T-Cell Signaling Regulated by the Tec Family Kinase, Itk
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T-Cell Signaling Regulated by the Tec Family
Kinase, Itk
Amy H. Andreotti1, Pamela L. Schwartzberg2, Raji E. Joseph1, and Leslie J. Berg3
1
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011
2
National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20814
3
Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts 01655
Correspondence: amyand@iastate.edu
The Tec family tyrosine kinases regulate lymphocyte development, activation, and differen-
tiation. In T cells, the predominant Tec kinase is Itk, which functions downstream of the T-cell
receptor to regulate phospholipase C-g. This review highlights recent advances in our
understanding of Itk kinase structure and enzymatic regulation, focusing on Itk protein
domain interactions and mechanisms of substrate recognition. We also discuss the role of
Itk in the development of conventional versus innate T-cell lineages, including both ab
and gd T-cell subsets. Finally, we describe the complex role of Itk signaling in effector
T-cell differentiation and the regulation of cytokine gene expression. Together, these data
implicate Itk as an important modulator of T-cell signaling and function.
he Tec family nonreceptor tyrosine kinases, that Itk is the predominant Tec kinase in T cells.
T Tec, Btk, Itk/Emt/Tsk, Rlk/Txk, and Bmx/
Etk, are expressed primarily in hematopoietic
In this review, we will cover recent findings that
highlight the critical role of Itk in T-cell signal-
cells and serve as important mediators of anti- ing and function.
gen receptor signaling in lymphocytes (Berg
et al. 2005; Felices et al. 2007; Readinger et al.
2009). The demonstration that the human STRUCTURE AND REGULATION OF
B-cell immunodeficiency, X-linked agamma- ITK ACTIVITY
globulinemia (XLA), is caused by mutations
Tec Kinase Domain Structure
in Btk first underscored the importance of this
tyrosine kinase family in lymphocyte develop- Itk (inducible T-cell kinase; also known as Emt,
ment and antigen receptor signaling (Rawlings expressed in mast cells and T lymphocytes,
et al. 1993; Thomas et al. 1993; Tsukada et al. and Tsk, T-cell-specific kinase) was cloned in
1993; Vetrie et al. 1993). T lymphocytes express the early 1990s (Siliciano et al. 1992; Gibson
three Tec kinases: Itk, Rlk and Tec. To date, only et al. 1993; Heyeck and Berg 1993; Tanaka
Itk has been found to have a clearly defined et al. 1993; Yamada et al. 1993). The domain
function in T cells, leading to the conclusion organization of Itk and related Tec kinase family
Editors: Lawrence E. Samelson and Andrey Shaw
Additional Perspectives on Immunoreceptor Signaling available at www.cshperspectives.org
Copyright # 2010 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a002287
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A.H. Andreotti et al.
members shares similarities with other tyrosine sensus sequence of an SH3 ligand. Instead of
kinase families but also reveals unique features the PH-BH region, Txk (also known as Rlk)
(Fig. 1A). Shared features include the com- contains a cysteine string motif at its amino
mon SH3-SH2-kinase cassette also found in terminus.
the Src, Csk, and Abl kinases (Williams et al.
1998; Tsygankov 2003). Four of the five Tec tyro-
Itk Activation Downstream of the TCR
sine kinases (Itk, Btk, Tec, and Bmx) contain an
amino-terminal region that includes a pleck- The Tec family kinases are involved in multiple
strin homology (PH) domain, followed by a receptor-mediated signaling pathways in many
Znþþ binding region termed the Btk homology cell types; several recent reviews have covered
(BH) motif, and (except for Bmx) a proline these in depth (Berg et al. 2005; Felices et al.
rich region (PRR) that conforms to the con- 2007; Gomez-Rodriguez et al. 2007; Mihara
A SH2-kinase
PRR linker
Itk PH-BH SH3 SH2 Kinase
B
APC
pMHC
TCR
CD4 PIP2 DAG
CD3
T Cell
2+
PIP3 PIP3 PIP3 hydrolysis O
Zn pY PI3K OH
YY YYYY –
O P O
ITAMS
pY
2+
YY YYYY pY OH
O O Ca
Lck Vav
YY Itk pY HO
OH
O P O
YY
PXXP pY Nck O 5
4 PKC Ras/GRP
YY PLCg1 –
O
O– O P O
YY
– O–
Zap-70 O P O
OH O–
SIp76 1 O OH
PXXP HO
4 O P O
O 5
O–
–
O P O
ADAP PXXP GADS
pY
O– IP3
HPK1 2+
Ca
pY
Grb2
LAT
SOS
IP3 Sensitive
2+
Actin Ca channel
Arp2/3
cdc42
WASP Actin reorganization/cell adhesion Erk 1/2 Transcription
Figure 1. Itk domain organization and it’s role in T cell signaling. (A) The domain structure of Itk. (B) Schematic
of T-cell signaling emphasizing the proximal signaling complex downstream of the TCR as described in the
text. The trio of tyrosine kinases, Zap-70, Lck, and Itk that initiate signaling following TCR engagement are
shown as gray, green, and brown circles, respectively. Arrows indicate early phosphorylation events on TCR/
pMHC recognition. The amino-terminal region of Lck binds to the cytoplasmic tail of CD4 via coordination
of Znþþ (Huse et al. 1998). Active Lck phosphorylates the ITAMs, Zap-70, and Itk, which in turn
phosphorylate their targets Slp-76, LAT (by Zap-70) and PLCg1 (by Itk). Activated PLCg1 then hydrolyzes
PIP2 (dashed arrow) to produce DAG and IP3 leading to increased calcium flux. Multiple copies of these
signaling molecules likely cooperate in the phosphorylation cascade and so stoichiometry as shown is overly
simplified. Moreover, negative regulation of the signaling cascade in the form of phosphatases, kinases and
other types of molecules also plays a significant role in T-cell function but are not represented here.
