tec-family kinases: regulators of t-helper-cell differentiation - Nature

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TEC-FAMILY KINASES: REGULATORS OF T-HELPER-CELL DIFFERENTIATION Pamela L. Schwartzberg, Lisa D. Finkelstein and Julie A. Readinger Abstract | The TEC-family protein tyrosine kinases ITK, RLK and TEC have been identified as key components of T-cell-receptor signalling that contribute to the regulation of phospholipase C-γ, the mobilization of Ca2+ and the activation of mitogen-activated protein kinases. Recent data also show that TEC kinases contribute to T-cell-receptor-driven actin reorganization and cell polarization, which are required for productive T-cell activation. Functional studies have implicated TEC kinases as important mediators of pathways that control the differentiation of CD4+ T helper cells. Here, we review studies of signalling pathways that involve TEC kinases and how these pathways might contribute to the regulation of T-helper-cell differentiation and function. X-LINKED AGAMMAGLOBULINAEMIA

(XLA). A human immunodeficiency that is caused by mutations in the gene encoding Bruton’s tyrosine kinase (BTK), which is located on the X chromosome.These mutations result in a block in B-cell maturation and in poor antibody production. A naturally occurring mouse mutant of BTK, X-linked immunodeficiency (XID), is associated with less severe disease.

National Human Genome Research Institute, National Institutes of Health, 4A38/ 49 Convent Drive, Bethesda, Maryland 20892, USA. Correspondence to P.L.S. e-mail: [email protected] doi:10.1038/nri1591

284

In 1993, several research groups discovered that mutations affecting a novel protein tyrosine kinase, BTK (Bruton’s tyrosine kinase), were associated with the severe B-cell immunodeficiency X-LINKED AGAMMAGLOBULINAEMIA (XLA) and the mouse X-linked immunodeficiency (XID)1–5. It was subsequently recognized that BTK is a member of a related family of NON-RECEPTOR PROTEIN TYROSINE KINASES of which TEC was the first to be defined (FIG. 1A). The TEC family now consists of five members, which are expressed mainly by haematopoietic cells: TEC (tyrosine kinase expressed in hepatocellular carcinoma), BTK, ITK (interleukin-2 (IL-2)-inducible T-cell kinase; also known as EMT or TSK), RLK (resting lymphocyte kinase; also known as TXK) and BMX (bone-marrow tyrosine kinase gene on chromosome X; also known as ETK)6. T cells express three TEC kinases — ITK, RLK and TEC — all of which are activated downstream of the T-cell receptor (TCR)7 and have been shown to be involved in signalling through the TCR8–16. Although no human disease has been associated with mutations of the TEC kinases that are expressed by T cells, ITK-deficient mice have specific defects in T HELPER 2 (T 2)-CELL RESPONSES and reduced pathology in models of allergic asthma11,15,17. Conversely, ITK expression is increased in T cells from patients with ATOPIC DERMATITIS, a TH2-cell-mediated disease18. These studies, combined with the recent finding that specific ITK inhibitors reduce disease in a mouse H

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model of allergic asthma19, have increased interest in understanding the roles of TEC kinases in T-cell function. In this Review article, we discuss the biology of the T-cell-specific TEC kinases, new findings about the cellular pathways that they regulate and clues to how these signalling pathways might contribute to the control of TH-cell function. TEC-family kinases

The TEC-family kinases are characterized by a common domain organization: they have an amino-terminal PHOSPHATIDYLINOSITOL-3,4,5-TRISPHOSPHATE (PtdIns(3,4,5)P )3 binding PLECKSTRIN-HOMOLOGY DOMAIN, which is followed by a TEC-homology domain that contains one or two proline-rich regions (PRRs), then SRC homology 3 (SH3) and SH2 protein-interaction domains, and a carboxyterminal kinase domain (FIG. 1A). The TEC kinases are the only tyrosine kinases that have pleckstrin-homology domains, which inducibly recruit TEC-family members to the plasma membrane by binding the phosphatidylinositol 3-kinase (PI3K) product PtdIns(3,4,5)P3, thereby promoting their activation. Membrane localization and activation of the pleckstrin-homologydomain-containing TEC kinases are therefore regulated by PI3K and the lipid phosphatases PTEN (phosphatase and tensin homologue) and SHIP (SH2-domaincontaining inositol-5-phosphatase), which catalyse the breakdown of PtdIns(3,4,5)P3 (REFS 20–23) (FIG. 1B). www.nature.com/reviews/immunol

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NON-RECEPTOR PROTEIN TYROSINE KINASES

Proteins that lack a transmembrane or extracellular domain, have no ligand, are found intracellularly and add a phosphate group to tyrosine residues in proteins. Tyrosine phosphorylation leads to a change in the ability of the phosphorylated protein to bind and activate downstream molecules.

A

TH PH

BH PRR

The atypical TEC kinase RLK lacks a pleckstrin-homology domain and, instead, has a palmitoylated string of cysteine residues, which leads to constitutive membrane association of RLK, independent of PI3K activity24,25. Activation of TEC-family kinases requires several interrelated steps: first, recruitment to the plasma membrane through interactions between their pleckstrinhomology domains and the products of PI3K and/or other proteins; second, phosphorylation by SRC-family kinases; and third, interactions with other proteins that bring the TEC-family kinases into antigen-receptorsignalling complexes7 (FIG. 1B). In addition, TEC-family kinases are thought to be regulated by conformational changes directed by intra- and intermolecular interactions involving their SH2 domains, SH3 domains and

PRRs7. These have been best characterized for ITK, for which nuclear-magnetic-resonance studies have shown that the PRR binds the SH3 domain within the same molecule26. An additional interaction between the SH2 domain of one ITK molecule and the SH3 domain of another ITK molecule can also be detected. These interactions are thought to be inhibitory and to prevent interactions with other proteins. The PEPTIDYLPROLYL ISOMERASE CYCLOPHILIN A contributes to ITK activation through isomerization of a proline residue in the SH2 domain of ITK, which alters the specificity of the protein interactions of the SH2 domain so that the cis form favours intramolecular interactions with the SH3 domain and the trans form favours interactions with other proteins in TCR-signalling complexes and activation of its own kinase activity 27.

P287 (ITK)

Roles for TEC kinases in T cells

SH3

SH2

Kinase

ITK, TEC and BTK

Cys

PRR SH3

SH2

Kinase

RLK

PH

BH

SH2

Kinase

BMX

SH3

B TCR CD4 PtdIns(3,4,5)P3

CCCC RLK

a

PtdIns(4,5)P2

PI3K ITK or TEC

P

b

c

P

GADS

SLP76

b

Plasma membrane

PTEN or SHIP

LCK

FYN

a

LAT

PtdIns(4,5)P2

P

Figure 1 | Structure and activation of TEC-family kinases. A | The five TEC-family kinases are depicted by a schematic that shows their structural domains. TEC kinases expressed by T cells are indicated in bold. The proline residue at position 287 (P287) is unique to ITK (interleukin-2-inducible T-cell kinase) and is targeted by the peptidylprolyl isomerase cyclophilin A. B | After T-cell receptor (TCR) engagement, ITK and TEC are recruited to the plasma membrane through interaction of their pleckstrin homology (PH) domain with phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3), which is generated from phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) by phosphatidylinositol 3-kinase (PI3K). SHIP (SRC homology 2 (SH2)-domain-containing inositol-5phosphatase) and PTEN (phosphatase and tensin homologue) can reduce the levels of PtdIns(3,4,5)P3, thereby decreasing membrane association and activation of certain TEC kinases. RLK (resting lymphocyte kinase) is constitutively associated with membranes because it contains a palmitoylated cysteine-string motif (a). SRC-family kinases (such as LCK) phosphorylate a tyrosine residue in the kinase domain of ITK, RLK and TEC (b). Changes in the conformation of ITK promote the formation of a complex between LAT (linker for activation of T cells), SLP76 (SH2-domaincontaining leukocyte protein of 76 kDa) and GADS (growth-factor-receptor-bound protein 2 (GRB2)-related adaptor protein) that allows activation of downstream effectors (c). BH, BTK homology; BMX, bone-marrow tyrosine kinase gene on chromosome X; BTK, Bruton’s tyrosine kinase; Cys, cysteine-string motif; PRR, proline-rich region; TH, TEC homology.

