might not fully indicate the contribution of CD28-asociated PI3K in normal T-cell responses to moderate- or low-affinity antigens. Further work is, therefore, required in responses where TCRinduced production of PtdInsP 3 is limited that is, with low-to -moderate-affinity peptide antigens. In these circumstances, it is probable that additional phospholipids from CD28 and other co-receptors would be required. As outlined in the next section, one clear pathway would involve PI3K-dependent inactivation of glycogen synthase kinase 3 (GSK3) and its abililty to upregulate the production of IL-2 by retaining NFAT in the nucleus. It is also noteworthy that the (Tyr Phe)-Met-Asn-Met mutation used in these studies does not selectively abrogate PI3K binding. Instead, it might interfere with the binding of any SH2-containing protein with unknown consequences. For example, the binding of GRB2 and GADS is also disrupted. Indeed, the loss of binding of GRB2 (or GADS) alone due to mutation of the Asn residue in the plus two position, which retains the association of PI3K, abrogates CD28-dependent production CD28 TCR/CD3 P PH PKB BAD IκB FOXO mtor RAF PtdInsP 3 PtdInsP 3 PH p85 GRB2 PDK1 PKC PI3K? LCK RSKs SLP76 PH S6K DH VAV GSK3? Cbl-b Nucleus CDC42 RAC1 p110 Actin remodelling PAK NFAT AP1 IL-2 JNK PI3K PDK1 PKB pathway VAV JNK pathway Figure 2 CD28 PI3K PDK1 PKB and CD28 VAV pathways. Binding of phosphatidylinositol 3-kinase (PI3K) to CD28 generates PtdInsP 3 that anchors proteins that contain a pleckstrin-homology domains to the plasma membrane. In one pathway, PI3K is required for activation of phosphoinositide-dependent kinase 1 (PDK1) and protein kinase B (PKB), which in turn regulate many downstream targets that are involved in functions as diverse as protein translation and cellular metabolism 162. Targets include PDK1-mediated activation of S6 kinase (S6K), protein kinase A (PKA) and others, whereas PKB phosphorylates glycogen synthase kinase 3 (GSK3), mtor (the target of rapamycin) and CREB (camp responsive element binding protein 1). PKB-mediated inactivation of GSK3 by phosphorylation should enhance the production of IL-2 by prolonging the nuclear residency of nuclear factor of activated T cells (NFAT). Despite the importance of the CD28 PI3K PDK1 PKB pathway, binding of PI3K alone is insufficient to account for the full effects of CD28. One promising candidate protein is the GTP GDP exchange factor VAV1 that probably connects CD28 with the activation of RAC and cell-division cycle 42 (CDC42) and subsequent activation of MEK kinase 1 (MEKK1) and JUN N-terminal kinase (JNK). JNK phosphorylates and activates the activator protein 1 (AP1) complex (JUN/FOS) a transcription complex that is required for transcription of IL-2. Activation of RAC, CDC42 and PAK also can induce cytoskeleton re-modelling. BAD, BCL2-antagonist of cell death; GRB2, growth-factor receptor-bound protein; IκB, inhibitor of nuclear factor-κb; LCK, lymphocyte-specific protein tyrosine kinase; PAK, p21/cdc42/rac1-activated kinase; PH, pleckstrin-homology domain; SLP76, SH2-domain containing leukocyte protein of 76 kda. NATURE REVIEWS IMMUNOLOGY VOLUME 3 JULY

5 Box 2 Targets of the PDK1 PKB and GSK3 kinases Binding of CD28, inducible co-stimulatory molecule (ICOS) and cytotoxic T lymphocyte antigen 4 (CTLA4) to phosphatidylinositol 3-kinase (PI3K) indicates that they share many downstream pathways. This includes the kinases phosphoinositide-dependent kinase 1 (PDK1), protein kinase B (PKB) and glycogen synthase kinase 3 (GSK3). The serine/threonine kinase PDK1 consists of an amino-terminal kinase domain and a carboxy-terminal pleckstrin-homology domain. Once recruited to the inner face of the membrane by binding of the pleckstrin-homology domain to PtdInsP 3, PDK1 is activated by phosphorylation of the Ser residue at position 241 and by binding to proteins such as protein kinase C (PKC)-related kinase-1/2 (PRK1/PRK2) 162. PDK1 then phosphorylates and activates another serine/threonine kinase, PKB/AKT, by phosphorylation of the Thr308 residue. Although the activation of PDK1 by CD28 has not been reported, CD28 increases the activation of PKB in a PI3K-dependent manner 170, which indirectly points to PDK1 activation. Activation of these kinases implies a connection to a diverse array of downstream pathways and functions. PDK1 activates protein kinase A (PKA), PKC isoforms, S6 kinase (S6K) and others. S6K