TREIMM-1014; No.

of Pages 13

Review

A new look at T cell receptor signaling to nuclear factor-kB

Suman Paul1 and Brian C. Schaefer1,2
1 2

Department of Microbiology and Immunology, Uniformed Services University, Bethesda, MD 20814, USA Center for Neuroscience and Regenerative Medicine, Uniformed Services University, Bethesda, MD 20814, USA

Antigen stimulation of T cell receptor (TCR) signaling to nuclear factor (NF)-kB is required for T cell proliferation and differentiation of effector cells. The TCR-to-NF-kB pathway is generally viewed as a linear sequence of events in which TCR engagement triggers a cytoplasmic cascade of proteinprotein interactions and posttranslational modications, ultimately culminating in the nuclear translocation of NF-kB. However, recent ndings suggest a more complex picture in which distinct signalosomes, previously unrecognized proteins, and newly identied regulatory mechanisms play key roles in signal transmission. In this review, we evaluate recent data and suggest areas of future emphasis in the study of this important pathway. The current consensus model of TCR signaling to NF-kB In the past decade, much progress has been made in dening molecular mechanisms by which the TCR (see Glossary) activates the NF-kB transcription factor. Most of the key mediators in this cascade are now dened, and many key signal transmission mechanisms have been elucidated [14] (Figures 1 and 2). The general consensus understanding is that engagement of the TCR by major histocompatibility complex (MHC) plus antigen initiates downstream CD3 immunotyrosine activation motif (ITAM) phosphorylation by the Src family kinases, FYN and leukocyte C-terminal src kinase (LCK). Phosphorylated CD3 activates the T cell specic tyrosine kinase, zeta-chain associated protein kinase (ZAP-70), which phosphorylates the adapter proteins linker for activation of T cells (LAT) and SH2 domain containing leukocyte protein of 76 kDa (SLP-76), causing SLP-76 to bind to VAV1. The VAV1SLP76IL-2-inducible T cell kinase (ITK) complex activates phospholipase (PL)Cg1, generating inositol 1,4,5-tripohosphate (IP3) and diacylglycerol (DAG), which ultimately trigger calcium release and protein kinase (PK)C activation, respectively. Activation of a specic PKC isoform, PKCu, connects the above described TCR proximal signaling events to distal events that ultimately lead to NF-kB activation. Importantly, PKCu activation is also driven by engagement of the T cell co-stimulatory receptor CD28 by B7 ligands on antigenpresenting cells (APCs). This molecular interaction activates phosphoinositide 3-kinase (PI3K), inducing
Corresponding author: Schaefer, B.C. (Brian.Schaefer@usuhs.edu). 1471-4906/$ see front matter . Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.it.2013.02.002

recruitment of phosphoinositide-dependent kinase (PDK1) and AKT to the plasma membrane. At the immune synapse (IS), PDK1 phosphorylates and activates PKCu. PKCu-mediated phosphorylation of caspase recruitment domain (CARD)-containing MAGUK protein (CARMA1)

Glossary
CD28: a cell surface receptor providing a co-stimulatory signal required in conjunction with TCR signaling for productive activation of na ve T cells. Effector T cells: short-lived differentiated T cells with immunological activities that can include cytokine production and cytolytic activity. Activation requirements are less stringent than for na ve T cells, requiring shorter TCR engagement ($1 h) and no co-stimulation. IKK: IkB kinase. A trimeric protein complex composed of two catalytic subunits, IKKa and IKKb, and a homodimer of the regulatory subunit, IKKg/NF-kB essential modier (NEMO). The canonical NF-kB activation pathway converges on IKK activation, resulting in phosphorylation and subsequent degradation of IkB, the protein responsible for NF-kB cytosolic sequestration. IS (immunological synapse)/SMAC (supramolecular activation cluster): microclusters fuse to form the micrometer sized IS between the APC and T cell. The IS is composed of concentric rings of signaling molecules: in general, c-SMAC contains the TCR and signaling partners, and the peripheral SMAC (p-SMAC) contains molecules primarily involved in T cellAPC adhesion. ITAM: immunotyrosine activation motif. Conserved amino acid sequence containing tyrosine residues located in cytosolic regions of certain cell surface receptors, including CD3 chains of T cells. Phosphorylated tyrosines serve as binding site for downstream signaling proteins. K48- vs K63-polyubiquitination: distinct polyubiquitin chain conjugations in which polymeric linkages are via lysine 48 or lysine 63, respectively. K48-polyubiquitinated proteins are generally degraded by the proteasome. The consequences of K63-polyubiquitination are more diverse, ranging from modication of protein function to targeting proteins for degradation by the autophagy-lysosomal pathway. Memory T cells: long-lived differentiated T cells that persist in a resting state until re-encounter with specic antigen. Activating requirements are similar or identical to effector cells. upon restimulation, memory cells proliferate and differentiate into secondary effector and memory populations. Memory cells may be directly derived from stimulated na ve cells (divergent development model), or surviving effector cells (linear development model) (reviewed in [109,110]) Microclusters: nanometer-sized clusters of ligand-engaged TCR that initiate signal transduction. The cytoplasmic face of microclusters is enriched in specic signaling molecules and phosphotyrosine. Na ve T cells: T cells that have never encountered antigen. Entry into cell cycle and concomitant effector/memory differentiation requires sustained stimulation from hours to days by engagement of TCR and CD28 molecules. NF-kB: nuclear factor-kB. A ve-member family of transcription factors that exist in homo- or heterodimers. Members are RELA, RELB, cREL, p100/p52 and p105/ p50. NF-kB is generally used to refer to the RELAp50 activating heterodimer. In unstimulated T cells, NF-kB is sequestered in the cytosol by its binding partner IkB. IkB is phosphorylated by IKK, triggering proteasomal degradation, freeing NF-kB to translocate to the nucleus and drive transcription of target genes. Signalosome: an organized cluster of proteins that coordinately controls cell signal transmission and regulation. T cell receptor (TCR) complex: heteromeric complex comprised of the T cell receptor a/b heterodimer and CD3 complex. TCR recognizes peptide antigens presented by MHC proteins, whereas the CD3 chains are the signal-transducing subunits.

Trends in Immunology xx (2012) 113

TREIMM-1014; No. of Pages 13

Review
TCR CD28

Trends in Immunology xxx xxxx, Vol. xxx, No. x

Ca2
Calmodulin

SLP-76

PI3K

TCR-dependent PKC acvaon CBM complex formaon TAK1 recruitment CBM complex formaon CARMA1 phosphorylaon CARMA1 phosphorylaon CARMA1 and BCL10 phosphorylaon BCL10 dephosphorylaon IKK recruitment and ubiquinaon

GLK

PDK1

CD28-dependent PKC acvaon

ADAP CK1 HPK1

CaMKII

PKC

BCL10

T cell adhesion JNK acvaon

CARMA1
CYLD

BCL10 MIB2 MALT1 TRAF6

A20 RELB

NF-B acvaon

Calcineurin

Caspase 8

IKK recruitment cFLIP cleavage

IKK IKK IKK Acn regulaon

Homer3

FLIP P TRAF2

cFLIP

IKK

IKK ubiquinaon

IB NF-B NF-B

TAK1

Key:
Acvaon signal Calcium-mediated signal MALT1-mediated acons P Acvang phosphorylaon U K63-polyubiquinaon
TRENDS in Immunology

NF-B

Gene transcripon

Figure 1. New developments in the T cell receptor (TCR)-to-nuclear factor (NF)-kB signaling pathway. The TCR transmits signals through the linker for activation of T cells (LAT)SH2 domain containing leukocyte protein of 76 kDa (SLP76) complex, and possibly through glucokinase (GCK)-like kinase (GLK) to activate protein kinase (PK)Cu. CD28 signals through phosphoinositide 3-kinase (PI3K) and phosphoinositide-dependent kinase (PDK1) to activate PKCu. Activated PKCu induces formation of the CARDcontaining MAGUK protein (CARMA1), B cell leukemia/lymphoma (BCL10), mucosa-associated lymphoid tissue lymphoma translocation protein (MALT1) (CBM) complex. Newly identified proteins acting on CARMA1 and BCL10 are shown on the left. MALT1 enzymatic protein cleaving functions and caspase-8 recruitment are shown on the right. The CBM complex transmits activating signals to IkB kinase (IKK) through the ubiquitin ligases TNF receptor-associated factor (TRAF6), TRAF2 and/or mind bomb-2 (MIB2). IKK phosphorylates inhibitor of kB (IkBa) leading to IkBa ubiquitination and degradation, allowing NF-kB nuclear translocation and gene transcription.

triggers a conformational change, causing CARMA1 to bind to B cell leukemia/lymphoma (BCL10) and mucosaassociated lymphoid tissue lymphoma translocation protein (MALT1), forming the CARMA1-BCL10-MALT1 (CBM) complex. Through a mechanism that may involve TNF receptor-associated factor (TRAF6), both BCL10 and MALT1 become polyubiquitinated. The IkB kinase (IKK) complex is then recruited to the CBM complex via the IKKg polyubiquitin binding motif. This association leads
2

to polyubiquitination of IKKg and phosphorylation of IKKb by TGF-b activated kinase (TAK1), activating IKKb. IKKb then phosphorylates inhibitor of kB (IkBa), triggering its proteasomal degradation, enabling nuclear translocation of canonical NF-kB heterodimers comprised of p65 reticuloendotheliosis viral oncogene homolog A (RELA) and p50 proteins. Once in the nucleus, NF-kB governs the transcription of numerous genes involved in T cell survival, proliferation, and effector functions.