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Tec Kinase Itk in T-Cell Signaling
and Suzuki 2007; Gilfillan and Rivera 2009; Itk autophosphorylation may serve as a trigger
Koprulu and Ellmeier 2009; Prince et al. 2009; to alter protein interaction partners or locali-
Readinger et al. 2009). For Itk, significant atten- zation either before or after PLCg1 activation.
tion has been given to activation via T-cell recep- PLCg1, once activated, hydrolyzes PIP2 to
tor (TCR) stimulation (see Fig. 1B). Interaction produce the second messengers IP3 and DAG
of the TCR with peptide-MHC complexes on (Rhee 2001). Downstream consequences in-
antigen presenting cells (APC) activates the Src clude Ca2þ flux (Lewis and Cahalan 1995),
kinase Lck, leading to phosphorylation of the Erk activation (Schaeffer et al. 1999; Schaeffer
CD3 immunoreceptor tyrosine-based activation et al. 2000; Miller and Berg 2002), transcrip-
motifs (ITAMs). Zap-70 then binds to the phos- tion (Crabtree and Olson 2002), cytokine re-
phorylated ITAMs and is phosphorylated by lease (Liao and Littman 1995; Liu et al. 1998),
Lck, resulting in Zap-70 activation and subse- and actin reorganization (Labno et al. 2003),
quent phosphorylation of the adaptors LAT all of which are impaired in the absence of Itk.
and SLP-76 (Pitcher and van Oers 2003; Hout- Importantly, these downstream consequences
man et al. 2005; Au-Yeung et al. 2009; Smith- of TCR activation are not ablated by a deficiency
Garvin et al. 2009). Following activation of in Itk, but instead, are substantially reduced,
PI3K and accumulation of phosphatidylinositol leading to altered development and differentia-
(3,4,5) trisphosphate (PIP3) in the plasma mem- tion of distinct T-cell lineages, as discussed later.
brane, Itk is recruited to the membrane via its PH
domain (August et al. 1997; Ching et al. 1999;
The Itk Signaling Complex
Yang et al. 2001). There, Itk interacts with the
phosphorylated SLP-76/LAT adapter complex The Itk/SLP-76 interaction likely involves the
via Itk’s Src homology (SH3 and SH2) domains SH3 and SH2 domains of Itk targeting a
(Bubeck Wardenburg et al. 1996; Zhang et al. proline-rich stretch within SLP-76 (residues
1998; Shan and Wange 1999; Su et al. 1999; 184-195) and an adjacent phosphorylated tyro-
Bunnell et al. 2000; Ching et al. 2000; Sommers sine (Y145) in SLP-76, respectively (Su et al.
et al. 2004; Koretzky et al. 2006), permitting 1999; Bunnell et al. 2000). In an elegant knockin
phosphorylation of Itk on its activation loop mouse study, tyrosine 145 of SLP-76 was
(Y511) by Lck (Heyeck et al. 1997). replaced with the nonphosphorylatable phe-
Once activated, Itk undergoes cis autophos- nylalanine to probe the contribution of the
phorylation on Y180 in its SH3 domain (Wilcox ItkSH2/pY145 interaction to signaling (Jordan
and Berg 2003; Joseph et al. 2007a). Itk also et al. 2008). Mutation of Y145 resulted in mark-
interacts with and directly phosphorylates its edly diminished PLCg1 phosphorylation, dec-
downstream target, PLCg1 (Perez-Villar and reased Ca2þ flux, lowered phospho-ERK levels
Kanner 1999; Joseph et al. 2007c), resulting and gave rise to T cells that resembled those
in phospholipase activation (Liu et al. 1998; lacking Itk, as discussed later. This observation
Schaeffer et al. 1999; Wilcox and Berg 2003; (Jordan et al. 2008) is consistent with findings
Bogin et al. 2007). The kinetics characterizing that the Itk/SLP-76 interaction is required to
many of these early phosphorylation events activate Itk kinase activity in Jurkat cells (Bogin
have been delineated, providing an under- et al. 2007). Intriguingly, the majority of the cat-
standing of the sequential nature of signaling alytically active Itk in the cell consists of only the
following TCR engagement (Houtman et al. small fraction of Itk that is bound to SLP-76
2005). It is not known, however, whether Itk (Bogin et al. 2007). Surprisingly, the SLP-76
autophosphorylation occurs prior to, or follow- Y145F mutation did not lead to loss of the
ing, phosphorylation of PLCg1. Because auto- Itk/SLP-76 association (Jordan et al. 2008)
phosphorylation on Y180 does not affect Itk despite altered signaling. This finding suggests
catalytic activity but instead modulates binding that cooperative multivalent interactions pre-
of the Itk SH3 domain to different targets serve Itk localization in this signaling complex
(Wilcox and Berg 2003; Joseph et al. 2007a), even when a single contact site is disrupted.
Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002287 3Downloaded from http://cshperspectives.cshlp.org/ on July 26, 2021 - Published by Cold Spring Harbor Laboratory Press
A.H. Andreotti et al.
The importance of the Itk PH domain/PIP3 Tec kinases lack the carboxy-terminal regula-
interaction (Fukuda et al. 1996; August et al. tory tail that plays an important role in Src
1997) was also recently probed by inhibition regulation (Brown and Cooper 1996; Xu et al.
of phosphatidylinositol-3 kinase (PI3K) activ- 1997; Xu et al. 1999; Cowan-Jacob et al. 2005).
ity in human peripheral blood T cells (Cruz- Moreover, removal of the noncatalytic domains
Orcutt and Houtman 2009). In a manner from the Src kinase domain yields an active
similar to SLP-76 Y145F, loss of the Itk PH enzyme, whereas the isolated Itk and Btk kinase
domain/PIP3 interaction resulted in impaired domains show poor catalytic activity (Fig. 2A)
downstream signaling but did not affect the (LaFevre-Bernt et al. 1998; Hawkins and Marcy
ability of Itk and other PH domain containing 2001; Guo et al. 2006; Joseph et al. 2007b).