regulates protein synthesis by phosphorylating the S6 ribosomal complex. Similarly, PKB can regulate apoptotic proteins such as BAD, caspase-9, transcription factors, such as CREB (camp responsive element binding protein 1) and Forkhead, the ataxia telangiectasia mutant (ATM)-related kinase, mtor (the target of rapamycin), which are required for translation, as well as the cell-cycle regulators G1 cyclins and cyclin-dependent protein kinases (CDKs). PKB also regulates the activation status of GSK3. Phosphorylation by PKB leads to its inactivation. Active GSK3 is involved in glycogen and protein synthesis by activating glycogen synthase and the eukaryotic initiation factor 2B (EIF2B). DEATH-INDUCING SIGNALLING COMPLEX (DISC). A complex that is recruited by the death effector molecule FAS (CD95)-associated death domain protein (FADD), which binds to the cytoplasmic tail of CD95. Leads to the activation of caspase-8 and ultimately induces apoptosis. of IL-2 (REF. 36). Instead, mutation of the Met residue in the plus three position of the Tyr-Met-Asn-Met motif causes the loss of PI3K binding without affecting GRB2 binding 36,53,56. Further transgenic studies with this mutant will be important in resolving the role of the CD28 PI3K interaction in cytokine production and proliferation. Although, most work on the function of co-receptorassociated PI3K has focused on CD28, ICOS and CTLA4 recruit PI3K in the same manner as CD28 and can activate the kinase, and as such are connected to many downstream signalling pathways. CD28 and the PI3K PDK1 PKB pathway One important PI3K-dependent pathway involves activation of the serine/threonine kinases phosphoinositidedependent kinase 1 (PDK1), protein kinase B (PKB) and GSK3 (FIG. 2 and BOX 2). Briefly, each has a pleckstrin-homology domain that binds to PtdInsP 3 at the inner face of the plasma membrane leading to the activation of PDK1, followed by its activation of PKB, which in turn inactivates GSK3. PDK1 phosphorylates various proteins, such as protein kinase C (PKC) and S6 kinase (S6K), whereas the targets of PKB include proapoptotic molecules, transcription factors and cell-cycle regulators (FIG. 2 and BOX 2). This PI3K-dependent pathway implicates CD28, ICOS and CTLA4 in the regulation of numerous events from protein synthesis to cellular metabolism. Apoptosis is one clear example. In contrast to cytokine production, the induction of apoptosis depends on the interaction between CD28 and PI3K. The threshold levels of D3 lipids required for cytokine production and apoptosis might therefore differ. The anti-apoptotic activity of PI3K was first shown by Cooper and colleagues 60, who showed that binding of PI3K to the nerve growth-factor receptor (NGFR) was essential to rescue cells from apoptosis. Similarly, T cells that express the (Tyr Phe)-Met-Asn-Met mutant CD28 show a clear defect in the rescue of cells from death that is normally mediated by B-cell lymphoma protein 2 (BCL2)-family members 58,59. This might be accomplished by increasing the level of BCL-X L and altering the composition of the FAS (CD95)-associated DEATH-INDUCING SIGNALLING COMPLEX (DISC). T cells that express active PKB have reduced activation of caspase-8, the pro-apoptotic protein BID (BH3-interacting domain death agonist) and caspase-3 due to the impaired recruitment of pro-caspase-8 to the DISC 61. PKB-mediated inactivation of the pro-apopotic transcription factors of the FOXO family is also likely to prevent cell death through an alternative pathway. However, despite the importance of apoptosis, it alone cannot account for the phenotype of CD28-deficient mice, as overexpression of BCL-X L in these mice does not re-constitute a normal phenotype 62. Another important CD28 PI3K PKB connection involves the regulation of cellular metabolism. Increased cellular glycolysis is required to provide energy for the activation and transition from a resting T cell to an effector T cell. An exciting study from Thompson and colleagues 63 showed that the increase in glucose transport owing to upregulation of expression of the Glut4 receptor and glycolysis that was dependent on CD28. Furthermore, these events are PI3K dependent, and constitutively active PKB could increase glycolysis. Although blockade of CD28-mediated upregulation of glycolysis by a dominant-negative form of PKB was not shown, this study strongly argues that the CD28 PI3K interaction influences glycolysis