TREIMM-1014; No. of Pages 13

Review
TCR CD28

Trends in Immunology xxx xxxx, Vol. xxx, No. x

L A T
P

SLP-76
P

ZAP70
P

LCK
P

PI3K

PDK1
P

PKC

Early

?
La t e

CARMA1 P MALT1 BCL10

GAKIN

CARMA1 PP2A CK1

P

STS Endosome-bound signalosome ZAP70 L A T

P

SHP1 PKC LCK CARMA1

P

Membrane-associated signalosome

CRADD BCL10

CARMA1

CARMA1

MALT1 BCL10
U U

SLP-76
P

TRAF6 PLC IP3 A20 DAG

U U

p62 BCL10
U

P U

Cytosolic signalosome

c-Cbl

HPK1

p62

Autophagosome

SLP-76
P

IKK IKK IKK IKK

P

TAK1
U

Proteasome

L A T
P U

CYLD IB P NF-B NF-B

U

PP4c

Key:
Acvaon signal Inhibion signal P Acvang phosphorylaon P Inhibitory phosphorylaon U K48-polyubiquinaon U K63-polyubiquinaon U Ubiquinaon, undetermined form

IB NF-B NF-B Gene transcripon

Unknown mediator

TRENDS in Immunology

Figure 2. Negative regulation of T cell receptor (TCR)-to-nuclear factor (NF)-kB signaling. The TCR/CD28 activation signal, shown by green arrows, passes through multiple intermediate signaling proteins, ultimately causing NF-kB activation. Negative modulation activities are depicted by red arrows. Yellow ovals indicate membrane proximal and cytosolic signalosomes described in detail in the text.

Recent data suggest that aspects of the consensus model for TCR signaling are overly simplistic, and that additional molecules play a role in the TCR-to-NF-kB cascade. Here, we summarize data suggesting that multiple signalosomes participate in TCR activation of NF-kB, and describe the negative regulatory mechanisms that control this pathway. We also discuss evidence for connections between control of NF-kB activation and other cellular processes, such as actin remodeling. Overall, the emerging picture is that the TCR-to-NF-kB signaling cascade is a crucial nexus that both governs and is regulated by a diverse network of T cell biological processes. New developments in the TCR-to-NF-kB pathway Deletion of the genes encoding PKCu and CBM complex proteins results in impaired TCR-induced NF-kB activation. Recent work has also identied several additional molecules that regulate this pathway (Figure 1). PKCu Phosphorylated PKCu connects LAT and SLP76 with the CBM complex [4,5]. The protein kinase PDK1 is considered essential for PKCu activation as PDK1-decient Jurkat and primary CD4 T cells show a defect in PKCu

phosphorylation and NF-kB activation [6,7]. However, there is a lack of in vitro evidence that PDK1 directly phosphorylates PKCu. Moreover, PDK1 activation is dependent on CD28 engagement, whereas PKCu IS translocation and NF-kB activation can occur in a purely CD3-dependent manner, without participation of CD28 [6,810]. These observations suggest that another kinase links the TCRCD3 complex with PKCu. Indeed, GCK-like kinase (GLK), a SLP76-regulated kinase, was recently reported to phosphorylate directly PKCu both in vitro and in primary T cells and T cell lines in response to TCR stimulation [11]. Additionally, GLK-decient murine lymph node cells exhibit reduced PKCu and IKK phosphorylation, correlating with reduced cytokine and antibody production. Collectively, these data suggest that PDK1 and GLK1 might function together to induce PKCu phosphorylation and activate NF-kB (Figure 1). Alternatively, GLK and/or PDK1 may be utilized in an exclusive manner to phosphorylate PKCu, depending on the activation and/or differentiation state of the T cells, the type of antigen-bearing stimulatory cell, etc. Elucidation of such details will require a careful comparison of the GLK and PDK1 knockout models under a variety of T cell activation paradigms.
3

TREIMM-1014; No. of Pages 13

Review
CARMA1 CARMA1, a critical target of PKCu phosphorylation, resides in lymphocytes in an inactive state. Extensive CARMA1 mutagenesis data suggest that this inactive state is maintained by intramolecular interactions that prevent the CARMA1 CARD from interacting with the CARD of BCL10 [12,13]. PKCu phosphorylates human CARMA1 at three serine residues, S552, S645 and S637 (S564, S657, S649 in mice) [1]. Phosphorylation at S552 and S645 is critical for the CARMA1 conformational change that enables binding to BCL10MALT1, leading to further signal transmission [14,15] (CBM complex post-translational modications are reviewed in [1]). By contrast, S649 phosphorylation suppresses CARMA1mediated NF-kB and C-jun N-terminal kinase (JNK) activation [16]. Although PKCu induces CARMA1 S649 phosphorylation in vitro and PKC inhibitors block the phosphorylation event in vivo, there is to date little evidence that PKCu directly phosphorylates CARMA1 at S649 in living cells. Also, the phosphorylation events of opposing function do not occur simultaneously; the activating phosphorylation at S552/S645 is early and transient, whereas the inhibitory phosphorylation at S649 is delayed, but sustained for a longer duration. Considering both the absence of evidence of direct PKCu phosphorylation of CARMA1 S649 and the rapid PKCu activation kinetics post-TCR activation, it remains a strong possibility that the delayed CARMA1 S649 phosphorylation is mediated by an unidentied PKCu-dependent kinase that is activated and/or interacts with CARMA1 in a delayed manner (Figure 2). Apart from PKCu, three additional protein kinases, hematopoietic progenitor kinase (HPK-1), casein kinase (CK1a) and calcium/calmodulin-dependent protein kinase (CaMKII) are capable of phosphorylating CARMA1 and regulating CBM complex activity (Figure 1). In Jurkat T cells, siRNA knockdown of HPK-1 reduces IKK enzymatic activity, NF-kB nuclear translocation, and T cell survival [17]. HPK-1 overexpression produces the opposite effect [18]. Demonstration of TCR-regulated interaction between HPK-1 and CARMA1 explains the ability of HPK-1 to stimulate NF-kB activation. These authors also provided evidence that HPK-1 phosphorylates CARMA1 at S551, a site distinct from the residues modied by PKCu [18]. However, the overall role of HPK-1 in lymphocyte function remains a hotly debated issue, because mouse knockout data have demonstrated that HPK-1 suppresses lymphocyte activation, due to its inhibitory effect on the proximal signaling protein SLP76 [19,20] (see Negative regulation of TCR signaling to NF-kB, below). CK1a, another kinase that acts via modication of CARMA1, has been identied by mass spectrometry as a CARMA1 binding partner. CK1a induces TCR-mediated NF-kB activation in Jurkat cells and interleukin (IL)-2 secretion in primary human T cells [21]; probably by recruiting activated IKKb to the CBM complex. Although CK1a phosphorylates CARMA1 at S608, limiting NF-kB activation, the positive CK1a function supercedes its negative role. [21]. Thus CK1a enzymatic activity opposes its IKK recruiting function, indicating that CK1a is also a bifunctional regulator of the TCR-to-NF-kB pathway.
4

Trends in Immunology xxx xxxx, Vol. xxx, No. x

Accumulating data suggest that the TCR-to-NF-kB pathway is also regulated by the protein kinase, CaMKII. TCR stimulation results in a rapid increase in T cell cytosolic calcium, which binds CaM, triggering activation of CaMKII and the phosphatase, calcineurin (see also BCL10 section below) (Figure 1). Both enzymes inuence the TCR-to-NF-kB pathway. In Jurkat cells, following APC+ antigen or CD3+CD28 stimulation [22,23], CaMKII is recruited to the IS. CaMKII inhibition by siRNA knockdown (g or d isoform) or by the pharmacological inhibitor, KN-93, reduces NF-kB activation [23]. However, the mechanism of CaMKII action is unclear. In a cell-free system, CaMKII can phosphorylate CARMA1 at S109 [23] and BCL10 at S138 [24] or at S48 and T91 [22]. Studies in Jurkat cells have shown CARMA1 S109 phosphorylation assists in CARMABCL10 binding [23], and BCL10 T91 phosphorylation promotes K63-polyubiquitination of BCL10 and signaling to IKK [22]. By contrast, there is evidence that BCL10 phosphorylation at S138 might have a negative effect on NF-kB activation [24]. Considering the above data, the mechanism by which CaMKII connects calciumCaM signals to NF-kB remains largely unclear. Future studies using genetic deletion or RNAi silencing of CaMKII in primary T cells will be required to clarify the role of CaMKII in TCR activation of NF-kB. Interestingly, CARMA1 phosphorylation is necessary but not sufcient for CBM complex formation. An additional essential step involves interaction with the integrin receptor regulatory protein adhesion and degranulationpromoting adapter protein (ADAP) (Figure 1). ADAP-decient T cells show impaired TCR- and CD28-dependent proliferation and reduced cytokine secretion. ADAP binds both CARMA1, facilitating CBM complex assembly [25], and TAK1, promoting IKK activation [26], and these actions might account for the effect of ADAP on NF-kB activation and cytokine secretion. The molecular mechanism by which ADAP enhances CBM complex assembly remains unclear. The ADAPCARMA1 interaction is also critical for cyclin-dependent kinase (CDK)2 and cyclin E expression [27]. As cyclins regulate progression through the cell cycle, this observation suggests a molecular basis for the impaired T cell proliferation observed in ADAP deciency. BCL10 As a result of the above modications and interactions of CARMA1, the constitutively associated BCL10MALT1 complex associates with CARMA1, forming the CBM complex [3,4]. Immediately following TCR stimulation, BCL10 is phosphorylated and ubiquitinated. BCL10 post-translational modication is complex, with many reported sites of modication. The regulation and signicance of many modications remain poorly understood (see [1,5] for review). To date, evidence suggests that IKKb is one of the principal kinases responsible for BCL10 phosphorylation (Figure 2) (see above for discussion of BCL10 phosphorylation by CaMKII). Initially, IKKb-phosphorylation of BCL10 stabilizes the CBM complex, but subsequent IKKb phosphorylation of BCL10 triggers BCL10 dissociation from MALT1 [28] and/or BCL10 degradation [29]. Thus BCL10 phosphorylation may be an important step for