signaling proteins to assemble into the signaling Mutation of a well-conserved tryptophan resi-
complex (Cruz-Orcutt and Houtman 2009). due in the Itk and Btk SH2-kinase linkers
In a separate study, soluble IP4 was found to (W355 in Itk, W395 in Btk) results in a signifi-
promote binding of the Itk PH domain to cant decrease in kinase activity (Lin et al. 2005;
PIP3 (Huang et al. 2007). Reminiscent of the Guo et al. 2006; Joseph et al. 2007b) whereas
Itk/SLP-76 interaction (Bogin et al. 2007), these mutation of the corresponding tryptophan in
studies also showed that only a limited pool the Src kinase Hck leads to a dramatic increase
of Itk in the cell is capable of binding PIP3 in the activity of that kinase (LaFevre-Bernt
even in the presence of excess PIP3 (Huang et al. 1998). In this regard, Itk is similar to Csk
et al. 2007). These separate findings suggest (carboxy-terminal Src kinase), another tyrosine
that multiple interaction sites exist to properly kinase comprised of the common SH3-SH2-
localize Itk following TCR stimulation (Graef Kinase cassette and regulated in a manner
et al. 1997; Garcon and Nunes 2006; Beach opposite that of Src (Huang et al. 2009). The
et al. 2007). Furthermore, part of the function isolated Csk kinase domain shows negligible
of Itk and other proteins in the LAT/SLP-76 catalytic activity (Fig. 2A) and mutation of the
complex may be to function as adaptors and conserved tryptophan (W188) decreases Csk
stabilize proteins in the complex. Kinase-inde- activity (Lin et al. 2005). Based on similar bio-
pendent functions for Itk have been observed chemical profiles, a model for active Itk can
in the regulation of actin polymerization be developed using the available structure of
(Dombroski et al. 2005) and activation of the active Csk (Fig. 2B).
transcription factor, SRF (Hao et al. 2006); these In agreement with the established interac-
functions may be secondary to effects on re- tion between active Itk and SLP-76 (Jordan
cruitment of the guanine-nucleotide exchange et al. 2008; Bogin et al. 2007), the distance
factor Vav to this complex (Labno et al. 2003). between the SH3 and SH2 binding pockets
in the high-resolution structure of Csk closely
matches the distance between the phosphotyro-
Regulation of Itk Activity
sine motif and proline-rich region in SLP-76
The regulatory mechanisms that control the (residues 143– 195). Moreover, the Itk PRR
activity of Lck and Zap-70 during TCR signal- region adjacent to the Itk SH3 domain in the
ing have been the subject of intense investiga- model of active Itk (Fig. 2B) could be available
tion, leading to detailed insights into function for binding to an SH3 domain from an exoge-
(Palacios and Weiss 2004; Au-Yeung et al. nous signaling partner. The precise binding
2009). In contrast, a mechanistic understanding partner of the Itk PRR is not yet clear but can-
of Itk regulation remains elusive, as Itk and didates include the SH3 domains of Vav, Fyn,
other Tec kinases have, to date, resisted crystal- Src, PLCg1 or Grb2 (Yang et al. 1995; Perez-
lization in their full-length form. Comparing Villar and Kanner 1999; Bunnell et al. 2000;
the Tec and Src kinase families, it is evident Chamorro et al. 2001). Csk lacks the PH-BH
that regulatory differences must exist. In spite region of the Tec kinases and so comparative
of the shared SH3-SH2-Kinase cassette, the model building for this region of Itk becomes
4 Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002287Downloaded from http://cshperspectives.cshlp.org/ on July 26, 2021 - Published by Cold Spring Harbor Laboratory Press
Tec Kinase Itk in T-Cell Signaling
A D
SH3-SH2-kinase Full-length
PRR Itk fragment Itk
Itk PH-BH SH3 SH2 Kinase Itk Kinase * *
* *
*
* *
Csk SH3 SH2 Kinase Csk Kinase
* *
Src myristoylation SH3 SH2 Kinase Y Src Kinase Y P
*
B C E
BH
PH
N
(SH3?) PIP3 PIP3 PIP3
PIP3
PRR SH3 BH BH BH
PRRBH N N N
PH PH PH
Slp76 PH N
SH3-SH2 SH3 SH3 SH3
SH3 linker SH2
SH2 SH2 SH2
SH2
Kinase SH2-Kinase Kinase Kinase Kinase Kinase
linker
C C C C
(contains W355)
C
Figure 2. Activation/inactivation of Itk. (A) Comparison of the domain structures of Itk, Csk and Src kinases.
The domains are colored as follows: PH-BH is brown, PRR is yellow, SH3 is green, SH2 is orange, SH2-Kinase
linker and Kinase domain are blue. All three kinases share the SH3-SH2-kinase cassette but differ with respect to
resulting catalytic activity of the isolated kinase domain. Itk and Csk kinase domains alone show very poor
catalytic activity whereas the Src kinase domain readily phosphorylates substrates. (B) Model of active Itk
based on biochemical data and the high-resolution structure of Csk (PDB code: 1K9A). The SH2-Kinase
linker and SH3-SH2 linker are both arranged along the back of the small kinase lobe. Activating interactions
between the kinase domain and the SH2-kinase linker in particular are also supported by a recent structure of
Btk (Marcotte et al. 2010). SLP-76 is depicted by the black line and is shown binding to both Itk SH3 and
SH2. The PH-BH domain is placed so that its carboxy-terminus is close in space to the amino-terminus of
the SH3 domain. The intervening sequence is the PRR, which would be available in this model for binding an
SH3 domain from another protein. PH domain is shown bound to its PIP3 ligand. (C) Model of Itk adopting
the extended conformation seen for Btk in SAXS studies (Marquez et al. 2003). Of note is the fact that the
intramolecular PRR/SH3 interaction is sterically allowed and that the SH2-Kinase linker region is likely in an
extended conformation and not the active conformation shown in (B). (D) Native gel electrophoresis for the
SH3-SH2-Kinase fragment of Itk and for full length Itk showing ladders indicative of mulitmerization.
Sample concentration for both purified proteins is approximately 5 micromolar. (E) Model of Itk
multimerization based on the structure of the binary Itk SH3/SH2 complex (PDB code: 2K79). Doubled
headed arrows indicate PH domain self-association. Whether Itk multimerization occurs exclusively at the
membrane remains to be determined. It is of note that the SH3/SH2 interface defined in PDB 2K79 is
accessible in the models shown in both (B) and (C) suggesting that either configuration could multimerize.