through the regulation of PKB. Finally, another important connection involves CD28-mediated inactivation of GSK3 (REFS 64,65) (H.S. and C.E.R., unpublished observations). Unlike most kinases, GSK3 is active in resting cells, in which it targets various substrates including glycogen synthase, JUN, c-myc, eukaryotic translation initiation factor 2B (EIF2B) and NFAT (BOX 2). Phosphorylation of NFAT promotes its exit from the nucleus, thereby attenuating transcription 66. PKB-mediated phosphorylation of GSK3 on Ser residues (residue 9 for GSK3β and residue 21 for GSK3α) inactivates the enzyme. Therefore, inactivation of GSK3 by the CD28 PI3K PKB pathway would be expected to prolong the residency of NFAT in the nucleus and enhance transcription of the gene encoding IL-2. Although it is yet to be established, this pathway might account for the ability of constitutively active PKB to enhance the production of IL-2 (REF. 67). Overall, the shared ability of CD28, ICOS and CTLA4 to recruit PI3K through classic SH2-domain binding connects these co-receptors to the regulation of multiple shared pathways. More than fifty proteins with a pleckstrin-homology domain are expressed by T cells. PDK1 and PKB influence events as diverse as protein synthesis and cellular metabolism. The issue of whether these events are regulated by co-receptors is just beginning to be explored. Nevertheless, the regulation of events such as protein synthesis would be expected to 548 JULY 2003 VOLUME 3

6 CARD CARD CARD REVIEWS LIPID RAFTS Micro-aggregates of cholesteroland sphingomyelin-enriched microdomains that are thought to occur in the plasma membrane. Also described as glycolipid-enriched microdomains (GEMs) or detergent-insoluble glycosphingolipid-enriched membrane microdomains (DIGs). They function as platforms that compartmentalize crucial components that are involved in signalling. PtdInsP 3 PH DH VAV CARMA 1 GUK Coiled-coil PDZ SH3 CARD DD Ig Ig Casp-like MALT1 BCL10 DD Ig Ig Casp-like affect most functions in T cells. Ultimately, this would be predicted to depend on the efficacy of TCR signalling and the production of D3 lipids. The one paradox and caveat concerns CTLA4, where the presence of an additional inhibitory molecule might limit signalling through this pathway. However, even in this example, the co-receptor retains an ability to induce D3 lipids and the PKB GSK3 pathway (H.S. and C.E.R., unpublished observations). Engagement of co-receptors would be expected to complement and/or substitute for each other at different stages of T-cell activation. CD28 and the VAV MEKK1 JNK pathway Despite these important connections, the CD28 PI3K interaction is insufficient to account for the full costimulatory effect of CD28. This is evident from the fact that other PI3K-binding receptors, such as CD7, CD19 and CTLA4, cannot fully substitute for CD28 (REFS 33,68), and CD28 can co-stimulate Jurkat T cells with an abundance of D3 lipids 31,32,69. The nature of this additional component is still a mystery, and a subject of great interest. Candidate contributors to this signal include LCK, ITK, docking protein 1 (DOK1) CD28 PI3K DD Ig Ig Casp-like p85 p110 MAPKKKs (MEKK1, COT etc) IKKγ IKKα IKKβ IKK Complex Nucleus PH PDK1?? Ub NF-κB PtdInsP 3 P P PH PKB Proteasomal degradation IκB NF-κB CBP/p300 Figure 3 CD28 and the regulation of NF-κB Pathway. In addition to nuclear factor of activated T cells (NFAT) and activator protein 1 (AP1), nuclear factor-κb (NF-κB) has a crucial role in the regulation of gene transcription of the IL-2 promoter and anti-apoptotic factors. The two CD28-responsive elements in the IL-2 promoter are NF-κB binding sites. CD28 interfaces with this pathway by both enhancing the degradation of inhibitors of NF-κB (IκBs) and by increasing the activity of IκB kinase (IKK). NF-κB dimers are normally retained in cytoplasm by binding to inhibitory IκBs. Phosphorylation of IκB initiates its ubiquitination and degradation, thereby freeing dimers to translocate to the nucleus. Phosphorylation is mediated by IKK. CD28 impinges on the pathway through protein kinase B (PKB) and members of the MAPKKK family (MEKK1 and COT, for example) that phosphorylate and activate IKKs. Dominant-negative forms of MEKK1 and NF-κB-inducing kinase (NIK) interfere with CD28-mediated activation of IKKs. In another new pathway, membrane-associated guanylate kinase (MAGUK)-family members function between CD28 and