TREIMM-1014; No. of Pages 13

Review
terminating TCR signals to NF-kB. Interestingly, calcineurin, a calcium-dependent phosphatase, has been reported to dephosphorylate BCL10 [30,31], stabilizing the CBM complex and enhancing NF-kB activation (Figure 1). Calcineurin, primarily known for its role in TCR-induced activation of the nuclear factor of activated T cells (NFAT) transcription factor, is also involved in lysosome-dependent degradation of PLCg1 and PKCu upon T cell restimulation, resulting in T cell anergy [32]. As PKCu is essential for NF-kB activation, these seemingly conicting roles of calcineurin in TCR activation of NF-kB require further examination. An area of intense recent investigation involves elucidation of the mechanism and purpose of BCL10 ubiquitination. Reports indicate that BCL10 is modied by K63-polyubiquitination [33,34] or both K48- and K63polyubiquitination with K63- preceding K48-polyubiquitination [35]. Mutagenesis data suggest that both K48- and K63-polyubiquitin chains are conjugated exclusively to lysines 31 and 63 of BCL10 [35]. K63-polyubiquitination of Bcl10 is triggered by interaction with CARMA1 [12,13]. The identity of the BCL10 ubiquitin ligase is highly controversial, with neural precursor cell expressed, developmentally downregulated (NEDD4) and ITCH [36], cellular inhibitor of apoptosis (cIAP2) [37], b-transducin repeatscontaining proteins bTrCP [29], and TRAF6 [35] all implicated as E3 ligases targeting BCL10. These uncertainties aside, the emerging consensus is that BCL10 ubiquitination serves two important purposes: initial transmission of the NF-kB activation signal [33,35], followed by BCL10 degradation and signal termination [29,33,35,36] (see Negative regulation of TCR signaling to NF-kB, below). BCL10 ubiquitination is important for signal transmission,

Trends in Immunology xxx xxxx, Vol. xxx, No. x

because ubiquitin chains create binding sites for IKKg [35] and p62 [33] (Figure 2); two molecules critical for NF-kB activation. Interestingly, the requirement for p62 and certain other signaling molecules seems to depend on the stage of T cell differentiation (Box 1). Another emerging area of interest regarding the biology of BCL10 involves the relation between TCR-dependent post-translational modication of BCL10 and regulation of the T cell actin cytoskeleton [38]. Although current evidence suggests that actin cytoskeletal dynamics do not directly inuence NF-kB activation, actin polymerization has a profound inuence on the immunological synapse and TCR microclusters (see Cell membrane and cytosolic complexes in TCR signaling to NF-kB, below), indirectly linking regulation of the actin cytoskeleton to TCR signaling to downstream transcription factors [39,40]. Also, there are multiple points of intersection between TCR-to-NF-kB signaling and regulation of the actin cytoskeleton, suggesting an as-yet poorly understood mechanistic coupling of the regulation of these pathways. TCR stimulation alters the T cell actin cytoskeleton, enabling T cell spreading and conjugation with APCs. In Jurkat cells, successful actin remodeling post-TCR stimulation requires BCL10 S138 phosphorylation [38] and IKKbHomer3 association at the IS [41] (Figure 1). Interestingly, BCL10 S138 phosphorylation is also required for macrophage Fc receptor-induced actin polymerization, leading to phagosome formation [42]. Phosphorylation of S138 is unrelated to CARMA1BCL10 MALT1 mediated NF-kB activation in both T cells and macrophages [38,42]. Similarly, the IKKbHomer3 mediated actin regulation in T cells is also NF-kB independent [41]. The identity of the precise mechanisms that link phosphoBCL10 and Homer3 to actin remains to be determined.

Box 1. Differences in TCR-to-NF-kB signaling between na ve and effector/memory T cells

Compared to na ve T cells, effector and memory cells demonstrate a faster response to antigen stimulation. This accelerated response of effector/memory T cells may partly reflect differentiation-dependent changes in NF-kB signaling mechanisms. One proposed mechanism is that crucial signaling proteins in differentiated T cells are constitutively phosphorylated, allowing more rapid activation. However, investigations have failed to reveal differences in phosphorylation states of ZAP70 and PLC-g between na ve and memory cells [111]. A second possible mechanism is that signaling proteins in effector/ memory T cells may be constitutively associated with lipid rafts; cholesterol-rich regions in the cell membrane that are sites of signaling molecule aggregation. Studies have indeed demonstrated increased interaction of signaling proteins with lipid rafts in memory CD4 and CD8 T cells [112,113], but the functional consequences have not been established. A third potential mechanism is that key signaling proteins may be more highly expressed in effector/memory T cells. Single cell experimental studies and computer modeling suggest a correlation between increased signaling protein expression and T cell functional capacity [114]. Multiple studies have shown higher total ZAP70 protein expression in CD4 effector and memory T cells, compared to na ve T cells [115,116]. Furthermore, data show that IFN-g production occurs selectively from effector T cells expressing the highest levels of ZAP70, and that IFN-g expression can be reduced by silencing ZAP70 using siRNA. Another study has demonstrated higher levels of the adaptor protein BCL10 in both effector CD4 and memory CD8 T cells, compared to na ve T cells [33], although the functional importance of this difference remains unexplored. Importantly, elevations of signaling protein levels do not universally accompany T cell differentiation. For example, relative to na ve T cells, SLP-76 levels are increased in effector T cells but decreased in memory T cells [117]. A final possible mechanism is that differentiated T cells might utilize signaling molecules that are distinct from those involved in na ve cell signaling. Two studies have demonstrated that the scaffolding protein p62 is required for TCR-to-NF-kB signaling in effector T cells, but not in na ve T cells [33,118]. Consistent with these findings, p62 expression is dramatically increased concomitant with TCR stimulation and effector differentiation [119]. These studies have indicated that differentiation-dependent increases in expression of certain intermediate signaling proteins and utilization of distinct signaling proteins may enhance TCR signaling in effector/memory T cells, versus na ve T cells. Apart from changes in signal transduction, alterations in transcription factor utilization and chromatin modification contribute to the rapid responses of memory T cells to antigen stimulation. The increase in IFN-g production by memory CD4 T cells compared to na ve cells is associated with a switch from T-bet- to NF-kB-dependent transcription. Additionally, memory cells exhibit increased expression and promoter binding of the NF-kB subunit p50 [120]. Also, the kinetics of gene transcription may be accelerated in memory T cells by histone acetylation of the promoter regions of specific genes, including perforin and Eomes, facilitating transcription factor binding [121]. Thus, epigenetic modifications and differential transcription factor usage may contribute to the rapid transcriptional response of memory T cells.
5

TREIMM-1014; No. of Pages 13

Review
MALT1 MALT1, the third member of the CBM complex, plays a more nebulous role in TCR activation of NF-kB. Initial studies suggested that MALT1 functions as a scaffolding protein that participates in NF-kB activation by connecting the CBM complex with TRAF6. Later studies showed that MALT1 contributes less to TCR activation of NF-kB than BCL10 or PKCu in primary mouse T cells [43] and that silencing MALT1 expression in Jurkat cells reduces but does not completely block IkBa phosphorylation [44]. Additionally, in B cells, BCL10 is essential for RELA activation, whereas MALT1 is dispensable [45]. Thus, there is an emerging understanding of MALT1 as a protein which modies antigen receptor-mediated signaling to NFkB, without being strictly required for NF-kB activation. Moreover, the protease activity of MALT1 is associated with regulation of diverse cellular functions, most of which are distinct from NF-kB signal transduction. MALT1 cleaves target proteins after arginine residues preceded by a serine [44,46]. To date, four MALT1 substrates, BCL10, A20, cylindromatosis (CYLD) and reticuloendotheliosis viral oncogene homolog B (RELB), have been identied (Figure 1). MALT1 cleavage of BCL10 and CYLD regulates T cell adhesion [44] and JNK activation [47] respectively, with no detected effect on NF-kB signal transduction. Another target of MALT1 cleavage is A20, a known inhibitor of NF-kB activation [46]. However, MALT1 cleaves only a small fraction of the total cellular A20 pool [46], and blockade of MALT1 protease activity fails to limit IKK activation [48]. Thus, the signicance of A20 cleavage as a mechanism regulating TCR signaling to NF-kB remains uncertain. Among known MALT1 substrates, RELB is most compelling as an NF-kB signal mediator that is cleaved to regulate NF-kB activation. MALT1-dependent RELB cleavage and concomitant degradation leads to increased RELA and c-REL DNA binding [49,50]. How RELB degradation affects RELA or c-REL activity is not well understood, but it might reect competition between different NF-kB heterodimers for DNA binding sites, or inhibition of canonical activation by gene products of the noncanonical (i.e., RELB-dependent) pathway. Caspase-8 The protease caspase-8 is well known as a mediator of apoptosis. Impaired lymphocyte activation in caspase-8decient mice has also suggested a role of caspase-8 in the TCR-to-NF-kB pathway [51]; a nding now conrmed and extended by several additional studies (Figure 1). Caspase8 recruits IKK to activated BCL10MALT1 by a TRAF6dependent mechanism [51,52], and MALT1 activates caspase-8, causing caspase-8-mediated cleavage of the NF-kB inducer cellular FLICE (Caspase-8)-inhibitory protein, long form (c-FLIPL) [53] (reviewed in [54]). Additionally, caspase-8 proteolytic activity is required for CD3/CD28dependent NF-kB activation, because T cells treated with the caspase inhibitor Z-VAD-FMK or expressing the caspase-8 catalytically inactive mutant, C360S, fail to induce NF-kB [51]. Interestingly, in HEK293 cells, neither ZVAD-FMK nor caspase-8 C360S blocks caspase-8-induced NF-kB activation [55], suggesting that the mechanistic
6