In (B), (C) and (E) the amino and carboxy-termimi of full length Itk are indicated by N and C, respectively.
speculative. It is interesting to note however, affecting kinase activity (Joseph et al. 2007b)
that the PH-BH domain of Itk extends from (Fig. 2B).
the amino-terminus of the PRR region and,
within the context of the Csk-based model,
Mechanism of Itk Substrate Recognition
the PH-BH domain could pack between the
SH3 and SH2 domains making contact with Once activated and assembled within the
the small lobe of the kinase domain and/or the SLP-76/LAT signaling complex, Itk meets its
SH3-SH2/SH2-kinase linker regions potentially downstream substrate, PLCg1 (Liu et al. 1998;
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A.H. Andreotti et al.
Schaeffer et al. 1999; Wilcox and Berg 2003; kinase comes from small-angle X-ray scattering
Houtman et al. 2005; Bogin et al. 2007). Itk (SAXS) analysis of Btk (Marquez et al. 2003).
mediated phosphorylation of Y783 in PLCg1 These data indicate that an inactive preparation
requires a remote specificity-determining ele- of Btk (Marquez et al. 2003; Lin et al. 2009)
ment within the second SH2 domain of adopts an extended conformation in solution
PLCg1 (Joseph et al. 2007c). Mutation in the with negligible contacts between domains
PLCg1 SH2 domain that disrupts the Itk/ (Fig. 2C). Whether the fully extended con-
PLCg1 interaction results in decreased PLCg1 figuration derived from SAXS data represent
activation as measured by calcium flux in Jurkat a physiologically relevant state remains to be
T cells (Braiman et al. 2006; Min et al. 2009). determined but the available data suggest that
Earlier work also identified a TCR-regulated a conformational shift of the regulatory do-
association between Itk and PLCg1 involving mains away from the kinase domain could pro-
the SH3 domain of PLCg1 (Perez-Villar and vide one layer of negative regulation.
Kanner 1999). Although PLCg1 is the most In addition to negatively regulating the ca-
well established substrate of Itk, additional talytic domain, the configuration of the in-
candidate substrates have been described and active kinase might also serve to sequester its
include TFII-I (August 2009; Sacristan et al. ligand binding sites (Pawson and Kofler 2009).
2009), Tim-3 (van de Weyer et al. 2006) and The SH3/PXXP interaction, in particular, is
T-bet (Hwang et al. 2005); the molecular details constitutive and not controlled by transient
of Itk recognition of these substrates has not phosphorylation/dephosphorylation events. In
been elucidated. down-regulated Itk, the SH3 domain-binding
site might be masked in an intramolecular fash-
ion by the adjacent PRR (Andreotti et al. 1997;
Downregulation of Itk Kinase Activity
Laederach et al. 2003) (Fig. 2C). However, it is
Inactivation of kinase activity is an important experimentally difficult to probe the precise
regulatory feature that must appropriately bal- role of the PRR in kinase regulation because it
ance activating signals. Although few negative is also likely involved in activating interactions
regulators of Itk have been described, expression with SH3 domain-containing signaling part-
of PTEN, a lipid phosphatase, can decrease ners (Yang et al. 1995; Bunnell et al. 2000).
Itk activity by reducing PIP3 levels (Shan et al. Efforts that use deletion of the PRR region to
2000). Negative regulation of Itk by the peptidyl probe its regulatory role (Hao and August
prolyl isomerase, cyclophilin A, has been de- 2002) are therefore complicated by the dual
scribed (Brazin et al. 2002; Colgan et al. 2004). nature of the PRR.
Interestingly, another peptidyl prolyl isomerase,
Pin1 ( protein interacting with NIMA1), regu-
Regulation of Itk Activity by Multimerization
lates phosphorylation and steady state levels of
Btk (Yu et al. 2006). However, the mechanism Structural studies using different Tec kinase
of action remains poorly understood for both fragments indicate that the noncatalytic regula-
Pin1 and CypA (Joseph and Andreotti 2009; tory domains interact in an intermolecular
Mohamed et al. 2009). fashion driving multimerization in solution
As already described, the regulatory do- (Andreotti et al. 1997; Brazin et al. 2000; Hans-
mains of the Tec kinases exert a positive in- son et al. 2001; Laederach et al. 2002; Okoh and
fluence on the kinase domain in a manner Vihinen 2002; Pursglove et al. 2002; Laederach
similar to Csk (Fig. 2B) and so conformational et al. 2003; Severin et al. 2009). For Itk, multi-
rearrangements that uncouple the regulatory merization is mediated by self-association of
domains ( particularly the SH2-kinase linker the PH domain (Huang et al. 2007) and inter-
region) from the catalytic domain would down- molecular interactions between the SH3 and
regulate Itk activity. In this context, the only SH2 domains of different Itk molecules (Bra-
structural data available for any full length Tec zin et al. 2000; Severin et al. 2009). Native gel
6 Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002287Downloaded from http://cshperspectives.cshlp.org/ on July 26, 2021 - Published by Cold Spring Harbor Laboratory Press
Tec Kinase Itk in T-Cell Signaling
electrophoresis indicates that the full-length Itk observed for almost any domain arrangement
molecule, the SH3-SH2-kinase fragment, and of Itk; compact or extended. Thus, the fluores-
the Itk SH3-SH2 fragment all form multimers cence “ruler” used in these experiments is un-
(Severin et al. 2009) (Fig. 2D). The high resolu- likely to discriminate between different domain
tion structure of the Itk SH3/SH2 complex arrangements.