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Although ITK, RLK and TEC are all found in T cells, they are expressed at different levels and by different subpopulations. Evaluation of mRNA levels by realtime reverse-transcriptase PCR showed that ITK is the main TEC kinase expressed by naive mouse T cells, with Rlk mRNA expressed at 3–10-fold lower levels and Tec mRNA at ∼100-fold lower levels28,29. On T-cell activation, ITK expression is increased, particularly in TH2 cells, whereas RLK expression rapidly drops and is re-established only in TH1 cells30. TEC expression increases after several days of T-cell stimulation31. There is evidence indicating that all three of these kinases function downstream of TCR signalling. Phospholipase C-γ activation and gene transcription. Engagement of the TCR by peptide–MHC complexes, in conjunction with interaction with co-stimulatory molecules at the cell surface of antigen-presenting cells (APCs), leads to rapid activation of the SRC-family kinase LCK, phosphorylation of the ζ-chain of the TCRproximal signalling molecule CD3, and recruitment and activation of the protein kinase ZAP70 (ζ-chainassociated protein kinase of 70 kDa) and of PI3K32,33 (FIG. 2). ZAP70 then phosphorylates the adaptors LAT (linker for activation of T cells) and SLP76 (SH2domain-containing leukocyte protein of 76 kDa), which together function as a platform for the recruitment of several key signalling molecules, including the enzyme phospholipase C-γ (PLC-γ), the adaptors GRB2 (growth-factor-receptor-bound protein 2), NCK (non-catalytic region of tyrosine kinase) and ADAP (adhesion- and degranulation-promoting adaptor protein), and the guanine nucleotide-exchange factor VAV1. This TCR-signalling complex is crucial for downstream effector functions, including mobilization of Ca2+, activation of mitogen-activated protein kinases (MAPKs) and downstream transcription factors, and regulation of the actin cytoskeleton33 (FIGS 2,3). TEC kinases are activated through phosphorylation by SRC-family kinases, such as LCK, and recruitment to the plasma membrane through binding of PtdIns(3,4,5)P3, where they are brought into TCRsignalling complexes through interactions with SLP76,

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T-HELPER-2-CELL RESPONSES

(TH2-cell responses). There are two main effector subsets of CD4+ T cells, which are characterized by distinct cytokine profiles and by functional activity. TH2 cells produce interleukin-4 (IL-4), IL-5, IL-9, IL-10 and IL-13, leading to activation of humoral immune responses. By contrast, TH1 cells produce interferon-γ, IL-2 and lymphotoxin, which support cell-mediated immunity. Appropriate differentiation of T cells into these subsets is important for mounting immune responses to different pathogens, whereas an imbalance between these subsets is associated with diseases, including asthma, hypersensitivity and autoimmune disorders.

LAT and other molecules34–37. Early data showed a role for TEC kinases in antigen-receptor-induced phosphorylation and activation of PLC-γ 9,10,38,39. PLC-γ is an enzyme that catalyses phosphatidylinositol-4,5bisphosphate (PtdIns(4,5)P2) catabolism to generate both inositol-1,4,5-trisphosphate (Ins(1,4,5)P3), which is required for Ca2+ mobilization, and diacylglycerol, which activates diacylglycerol-binding proteins such as protein kinase C (PKC) and RASGRP (RAS guanylreleasing protein), which are important for activating MAPKs, as well as the downstream transcription factor AP1 (activator protein 1) and other effectors. Together, these signalling intermediates are crucial for the production of cytokines and the expression of activation markers by T cells40. Accordingly, T cells from mice that are deficient in TEC kinases show defective phosphorylation of PLC-γ, production of Ins(1,4,5)P3, influx of Ca2+ and activation of the Ca2+-sensitive transcription factors TCR CD4

NFATc1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1) and NFATc2 (REFS 9–11,15) (TABLE 1). Impaired activation of the MAPKs ERK (extracellular-signal-regulated kinase) and JNK (JUN aminoterminal kinase) and of the transcription factor AP1 are also observed in these T cells15,16 (TABLE 1; FIG. 2). Consistent with the expression patterns of TEC kinases, mice deficient in ITK show moderately severe defects in T-cell function, whereas relatively minor defects are observed in RLK-deficient mice; so far, there are no reported T-cell defects in TEC-deficient mice10,41 (TABLE 1). Combined disruption of ITK and RLK leads to more severe biochemical and cellular defects than does ITK deficiency alone, indicating that there might be some degree of redundancy between these two kinases, despite their structural differences10,42. Stimulation through the TCR of ITK-deficient T cells or T cells deficient in both RLK and ITK leads to graded defects in PtdIns(3,4,5)P3

PtdIns(3,4,5)P3

Ca2+

PtdIns(4,5)P2

PtdIns(4,5)P2

CD3

Plasma membrane

PLC-γ

ITK

LCK

P

P

P P

P

286

Ins(1,4,5)P3 receptor

P

+

P VAV1 P

CDC42

WASP ARP2/ ARP3

Ca2+ Ca2+ Ca2+

RAC NCK P GADS P ADAP

Intracellular calcium store

F-actin P

P

GRB2

P

Ca2+

DAG

Calcineurin

NF-κB

Adhesion and/or migration

PKC RASGRP

PKC-θ

SOS

IκB

PLECKSTRIN-HOMOLOGY DOMAIN

A protein–lipid interaction domain that usually consists of 100 amino-acid residues. Pleckstrin-homology domains have little overall sequence homology but have conserved motifs and tertiary structure. They are thought to be involved in anchoring of proteins to the membrane and have been found to bind the following: phospholipids (including phosphatidylinositol-4,5bisphosphate and phosphatidylinositol-3,4,5trisphosphate), proteins (including the β and γ subunits of heterotrimeric G proteins), phosphorylated serine or threonine residues, and membranes.

Ins(1,4,5)P3

P

SLP76

(PtdIns(3,4,5)P3). A product of phosphatidylinositol 3-kinase, which adds a phosphate group to phosphatidylinositol-4,5bisphosphate (PtdIns(4,5)P2) to yield PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 binds pleckstrinhomology-domain-containing proteins, resulting in membrane recruitment of these proteins and initiation of signalling cascades.