Trends in Immunology xxx xxxx, Vol. xxx, No. x

link between caspase-8 and NF-kB activation might be cell-type specic. Thus, additional mechanistic data are required to yield a clear understanding of how caspase-8 participates in this signaling pathway. IKK complex A key step in TCR activation of NF-kB is CBM complexdependent K63-polyubiquitination of IKKg. TRAF6 has been proposed as the ubiquitin ligase that performs this key function [1,3,54] (Figure 1). However, TRAF6-decient T cells do not show impaired NF-kB activation [56], suggesting either that TRAF6 does not play such a role, or that there are redundant ubiquitin ligases that compensate for loss of TRAF6. For example, TRAF2, an ubiquitin ligase recruited by the caspase-8FLIP complex is also capable of polyubiquitinating IKKg [53]. Another possible candidate is the ubiquitin ligase mind bomb-2 (MIB2), which binds to BCL10 and promotes IKKg ubiquitination and NF-kB activation in transfectionoverexpression experiments [57] (Figure 1). Thus, it is possible that several ubiquitin ligases contribute to K63-polyubiquitination of IKKg. It is also possible that there is a key role for modication of IKKg by M1 (linear head-to-tail linked) polyubiquitin chains. M1 polyubiquitination plays a key role in TNF receptor activation of NF-kB [58], but involvement in TCR-mediated activation of IKK is undetermined. Resolution of these lingering mechanistic questions will require compelling genetic and biochemical evidence using primary T cells to denitively demonstrate which ligases and which ubiquitin modications are essential. Activation of IKK requires a combination of IKKg ubquitination and IKKb phosphorylation. The latter process is mediated by the protein kinase TAK1 [59] (Figure 1). TAK1 activation seems to be dependent on PKCu, but not on the CBM complex members CARMA1 and BCL10 [60]. The adapter molecule ADAP is required for TAK1 recruitment to PKCu [26]. However, the precise mechanism of PKCumediated TAK1 activation is not well dened. The IKKb phosphorylation events can be countered by the protein phosphatase 4 regulatory subunit 1-protein phosphatase 4 regulatory subunit (PP4R1PP4c) phosphatase complex, limiting IKK activation and T cell function [61] (Figure 2). The all-or-none NF-kB response At a single cell level, signaling pathways may be broadly classied as digital or analog. Whereas analog signaling is a graded response that is proportional to stimulus intensity, digital signaling produces an all-or-none response, irrespective of intensity of the activation stimulus [62]. In T cells, increasing TCR stimulation strength leads to an increasing percentage of cells with IkBa phosphorylation and RELA activation, without altering per cell level of the two proteins [63]. This observation suggests a digital signaling mechanism similar to ndings regarding tumor necrosis factor (TNF)a-induced NF-kB signaling [64], and TCR-induced extracellular signal-regulated kinase (ERK) phosphorylation [65,66] or NFATc2 activation [67]. By contrast, bypassing the TCR via stimulation with phorbol 12-myristate 13-acetate (PMA+ionomycin) leads to a graded (analog) NF-kB response in T cells [63,67], indicating that signal digitization occurs at an early

TREIMM-1014; No. of Pages 13

Review
TCR-proximal step. The molecular switch enabling digitization of TCR signals remains to be determined. The above studies have demonstrated that, rather than the early understanding of TCR activation of NF-kB as a simple linear cascade (PKCu!CBM complex!IKK), there is a complex web of interconnected signaling molecules that link the TCR to NF-kB. In particular, there is accumulating evidence of multiple signaling inputs converging on key proteins, particularly CARMA1 and BCL10. Additionally, the mechanistic understanding of the crucial functions of certain mediators in transmitting the activating signal to NF-kB, for example, MALT1, is incomplete. Also, the precise roles and relative importance of kinases recently implicated as regulators of this pathway, including PDK1, GLK, HPK-1, and CK1a remain to be well dened. Crosstalk between the TCR-to-NF-kB pathway and other cellular processes, such as regulation of the actin cytoskeleton and T cellAPC interactions, is an emerging area of interest for which there is currently very limited mechanistic understanding. Cell surface and cytosolic complexes in TCR signaling to NF-kB Cell surface TCR microclusters and the IS The concept of the IS developed in the late 1990s when imaging revealed micrometer-sized clusters, composed of surface receptors and signaling proteins, at the T cellAPC intercellular contact site [68,69]. Initially, the IS was proposed as the site at which intracellular signaling from the TCR is initiated and sustained. Later studies showed that initiation of TCR signaling and tyrosine kinase activation can be detected within seconds following stimulation and before IS formation, in peripheral TCR-rich regions termed microclusters [70,71]. Current data suggest that the earliest signaling events occur in these peripheral microclusters, which move centripetally and eventually fuse to form the mature IS. Recent data also cast doubt on the model that the IS represents the primary site of sustained signaling to downstream mediators. Specically, TCR signaling to NF-kB can occur in the absence of an IS [72,73]. Also, as detailed below, accumulating data point towards the existence of cytosolic signaling clusters/signalosomes that may sustain and regulate TCR signal transduction. Although the IS might not be involved in signal initiation or maintenance, the IS has certain other critical functions. Experimental studies and computer modeling have revealed that at low antigenMHC concentrations, the IS amplies TCR activation by grouping together antigens, TCR, and signaling proteins. This allows the TCR to engage rapidly the small number of stimulatory ligands, resulting in efcient signal transduction [74]. Also, the IS may serve as a site of TCR internalization and degradation, serving to limit signal strength and/or duration [75]. Interestingly, ubiquitination of as-yet-undened central supramolecular activation cluster (c-SMAC) proteins leads to interaction with the ubiquitin-binding protein, tumor susceptibility gene (TSG101), a component of the endosomal sorting complex required for transport (ESCRT). This interaction is required for c-SMAC formation and organization, as well as for termination of signaling by microclusters and TCR degradation [76]. These

Trends in Immunology xxx xxxx, Vol. xxx, No. x

observations suggest that TCR signaling and signal termination by TCR degradation are intimately connected. Cytosolic T cell signalosomes Emerging data suggest that several distinct cellular sites may together coordinate TCR signaling to NF-kB. Immediately following TCR engagment, the integral membrane protein LAT and the cytosolic protein SLP76 are recruited to the region immediately below the TCR microclusters [3]. ZAP70-mediated phosphorylation of LAT and SLP76 enables these adaptor proteins to bind downstream mediators, transmitting the activation signal. Data suggest that following initial recruitment, LAT and SLP76 dissociate from TCR microclusters in endosomes [77] (Figure 2). SLP76 remains on the the outer cytosolic side of the endosome, and its attachment to endosomes is mediated indirectly through LAT. Endosome-associated LAT and SLP76 remain phosphorylated, indicating that these adaptors are capable of binding effector molecules and continuing the signaling cascade. LATSLP76 endosomes might represent sites of continous signaling to downstream mediators, similar to the signaling complexes on intracellular endosomal membranes observed downstream of the epidermal growth factor receptor (EGFR) [78,79]. LAT SLP76 endosomes might also have a role in termination of TCR signaling, as inhibition of microcluster movement results in enhanced TCR signaling [80,81]. There is also evidence for cytosolic signalosomes containing BCL10 and MALT1 that specically sustain NFkB-activation. Upon PKCu activation, CARMA1 is recruited to the plasma membrane, apparently undergoing a conformational change that opens access to binding sites for BCL10 and MALT1, leading to activation of IKK [3,4] (Figure 2). However, the mechanism that links the membrane bound CBM complex to IKK activation is not well dened. One possibility is that BCL10MALT1 clusters interact transiently with membrane bound CARMA1, followed by redistribution to a cytosolic site at which these proteins transmit the signal to IkBaNF-kB. Biochemical analysis of Jurkat cells has revealed the existence of two complexes [34]: an early membrane-bound CBM complex (Figure 2), and a late BCL10MALTIKK complex, and this late complex has been shown to interact inducibly with IkBa. Additionally, in a cell-free system, puried recombinant BCL10 and MALT1 proteins along with TRAF6, ubiquitin-conjugating enzyme (UBC13), and TAK1 were sufcient for IKK activation, demonstrating that the BCL10MALT1 complex can activate IKK in a manner that is independent of concurrent physical association with CARMA1 [48] (i.e., although CARMA1 is required for transmitting the activating signal to BCL10, it does not need to remain associated with BCL10MALT1). Importantly, IKK activation is mediated by a small fraction of the total BCL10 and MALT1 pool that exists as high molecular weight oligomers, providing biochemical evidence of BCL10MALT1 signalosomes. However, it is also important to note that there are as yet no published biochemical data showing that similar signaling-competent oligomers of BCL10MALT1 exist in intact cells or cellular lysates. Imaging studies in primary mouse T cells and D10 T cells have demonstrated the presence of TCR-induced
7

TREIMM-1014; No. of Pages 13

Review
cytosolic BCL10MALT1 clusters, termed POLKADOTS [9,82], at locations that are distinct from membrane bound PKCu, a marker for the IS. Furthermore, these BCL10 MALT1 clusters are also enriched in the downstream signaling protein TRAF6 [82]. Formation of the POLKADOTS clusters is dependent both on expression of the autophagy adaptor protein p62 and K63-polyubiquitination of BCL10 [33]. Interaction between BCL10MALT1 and p62 and the resultant signal transmission to IKK is driven by BCL10 K63-polyubiquitination (Figure 2). The formation of the proposed POLKADOTS signalosome is strongly correlated with IKKa/b phosphorylation [33] and RelA nuclear translocation [63], supporting a direct role in NF-kB activation. The above studies provide compelling evidence that TCR signaling to NF-kB involves several organized signalosomes that concentrate groups of interacting signaling proteins. Interestingly, these signalosomes are not universally required for TCR signaling to NF-kB. Specically, NF-kB activation can occur in the absence of the IS, and the POLKADOTS signalosome apparently plays no role in na ve T cells (Box 1). Although early data suggest that such signalosomes represent cellular platforms for coordinating and precisely regulating positive and negative signals to NF-kB, much further work is needed to dene fully the components and functions of these intriguing macromolecular structures. Areas for future focus include: determining how TSG101 and the ESCRT complex interact with microclusters and c-SMAC constitutents to regulate signal transmission by the TCR; better establishing the role of endosomal LATSLP76 in activation of downstream transcription factors; establishing the mechanistic connection between the IS and POLKADOTS signalosome; and dening in molecular detail how mechanistic requirements for NF-kB activation change with T cell differentiation (Box 1). Negative regulation of TCR signaling to NF-kB TCR activation of NF-kB is critical for T cell proliferation and differentiation. However, unrestricted and persistent NF-kB activation can lead to development of autoimmune diseases and neoplasms [83], cellular senescence [84], or apoptosis [85]. In order to strike a balance between productive T cell activation and deleterious consequences of excessive NF-kB activation, TCR signaling has to be precisely regulated. After T cell activation, negative regulation starts at the level of cell surface receptors (e.g., by TCR endocytosis and degradation) and continues at multiple steps of cytoplasmic and nuclear signaling. In this review, we focus on cytoplasmic mechanisms that limit TCR-toNF-kB signaling (cell surface receptor and nuclear regulation mechanisms are reviewed in [2,3] and [86], respectively). Cytosolic negative regulators and mechanisms of action are listed in Table 1 and illustrated in Figure 2. TCR-proximal regulatory mechanisms Successful transmission of the activation signal from the TCR to NF-kB requires dynamic regulation at multiple intermediate steps. TCR engagement results in the phosphorylation and/or ubiquitination of many intermediates, and these post-translational modications activate or
8