has been solved and provides insight into how Intermolecular fluorescence complementa-
full length Itk might self-associate (Severin tion between differentially tagged Itk molecules
et al. 2009) (Fig. 2E). Functionally, disruption is consistently observed only at the cell mem-
of the SH3/SH2 interface by mutation reduced brane and not in the cytosol (Qi et al. 2006;
oligomerization and increased Itk kinase activ- Qi and August 2009). Although this data alone
ity both in vitro and in T cells (Severin et al. does not rule out multimerization in the cyto-
2009; Min et al. 2010). Moreover, both binding sol, the spatial segregation of the stronger fluo-
experiments and structural data indicate that rescence signal to the membrane is certainly
intermolecular association of Itk is mutually interesting and suggests that Itk clusters are
exclusive with binding of SLP-76 to the SH3 at least stabilized upon or after membrane lo-
and SH2 domains (Brazin et al. 2000; Pletneva calization. Notably, Itk autophosphorylation
et al. 2006; Severin et al. 2009). Thus, down- enhances the affinity between the SH3 and
regulation of Itk activity might involve intermo- SH2 domains (Joseph et al. 2007a), possibly
lecular self-association whereas multimeriza- triggering sequestration of Itk into clusters
tion is disfavored for active, SLP-76 bound and down-regulation of kinase activity. Release
Itk. The interplay between specific clustering of Itk from the membrane, promoted by de-
of signaling molecules (Houtman et al. 2006), phosphorylation of PIP3 and the SLP-76/LAT
the formation of TCR microclusters (Bunnell complex (Shan et al. 2000; Baker et al. 2001;
et al. 2006; Seminario and Bunnell 2008) and Yang et al. 2001; Tomlinson et al. 2004), would
the specific role of Itk multimerization during then result in decreased local Itk concentration,
T-cell signaling certainly deserve continued which could cause Itk oligomers to disassociate
attention. into inactive, monomeric species in the cytosol.
A split YFP/fluorescence complementation Regardless of aggregation state, the inactive spe-
approach has been used to probe the different cies would adopt a conformation that decouples
conformational and oligomeric states of Itk the regulatory region (in particular the SH2-
in cells (Qi et al. 2006; Qi and August 2009). kinase linker) from the Itk kinase domain.
Itk that is doubly tagged at the amino- and
carboxy-termini results in fluorescence comple-
mentation in the cytosol and at the membrane. ITK REGULATION OF T-CELL FUNCTION
The authors suggest that the observed fluores-
Naı̈ve T-Cell Activation is Impaired in the
cence complementation within a single Itk mol-
Absence of Itk
ecule is indicative of a folded compact structure.
Taking the linker lengths between Itk and each The central role of Itk in the LAT-SLP-76 com-
of the split YFPs (two linkers each 40 Å) plex suggests that Itk is an important com-
and the diameter of YFP itself (30 Å) into ponent of TCR signaling. Early studies using
account, fluorescence complementation in this primary murine Itk -/- T cells showed that Itk
system would occur for distances of approxi- is required for robust T-cell activation in re-
mately 110 Å between the Itk termini. It should sponse to TCR plus costimulatory receptor sig-
be noted that the distance between the amino- naling. However, these studies also revealed that
and carboxy-termini of Btk in the fully extended Itk is not absolutely required for all responses
model derived from SAXS data is 125 Å and downstream of the TCR (Liao and Littman
given the flexibility inherent in the extended 1995; Liu et al. 1998; Schaeffer et al. 1999).
model (Marquez et al. 2003), it seems likely One aspect of this modulatory role for Itk
that fluorescence complementation would be is the partial redundancy between Itk and a
Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002287 7Downloaded from http://cshperspectives.cshlp.org/ on July 26, 2021 - Published by Cold Spring Harbor Laboratory Press
A.H. Andreotti et al.
second Tec kinase family member, Rlk. Thus, a Leishmania major infection typically seen in
combined deficiency in both Itk and Rlk results wild-type Balb/c mice and instead, mounted a
in TCR signaling defects that are significantly protective TH1 response that cleared the infec-
more profound than is seen in T cells lacking tion (Fowell et al. 1999). These results high-
Itk alone (Schaeffer et al. 1999). Overall, the lighted the importance of Itk signaling in the
message from these experiments was that Itk generation of TH2 responses, a function of Itk
functions to amplify TCR signaling, rather that has been studied in more detail both in
than as an all-or-nothing component of the vitro and in vivo.
TCR signaling pathway.
Itk Promotes TH2 Differentiation and
T-Cell Differentiation and Effector Functions Cytokine Production
are Regulated by Itk
The initial findings that Itk-deficient mice were
In spite of the relatively modest impact of the Itk unable to mount a protective TH2 response to
deficiency on naı̈ve T-cell activation, numerous infection were verified in a number of distinct
studies have shown that Itk signaling plays a infectious disease systems including Leishmania
critical role in regulating T-cell differentiation major in Itk -/- Balb/c mice, as well as Nippo-
and T-cell effector functions (Fig. 3). The first strongylus brasiliensis and Schistosoma mansoni,
of these studies showed that Itk -/- Balb/c mice two parasites that are eliminated by TH2-depen-
were unable to generate the TH2 response to dent recruitment and activation of granulocytes
T helper cell development ITK-deficiency
STAT1/4
T-bet T-bet
IFN-g IFN-g
Th1 Th1
TNF-µ TNF-µ
IL-12
LT LT
IFN-g
IL-18 RLK RLK
ITK ITK
Naive Naive
CD4+ GATA-3 CD4+
STAT6 GATA-3
TH cell TH cell
C-MAF C-MAF
IL-4 IL-4
IL-4 NFATc1 IL-5
NFATc1
ITK Th2 IL-10 ITK Th2
TGF-b IL-13
IL-6
IL-1
RORgT
RORµ RORgT
STAT3 RORµ
ITK ITK
Th17 IL-17A Th17 IL-17A
NFATc1
IL-17F IL-17F
IL-21 IL-21
IL-22 IL-22
Figure 3. Itk function in T helper cell differentiation. At the left, the three major lineages of effector T cells are
shown, along with the cytokine signals and transcription factors that regulate their differentiation from naı̈ve
CD4þ T cells. Unlike TH1 cells, which coexpress Itk and Rlk, TH2 cells express only Itk. The status of Rlk
expression in TH17 cells is currently unknown. In the absence of Itk (right panel), NFATc1 activity is greatly
reduced, leading to impaired production of IL-4 by TH2 cells and IL-17A by TH17 cells. Itk -/- TH1 cells,
which continue to express Rlk, have a more modest defect in effector cytokine production.