P

PI3K

LAT

PHOSPHATIDYLINOSITOL-3,4,5TRISPHOSPHATE

P ZAP70

A disease that is characterized by a scaly, itchy rash that is caused by inflammation of the skin and by increased production of T-helper-2 cytokines.

FYN

ATOPIC DERMATITIS

JNK

ERK1/ERK2

AP1

Transcription

P P NFAT

NFAT

Nucleus

Figure 2 | TEC-family kinases in T-cell-receptor-signalling pathways. TEC-family kinases have a central role in the propagation of the signal induced by engagement of the T-cell receptor (TCR). ITK (interleukin-2-inducible T-cell kinase) forms a complex with several signalling molecules that are nucleated by the adaptors LAT (linker for activation of T cells) and SLP76 (SRC-homology-2domain-containing leukocyte protein of 76 kDa). Activation of phospholipase C-γ (PLC-γ) by ITK leads to the generation of inositol1,4,5-trisphosphate (Ins(1,4,5)P3) (which is required for Ca2+ flux within the cell) and diacylglycerol (DAG) (which activates members of the protein kinase C (PKC) family and RAS guanyl-releasing protein, RASGRP). This results in the downstream activation of mitogen-activated protein kinases — such as JNK (JUN amino-terminal kinase), ERK1 (extracellular-signal-regulated kinase 1) and ERK2 — and other effectors that direct gene transcription. In addition to activation by DAG, PKC-θ is activated through a VAV1- and RAC-mediated pathway. The LAT–SLP76 complex also functions as a platform for the accumulation of molecules — including VAV1, RAC, CDC42 (cell-division cycle 42), WASP (Wiskott–Aldrich syndrome protein), ARP2 (actin-related protein 2 homologue) and ARP3 — that regulate the polymerization of F-actin. These molecules, together with other downstream effectors, control TCR-mediated T-cell polarization, adhesion and migration. ADAP, adhesion- and degranulation-promoting adaptor protein; AP1, activator protein 1; GADS, GRB2-related adaptor protein; GRB2, growth-factor-receptor-bound protein 2; IκB, inhibitor of NF-κB; NCK, non-catalytic region of tyrosine kinase; NFAT, nuclear factor of activated T cells; NF-κB, nuclear factor-κB; PI3K, phosphatidylinositol 3-kinase; PtdIns(3,4,5)P3, phosphatidylinositol-3,4,5-trisphosphate; PtdIns(4,5)P2, phosphatidylinositol4,5-bisphosphate; SOS, son of sevenless homologue; ZAP70, ζ-chain-associated protein kinase of 70 kDa.

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TCR CD4 CD3

αi

P

LCK

Chemokine receptor

Plasma membrane

β γ G protein

ZAP70

P P P

P

TEC kinases

VAV1

RHO-family GTPases

PLC-γ

DAG

Ins(1,4,5)P3

PKC

Migration

Actin reorganization

Adhesion

Gene expression

Ca2+ mobilization

Figure 3 | T-cell receptors and chemokine receptors signal through TEC-family kinases. After engagement of the T-cell receptor (TCR) or chemokine receptors, TEC-family kinases are recruited to the membrane and become activated. Subsequently, many signalling molecules take part in downstream pathways that lead to actin reorganization, adhesion, migration, Ca2+ mobilization and gene expression. These downstream effects are interrelated in that migration, adhesion and gene expression all depend on VAV1 and on proper reorganization of actin in the cell. Similarly, Ca2+ mobilization regulates both adhesion and gene expression. DAG, diacylglycerol; Ins(1,4,5)P3, inositol-1,4,5-trisphosphate; PKC, protein kinase C; PLC-γ, phospholipase C-γ; ZAP70, ζ-chain-associated protein kinase of 70 kDa.

TCR-induced proliferation and IL-2 production, impaired immunity to infection with Toxoplasma gondii and reduced ACTIVATION-INDUCED CELL DEATH (AICD), implicating these kinases in the regulation of immune responses8–10,16. SH3

(SRC homology 3). A proteininteraction domain that is commonly found in signaltransduction molecules. It specifically interacts with certain proline-containing peptides (containing either (R/K)XXPXXP or PXXPXR motifs, where X denotes any amino acid) to facilitate protein– protein interactions that are required for protein function or subcellular localization. SH2

(SRC homology 2). A proteininteraction domain that is commonly found in signaltransduction molecules. It specifically interacts with phosphotyrosine-containing peptides.

Actin reorganization. When T cells are stimulated by APCs, they become rapidly polarized, with recruitment of F-actin and signalling molecules to the site of TCR stimulation, where these molecules are organized into a structure known as the immunological synapse43,44. Although the function of the immunological synapse is not well understood, one proposed function is to stabilize T-cell interactions with APCs through recruitment of signalling molecules, and integrins and other adhesion molecules. TCR-induced rearrangement of the actin cytoskeleton is controlled, in part, by the guanine nucleotide-exchange factor VAV1, which is tyrosine phosphorylated during signalling through the TCR and, in turn, activates the RHO family of small GTPases — RAC, RHO and CDC42 (cell-division cycle 42) — which are crucial for actin reorganization45–49. Although TEC kinases are best recognized for their roles in PLC-γ regulation and Ca2+ mobilization, data

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now indicate that TEC kinases also contribute to TCR-induced actin rearrangement. ITK-deficient T cells have reduced F-actin polarization after TCR stimulation12,13. This phenotype is recapitulated in both human peripheral-blood T cells and cells of the Jurkat T-cell line that are treated with SMALL INTERFERING 50 RNA (siRNA) directed against ITK . Intriguingly, regulation of the actin cytoskeleton might not require the kinase activity of ITK; kinase-dead mutants fail to block actin polarization and can rescue actin defects in cells treated with siRNA directed against ITK13,50,51. Instead, defects in actin polarization in ITK-deficient cells correlate with defective recruitment of VAV1 to the site of TCR stimulation12,50 and with impaired association of VAV1 with SLP76 (REF. 50), despite relatively normal tyrosine phosphorylation of VAV1 (REF. 52). These results indicate that ITK might be required to stabilize the interactions of VAV1 in TCR-signalling complexes. Importantly, expression of a prenylated, constitutively membrane-targeted mutant of VAV1 rescues the actinpolarization defect in cells treated with siRNA directed against ITK50, supporting the idea that VAV1 is an important contributor to this phenotype. However, it should be noted that ITK activation is also impaired in VAV1-deficient cells, owing to decreased PI3K activity53, which underscores the extent of cross-regulation between molecules in these signalling complexes. Consistent with these observations, many of the biochemical and cellular defects in Itk –/– and Rlk –/–Itk –/– T cells, although less severe, resemble those of cells that lack VAV1 (TABLE 1). Both Itk –/– and Vav1–/– T cells show relatively normal gross patterns of early TCR-induced tyrosine phosphorylation, yet they have decreased activation of PLC-γ, ERK and downstream transcription factors9–11,15,46,54,55. VAV1 deficiency prevents activation of the RHO-family GTPases RAC1 and CDC42 (REFS 53,56). Similarly, a defect in localized activation of CDC42 is observed in Itk –/– and Rlk –/–Itk –/– T cells stimulated with peptide-loaded APCs12. Vav1–/– T cells also show defective cell adhesion, which correlates with impaired clustering of the integrin LFA1 (lymphocyte function-associated antigen 1) to the site of TCR stimulation57. Similarly, mutations that affect ITK block TCR-mediated activation of β1- and β2-integrin adhesion and recruitment of LFA1 to the site of TCR stimulation (REF. 14, and L.D.F. and P.L.S., unpublished observations). Finally, VAV1 and RAC1 are required for recruitment of PKC-θ, a key marker of the immunological synapse58. Defective recruitment of PKC-θ is also observed in Itk –/– and Rlk –/–Itk –/– T cells stimulated with TCR-specific-antibody-coated beads (L.D.F. and P.L.S., unpublished observations), highlighting the similarities between the phenotypes of T cells deficient in TEC kinases and VAV1. ITK, therefore, seems to be a crucial component of pathways that are required for the reorganization of actin, the induction of cell polarization and the recruitment of molecules to the immunological synapse (including PKC-θ and LFA1, a key adhesion molecule that helps to stabilize contacts with the APC), leading to productive T-cell activation.