Trends in Immunology xxx xxxx, Vol. xxx, No. x

repress signal transmission. The TCR-proximal signaling events involving ZAP70, LCK, LAT, and SLP-76 are dynamically regulated by multiple protein phosphatases and kinases (Figure 2). Data suggest that Src homology region 2 domain-containing phosphatase (SHP-1), well known for its ability to regulate T cell signaling, dephosphorylates LCK [87], ZAP70 [88], and SLP-76 [89]. Mice with T cellspecic deletion of SHP-1 exhibit increased expansion of CD8 effector (but not memory) T cells in response to viral infection [90]. In addition to SHP-1, ZAP70 can also be dephosphorylated by suppressor of T cell receptor signaling Sts-1 and Sts-2, limiting TCR stimulation. Deletion of both Sts-1 and Sts-2 causes T cell hyper-responsiveness, with augmented cytokine secretion in response to antigen stimulation [91]. In stimulated T cells, Sts-1 and -2 deletion results in accumulation of hyper-phosphorylated and ubiquitinated proteins [92]. Deletion of either Sts-1 or Sts2 alone does not produce any detectable phenotype, thus, there is likely a wide overlap in the function of these related molecules [93]. LCK activity is controlled by two opposing enzymes: c-src tyrosine kinase (CSK), a protein kinase, and CD45, a protein phosphatase. Data show that CSK-mediated inhibitory phosphorylation of LCK diminishes TCR activation (Figure 2), whereas CSK silencing amplies TCRinduced IL-2 production [94]. CD45 removes this inhibitory phosphate group from LCK, allowing LCK to participate in TCR signaling [95]. SLP-76 activity is also limited by phosphorylation. After the initial transient ZAP70-mediated activating phosphorylation of SLP-76 (at Y112, Y128 and Y145; reviewed in [96]), HPK-1 adds a phosphate group to SLP-76 at S376 (Figure 2), causing increased binding to the inhibitory protein 14-3-3 [19,20]. T cells lacking HPK-1 demonstrate CD3-induced hyperproliferation and increased cytokine secretion, although it is unclear whether this effect is mediated by the effect of HPK-1 on NF-kB or on other T cell transcription factors (e.g., NFAT and activator protein AP-1). TCR-distal regulatory mechanisms In the case of the CBM complex and other TCR-distal mediators of NF-kB activation, these proteins were identied relatively recently, and regulatory mechanisms are therefore only now being recognized. One protein newly identied as a CARMA1 regulator is the serinethreonine phosphatase, PP2A (Figure 2). As discussed above, PKCu phosphorylation of CARMA1 at Ser645 is critical for assembly of the CBM complex [1]. PP2A removes the phosphate group from Ser645, destabilizing the CARMA1 BCL10 interaction and reducing NF-kB activation. Further, siRNA silencing of PP2A results in higher TCR-induced IL-2 and interferon (IFN)-g production [97]. However, it is also important to note that PP2A directly deactivates the IKK complex [98], making it unclear whether inhibition of NF-kB activation is primarily due to PP2A effects on CARMA1 or IKK. Also discussed above, CARMA1 phosphorylation at S649 and S608 by PKCu (directly or indirectly) [16] and CK1a [21], respectively, impairs NF-kB signaling. A phosphorylation-independent mechanism of CARMA1 regulation involves binding of CARMA1 to the kinesin motor

TREIMM-1014; No. of Pages 13

Review

Trends in Immunology xxx xxxx, Vol. xxx, No. x

Table 1. Cytosolic proteins involved in negative regulation of TCR-to-NF-kB signaling.

Protein mediator SHP-1 (phosphatase) Proposed mechanism Dephosphorylates LCK, ZAP70, SLP-76 Gene deletion or silencing phenotype me/me moth-eaten mouse TCR-induced thymocyte hyperproliferation, increases IL-2 production Increased CD8 effector cell expansion with viral infection Sts1/ Sts2/ double knockout T cell hyperproliferation Increased IL-2, -4, -5, -10, IFN-g secretion Increased susceptibility in murine multiple scelerosis model siRNA silencing: increased TCR-induced IL-2 secretion Cbl-b/: increased lymphocyte proliferation and secretion of IL-2 and antibody c-Cbl/: lymphoid hyperplasia Map4k1/: T cell hyperproliferation, increased T cell cytokines and B cell antibody production Increased susceptibility to autoimmune encephalitis siRNA silencing: increased IL-2 and IFN-g secretion TAX mediated PP2A inhibition: constitutive IKK activation CK1a inactive kinase mutant: increased NF-kB activity CK1a silencing: reduced NF-kB activity, T cell IL-2 secretion and proliferation (contradictory results suggest CK1a is a bifunctional regulator; dominant effect is positive regulation) Mutation of S649 phosphorylation site increases IKK and NF-kB activation in Jurkat cells (PKCu knockout blocks NF-kB activation;dominant effect is positive regulation) shRNA silencing: increased IKK activation and IL-2 secretion Cradd/: increased T cell CARMA1BCL10 binding, RELA activation and cytokine secretion Autophagy-decient T cells: increased IL-2 secretion, CD25 expression; decreased survival Inhibition of BCL10 phosphorylation: increased secretion of IL-2 and TNFa Tnfaip3/: multiorgan inammation, leading to death B cell conditional knock out: lupus-like disease, increased antibody secretion Cyld/: increased IKKb phosphorylation Hyper-responsive T and B cells Spontaneous colonic inltration PP4R1-silenced T cells demonstrate enhanced IKK phosphorylation and NF-kB activation Refs [8790,122]

STS-1, STS-2 (phosphatase)

Dephosphorylates ZAP-70

[91]

CSK (protein kinase) CBL-B, C-CBL (E3 ubiquitin ligase) HPK-1 (protein kinase)

LCK inhibitory phosphorylation C-CBL: LAT trafcking to endosomes SLP-76 inhibitory phosphorylation increases 14-3-3 binding Dephosphorylates CARMA1 and IKK Phosphorylates CARMA1

[94] [77,81,107]

[19,20]

PP2A (phosphatase) CK1a (protein kinase) a

[97,98] [21]

PKCu (protein kinase)a (direct or indirect effect)

Late CARMA1 phosphorylation at S649

[16]

GAKIN (kinesin 13B) CRADD (adaptor protein) Autophagy pathway, proteasomes

Competes with BCL10 for CARMA1 binding Competes with CARMA1 to bind BCL10 BCL10 degradation

[99] [100] [28,29,33,36,108]

A20 (deubiquitinase and ubiquitin ligase) CYLD (deubiquitinase)

Removal of K63-polyubiquitin from MALT1 Removal of K63-polyubiquitin from TAK1 IKKa/b dephosphorylation

[101,102]

[104,105]

PP4R1PP4c (phosphatase)
a

[61]

Bifunctional NF-kB regulator with stimulatory effect over-riding negative function.

protein Guanylate kinase-associated kinesin (GAKIN) [99]. This interaction results in CARMA1 movement away from PKCu at the IS and reduces CARMA1 interaction with BCL10 (Figure 2). Post-TCR stimulation CARMA1 BCL10 binding is also interrupted by caspase and receptor interacting protein adaptor with death domain (CRADD), which competes with CARMA1 to bind BCL10 (Figure 2). Accordingly, CRADD-decient murine T cells show increased CARMA1BCL10 binding and RELA activation. CRADD deletion results in increased T cell cytokine production with concanavalin A stimulation, but does not yield any identied autoimmune phenotype [100]. Regulation of TCR signaling by ubiquitin-modifying enzymes The ubiquitin-editing enzyme, A20, has been long known as an inhibitor of TNF-dependent NF-kB activation. A20 can both remove K63-polyubiquitin chains from and add K48-polyubiquitin chains to target proteins [101]. A20 has a high basal expression in T cells, and it can limit TCRinduced NF-kB activity by removing K63-polyubiquitin

chains from the CBM complex protein, MALT1 [102] (Figure 2). Interestingly, MALT1 can cleave A20 in a TCR-dependent manner, and this proteolytic cleavage has been suggested to disrupt the ability of A20 to limit TCR activation of NF-kB [46]. A20 is also capable of cleaving K63-polyubiquitin chains from receptor-interacting serine/threonine-protein kinase (RIP1), TRAF6, and IKKg, limiting TNF-, IL-1-, and lipopolysaccharide (LPS)dependent NF-kB activation [103]. However, there is so far no evidence that these specic mechanisms regulate the TCR-to-NF-kB pathway. A20 activity is critical for downregulating NF-kB activity in numerous cell types, therefore, A20-decient mice (Tnfaip3/) die shortly after birth due to multiorgan inammation [101]. Although conditional inactivation of A20 in B cells results in autoantibody production and lupus-like disease [101], the in vivo role of A20 in T cells remains uncertain. A second ubiquitin-modifying enzyme, CYLD, has been reported as a modulator of JNK and NF-kB activation [101]. Cyld/ mice develop spontaneous autoimmune colitis. Biochemical studies of Cyld/ peripheral T cells
9