8 Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002287Downloaded from http://cshperspectives.cshlp.org/ on July 26, 2021 - Published by Cold Spring Harbor Laboratory Press
Tec Kinase Itk in T-Cell Signaling
(Fowell et al. 1999; Schaeffer et al. 2001). Anal- agreement with an independent set of exper-
yses of cytokine responses of the parasite-spe- iments examining T-cell-mediated airway hy-
cific T cells from these infected mice indicated perresponsiveness in Itk -/- mice (Mueller and
that Itk -/- T cells produced decreased IL-4, August 2003). In this system, in addition to
IL-5, and IL-10 (Schaeffer et al. 2001). Similarly, impaired TH2 cytokine production, T-cell
in vitro experiments showed that Itk -/- Balb/c recruitment to the lungs was also impaired fol-
CD4þ T cells stimulated under TH2-polarizing lowing inhaled antigen challenge in Itk -/- mice
conditions produced much less IL-4 than their compared to controls. Interestingly, a partial
wild-type counterparts (Fowell et al. 1999). inhibition of Itk kinase activity was able to block
These cytokine responses correlated with de- T-cell migration to the lung in this model sys-
fective TCR-induced activation of the calcium- tem, but had no effect on the priming of TH2
dependent transcription factor, NFAT, by Itk -/- effector responses in secondary lymphoid or-
T cells, providing a potential explanation for gans (Sahu et al. 2008a). These findings indicate
the reduced Il4 transcription (Fig. 3). roles for Itk in both T-cell priming and T-cell
However, it is difficult to infer from these migration, findings that are supported by studies
experiments the precise nature of the T-cell showing a role for Itk in signaling downstream of
defect resulting in impaired protection to para- chemokine receptors (Fischer et al. 2004; Take-
sitic infections. Reductions in T-cell activation, sono et al. 2004).
T-cell differentiation, T-cell migration, or elici- These in vivo experiments have been com-
tation of recall responses at the site of infection plemented by a series of in vitro experiments
could each contribute to the poor protection performed with naı̈ve Itk -/- CD4þ T cells. All
observed in Itk -/- mice. An additional compli- of these studies agreed that Itk -/- CD4þ T cells
cation is the presence of a large population of produced substantially less IL-4, IL-5, and
previously activated/memory CD4þ T cells in IL-13 than wild-type T cells, even after differen-
Itk -/- mice; global gene expression analysis has tiation in a powerful TH2 polarizing environ-
confirmed that Itk -/- CD4þ T cells are not com- ment (Fowell et al. 1999; Schaeffer et al. 2001;
parable to wild-type CD4þ T cells prior to their Miller et al. 2004; Au-Yeung et al. 2006). In these
activation (Blomberg et al. 2009), and thus, studies, the defects in effector cytokine produc-
might respond differently than naı̈ve T cells to tion were not because of impaired TH2 differen-
parasite infections. tiation, as molecular analyses indicated that
Nonetheless, useful information has been Itk -/- T cells polarized into the TH2 lineage
gleaned from direct infection of Itk -/- mice showed all of the hallmarks of bona fide TH2
with pathogenic microorganisms. One elegant effector cells (Schaeffer et al. 2001; Miller et al.
approach to evaluating T cell defects in the 2004; Au-Yeung et al. 2006). Interestingly, the
absence of Itk was taken by Au-Yeung and colle- requirement for Itk in effector cytokine produc-
agues, who crossed Balb/c Itk -/- mice to re- tion is shared by abTCRþ NKT cells specific
porter mice that express GFP under the control for a-galactosyl ceramide bound to CD1d; in
of the IL-4 promoter (Au-Yeung et al. 2006). the absence of Itk, NKT cells stimulated in
Following infection of these mice with Leishma- vivo or in vitro produce substantially less IL-4
nia major, equal numbers of CD4þGFPþ T cells or IFNg than wild-type NKT cells (Au-Yeung
were found at the sites of infection in wild-type and Fowell 2007; Felices and Berg 2008).
versus Itk -/- mice, indicating that Itk was not
required for the initiation of the TH2 response
TH1 Differentiation and Effector
or for the migration of IL-4-competent T cells
Functions are Modestly Impaired
to these sites. Instead, the effector T-cell recall
in the Absence of Itk
response at the sites of infection were impaired
in the absence of Itk, leading to reduced IL-4 Unlike the essential role for Itk in protective
production by Itk -/- T cells following restimula- immunity to helminthic parasites, Itk signaling
tion. However, these studies are not in complete in T cells is largely dispensible for protective
Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002287 9Downloaded from http://cshperspectives.cshlp.org/ on July 26, 2021 - Published by Cold Spring Harbor Laboratory Press
A.H. Andreotti et al.
CD4þ TH1 responses to intracellular pathogens. pro-inflammatory cytokines IL-17A, IL-17F,
Thus, Itk -/- mice clear infections of Leishmania IL-21 and IL-22 (Korn et al. 2009). TH17 cells
major, and are only modestly more susceptible are important for antimicrobial activity, partic-
to sublethal doses of Toxoplasma gondii than ularly in the gastrointestinal system, but are also
wild-type mice (Fowell et al. 1999; Schaeffer hallmarks of inflammation involved in multiple
et al. 1999). These observations may reflect the autoimmune disorders.