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Table 1 | T-cell defects in mice deficient in T-cell signalling proteins* Genotype Defects Defects in PLC-γ in Ca2+ activation flux

Defects in ERK activation

Defects Defects Defects in in NFAT in IL-2 proliferation activation production

Defects in Defects in actin polym- adhesion erization

Defects in chemokine responses

Tec–/–

ND

ND

Rlk –/–

No

No

Itk –/–

References

ND

ND

No

No

ND

ND

ND

41

No

No

Yes (mild)

No

No

No‡

No

10,12,15,60

Yes

Yes Yes (moderate)

Yes

Yes

Yes (moderate)

Yes

Yes‡

Yes

8–13,15–17, 59,60

Rlk –/–Itk –/–

Yes

Yes (severe)

Yes

Yes

Yes

Yes (severe)

Yes

Yes‡

Yes

10,12,15,60

Vav1–/–

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

No

46–49,55, 57,85

Pkc-θ –/–

Yes

Yes

No

Yes/No

Yes

Yes

Yes

ND

ND

83,84

LATY136F

Yes

Yes

No

ND

Yes

ND

ND

ND

ND

102,103

*Modified with permission from REF. 7  (2005) Annual Reviews. ‡L.D.F. and P.L.S., unpublished observations. ERK, extracellular-signal-regulated kinase; IL-2, interleukin-2; Itk, IL-2-inducible T-cell kinase; LATY136F, linker for activation of T cells mutant in which tyrosine is replaced by phenylalanine at position 136; ND, not determined; NFAT, nuclear factor of activated T cells; Pkc-θ, protein kinase C-θ; PLC-γ, phospholipase C-γ; Rlk, resting lymphocyte kinase.

PEPTIDYLPROLYL ISOMERASE

An enzyme that converts the peptide bond that is amino terminal to proline residues between cis and trans conformations. Cyclophilin A is a peptidylprolyl isomerase that acts on a proline residue present in ITK (interleukin-2-inducible T-cell kinase). CYCLOPHILIN A

A well-known binding partner of the immunosuppressant cyclosporin. The cyclosporin– cyclophilin complex inhibits activation of calcineurin, a phosphatase that activates the NFAT (nuclear factor of activated T cells) family of transcription factors. Cyclophilin A is also a peptidylprolyl isomerase, an enzyme that can affect the activity of ITK (interleukin-2inducible T-cell kinase). ACTIVATION-INDUCED CELL DEATH

(AICD). Apoptotic cell death that results from engagement of receptors at the cell surface of a lymphocyte to control clonal expansion. Defects in AICD result in lymphoproliferative disorders. SMALL INTERFERING RNA

(siRNA). Short double-stranded RNAs of 19–23 nucleotides that induce RNA interference (RNAi), a post-transcriptional process that leads to gene silencing in a sequence-specific manner. CHEMOKINES

Small, inducibly secreted proteins that induce activation and migration of lymphocytes.

288

Chemokine-mediated signalling. Recent data from several cell types show that TEC kinases are also important for the actin reorganization that occurs after stimulation with CHEMOKINES, which interact with G-protein-coupled receptors to initiate cell polarization and migration. Treatment of several cell types with either the chemoattractant fMLP (N-formyl-methionyl-leucyl-phenylalanine) or chemokines, including CXC-chemokine ligand 12 (CXCL12; also known as SDF1α), leads to membrane recruitment of multiple TEC kinases and to phosphorylation of ITK, TEC and RLK59–62. Moreover, T cells from Itk –/– and Rlk –/–Itk –/– mice show defective actin polarization, adhesion and migration in response to many chemokines, including the widely expressed CXCL12 (REFS 59,60). Importantly, Itk –/– mice have impaired T-cell recruitment to the lungs in response to inhaled CXCL12, confirming that these defects occur in vivo59. Furthermore, Jurkat T cells that express mutant versions of ITK show defective CXCL12-induced activation of CDC42 and RAC60. Therefore, TEC kinases influence actin reorganization and cell polarization downstream of both the TCR and chemokine receptors (FIG. 3). Consequences for T-cell function. As a result of these biochemical and cellular defects, T cells from Itk –/– and Rlk –/–Itk –/– mice show multiple developmental and functional defects. These include impaired thymic selection (with decreased positive selection)8,52,63, altered ratios of CD4+ to CD8+ T cells10, and decreased proliferation of, and IL-2 production by, mature T cells8,10,16. Many of these defects have previously been attributed to impaired TCR-mediated regulation of PLC-γ and downstream transcription factors that are required for T-cell development and function. However, it should be noted that these phenotypes might also result from impaired actin polarization and integrin-mediated adhesion, which might decrease the duration of signalling in the immunological synapse. Although ITK and RLK are important intermediates in T-cell signalling, it should also be noted that mutations of these TEC kinases do not completely block either TCR- or chemokine-receptor-mediated responses. Unlike mutations that affect the more

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proximal protein kinases LCK and ZAP70 or the adaptors LAT and SLP76 (REF. 33), mutations that affect TEC kinases do not prevent TCR signalling or T-cell development; instead, they alter functional outcomes. Whether these findings are solely a consequence of functional redundancy between the TEC kinases remains unclear. Nonetheless, these findings have given rise to the view that TEC kinases are modulators or amplifiers of T-cell signalling 42,64. The development of T cells in Itk –/– and Rlk –/–Itk –/– mice has allowed the examination of mature T-cell function. Importantly, studies of T cells from these mice have revealed intriguing roles for TEC kinases in the regulation of TH-cell differentiation. RLK and ITK in TH-cell differentiation

After stimulation with antigen, naive CD4+ TH cells differentiate into two distinct subsets — TH1 and TH2 cells — which are responsible for cell-mediated and humoral immune responses, respectively65 (FIG. 4A). These subsets are defined mainly by their unique cytokine profiles. TH1 cells express interferon-γ (IFN-γ), IL-2 and lymphotoxin, whereas TH2 cells produce IL-4, IL-5, IL-9, IL-10 and IL-13. The balance of these two subsets is crucial for proper immune responses to pathogens, and conversely, imbalances between these subsets have been associated with disease states, including autoimmunity (an excess of TH1 cells) and hypersensitivity (an excess of TH2 cells). Two subset-specific transcription factors are known to be important for dictating TH-cell lineage commitment65. The transcription factor T-bet directs IFN-γ expression by CD4+ T cells and is required for differentiation into TH1 cells. GATA-binding protein 3 (GATA3) is considered to be the master regulator of differentiation into TH2 cells, as it is required to remodel chromatin at the locus that encodes IL-4, IL-5 and IL-13. Expression of these transcription factors is regulated by both cytokines and signals from the TCR. How TCR signalling regulates TH-cell differentiation remains poorly understood. Nonetheless, studies from many research groups have implicated several T-cell signalling molecules (including LCK, ERKs, JNKs and