TREIMM-1014; No. of Pages 13

Review
and B cells showed constitutively phosphorylated IKKb and activated NF-kB [104,105]. This is explained by loss of CYLD-mediated TAK1 K63-deubiquitination [106], which suppresses TAK1 activation in wild type lymphocytes (Figure 2). Curiously, in CD3+CD28-stimulated Jurkat T cells, MALT1 cleaves CYLD increasing activation of JNK, but not NF-kB [47]. Further studies of A20/ and Cyld/ T cells will be required to determine the relative importance of these regulators of protein ubiquitination in TCRto-NF-kB signaling, and to assess whether there is any redundancy in their activities. Regulation of TCR signaling by molecular sequestration Emerging data suggest a novel mechanism of TCR signal transduction regulation, whereby cytosolic mediators are selectively sequestered in cytosolic vesicular compartments, making them unavailable for signal transmission. The ubiquitin ligases of the Casitas B cell lymphoma (CBL) family, CBL-B and C-CBL, negatively regulate T cell activation. Both CBL-B and C-CBL knockout results in higher NF-kB activation, lymphoid hyperplasia, and increased IL2 secretion from T cells [107]. Reports indicate that CBL activity might depend on promotion of TCR trafcking to lysosomes [107] and/or on ubiquitination of the adaptor protein LAT, targeting LAT for endosomal sequestration [81] (Figure 2). A similar mechanism involving the distal TCR signaling adaptor BCL10 has also recently been reported. Data suggest that BCL10 is selectively targeted for sequestration within autophagosomes [33], followed by lysosomal degradation [33,36]. The proteasome also contributes to TCR-dependent BCL10 degradation via an unclear mechanism [29,33]. The role of autophagy in limiting TCR activation of NF-kB is further supported by the observation that T cell-specic inactivation of the autophagy genes ATG7 [108] or ATG3 [33] results in increased IL-2 secretion and CD25 expression. BCL10 autophagy is highly selective, because the BCL10 binding partner MALT1 is neither detected inside autophagosomes nor degraded [33]. The process whereby MALT1 is separated from BCL10, protecting MALT1 from degradation, might depend on autophagosome formation [33] and/or IKKbmediated BCL10 phosphorylation [28,29]. Data showing that phosphorylation of BCL10 by IKKb causes BCL10 to dissociate from the CBM complex [28] suggest that IKKb has a dual function, both positively and negatively regulating the TCR-to-NF-kB pathway. Interestingly, the process of BCL10 autophagy occurs at the POLKADOTS signalosome, requiring the same molecular interactions that play a central role in TCR activation of NF-kB. Specically, autophagy of BCL10 requires interaction between p62 and K63-polyubiquitinated BCL10 [33,35,36] (Figure 2). Thus, p62 is an additional bifunctional regulator of TCR signaling to NF-kB, controlling both assembly of the POLKADOTS signalosome and degradation of its key signaling component, BCL10. How NFkB activation and BCL10 autophagy (a signal limiting mechanism) are coordinately regulated at the POLKADOTS signalosome will require further investigation. The above studies illustrate that negative regulatory mechanisms act at multiple points in the TCR-to-NF-kB signaling cascade. Interestingly, diverse independent
10

Trends in Immunology xxx xxxx, Vol. xxx, No. x

mechanisms including phosphorylation, dephosphorylation, ubiquitination, deubiquitination, protein trafcking, and protein degradation contribute to limiting activation of TCR signaling. These data suggest that negative regulation of this pathway is critical for T cell function and/or survival, in line with emerging data that unrestrained NF-kB activation has a spectrum of possible deleterious outcomes. Concluding remarks Recent ndings have signicantly altered our views of TCR signaling to NF-kB. Certain mediators, such as PKCu and CARMA1, are phosphorylated by multiple kinases, dispelling early models that postulated a simple linear cascade by which the TCR transmits activating signals to NF-kB. Studies have also revealed that cytosolic mediators in this pathway do not all coalesce in a single TCR-proximal signalosome at the IS. Rather, cytosolic signalosomes far removed from the TCR are also integral to NF-kB activation. Despite these new complexities, precise molecular mechanisms of signal transmission are gradually being revealed, with several essential post-translational modications and their downstream effects now identied. For example, BCL10, once thought to be a simple adaptor connecting CARMA1 to MALT1, is now known to undergo K63-polyubiquitination, triggering association with p62 and activation of IKK. Additionally, numerous mechanisms of negative regulation have been recognized, indicating that the TCR-to-NF-kB pathway is tightly modulated. Four kinases (PKCu, HPK-1, CK1a, and IKKb) and two ubiquitin-binding proteins (TSG101 and p62) play a dual role in signaling to NF-kB, with both activating and signal limiting activities. Precise control of NF-kB activation may guarantee production of exact levels of cell cycle

Box 2. Directions for future research

 Elucidation of differences between na ve and effector T cells in the TCR-to-NF-kB pathway. In particular, it is unclear why effector/ memory T cells require a cytosolic signalosome to successfully activate NF-kB (Box 1), and why levels of signaling proteins differ between na ve and effector/memory cells.  Defining how the CBM complex and the POLKADOTS signalosome are mechanistically connected to IKK activation. Related questions include how BCL10 is physically separated from MALT1, allowing selective BCL10 degradation, and whether MALT1 function changes after its signaling partner BCL10 is degraded.  Revealing the mechanism and purpose of coupling NF-kB activation with other T cell signaling cascades. In particular, it is currently unclear why JNK activation and actin polymerization are closely networked to the TCR-to-NF-kB cascade.  Exploring the role of miRNAs in TCR-to-NF-kB signaling. miRNAs are emerging as key regulators of T cell activation, with likely effects on signaling to NF-kB. Indeed, recent data show mir-146 and mir-155 affect mediators utilized by the TCR-to-NF-kB cascade and alter T cell behavior [123126].  Determining the role of atypical ubiquitination in TCR-to-NF-kB signaling. To date, most research in this pathway has focused on typical K48- and K63-polyubiquitination, due to the existence of highly specific tools that enable detailed study of these modifications. The six atypical ubiquitins have demonstrated function in NF-kB activation [58] but there are few data to date regarding how these under-studied ubiquitin polymers modulate TCR signaling to NF-kB.

TREIMM-1014; No. of Pages 13

Review
proteins, thereby avoiding failed cell division and apoptosis. Emerging data also suggest that signaling to NF-kB is coupled to seemingly unrelated biological processes such as regulation of the actin cytoskeleton. Additionally, there may be considerable differences in mechanisms of TCR activation of NF-kB utilized by T cells at distinct stages of differentiation. Despite these recent advances in our understanding of this pathway, there remain many mechanistic details that are poorly understood. Important questions for the eld are outlined in Box 2.
Acknowledgments
The authors thank A. Snow, C. Gray, K. McCorkell, M. May, and C-Z. Giam for critical reading of the manuscript. Supported by grants from the US National Institutes of Health (AI057481 to B.C.S.), the Center for Neuroscience and Regenerative Medicine (CNRM) (to B.C.S.), and predoctoral fellowships (to S.P.) from the American Heart Association (10PRE3150039) and the Henry M. Jackson Foundation. The views expressed are those of the authors and do not necessarily reect those of the Uniformed Services University or the Department of Defense. The authors declare no competing nancial interests.

Trends in Immunology xxx xxxx, Vol. xxx, No. x

References
1 Thome, M. et al. (2010) Antigen receptor signaling to NF-kappaB via CARMA1, BCL10, and MALT1. Cold Spring Harb. Perspect. Biol. 2, a003004 2 Vallabhapurapu, S. and Karin, M. (2009) Regulation and function of NF-kappaB transcription factors in the immune system. Annu. Rev. Immunol. 27, 693733 3 Smith-Garvin, J.E. et al. (2009) T cell activation. Annu. Rev. Immunol. 27, 591619 4 Schulze-Luehrmann, J. and Ghosh, S. (2006) Antigen-receptor signaling to nuclear factor kappa B. Immunity 25, 701715 5 Thome, M. and Weil, R. (2007) Post-translational modications regulate distinct functions of CARMA1 and BCL10. Trends Immunol. 28, 281288 6 Park, S.G. et al. (2009) The kinase PDK1 integrates T cell antigen receptor and CD28 coreceptor signaling to induce NF-kappaB and activate T cells. Nat. Immunol. 10, 158166 7 Lee, K.Y. et al. (2005) PDK1 nucleates T cell receptor-induced signaling complex for NF-kappaB activation. Science 308, 114118 8 Cartwright, N.G. et al. (2011) An active kinase domain is required for retention of PKCtheta at the T cell immunological synapse. Mol. Biol. Cell 22, 34913497 9 Schaefer, B.C. et al. (2004) Complex and dynamic redistribution of NFkappaB signaling intermediates in response to T cell receptor stimulation. Proc. Natl. Acad. Sci. U.S.A. 101, 10041009 10 Huang, J. et al. (2002) CD28 plays a critical role in the segregation of PKC theta within the immunologic synapse. Proc. Natl. Acad. Sci. U.S.A. 99, 93699373 11 Chuang, H.C. et al. (2011) The kinase GLK controls autoimmunity and NF-kappaB signaling by activating the kinase PKC-theta in T cells. Nat. Immunol. 12, 11131118 12 Lamason, R.L. et al. (2010) Oncogenic CARD11 mutations induce hyperactive signaling by disrupting autoinhibition by the PKCresponsive inhibitory domain. Biochemistry 49, 82408250 13 Chan, W. et al. (2013) A quantitative signaling screen identies CARD11 mutations in the CARD and LATCH domains that induce Bcl10 ubiquitination and human lymphoma cell survival. Mol. Cell. Biol. 33, 429443 14 Sommer, K. et al. (2005) Phosphorylation of the CARMA1 linker controls NF-kappaB activation. Immunity 23, 561574 15 Matsumoto, R. et al. (2005) Phosphorylation of CARMA1 plays a critical role in T cell receptor-mediated NF-kappaB activation. Immunity 23, 575585 16 Moreno-Garcia, M.E. et al. (2009) Serine 649 phosphorylation within the protein kinase C-regulated domain down-regulates CARMA1 activity in lymphocytes. J. Immunol. 183, 73627370 17 Brenner, D. et al. (2005) Activation or suppression of NFkappaB by HPK1 determines sensitivity to activation-induced cell death. EMBO J. 24, 42794290