fact that IFNg production by Itk -/- CD4þ T cells Although the requirements for cytokine
is not reduced to the same extent as production signals for TH17 differentiation have been
of TH2 cytokines (Fowell et al. 1999; Schaeffer extensively examined, the contribution of TCR
et al. 2001; Miller et al. 2004; Au-Yeung et al. signaling to the regulation of TH17 cytokine
2006). Indeed, under certain conditions that production was unknown. To evaluate this
normally lead to TH2 cytokine production, question, naı̈ve CD4þ T cells from Itk -/- and
such as stimulation with altered peptide ligands, Rlk -/-Itk -/- mice were differentiated under TH1
Itk -/- CD4þ T cells produce IFN-g (Miller et al. and TH17 polarizing conditions, and cytokine
2004). A second aspect of this differential re- production evaluated by intracellular stain-
quirement for Itk in TH1 versus TH2 effector re- ing (Gomez-Rodriguez et al. 2009). Although
sponses is the interesting expression pattern of both cell types could generate large percentages
the two predominant Tec kinases in T cells, Itk of IFNg producing TH1 cells, Itk -/- and even
and Rlk (Fig. 3). Unlike naı̈ve T cells which ex- more dramatically Rlk -/-Itk -/- T cells showed
press both Itk and Rlk, TH2 effector cells lose reduced IL-17A production. These defects did
Rlk and express only Itk; in fact, the levels of not appear to be because of altered thymic
Itk are several-fold increased in TH2 cells com- development, because they could be reversed
pared to naı̈ve T cells (Miller et al. 2004). In con- by re-introduction of Itk by retroviral transduc-
trast, TH1 effector cells continue to express both tion before TH17 differentiation. Further analy-
of these Tec kinases. As a consequence, whereas ses showed decreased Il17a message in Itk -/- T
Itk -/- TH1 cells continue to express Rlk, Itk -/- cells (Fig. 3). However, surprisingly, expression
TH2 cells lack both Itk and Rlk, thus accounting of other TH17 cytokines including the closely
for the more severe functional defect observed linked Il17f gene, was relatively intact, as was
in Itk -/- TH2 responses. Corroborating this no- the expression of genes encoding the master reg-
tion, ectopic expression of Rlk in effector TH2 ulators RORgt and RORa. Although phosphor-
cells restores wild-type T cell responses in ylation of STAT3 in response to IL-6 was slightly
Itk -/- mice injected with Schistosoma mansoni attenuated in Itk-deficient T cells, re-expression
eggs or immunized to induce allergic airway of a constitutively activated STAT3 construct
hypersensitivity (Sahu et al. 2008b). did not restore normal IL-17A production.
The selective role for Itk in IL-17A produc-
tion resulted from a requirement for robust cal-
Itk Is Required for IL-17A Production in
cium signaling, leading to NFAT activation, in
TH17 Effector Cells
the transcription of the Il17a gene (Fig. 3).
In addition to the global defects in effector Thus, differentiation of Itk -/- T cells in the pres-
T-cell responses described above for TH2, and ence of anti-CD3 plus ionomycin, which can
to a lesser extent, TH1 responses, T cells lacking rescue Ca2þ mobilization in Itk-deficient T cells
Itk display more selective alterations in cytokine (Liu et al. 1998), did restore IL-17A production,
production that highlight the complex nature directly implicating defective TCR-driven
of the signaling pathways that regulate T-cell Ca2þ mobilization in this phenotype (Gomez-
effector responses. One recent example is the Rodriguez et al. 2009). Supporting this idea,
role of Itk in the regulation of cytokine produc- stimulation of wild-type T cells with decreas-
tion by TH17 cells, a recently recognized CD4 ing amounts of anti-CD3 during differen-
effector T-cell lineage that differentiates in tiation preferentially reduced expression of IL-
response to IL-6 and TGF-b and expresses the 17A, whereas leaving IL-17F relatively spared.
10 Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002287Downloaded from http://cshperspectives.cshlp.org/ on July 26, 2021 - Published by Cold Spring Harbor Laboratory Press
Tec Kinase Itk in T-Cell Signaling
Similarly, differentiation of wild-type T cells the exact mechanism of this defect awaits fur-
in the presence of low-dose CyclosporinA or ther study, these detailed investigations of Itk-
FK506, two inhibitors of Calcineurin that pre- deficient T lymphocytes have revealed another
vent NFAT activation (Winslow et al. 2003), feature of how TCR signaling can affect the dif-
also preferentially decreased IL-17A expression. ferentiation of distinct cytokine producing cells.
Conversely, expression of a constitutively active
mutant of NFATc1 (Neal and Clipstone 2003)
TCR Signaling in CD8þ T Cells Requires Itk
restored IL-17A production by Itk -/- TH17 cells,
suggesting that Itk-mediated activation of Although fewer studies have addressed the role
NFAT contributes to the regulation of IL-17A. of Itk in CD8þ T-cell responses, the conclusions
Sequence analyses showed a conserved NFAT thus far indicate that primary responses to viral
binding site located 3.5 kb upstream of the start infection are largely intact in the absence of
site of the Il17a gene and ChIP analyses showed Itk, or Itk and Rlk (Bachmann et al. 1997;
that this site was occupied in wild-type but not Atherly et al. 2006). Biochemical studies show
Itk-deficient cells differentiated under TH17 that Itk -/- and Rlk -/-Itk -/- CD8þ T cells share a
conditions. Although potential NFAT binding similar deficiency in TCR-induced PLC-g1 acti-
sites were found upstream of the Il17f gene, vation, calcium mobilization, ERK activa-
none were conserved cross-species. Nonethe- tion, and cytokine production as CD4þ T cells
less, conserved noncoding sequences in the re- lacking the Tec kinases (Atherly et al. 2006).