the activation of IKKs. CARMAI interacts with BCL10, which in turn dimerizes mucosaassociated lymphoid tissue protein 1 (MALT1), and event that is sufficient for the activation of IKK. GUK, guanylate kinase; PH, pleckstrin-homology domain. NIK and VAV 42,43. Activation of LCK by binding to CD28 (REF. 43), and its upregulation of PI3K binding, support a role in CD28-mediated co-stimulation 46.However, data are lacking on whether LCK (or CD4/CD8 LCK interactions) can substitute for the complete array of CD28 signals. By contrast, T cells that are deficient in ITK are actually hyperresponsive to antigen, indicating that ITK might have a negative role in co-stimulation 70. Similarly, DOK1, which is phosphorylated after ligation of CD28, is generally associated with negative signalling 71. In this light, the most compelling candidate, so far, is VAV1 (FIG. 2). The DBL-homology domain of this protein has GDP GTP-exchange activity for activation of the GTP-binding proteins RAC1 and cell-division cycle 42 (CDC42) 72,73. A relationship between VAV and PI3K has been shown, with PI3K positioned upstream of VAV (possibly through recruitment to the membrane) or with VAV upstream of PI3K by means of RAC binding to the GTP-activating (GAP) domain 74,75. CD28 can enhance the phosphorylation of VAV, whereas low levels of overexpression of VAV and the adaptor SLP76 can convert CD28 into a receptor that can induce the production of IL-2 in primary T cells 69. This occurs independently of TCR ligation and might account for the trans function of the co-receptor. SLP76 binds to VAV and, as such, might facilitate the binding of VAV to the plasma membrane and possibly influence its GDP GTP-exchange activity. In addition, VAV alone could promote the CD28-mediated induction of IL-2 when expressed at higher levels 76. Moreover, CD28, VAV and SLP76 were shown to cooperate with RAC1 to remodel the actin cytoskeleton, providing a pathway for CD80 CD28-induced cytoskeletal rearrangements 69,77. Furthermore, T cells that are deficient in Cbl-b an E3 ligase of the ubiquitination pathway show a correlation between enhanced phosphorylation of VAV and increased levels of CD28-mediated proliferation, resulting in autoimmunity 78,79. Another reason for implicating VAV in CD28 signalling involves the regulatory effect that both proteins have on the activation of JUN N-terminal kinase (JNK) (FIG. 2). CD28 provides an essential signal that cooperates with the TCR to activate JNK 80 (FIG. 2). Similarly, VAV can activate RAC and CDC42, which in turn activate a cascade that leads from MEK kinase 1 (MEKK1) to JNK through MEK4 and MEK7 (FIG. 2). CD28 can also induce the formation of GTP RAC 81, whereas VAV mutants that lack GTP GDP-exchange activity can block CD28-mediated activation of JNK 82,83. Similarly, CD28 can activate MEKK1, whereas a dominant-negative mutant MEKK1 can block the activation of JNK 77,82,83.PKC-θ might also be involved in the CD28 VAV JNK pathway. VAV is required for the translocation of PKC to the membrane 84, and the association of PKC-θ with LIPID RAFTS is needed for T-cell activation 85. A dominant-negative mutant PKC-θ can block the activation of JNK in Jurkat T cells 83,86, although this connection might be unique to Jurkat T cells as activation of JNK by CD28 is unaffected in PKC-θ-deficient mice 87. NATURE REVIEWS IMMUNOLOGY VOLUME 3 JULY

7 MEMBRANE-ASSOCIATED GUANYLATE KINASE FAMILY MAGUK proteins are defined by a basic core of three domains: a SRC-homology 3 (SH3) domain, a domain with homology to the enzyme guanylate kinase (GUK) and a PDZ domain. They are required for several types of cell-junction formation, contribute to cell shape and seem to hold together elements of individual signalling pathways. CD40L T H 2 unique pathway? + ICOS Increased IL-10, IL-4 and IFN-γ B7H p85 The identity of the proximal interaction that connects CD28 to VAV and JNK is an area of active interest. The loss of GRB2 binding by mutation of the Asn residue in the Tyr-Met-Asn-Met motif results in the loss of TCR-induced phosphorylation of VAV and activation of JNK, leading to reduced IL-2 production 36. Consistent with a role for GRB2 JNK activation, ICOS, which lacks an Asn residue in its Tyr-Met-Phe-Met motif, activates JNK poorly 88. Indeed, GRB2 binds to VAV through SH3 SH3 domain interactions 89. CTLA4, which also lacks the crucial Asn residue in its Tyr-Val- Lys-Met motif, can cooperate with