18 Brenner, D. et al. (2009) Phosphorylation of CARMA1 by HPK1 is critical for NF- B activation in T cells. Proc. Natl. Acad. Sci. U.S.A. 106, 1450814513 19 Shui, J.W. et al. (2007) Hematopoietic progenitor kinase 1 negatively regulates T cell receptor signaling and T cell-mediated immune responses. Nat. Immunol. 8, 8491 20 Di Bartolo, V. et al. (2007) A novel pathway down-modulating T cell activation involves HPK-1-dependent recruitment of 14-3-3 proteins on SLP-76. J. Exp. Med. 204, 681691 21 Bidere, N. et al. (2009) Casein kinase 1alpha governs antigenreceptor-induced NF-kappaB activation and human lymphoma cell survival. Nature 458, 9296 22 Oruganti, S.R. et al. (2011) CaMKII targets Bcl10 in T-cell receptor induced activation of NF-kappaB. Mol. Immunol. 48, 14481460 23 Ishiguro, K. et al. (2006) Ca2+/calmodulin-dependent protein kinase II is a modulator of CARMA1-mediated NF-kappaB activation. Mol. Cell. Biol. 26, 54975508 24 Ishiguro, K. et al. (2007) Bcl10 is phosphorylated on Ser138 by Ca2+/ calmodulin-dependent protein kinase II. Mol. Immunol. 44, 20952100 25 Medeiros, R.B. et al. (2007) Regulation of NF-kappaB activation in T cells via association of the adapter proteins ADAP and CARMA1. Science 316, 754758 26 Srivastava, R. et al. (2010) NF-kappaB activation in T cells requires discrete control of IkappaB kinase alpha/beta (IKKalpha/beta) phosphorylation and IKKgamma ubiquitination by the ADAP adapter protein. J. Biol. Chem. 285, 1110011105 27 Srivastava, R. et al. (2012) ADAP regulates cell cycle progression of T cells via control of cyclin E and Cdk2 expression through two distinct CARMA1-dependent signaling pathways. Mol. Cell. Biol. 32, 1908 1917 28 Wegener, E. et al. (2006) Essential role for IkB kinase b in remodeling Carma1-Bcl10-Malt1 complexes upon T cell activation. Mol. Cell 23, 1323 29 Lobry, C. et al. (2007) Negative feedback loop in T cell activation through IkB kinase-induced phosphorylation and degradation of Bcl10. Proc. Natl. Acad. Sci. U.S.A. 104, 908913 30 Palkowitsch, L. et al. (2011) The Ca2+-dependent phosphatase calcineurin controls the formation of the Carma1-Bcl10-Malt1 complex during T cell receptor-induced NF-kB activation. J. Biol. Chem. 286, 75227534 31 Frischbutter, S. et al. (2011) Dephosphorylation of Bcl-10 by calcineurin is essential for canonical NF-kappaB activation in Th cells. Eur. J. Immunol. 41, 23492357 32 Heissmeyer, V. et al. (2004) Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins. Nat. Immunol. 5, 255265 33 Paul, S. et al. (2012) Selective autophagy of the adaptor protein Bcl10 modulates T cell receptor activation of NF-kappaB. Immunity 36, 947958 34 Carvalho, G. et al. (2010) Interplay between BCL10, MALT1 and IkappaBalpha during T-cell-receptor-mediated NFkappaB activation. J. Cell Sci. 123, 23752380 35 Wu, C.J. and Ashwell, J.D. (2008) NEMO recognition of ubiquitinated Bcl10 is required for T cell receptor-mediated NF-kappaB activation. Proc. Natl. Acad. Sci. U.S.A. 105, 30233028 36 Scharschmidt, E. et al. (2004) Degradation of Bcl10 induced by T-cell activation negatively regulates NF-kappa B signaling. Mol. Cell. Biol. 24, 38603873 37 Hu, S. et al. (2006) cIAP2 is a ubiquitin protein ligase for BCL10 and is dysregulated in mucosa-associated lymphoid tissue lymphomas. J. Clin. Invest. 116, 174181 38 Rueda, D. et al. (2007) Bcl10 controls TCR- and FcgammaR-induced actin polymerization. J. Immunol. 178, 43734384 39 Gomez, T.S. and Billadeau, D.D. (2008) T cell activation and the cytoskeleton: you cant have one without the other. Adv. Immunol. 97, 164 40 Beemiller, P. and Krummel, M.F. (2010) Mediation of T-cell activation by actin meshworks. Cold Spring Harb. Perspect. Biol. 2, a002444 41 Yatherajam, G. et al. (2010) Cutting edge: association with I kappa B kinase beta regulates the subcellular localization of Homer3. J. Immunol. 185, 26652669 42 Marion, S. et al. (2012) The NF-kappaB signaling protein Bcl10 regulates actin dynamics by controlling AP1 and OCRL-bearing vesicles. Dev. Cell 23, 954967
11

TREIMM-1014; No. of Pages 13

Review
43 Kingeter, L.M. and Schaefer, B.C. (2008) Loss of protein kinase C theta, Bcl10, or Malt1 selectively impairs proliferation and NF-kappa B activation in the CD4+ T cell subset. J. Immunol. 181, 62446254 44 Rebeaud, F. et al. (2008) The proteolytic activity of the paracaspase MALT1 is key in T cell activation. Nat. Immunol. 9, 272281 45 Ferch, U. et al. (2007) MALT1 directs B cell receptor-induced canonical nuclear factor-kappaB signaling selectively to the c-Rel subunit. Nat. Immunol. 8, 984991 46 Coornaert, B. et al. (2008) T cell antigen receptor stimulation induces MALT1 paracaspase-mediated cleavage of the NF-kappaB inhibitor A20. Nat. Immunol. 9, 263271 47 Staal, J. et al. (2011) T-cell receptor-induced JNK activation requires proteolytic inactivation of CYLD by MALT1. EMBO J. 30, 17421752 48 Sun, L. et al. (2004) The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes. Mol. Cell 14, 289301 49 Hailnger, S. et al. (2011) Malt1-dependent RelB cleavage promotes canonical NF-kB activation in lymphocytes and lymphoma cell lines. Proc. Natl. Acad. Sci. U.S.A. 108, 1459614601 50 Hailnger, S. et al. (2009) Essential role of MALT1 protease activity in activated B cell-like diffuse large B-cell lymphoma. Proc. Natl. Acad. Sci. U.S.A. 106, 1994619951 51 Su, H. et al. (2005) Requirement for caspase-8 in NF-kappaB activation by antigen receptor. Science 307, 14651468 52 Bidere, N. et al. (2006) Caspase-8 regulation by direct interaction with TRAF6 in T cell receptor-induced NF-kappaB activation. Curr. Biol. 16, 16661671 53 Misra, R.S. et al. (2007) Caspase-8 and c-FLIPL associate in lipid rafts with NF-kappaB adaptors during T cell activation. J. Biol. Chem. 282, 1936519374 54 Kingeter, L.M. and Schaefer, B.C. (2010) Malt1 and cIAP2-Malt1 as effectors of NF-kappaB activation: kissing cousins or distant relatives? Cell. Signal. 22, 922 55 Chaudhary, P.M. et al. (2000) Activation of the NF-kappaB pathway by caspase 8 and its homologs. Oncogene 19, 44514460 56 King, C.G. et al. (2006) TRAF6 is a T cell-intrinsic negative regulator required for the maintenance of immune homeostasis. Nat. Med. 12, 10881092 57 Stempin, C.C. et al. (2011) The E3 ubiquitin ligase mind bomb-2 (MIB2) protein controls B-cell CLL/lymphoma 10 (BCL10)dependent NF-kB activation. J. Biol. Chem. 286, 3714737157 58 Kulathu, Y. and Komander, D. (2012) Atypical ubiquitylation the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages. Nat. Rev. Mol. Cell Biol. 13, 508523 59 Wang, C. et al. (2001) TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412, 346351 60 Shambharkar, P.B. et al. (2007) Phosphorylation and ubiquitination of the IkappaB kinase complex by two distinct signaling pathways. EMBO J. 26, 17941805 61 Brechmann, M. et al. (2012) A PP4 holoenzyme balances physiological and oncogenic nuclear factor-kappa B signaling in T lymphocytes. Immunity 37, 697708 62 Coward, J. et al. (2010) Perspectives for computer modeling in the study of T cell activation. Cold Spring Harb. Perspect. Biol. 2, a005538 63 Kingeter, L.M. et al. (2010) Cutting edge: TCR ligation triggers digital activation of NF-kappaB. J. Immunol. 185, 45204524 64 Tay, S. et al. (2010) Single-cell NF-kappaB dynamics reveal digital activation and analogue information processing. Nature 466, 267271 65 Das, J. et al. (2009) Digital signaling and hysteresis characterize ras activation in lymphoid cells. Cell 136, 337351 66 Altan-Bonnet, G. and Germain, R.N. (2005) Modeling T cell antigen discrimination based on feedback control of digital ERK responses. PLoS Biol. 3, e356 67 Podtschaske, M. et al. (2007) Digital NFATc2 activation per cell transforms graded T cell receptor activation into an all-or-none IL2 expression. PLoS ONE 2, e935 68 Monks, C.R. et al. (1998) Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 8286 69 Dustin, M.L. et al. (1998) A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts. Cell 94, 667677 70 Lee, K.H. et al. (2002) T cell receptor signaling precedes immunological synapse formation. Science 295, 15391542
12