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A T-helper-cell development

B RLK overexpression

Naive CD4+

Naive CD4+ TH cell

TH cell IL-12 IFN-γ IL-18 T-bet RLK

TH1

IFN-γ, IL-2 and lymphotoxin

ITK NFATc1 VAV1 PKC-θ

IL-4 GATA3 MAF

TH2

IFN-γ RLK

TH1

TH2

IL-4, IL-5, IL-9, IL-10 and IL-13

C ITK deficiency

D RLK and ITK deficiency

Naive CD4+ TH cell

Naive CD4+ TH cell

T-bet

T-bet? RLK

TH1

ITK

ITK NFATc1

RLK

TH2

TH1

ITK NFATc1

GATA3 (Failure to repress)

TH2

Figure 4 | RLK and ITK in T-helper-cell development. A | Multiple cytokines and transcription factors are involved in T helper (TH)-cell development. Polarizing cytokines are indicated in bold. RLK (resting lymphocyte kinase) and ITK (interleukin-2 (IL-2)inducible T-cell kinase) have been implicated in differentiation into TH1 and TH2 cells, respectively. B | RLK overexpression increases interferon-γ (IFN-γ) production and shifts T cells towards TH1-cell development. C | ITK deficiency results in defective NFATc1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1) activation, increased T-bet expression and an inability to mount an effective TH2-cell response. D | Deficiency in both RLK and ITK leads not only to defective NFATc1 activation but also to increased GATA3 (GATA-binding protein 3) levels, owing to a failure to repress GATA3 expression transiently after signalling through the T-cell receptor. TH2-cell responses remain intact, but these mice fail to mount an effective TH1-cell response. The effect of this double mutation on T-bet expression is unknown at present. Dashed lines indicate a defective pathway. Bold lines indicate exaggerated differentiation. PKC-θ, protein kinase C-θ.

PKC-θ) and TCR-ligation-induced transcription factors (including NFATs and AP1) in the regulation of TH-cellsubset polarization66. Given the role of TEC kinases in TCR-signalling pathways, it is not surprising that TEC kinases are implicated in the regulation of TH1 and TH2 cells. Intriguingly, data indicate that RLK and ITK might have distinct roles in these pathways (FIG. 4).

ANTISENSE OLIGONUCLEOTIDES

Short, gene-specific sequences of nucleic acids that are of the opposite strand (complementary) to the targeted mRNA. Classical antisense oligonucleotides target specific strands of RNA within a cell, thereby preventing translation of these RNAs.

RLK and TH1 cells. Initial studies of RLK in mice showed preferential expression of RLK by TH1-cell clones compared with TH2-cell clones67, findings that were later confirmed in primary CD4+ T cells cultured under TH1- or TH2-cell-skewing conditions30. Although mRNA encoding RLK is rapidly downregulated after TCR stimulation, Rlk mRNA and RLK protein are re-expressed by TH1 cells but not by TH2 cells30. Furthermore, TXK, the human homologue of RLK, is found in human TH1 cells but not TH2 cells 68. The link between RLK and TH1-cell function was strengthened by a study showing that overexpression of human RLK by Jurkat T cells resulted in increased production of IFN-γ, whereas IL-2 and IL-4 levels remained unchanged68 (FIG. 4B). Conversely, treatment

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of human peripheral-blood T cells with ANTISENSE OLIGONUCLEOTIDES directed against RLK decreased IFN-γ expression without altering levels of IL-2 and IL-4. Studies of RLK localization have shown that antigenreceptor stimulation leads to nuclear translocation of a high proportion of intracellular RLK molecules24. It is now recognized that a small proportion of ITK and BTK molecules also traffic to the nucleus, implying that TEC kinases might have direct nuclear effects69,70. Intriguingly, nuclear translocation of RLK was required for the induction of IFN-γ production68, indicating that RLK might be directly involved in transcriptional control of the gene encoding IFN-γ. Indeed, RLK binds a region (–53 to –39 base pairs) upstream of the transcriptional start site of the IFN-γ gene71. This DNA element is also found in the promoters of the CC-chemokine receptor 5 (CCR5) gene and the tumour-necrosis-factor gene, two TH1-cell-associated genes, to which RLK also specifically binds. As an extension of these studies, RLK was overexpressed in mice by intravenous administration of an RLK-encoding plasmid72. Splenic T cells from these mice showed increased antigen-specific IFN-γ production,

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REVIEWS again without any change in IL-2 or IL-4 expression. In addition, reductions in the level of serum IgE and the ratio of the IL-4-induced immunoglobulin isotype IgG1 to the IFN-γ-induced isotype IgG2a were observed. So, in vivo overexpression of RLK led to a shift towards a TH1-cell profile (FIG. 4B). Nonetheless, RLK-deficient mice show only minor defects in responses to T. gondii, a strong TH1-cellinducing pathogen, and they have relatively normal TH1-cell cytokine production10,15. These differences could result from compensatory mechanisms that might occur in Rlk –/– mice. Alternatively, results from the transfection studies discussed might not be specific, because the effects on overexpression or inhibition of other protein kinases were not examined in these experiments. The effects on RLK on TH-cell differentiation into TH1 cells therefore require further evaluation.

ALTERED PEPTIDE LIGANDS

Analogues that are derived from the original antigenic peptide. They commonly have aminoacid substitutions at T-cell receptor (TCR)-contact residues. TCR engagement by these altered peptide ligands usually leads to partial or incomplete T-cell activation. Some of these altered peptide ligands (antagonists) can specifically antagonize and inhibit T-cell activation that is induced by the wild-type antigenic peptide.

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ITK and TH2 cells. In contrast to RLK, which is not expressed by TH2 cells, Itk mRNA and ITK protein expression are increased during differentiation into TH2 cells compared with the levels in naive T cells30. Analysis of the Itk promoter region revealed several binding sites for the TH2-cell transcription factor GATA3, and these sites are absent in the Rlk promoter30. These two TEC kinases therefore have distinct patterns of expression in TH cells. A role for ITK in differentiation into TH2 cells is strongly supported by studies of ITK-deficient mice. Naive ITK-deficient CD4+ T cells have decreased production of IL-4 (REFS 11,30). Indeed, under conditions of low antigen dose or stimulation with ALTERED PEPTIDE –/– LIGANDS, which normally induce IL-4 production, Itk 30 T cells produce IFN-γ instead (FIG. 4C). IL-4 expression was restored by retroviral expression of ITK in ITKdeficient T cells, indicating that the defect in cytokine production is not secondary to altered development of ITK-deficient T cells11. Importantly, Itk –/– mice cannot mount effective TH2-cell responses to infection with many pathogens that are used to evaluate TH2-cell differentiation, including Nippostrongylus brasiliensis, Schistosoma mansoni and Leishmania major 11,15. For infection with L. major, the type of immune response generated depends on the genetic background of the mice73. Wild-type C57BL/6 mice produce TH1 cytokines and heal L. major lesions, whereas wild-type BALB/c mice have non-healing responses to infection with L. major that are associated with TH2-cytokine production. For ITK-deficient mice on the C57BL/6 background, the development of TH1-cell responses to L. major remained intact11. By contrast, on the BALB/c background, T cells from Itk –/– mice produced TH1 cytokines rather than TH2 cytokines in response to infection with L. major 11, which parallels findings in cell culture30. Similar results were seen for infection with S. mansoni 15. TH2-cell responses have been implicated in the pathology of allergic asthma, which is characterized by an increased number of TH2 cells in the lungs, increased TH2-cytokine production, increased mucus production in the lungs and inflammation of the airways74. In a