TCR signals in the activation of JNK in certain cells, albeit less efficiently 90. This indicates the presence of an alternative pathway of JNK activation, possibly involving PI3K. Ultimately, activated JNK phosphorylates transcription factors such as JUN, thereby activating the AP1 complex (JUN FOS) that leads to transcription of the gene encoding IL-2. AP1 cooperates with NFAT in the transcriptional activation of the IL-2 promoter. Overall, increasing evidence points to a special relationship between CD28 and VAV in the regulation of cytokine transcription, at least in part owing to its activation of JNK and the AP1 complex. Effects on the cytoskeleton have also been noted. Present studies indicate that this is associated with the presence of the unique Asn residue in the Tyr-Met-Asn-Met motif a residue that is not found in the other co-receptors. This p110 PDK1 PKB PI3K Figure 4 ICOS signalling pathways. Although less is understood about the mechanism of inducible co-stimulatory molecule (ICOS) signalling, the co-receptor shares with CD28 an ability to bind phosphatidylinositol 3-kinase (PI3K) and activate the kinases phosphoinositide-dependent kinase 1 (PDK1) and protein kinase B (PKB). Binding of PI3K implicates ICOS in the activation of the PDK1 PKB GSK3 (glycogen synthase kinase 3) pathway, and an involvement in events such as protein translation, prevention of cell death and the upregulation of cellular metabolism (see FIG. 2 and BOX 2). Its ineffective upregulation of IL2 transcription might be due to its inability to engage growth-factor receptor-bound protein 2 (GRB2), although in secondary responses, it might, nonetheless, provide CD28 substitute signals in the regulation of T-cell function. A remaining question is whether ICOS can provide a unique set of signals that are linked to the function of T helper 2 (T H 2) cells. CD40L, CD40 ligand; IL, interleukin; IFN-γ, interferon-γ.? possibly links GRB2 (or GADS) to VAV, the GTP-binding proteins RAC and CDC42, and their activation of JNK. However, the issue of low stoichiometric binding between CD28 and GRB2 needs to be resolved before a clear causal link can be made between CD28, VAV and JNK. Overall, CD28-mediated induction of JNK activity would fit well with the role of this serine/threonine kinase in increasing the stability of messenger RNA for IL-2 a crucial factor in determining whether T cells become anergic or activated. Prevention of anergy is one of the unique functions that is regulated by CD28. CD28 and NF-κB regulation In addition to NFAT, CD28 regulates the nuclear factor-κb family 91 (FIG. 3). The IL-2 promoter has two NF-κB-binding sites that depend on CD28 co-stimulatory signals that is, CD28-responsive elements (CD28REs) In addition, NF-κB has a crucial role in the regulation of apoptosis by controlling the expression of various anti-apoptotic factors, such as BCL-X L. NF-κB-mediated regulation involves phosphorylation by IκB kinase (IKK) a complex that is composed of two kinase subunits, IKKα and IKKβ and a non-catalytic subunit NEMO/IKKγ 91. Phosphorylation of IκB targets it for degradation, allowing NF-κB to enter the nucleus. Mice that lack NF-κB subunits have defective immune responses 96,97. Importantly, CD28 enhances the activity of IKK and the degradation of IκBs TCR CD28 coactivation is impaired in IKK-deficient cells 100, whereas disrupted CD28 binding abrogates the activation of IKK 101. Although ZAP70 and SLP76 that is, TCRspecific signals are required for the activation of NF-κB, there are specific connections of CD28 to NF-κB that involve PKB, MEKK1 and members of the MEMBRANE- ASSOCIATED GUANYLATE KINASE (MAGUK) FAMILY. A CD28 PI3K connection to the activation of NF-κB has been observed, with PKB-mediated activation of IKK in response to TCR ligation 102. Similarly, although the connection with PKB is unclear, MAPKKK family members (MEKK1, MEKK2 and MLK3) also activate IKKs 103,104 (FIG. 2) and CD28 can activate MEKK1 (REF. 101) (FIG. 3). Indeed, a dominant-negative MEKK1 mutant interferes with activation of the CD28 IKK pathway 101. The related kinases COT and NF-κB-inducing kinase (NIK) might be involved 104, as partial blockage of CD28- induced activation of IKK by a dominant-negative NIK mutant 101 and NIK deficiency in T cells impairs IL-2 production 105. Overall, dissection of the important kinases that are located upstream of IKK will be complicated by the number of related and redundant kinases with a conserved ability to phosphorylate the complex. By contrast, recent exciting data have implicated members of the MAGUK family in CD28-mediated regulation of NF-κB. MAGUKs have a distinctive structure that includes a PDZ domain and an SH3 domain followed by a region that is homologous to cytosolic guanylate kinase (GUK) (FIG. 3). They anchor to receptors through their PDZ domains and the remaining domains mediate additional protein protein interactions 106. Caspase-recruitment domain family members CARMA1 and related CARD11 are of particular interest due to their 550 JULY 2003 VOLUME 3