Trends in Immunology xxx xxxx, Vol. xxx, No. x

71 Varma, R. et al. (2006) T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster. Immunity 25, 117127 72 Purbhoo, M.A. et al. (2004) T cell killing does not require the formation of a stable mature immunological synapse. Nat. Immunol. 5, 524530 73 OKeefe, J.P. and Gajewski, T.F. (2005) Cutting edge: cytotoxic granule polarization and cytolysis can occur without central supramolecular activation cluster formation in CD8+ effector T cells. J. Immunol. 175, 55815585 74 Lee, K.H. et al. (2003) The immunological synapse balances T cell receptor signaling and degradation. Science 302, 12181222 75 Dustin, M.L. et al. (2010) Understanding the structure and function of the immunological synapse. Cold Spring Harb. Perspect. Biol. 2, a002311 76 Vardhana, S. et al. (2010) Essential role of ubiquitin and TSG101 protein in formation and function of the central supramolecular activation cluster. Immunity 32, 531540 77 Balagopalan, L. et al. (2009) Endocytic events in TCR signaling: focus on adapters in microclusters. Immunol. Rev. 232, 8498 78 Mills, I.G. (2007) The interplay between clathrin-coated vesicles and cell signalling. Semin. Cell Dev. Biol. 18, 459470 79 Sadowski, L. et al. (2009) Signaling from endosomes: location makes a difference. Exp. Cell Res. 315, 16011609 80 Nguyen, K. et al. (2008) T cell costimulation via the integrin VLA-4 inhibits the actin-dependent centralization of signaling microclusters containing the adaptor SLP-76. Immunity 28, 810821 81 Balagopalan, L. et al. (2007) c-Cbl-mediated regulation of LATnucleated signaling complexes. Mol. Cell. Biol. 27, 86228636 82 Rossman, J.S. et al. (2006) POLKADOTS are foci of functional interactions in T-cell receptor-mediated signaling to NF-kappaB. Mol. Biol. Cell 17, 21662176 83 Karin, M. and Greten, F.R. (2005) NF-kappaB: linking inammation and immunity to cancer development and progression. Nat. Rev. Immunol. 5, 749759 84 Zhi, H. et al. (2011) NF-kappaB hyper-activation by HTLV-1 tax induces cellular senescence, but can be alleviated by the viral antisense protein HBZ. PLoS Pathog. 7, e1002025 85 Krishna, S. et al. (2012) Chronic activation of the kinase IKKbeta impairs T cell function and survival. J. Immunol. 189, 12091219 86 Natoli, G. and Chiocca, S. (2008) Nuclear ubiquitin ligases, NFkappaB degradation, and the control of inammation. Sci. Signal. 1, pe1 87 Stefanova, I. et al. (2003) TCR ligand discrimination is enforced by competing ERK positive and SHP-1 negative feedback pathways. Nat. Immunol. 4, 248254 88 Plas, D.R. et al. (1996) Direct regulation of ZAP-70 by SHP-1 in T cell antigen receptor signaling. Science 272, 11731176 89 Binstadt, B.A. et al. (1998) SLP-76 is a direct substrate of SHP-1 recruited to killer cell inhibitory receptors. J. Biol. Chem. 273, 27518 27523 90 Fowler, C.C. et al. (2010) SHP-1 in T cells limits the production of CD8 effector cells without impacting the formation of long-lived central memory cells. J. Immunol. 185, 32563267 91 Carpino, N. et al. (2004) Regulation of ZAP-70 activation and TCR signaling by two related proteins, Sts-1 and Sts-2. Immunity 20, 3746 92 Carpino, N. et al. (2009) The Sts proteins target tyrosine phosphorylated, ubiquitinated proteins within TCR signaling pathways. Mol. Immunol. 46, 32243231 93 Carpino, N. et al. (2002) Identication, cDNA cloning, and targeted deletion of p70, a novel, ubiquitously expressed SH3 domaincontaining protein. Mol. Cell. Biol. 22, 74917500 94 Vang, T. et al. (2004) Knockdown of C-terminal Src kinase by siRNAmediated RNA interference augments T cell receptor signaling in mature T cells. Eur. J. Immunol. 34, 21912199 95 Hermiston, M.L. et al. (2003) CD45: a critical regulator of signaling thresholds in immune cells. Annu. Rev. Immunol. 21, 107137 96 Zou, T. et al. (2010) Understanding signal integration through targeted mutations of an adapter protein. FEBS Lett. 584, 49014909 97 Eitelhuber, A.C. et al. (2011) Dephosphorylation of Carma1 by PP2A negatively regulates T-cell activation. EMBO J. 30, 594605 98 Fu, D.X. et al. (2003) Human T-lymphotropic virus type I tax activates I-kappa B kinase by inhibiting I-kappa B kinase-associated serine/ threonine protein phosphatase 2A. J. Biol. Chem. 278, 14871493

TREIMM-1014; No. of Pages 13

Review
99 Lamason, R.L. et al. (2010) The dynamic distribution of CARD11 at the immunological synapse is regulated by the inhibitory kinesin GAKIN. Mol. Cell 40, 798809 100 Lin, Q. et al. (2012) Cutting edge: the death adaptor CRADD/RAIDD targets BCL10 and suppresses agonist-induced cytokine expression in T lymphocytes. J. Immunol. 188, 24932497 101 Harhaj, E.W. and Dixit, V.M. (2011) Deubiquitinases in the regulation of NF-kappaB signaling. Cell Res. 21, 2239 102 Duwel, M. et al. (2009) A20 negatively regulates T cell receptor signaling to NF-kappaB by cleaving Malt1 ubiquitin chains. J. Immunol. 182, 77187728 103 Shembade, N. and Harhaj, E.W. (2012) Regulation of NF-kappaB signaling by the A20 deubiquitinase. Cell. Mol. Immunol. 9, 123130 104 Reiley, W.W. et al. (2007) Deubiquitinating enzyme CYLD negatively regulates the ubiquitin-dependent kinase Tak1 and prevents abnormal T cell responses. J. Exp. Med. 204, 14751485 105 Jin, W. et al. (2007) Deubiquitinating enzyme CYLD regulates the peripheral development and naive phenotype maintenance of B cells. J. Biol. Chem. 282, 1588415893 106 Kovalenko, A. et al. (2003) The tumour suppressor CYLD negatively regulates NF-kappaB signalling by deubiquitination. Nature 424, 801805 107 Naramura, M. et al. (2002) c-Cbl and Cbl-b regulate T cell responsiveness by promoting ligand-induced TCR downmodulation. Nat. Immunol. 3, 11921199 108 Jia, W. et al. (2011) Autophagy regulates endoplasmic reticulum homeostasis and calcium mobilization in T lymphocytes. J. Immunol. 186, 15641574 109 Kalia, V. et al. (2006) Differentiation of memory B and T cells. Curr. Opin. Immunol. 18, 255264 110 Jameson, S.C. and Masopust, D. (2009) Diversity in T cell memory: an embarrassment of riches. Immunity 31, 859871 111 Farber, D.L. (2009) Biochemical signaling pathways for memory T cell recall. Semin. Immunol. 21, 8491 112 Watson, A.R. and Lee, W.T. (2004) Differences in signaling molecule organization between naive and memory CD4+ T lymphocytes. J. Immunol. 173, 3341

Trends in Immunology xxx xxxx, Vol. xxx, No. x

113 Kersh, E.N. et al. (2003) TCR signal transduction in antigen-specic memory CD8 T cells. J. Immunol. 170, 54555463 114 Feinerman, O. et al. (2008) Variability and robustness in T cell activation from regulated heterogeneity in protein levels. Science 321, 10811084 115 Chandok, M.R. et al. (2007) A biochemical signature for rapid recall of memory CD4 T cells. J. Immunol. 179, 36893698 116 Okoye, F.I. et al. (2007) Proximal signaling control of human effector CD4 T cell function. Clin. Immunol. 125, 515 117 Hussain, S.F. et al. (2002) Differential SLP-76 expression and TCRmediated signaling in effector and memory CD4 T cells. J. Immunol. 168, 15571565 118 Martin, P. et al. (2006) The signaling adapter p62 is an important mediator of T helper 2 cell function and allergic airway inammation. EMBO J. 25, 35243533 119 Yang, J.Q. et al. (2010) NBR1 is a new PB1 signalling adapter in Th2 differentiation and allergic airway inammation in vivo. EMBO J. 29, 34213433 120 Lai, W. et al. (2011) Transcriptional control of rapid recall by memory CD4 T cells. J. Immunol. 187, 133140 121 Araki, Y. et al. (2008) Histone acetylation facilitates rapid and robust memory CD8 T cell response through differential expression of effector molecules (eomesodermin and its targets: perforin and granzyme B). J. Immunol. 180, 81028108 122 Lorenz, U. et al. (1996) Lack of SHPTP1 results in src-family kinase hyperactivation and thymocyte hyperresponsiveness. Proc. Natl. Acad. Sci. U.S.A. 93, 96249629 123 Yang, L. et al. (2012) miR-146a controls the resolution of T cell responses in mice. J. Exp. Med. 209, 16551670 124 OConnell, R.M. et al. (2010) MicroRNA-155 promotes autoimmune inammation by enhancing inammatory T cell development. Immunity 33, 607619 125 Lu, L.F. et al. (2010) Function of miR-146a in controlling Treg cellmediated regulation of Th1 responses. Cell 142, 914929 126 van Loo, G. and Beyaert, R. (2011) Negative regulation of NF-kappaB and its involvement in rheumatoid arthritis. Arthritis Res. Ther. 13, 221

13