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mouse model of allergic asthma, Itk –/– mice have decreased IL-5 and IL-13 production, as well as reduced mucus production and T-cell infiltration in the lungs17. These findings indicate that ITK is a potential therapeutic target for asthma, an idea that has been supported by studies using two novel compounds that selectively inhibit the protein-kinase activity of ITK19. These inhibitors recapitulate the signalling defects that are observed for ITK-deficient T cells, including decreased tyrosine phosphorylation of PLC-γ and decreased mobilization of Ca2+, and one of these inhibitors significantly decreases the inflammation of the lungs that is observed after induction of allergic asthma. Consistent with a role for ITK in allergic responses, increased ITK expression has been seen in peripheralblood T cells from humans with atopic dermatitis18. Additional evidence that overactivation of ITK might be associated with increased TH2-cell responses comes from mice deficient in cyclophilin A (CYPA). These mice spontaneously develop allergic disease that is driven by TH2 cells, with concomitant increases in serum IgE levels and tissue infiltration by mast cells and eosinophils29. CYPA catalyses the isomerization of the proline residue at position 287 of ITK, which alters the conformation of the SH2 domain, promoting ITK dimerization and effectively decreasing ITK proteinkinase activity27. Although ITK activity was not examined in CYPA-deficient T cells, it would be predicted to be increased, leading to TH2-cell-subset polarization. Indeed, TH2 cells from these mice are hypersensitive to TCR stimulation, indicating that CYPA normally represses TCR signalling. CYPA-mediated regulation is unique to ITK, as other TEC kinases lack a homologous proline residue. How does ITK affect TH2-cell responses? Despite strong evidence that ITK deficiency impairs TH2-cell responses, the mechanisms by which ITK controls these responses are poorly understood. The observation that transfer of ITK-deficient CD4+ T cells to recombinationactivating gene (RAG)-deficient hosts recapitulates the T H2-cell defects indicates that the functions of ITK are at least partially intrinsic to T cells11,15. It is therefore notable that T cells from mice that lack other T-cell signalling proteins, including VAV1 and PKC-θ, also show markedly decreased levels of IL-4 production and diminished TH2-cell responses (REFS 75–77, and A. Altman, personal communication) (TABLE 2). Intriguingly, ITK, VAV1 and PKC-θ seem to be linked in TCR-signalling pathways. ITK and VAV1 regulate each other and are both required for cell polarization and recruitment of PKC-θ to the site of TCR stimulation (REFS 12,48–50,57,58, and L.D.F. and P.L.S., unpublished observations). Moreover, mature T cells that lack CD4, which also show defective TH2-cytokine production78–80, have defective immunological-synapse formation and PKC-θ recruitment81,82. Together, these phenotypic similarities raise the possibility that a pathway involving ITK, VAV1 and PKC-θ is crucial for TH2-cell development, in which recruitment of PKC-θ is a common requirement for TH2-cell responses. www.nature.com/reviews/immunol

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Table 2 | Functional phenotypes of mice deficient in T-cell signalling proteins* Genotype Defects in Defects thymocyte in TH2-cell ‡ development responses

Changes in cytokine production

Increase in serum IgG1 or IgE levels

Eosinophilia

Defects in NKT-cell development

CD4+ T cells Defects References with activated in AICD phenotype

Itk –/–

Yes

Yes

↓ IL-2, IL-4 and possibly IFN-γ §

IgG1: small IgE: 5-fold

Yes (mild)

Yes

Yes

Yes

8,10,11, 15–17,30, 52,63,105

Rlk –/–Itk –/–

Yes

No

Many are defective

IgG1: 5-fold IgE: 20-fold

ND

ND

Yes

Yes

10,15,52

Vav1–/–

Yes

Yes||

↓ IL-2 and IL-4||

ND

ND

Yes

ND

ND

46,107,119

Pkc-θ –/–

No

Yes

↓ IL-2 and IL-4

ND

ND

Yes

ND

ND

75,77,83, 84,106

Nfatc1–/–

Yes

ND

↓ IL-4 and IL-6

No

No

ND

No

No

66,87,88

Nfatc2

No

No, increased

↑ IL-4

Both increased

Yes

ND

Yes

Yes

66,98,99

Nfatc3–/–

Yes

ND

No¶

No

ND

ND

Yes

No, increased

Nfatc1–/– Nfatc2–/–

ND

ND

Many are defective

IgG1: 10-fold IgE: 500-fold

ND

ND

Yes

Yes

66,101

Nfatc2–/– Nfatc3–/–

ND

No, increased

↑ TH2 cytokines

IgG1: 400-fold IgE: 5,000-fold

Yes

ND

Yes

Yes

66,97

LATY136F

Yes

No, increased

↓ IL-2 and ↑ IL-4

IgG1: 200-fold IgE: 10,000-fold

Yes

ND

Yes

Yes

102,103

–/–

66,97

*Modified with permission from REF. 7  (2005) Annual Reviews. ‡Defects in thymocyte cellularity and/or repertoire selection. §Normal levels of interferon-γ (IFN-γ ) were observed in certain studies11,30. ||A. Altmon, personal communication. ¶Despite this, a constitutively active Nfatc3 (nuclear factor of activated T cells, cytoplasmic, calcineurindependent 3) transgene expressed by T cells blocks production of T helper 2 (TH2) cytokines. AICD, activation-induced cell death; IL, interleukin; Itk, IL-2-inducible T-cell kinase; LATY136F, linker for activation of T cells mutant in which tyrosine is replaced by phenylalanine at position 136; ND, not determined; NKT, natural killer T; Pkc-θ, protein kinase C-θ; Rlk, resting lymphocyte kinase.