8 MUCOSA-ASSOCIATED LYMPHOID TISSUE PROTEIN 1 (MALT1). A protein that was first indentified as a casual factor in the development of MALT lymphomas, owing to fusion of the MALT1 gene with the apoptosis inhibitor 2 (API2) gene. CTLA4 p85 PI3K p110 PDK1 PKB PtdInsP 3 + preferential expression by lymphocytes BIMP1 (BCL10-interacting MAGUK protein) has also been studied 108. Both have caspase-recruitment domains (CARDs) that dimerize with other CARD-containing proteins, in particular, BCL10 (REF. 106). Notably, CARMA1-deficient and BCL10-deficient T cells have defects in TCR CD28-induced activation of IKK and induction of NF-κB 109,111. Specificity for CARMA1 and BCL10 in TCR CD28 signalling is evident from the fact that activation of NF-κB by tumour-necrosis factor (TNF) and Toll-like receptor (TLR) pathways is unaffected in CARMA1- and BCL10-deficient cells. Dominant-negative BIMP1 mutants that lack a CARD can also block activation of IKK 108. The observation that expression of BIMP1 in BCL10-deficient embyronic fibroblasts fails to activate NF-κB indicates that BIMP1 and CARMA1 are positioned upstream of BCL10 (REF. 104). Further downstream, BCL10 can induce the oligomerization of MUCOSA-ASSOCIATED LYMPHOID TISSUE PROTEIN 1 (MALT1) through binding to its immunoglobulin-like domains 112 and, importantly, this seems sufficient for activation of NF-κB 112. PKC-family members are thought to function with CARMA1, as NF-κB activation is impaired in PKC-θ-deficient T cells 87. In fact, CARMA1 and PKCθ might bind each other (X. Lin, personnal communication). This pathway might operate differently to the activation of NF-κB mediated through COT and NIK 113. Other components that are involved might include CARD14 (CARMA2; human orthologue of BIMP2) 114 and the CARD-containing protein receptorinteracting protein 2 (RIP2 also known as CARDIAK or RICK) 115. RIP2-deficient mice respond poorly to stimulation through TCR and CD28 (REF. 115). AP2 Endocytosis AP1 PP2A SHP2 Lysosomal degradation TCR/CD3 Figure 5 CTLA4 signalling pathways. Cytotoxic T lymphocyte antigen 4 (CTLA4) binds to phosphatidylinositol 3-kinase (PI3K), the tyrosine phosphatase SHP2 and the serine/threonine phosphatase PP2A. SHP2 has been postulated to dephosphorylate T-cell receptor (TCR) signalling proteins, whereas PP2A targets phospho-serine/threonine residues and is known to interfere with the activation of protein kinase B (PKB). Binding to PI3K indicates that the co-receptor can generate positive signals in common with CD28 and inducible co-stimulatory molecule (ICOS), but in the context of an additional negative-signalling protein, such as PP2A and SHP2 that would be dominant by interfering with T-cell function. Finally, CTLA4 is unique in binding to the clathrin adaptor complexes activator protein 2 (AP2) and AP1 through its nonphosphorylated Tyr-Val-Lys- Met motif. AP2 regulates endocytosis of CTLA4, whereas AP1 controls the amount of intracellular CTLA4. The rapid endocytosis that is controlled by AP2 ensures that the cell-surface expression of CTLA4 is tightly regulated, and most resides in intracellular compartments. Whether this rapid endocytosis can disrupt the arrangement of other receptors on the cell surface of T cells that leads to an inhibition of signalling has yet to be explored. PDK1, phosphoinositide-dependent kinase 1. ICOS signalling: a case of restricted redundancy? Unlike CD28, ICOS is only expressed by activated cells and has been implicated in the regulation of various functions, particularly those of T H 2 cells 19,20. Although CD28 is required for the primary activation of naive T H 2 cells, this co-receptor has little, if any, role in secondary stimulation when ICOS is expressed. This indicates that ligation of ICOS by B7H can provide signals that substitute for CD28. This model also fits the finding that ICOS can bind to PI3K 19 and activate PDK1 and PKB 88 (FIG. 4). CD28 and ICOS, therefore, share a common pathway. Ligation of ICOS by B7H might occur in the absence of CD80 and CD86, and so provide the minimal CD28-like signals that are required for costimulation. This would allow the clonal expansion of T H 2 cells without concurrent activation of T H 1 cells through CD28 co-stimulation. ICOS might also operate together with CD28 to alter the threshold of signalling. Conversely, although ICOS and CD28 enhance the production of interferon-γ (IFN-γ), IL-4 and IL-10, ICOS only marginally induces IL-2 production 88. At the molecular level, ICOS can be distinguished from CD28 due to the absence of an Asn residue in the Tyr-Met-Phe-Met motif (FIG. 1). Absence of the Asn residue and an inability to bind to GRB2 correlates with an inability of ICOS to induce the production of IL-2 (REF. 116). An add-back substitution of Asn for Phe in the Tyr-Met-Phe-Met motif can enhance the induction of IL-2. This supports previous studies on CD28 that implicate the Asn residue in GRB2 binding, VAV phosphorylation, JNK activation and IL-2 production 36. Similarly, ligation of ICOS is less efficient than ligation of CD28 in the activation of JNK 88.The absence of the Asn residue might, therefore, contribute to the ineffective induction of IL-2. Furthermore, ICOS lacks Pro residues that are found in the cytoplasmic domain of CD28 and that interact with the SH3 domains in LCK and GRB2 (FIG. 1). In this way, present information indicates that ICOS is more limited in its generation of signals but, nevertheless, retains an ability to engage PI3K and its targets PDK1, PKB and GSK3. From this, ICOS would be predicted to have general effects on protein translation, cellular metabolism and apoptosis, as previously described for CD28. Consistent with the theme of limited redundancy, a comparison of CD28 and ICOS co-stimulation has shown that ICOS induces expression of fewer genes than does CD28 (REF. 117). For example, the CD28- inducible genes encoding IL-2, IL-9 and 3-methylaspartase were not induced by ICOS 117.However, surprisingly, a few genes, such as those encoding an unconventional myosin (MYL1) and T-lymphocyte maturation-associated protein (MAL), were induced by ICOS alone. MAL is a component of the protein machinery that is involved in apical transport in epithelial polarized cells, and it is linked to induction of the CD95 CD95L pathway. Whether ICOS can induce a unique set of signals that are linked to the unique biochemistry of T H 2 cells is still uncertain. CD28 shares with ICOS an ability to amplify the production of T H 2 cytokines 118, which indicates that NATURE REVIEWS IMMUNOLOGY VOLUME 3 JULY

9 Protein X CTLA4 PP2A SHP2 TCR/ CD3 LAT Raft CD28 Figure 6 Lipid rafts: integration of positive and negative co-signals. CD28 and cytotoxic T lymphocyte antigen 4 (CTLA4) target the cell-surface expression of membrane rafts in the modulation of T-cell function. CD28 enhances the release of membrane rafts from intracellular stores to the cell surface, whereas CTLA4 potently inhibits cell-surface raft expression that is induced by either ligation of T-cell receptors (TCRs) or combined TCR and CD28 ligation. In this model, the yin and yang of CD28 (positive) and CTLA4 (negative) signalling would operate by enhancing or restricting the release of intracellular rafts to the cell surface and so modulate the availability of crucial signal mediators that are required for efficient TCR signalling. The importance of raft modulation by CD28 and CTLA4 presumably operates during the formation of T-cell antigenpresenting cell (APC) conjugates that lead to the development of the immunological synapse. By modulating overall raft expression, CD28 and CTLA4 would be expected to modulate interaction with the TCR in the T-cell contact region. LAT, linker for activation of T cells; PP2A protein phosphatase 2A; SH2- domain-containing protein tyrosine phosphatase 2. their shared activation of the PI3K PDK1 PKB pathway might regulate these T H 2 cytokines. However, surprisingly, overexpression of constitutively active PKB has been reported to have no effect on the production of T H 2 cytokines 67. PKB signals alone, elicited by CD28 or ICOS might, therefore, be insufficient to upregulate expression of IL-4. Instead, this indicates the possible existence of a unique