It remains unclear, however, whether there is a common mechanism that controls TH2-cell development downstream of these molecules. Mutations that affect ITK, VAV1 or PKC-θ all decrease IL-2 production8–10,46,83–85 (TABLE 1), and IL-2 is required for IL-4 expression86. It seems, however, that neither diminished IL-2 production nor decreased proliferation is solely responsible for impaired TH2-cell development of Itk –/– T cells11, indicating that these cells have broader defects. Interestingly, ITK-, VAV1- and PKC-θ-deficient mice all have multiple downstream biochemical defects that are similar (TABLE 1). ITK, VAV1 and PKC-θ are all required for complete mobilization of Ca2+ and for activation of the calcium-sensitive NFAT transcription factors9–11,15,55,83. Mice deficient in NFATc1 have decreased TH2-cytokine production87,88 (TABLE 2), indicating that defective NFATc1 activation might provide a common mechanism for impaired TH2-cell responses. Whether the TH2-cell defect results from direct effects of NFATc1 on IL-4 gene expression or from secondary effects on GATA3, MAF (another IL-4-inducing transcription factor) or T-bet requires further examination. Notably, ITK-deficient CD4+ T cells show increased expression of T-bet under conditions that normally promote TH2-cell differentiation30. It remains to be shown whether altered T-bet expression is seen in T cells that lack VAV1 or PKC-θ. Finally, effects on other transcription factors need to be considered, including AP1, the activation of which is also defective in T cells that lack ITK, VAV1 or PKC-θ15,55,83,84. Moreover, although defects in nuclear factor-κB (NF-κB) activation have not been reported for ITK-deficient T cells11,15, given the role of VAV1 and

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PKC-θ in the regulation of NF-κB55,83,84 and given the influence of particular NF-κB-family members on TH2-cell differentiation89,90, this family of transcription factors should be further evaluated. Interpreting the role of TEC kinases in TH cells

Because ITK and RLK have unique structural features, it is intriguing to speculate that these differences contribute to their opposing roles in TH-cell regulation. However, examination of mice that are deficient in both ITK and RLK indicates that this dichotomy might be simplistic. Initial characterization of Rlk –/–Itk –/– mice showed that most of the TCR-signalling and cellular defects, as well as the impaired responses to T. gondii, were more severe than those observed for Itk –/– mice10 (TABLE 1). By analogy, one might expect Rlk –/–Itk –/– mice to have an even weaker response to TH2-cell-inducing stimuli than do Itk –/– mice. Surprisingly, the double-deficient mice mounted effective TH2-cell responses to infection with S. mansoni, with near normal levels of TH2-cytokine production, despite defective NFAT activation15 (FIG. 4D; TABLE 2). So, a mutation that affects RLK reverses the TH2-cell defect observed for ITK-deficient mice. The mechanism by which this rescue occurs is not clear, but several explanations have been put forward (BOX 1). Differential expression of ITK and RLK. One possible explanation for the restoration of TH2-cell responses in Rlk –/–Itk –/– mice could be that it results from the absence of the TH-cell-subset polarizing effects of both of these TEC kinases. In the absence of both RLK and ITK, perhaps TH2-cell responses develop as the default pathway. However, studies of RLK overexpression on the Itk –/–

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REVIEWS background further confound this issue. When RLK is expressed under the control of the Cd2 promoter in T cells of Itk –/– mice, TCR-stimulated Ca2+ mobilization is improved91, and defective responses to infection with S. mansoni are rescued (A. Venegas and P.L.S., unpublished observations). RLK therefore compensates for the lack of ITK in promoting an effective TH2-cell response, contradicting the idea that RLK specifically drives TH1-cell development. Instead, this finding might support the idea that differential expression of RLK and ITK in TH-cell lineages contributes to the requirement for ITK in TH2-cell responses. In the context of ITK deficiency, perhaps any TEC kinase can restore the normal balance of TH cells if it is ectopically expressed at high enough levels in developing TH2 cells. Such data also raise the question of whether ITK is required for differentiation into the TH2-cell lineage or for survival or clonal expansion of TH2 cells. Signal strength dictates TH-cell development: GATA3 regulation. Another explanation for the restoration of TH2-cell responses in Rlk –/–Itk –/– mice derives from their more severe T-cell-signalling defects. Although correlations between signal strength and TH-cell development remain controversial, studies based on the stimulation of T cells with low antigen doses or altered peptide ligands indicate that weak signals preferentially lead to TH2-cell development92–94. Rlk –/–Itk –/– mice might mount a TH2-cell response specifically because of the weak or decreased duration of signalling that occurs downstream of their TCR. But, how might this occur? Wild-type naive T cells express a low level of GATA3, which is transiently downregulated after T-cell activation. Although the mechanism of GATA3 repression is unclear, such repression might allow activated T cells to develop into either TH-cell lineage. Consistent with the idea that impaired TCR signalling leads to differentiation into TH2 cells, Rlk –/–Itk –/– T cells have defective TCR-stimulated repression of GATA3 (REF. 15), which might drive Rlk –/–Itk –/– T cells towards a TH2-cell pathway. Intriguingly, repression of GATA3 in Rlk –/–Itk –/– T cells is restored by treatment with CD3-specific antibody together with PMA (phorbol 12-myristate 13-acetate)15. Recent data show that the development of IL-4-producing cells in response to altered peptide ligands results from attenuated ERK activation and

Box 1 | Mechanisms for TH2-cell development in Rlk –/–Itk –/– mice • Absence of both polarizing kinases allows differentiation into T helper 2 (TH2) cells • Differential expression of RLK (resting lymphocyte kinase) and ITK (interleukin-2inducible T-cell kinase) in TH1 and TH2 cells • Very low signals through the T-cell receptor preferentially lead to differentiation into TH2 cells, by affecting the mobilization of Ca2+, the expression of GATA-binding protein 3 and the activation of nuclear factor of activated T cells • Effects of other cell types (such as natural killer T cells, mast cells, eosinophils and basophils) • Influence of other factors that affect T-cell function, including altered development, migration defects and co-stimulation

292

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altered AP1 complexes95. Because, in T cells, PMA activates ERK through RASGRP96, it is possible that reduced ERK activation contributes to the abnormal GATA3 regulation and the TH2-cell development seen in Rlk –/–Itk –/– mice. The effects of PMA on cytokine production by Rlk –/–Itk –/– T cells have not yet been examined. Effects of impaired NFAT activation. Altered NFAT activation might also contribute to the restoration of TH2-cell responses in Rlk –/–Itk –/– mice. Whereas Nfatc1–/– T cells fail to produce adequate levels of TH2 cytokines, T cells from mice that lack NFATc2 or both NFATc2 and NFATc3 show increased expression of cell-surface activation markers and increased TH2-cell development, and these mice show eosinophilia and increased levels of IgE and IgG1 (REFS 97–99) (TABLE 2). Interestingly, excessive nuclear accumulation of NFATc1 occurs in T cells from mice that are deficient in both NFATc2 and NFATc3 (REF. 97). A recent study has shown that low-potency TCR signals induce higher nuclear levels of NFATc1 than NFATc2, indicating that this imbalance is the cause of polarization towards TH2-cell development and transcription of Il-4 under these conditions100. Although this pattern of differential activation of NFAT-family members might not occur in Rlk –/–Itk –/– T cells15, these studies show the complex effects of NFATfamily members. Indeed, mice with haematopoietic cells deficient in NFATc1 and NFATc2 also show high levels of IgE and IgG1 in association with cell-surface markers of activated T cells, despite poor cytokine production by their T cells in vitro 101 (TABLE 2). The effects on TH-cell development of Ca2+-mobilization defects downstream of TCR signalling might therefore depend on the extent of the defect and the relative activation of distinct NFAT-family members, with more severe Ca2+-mobilization defects predisposing cells to a TH2 phenotype. Supporting this view, spontaneous T-cell activation associated with TH2-cytokine production and high levels of serum IgE were reported for mice homozygous for a mutation that affects the tyrosine residue at position 136 of LAT (LATY136F)102,103 (TABLE 2). This mutation specifically affects PLC-γ1 binding, and T cells from LATY136F mice have severely impaired TCR-stimulated Ca2+ mobilization103. Such results indicate that there might be a continuum of signals that helps to regulate TH-cell development by contributing to the balance of activation and repression of multiple transcription factors, depending on the stimulus. Indeed, although ITK-deficient animals fail to mount effective TH2-cell responses to infection, uninfected Itk –/– mice have increased IgE levels15, which implies a TH2-cell bias. A recent report shows that ITK phosphorylates T-bet on the tyrosine residue at position 525, promoting an interaction between T-bet and GATA3 that reduces GATA3 DNA binding and represses TH2-cell development104. Although these data are difficult to reconcile with the TH2-cell defects in Itk –/– animals, they might account for the TH2-cell bias of naive Itk –/– mice. Whether similar T H2-cell biases and altered cytokine patterns are present in naive Vav1–/– and Pkc-θ –/– mice is unknown. www.nature.com/reviews/immunol