pathway that is linked to ICOS. Mechanisms of CTLA4 function In contrast to CD28 and ICOS, CTLA4 negatively regulates many aspects of T-cell function 12,119.Several mechanisms have been proposed. These include ligand competition between CTLA4 and CD28, interference of TCR and/or CD28 signalling, as well as an effect on the formation of lipid rafts. The competition model. High-avidity binding of CTLA4 to CD80 and CD86 would be expected to compete for binding of CD28 in the limited space between T cells and APCs. Supporting this, expression of a mutant form of CTLA4 lacking a cytoplasmic tail in CTLA4-deficient mice can inhibit lymphocytic infiltration of organs 120. However, the same mutant did not prevent lymphoproliferaton, indicating that intracellular signalling can still contribute to the disease phenotype. Furthermore, CTLA4 can inhibit T-cell responses in the absence of CD28, when competition for ligand would not occur 121,122. In addition, CTLA4 function is not entirely cell autonomous, as shown in CTLA4-deficient mice, in which reconstitution with a combination of deficient and normal T cells failed to lead to lymphoproliferative disease Interference with TCR CD28 signals. The signalling mechanism that accounts for negative signalling is not clear. CTLA4 lacks a classic inhibitory immunoreceptor tyrosine-based inhibitory motif (ITIM) that is found in other inhibitory receptors. Instead, as outlined in FIG. 1, it has several shared and unique properties relative to CD28 and ICOS. The Tyr-Val-Lys-Met motif seems to be important given that swapping the Ser-Asp-Tyr- Met-Asn-Met motif present in CD28 for the Gly-Val- Tyr-Lys-Met motif can convert CTLA4 into a positive co-stimulatory receptor 124. Members of the TEC-kinase family and FYN have been reported to phosphorylate the motif 47,48. Furthermore, the region has the potential to bind to two phosphatases, SH2-domain-containing protein tyrosine phosphatase 2 (SHP2) and the serinethreonine phosphatase PP2A In both cases, the proposed model is analogous to that of killer inhibitory receptors (KIRs) that mediate signalling through an intracellular phosphatase. Indeed, expression of a KIR transgene in CTLA4-deficient mice partially restores a normal phenotype 128. Binding of SHP2 to CTLA4 depends on the Tyr-Val-Lys-Met motif 125,126, although it is likely to occur indirectly, possibly by binding to a bridging protein 129 (FIG. 5). By contrast, PP2A has been reported to require either the Tyr-Val-Lys-Met motif 127 or the membrane-proximal Lys residues of CTLA4 for binding 130. Both of the associated phosphatases dephosphorylate components that are required for signalling. The main difference is that SHP2 targets phosphotyrosine residues, whereas PP2A dephosphorylates serine and threonine residues. Supporting this, co-ligation of CTLA4 can reduce the phosphorylation of LAT 126, whereas various TCR signalling proteins are hyperphosphorylated in CTLA4-deficient mice 125,126. In the latter case, whether this is due to the absence of CTLA4- associated phosphatases or an unrelated consequence of hyperactivation is unclear. Furthermore, SHP2 has also been reported to associate with CD28, and has generally been connected to positive signalling 131. Disruption of the Tyr-Val-Lys-Met motif also has a null or weak effect on the function of CTLA4 (REFS 130, ), whereas in naive CD8 + T cells, CTLA4 is not inhibitory, even when associated with SHP2 (REF. 135). In this context, PP2A seems to be an attractive candidate for mediating CTLA4 function as it can inhibit the activity of PKB, and has well-established negative effects on differentiation and cell growth 136. This, in turn, also fits with the inhibitory effects of CTLA4 on PKB-mediated potentiation of glucose metabolism 63. By contrast, PP2A has also been found to associate with CD28, requiring that the phosphatase dissociates from CD28 during the activation process. Binding of PP2A to the Tyr-Val-Lys-Met motif also needs to be reconciled with the effects on T-cell inhibition of a mutant in which the Lys residues are substituted that is, substitution of Lys152, Lys155 and Lys156 for Ala. More than in the case of the Tyr mutation of the Tyr-Val-Lys-Met site, the Lys-less mutant had an attenuating effect on negative signalling 137,138. Whether this effect is directly related to the loss of binding of PP2A or another event still needs to be determined. 552 JULY 2003 VOLUME 3