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REVIEWS Other factors influencing TH-cell development. Taken together, these studies emphasize the high degree of complexity in the pathways that regulate TH1and TH2-cell polarization. Although we have mainly addressed biochemical T-cell-intrinsic signalling defects, it is still possible that other defects or effects on other cell types might contribute to the phenotypes of Rlk –/–Itk –/– mice (BOX 1). ITK-, VAV1- and PKC-θ-deficient mice all have abnormal development of natural killer T cells, which are responsible for IL-4 production early after infection105–107 (TABLE 2). These molecules are also all expressed by mast cells108–110, another important cell type that is involved in the development of asthma. The role of TEC kinases and other signalling molecules in eosinophils and basophils remains another unexplored area. Within the T-cell compartment, other factors might also contribute to the TH-cell phenotypes of mice that are deficient in TEC kinases (BOX 1). Both Itk –/– and Rlk –/–Itk –/– T cells show abnormal expression of cell-surface markers of activation and impaired AICD10,16,28,63 (TABLE 2), which is indicative of altered development or homeostasis. Both Itk –/– and Rlk –/–Itk –/– mice have impaired positive selection in the thymus. Rlk –/–Itk –/– mice also show defective negative selection, and they can positively select potentially self-reactive cells that would normally be deleted52. The involvement of these altered cell populations in TH2-cell responses remains to be evaluated. Finally, other signalling pathways might also contribute to these phenotypes. Both ITK and TEC bind, and are activated by, CD28, an important co-stimulatory molecule that is required for differentiation into TH2 cells111,112. However, both positive and negative effects of ITK have been reported, confounding interpretations of the requirement for ITK in signalling through CD28 (REFS 113,114). Other pathways that might contribute to altered TH-cell phenotypes are those downstream of chemokine receptors. Impaired migration of Itk –/– T cells to the lungs has been observed in response to allergens17, perhaps secondary to defective T-cell polarization of actin and responses to chemokines. Clearly, further studies are required to fully understand the mechanisms by which TEC kinases and the signalling molecules that they interact with help to regulate TH-cell differentiation. Potential roles for TEC in T cells

Although TEC is expressed at low levels by resting T cells, TEC expression is upregulated after T-cell activation and is higher in TH2 cells than in TH1 cells31. This observation indicates that TEC might participate in

1.

2.

3.

Thomas, J. D. et al. Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes. Science 261, 355–358 (1993). Tsukada, S. et al. Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia. Cell 72, 279–290 (1993). Vetrie, D. et al. The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein tyrosine kinases. Nature 361, 226–232 (1993).

4.

5.

effector T-cell function, and it raises the question of whether its presence allows the development of TH2 cells in Rlk –/–Itk –/– mice. Interestingly, TEC seems to have distinct localization and signalling attributes compared with other TEC kinases: TEC is localized in distinct punctate structures near the immunological synapse; overexpression of TEC, but not other TEC kinases, induces activation of NFAT and AP1 reporter genes; and only TEC is regulated by the SH2-domain-containing inositol phosphatases, SHIP1 and SHIP2 (REFS 21,31,115,116). TEC is also required for regulation of PLC-γ downstream of PKC-θ, and it is constitutively associated with PKC-θ in pre-activated T cells115. Although these data indicate that TEC might have a unique role in T cells, it remains to be determined whether TEC is involved in TH-cell development. Concluding remarks

Given the complex nature of TH-cell differentiation, the question remains whether TEC kinases are good therapeutic targets for diseases that are associated with imbalances in TH-cell subsets. For RLK, the answer is unclear — although overexpression studies or studies using antisense oligonucleotides indicate a role for RLK as a TH1-cell-inducing protein kinase, Rlk –/– animals fail to support these conclusions. Further analyses of the potential effects of RLK inhibition are required. For several reasons, ITK, however, might be an ideal therapeutic target for TH2-cell-mediated diseases, provided that the inhibitor has a high degree of specificity. First, if ITK is the main TEC kinase that is expressed by TH2 cells and if it is required for formation or maintenance of these cells, then selective inhibition of ITK would specifically affect this TH-cell lineage. Moreover, although mutations that affect ITK have relatively severe effects on TCR signalling in vitro, ITK-deficient mice have only minor defects in response to infection with viruses and have variable responses to infection with TH1-cell-inducing pathogens10,11,117. By contrast, inhibition of other molecules that are implicated in TH-cell cytokine production might have more severe effects on T-cell function, as shown by the marked immunosuppression that results from the inhibition of NFATs by cyclosporin118 and the more profound effects on T-cell development that are seen in VAV1deficient animals119. So, although the TEC-family kinases were first appreciated because of the crucial role of BTK in B-cell development and function, the T-cell-specific TEC kinases have now emerged as important modulators of T-cell function that have exciting therapeutic potential for the regulation of polarized T-cell responses.

Rawlings, D. J. et al. Mutation of unique region of Bruton’s tyrosine kinase in immunodeficient XID mice. Science 261, 358–361 (1993). de Weers, M., Mensink, R. G., Kraakman, M. E., Schuurman, R. K. & Hendriks, R. W. Mutation analysis of the Bruton’s tyrosine kinase gene in X-linked agammaglobulinemia: identification of a mutation which affects the same codon as is altered in immunodeficient xid mice. Hum. Mol. Genet. 3, 161–166 (1994).

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Smith, C. I. et al. The Tec family of cytoplasmic tyrosine kinases: mammalian Btk, Bmx, Itk, Tec, Txk and homologs in other species. Bioessays 23, 436–446 (2001). Berg, L. J., Finkelstein, L. D., Lucas, J. A. & Schwartzberg, P. L. Tec family kinases in T lymphocyte development and function. Annu. Rev. Immunol. 23, 549–600 (2005). Liao, X. C. & Littman, D. R. Altered T cell receptor signaling and disrupted T cell development in mice lacking Itk. Immunity 3, 757–769 (1995).

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Acknowledgements The authors thank R. Houghtling and members of the Schwartzberg laboratory for helpful discussions, and A. Altman for permission to cite unpublished results.

Competing interests statement The authors declare no competing financial interests.

Online links DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene BMX | BTK | GATA3 | ITK | MAF | NFATc1 | PKC-θ | RLK | T-bet | TEC | VAV1 Access to this interactive links box is free online.

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