gion around both the linked Il17a and Il17f Nonetheless, Itk -/- mice do mount protective
genes showed epigenetic modifications sugges- immune responses to LCMV, vaccinia virus,
tive of open chromatin conformations (Wilson and VSV, and Rlk -/-Itk -/- mice to LCMV,
et al. 2009). although kinetics of viral clearance were often
These results suggest that a TCR-driven slightly delayed. Tracking of virus-specific
pathway involving Itk-mediated regulation of CD8þ T cells indicated that Itk -/- and
Ca2þ and NFAT is required for full expression Rlk -/-Itk -/- T cells were unable to expand to
of IL-17A and that this pathway distinguishes the extent seen with wild-type T cells, resulting
expression of different TH17 cytokines. These in an overall deficit in the magnitude of the
data support the growing view that there are dis- CD8þ T-cell response. This reduction in T-cell
tinct subclasses of TH17 cells that produce over- expansion could not be accounted for by
lapping sets of cytokines based upon distinct impaired CD4þ T cell help in Itk -/- mice, as
inputs. Thus, TCR signaling may be particularly adoptive transfer of wild-type LCMV-immune
important for expression of IL-17A, which is T cells (CD4þ and CD8þ) into Itk -/- mice prior
more strongly proinflammatory than IL-17F to LCMV challenge was unable to rescue the
(Yang et al. 2008; Ishigame et al. 2009). This CD8þ T-cell defects. In addition, the impaired
regulation operates during the differentiation response of Itk -/- CD8þ T-cells was not because
of TH17 cells, because the defect in IL-17A pro- of the predominant population of “innate-like”
duction is seen even upon restimulation of cells CD8þ T-cells present in these mice (see the fol-
with PMA and ionomycin (Gomez-Rodriguez lowing), as OT-1 TCR transgenic Itk -/- CD8þ
et al. 2009). NFATc1 autoregulates its own ex- T-cells, which develop as conventional naı̈ve
pression and previous data showed defective T-cells, also shared the defect seen with polyclo-
NFATc1 induction in Itk-deficient cells (Nurieva nal Itk -/- CD8þ T-cells in non-TCR transgenic
et al. 2007), perhaps accounting for the require- mice. It still remains to be determined whether
ment for Itk during TH17 differentiation. Alter- Itk -/- CD8þ T-cells can generate functional
natively, other chromatin remodeling factors memory cells. Although virus-specific T-cells
that are recruited by transcription factors, persist in Itk -/- mice well after virus has been
including the BRG proteins, may not assemble cleared from the animals, the ability of these
properly on the IL-17A locus in the absence of cells to provide protection, and to mount an
efficient NFATactivation (Berg 2009). Although effective recall response, has not yet been tested.
Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002287 11Downloaded from http://cshperspectives.cshlp.org/ on July 26, 2021 - Published by Cold Spring Harbor Laboratory Press
A.H. Andreotti et al.
This issue is complex, as defects in Itk -/- CD4þ 2007). However, Rlk -/-Itk -/- T cells also showed
T-cells could impact the generation of robust a competitive advantage compared to WT cells
memory CD8þ T-cells; thus, appropriate stud- at earlier stages ( prior to DN3), perhaps reflect-
ies will be required to distinguish CD4þ T- ing a negative signaling role for Rlk in cell
cell-intrinsic from CD8þ T-cell-intrinsic con- survival/cytokine signaling pathways.
tributions to antiviral memory responses. Defects in positive selection of thymocytes
lacking Itk have been shown using a variety of
MHC Class I and Class II-restricted TCR trans-
TEC KINASES IN T-CELL DEVELOPMENT genes (Liao and Littman 1995; Schaeffer et al.
2000; Lucas et al. 2002; Lucas et al. 2003). The
Role of Itk and Rlk in Conventional T-cell
extent of these defects depended on the sel-
Development
ecting TCR, with the weakest selecting TCRs
TCR signaling plays a critical role in the devel- showing the largest effects (Lucas et al. 2002).
opment of T cells within the thymus, where Defects in positive selection were exacerbated
the strength or duration of TCR signaling helps in Rlk -/-Itk -/- mice, providing evidence that
determine the survival, maturation, and dif- these genes are partially redundant, and sup-
ferentiation of thymocytes into mature T cells. porting the idea that decreased TCR signaling
Double negative CD42CD82 (DN) cells that reduces positive selection of conventional T
have rearranged their TCRb chains must receive cells (Schaeffer et al. 2000). Supporting partial
competent signals through the pre-TCR to redundancy of these genes, overexpression of
progress through the DN3 stage and expand Txk/Rlk partially rescued positive selection
in the DN4 to double positive (DP) transition defects in Itk -/- mice (Sommers et al. 1999).
(Ciofani and Zuniga-Pflucker 2006). At the Itk-deficient animals also show reduced
double positive stage, TCR signaling regulates or delayed negative selection in multiple TCR
cell survival and differentiation of conventional transgenic systems (Liao and Littman 1995;
T cells (Jameson et al. 1995). Thymocytes that Schaeffer et al. 2000; Lucas et al. 2002). In
receive very poor or no TCR signals undergo Rlk -/-Itk -/- mice, negative selection is more
“death by neglect.” Thymocytes that receive clearly impaired, leading to positive selection
strong TCR signals, as from agonist peptides, of normally deleted HY TCR transgenic cells in
particularly in the context of costimulation, male Rlk -/-Itk -/- mice (Schaeffer et al. 2000).
undergo negative selection and also die within These data support a model where negative sel-
the thymus. Only thymocytes that receive ection is converted to positive selection in the
weak but adequate signals will progress onto context of impaired TCR signaling and may
CD4/CD8 selection, where the duration of account for the increased SP cells in Rlk -/-Itk -/-
TCR signaling, in large part dictated by the mice.
expression patterns of the co-receptors CD4
and CD8 upon TCR engagement, will deter-
Development of Innate T-Lymphocyte
mine their ultimate lineage as CD4 or CD8
Populations
single positive (SP) cells (Bosselut 2004).
It is therefore not surprising that Itk -/- and Recent data have revealed surprising new find-
Rlk Itk -/- T cells show altered thymocyte de-
-/-
ings on thymic selection in mice deficient in
velopment (Fig. 4). Itk -/- mice have smaller Itk and Rlk (Atherly et al. 2006; Broussard
thymi and show phenotypes consistent with et al. 2006; Dubois et al. 2006; Berg 2007; Hu
impaired pre-TCR signaling. Although these et al. 2007). Although Itk -/- and Rlk -/-Itk -/-
defects are relatively subtle, competitive bone mice show decreased positive selection of con-
marrow transfers using mixtures of WT and ventional T cells using both MHC Class I and
Itk -/- bone marrow indicated that both Itk -/- Class II-restricted TCR transgenes, surprisingly,
and Rlk -/-Itk -/- cells had a selective disadvan- Itk -/- and Rlk -/-Itk -/- mice have increased num-
tage at the DN-DP transition (Lucas et al. bers of CD8 SP cells (Liao and Littman 1995;
12 Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002287Downloaded from http://cshperspectives.cshlp.org/ on July 26, 2021 - Published by Cold Spring Harbor Laboratory Press
Tec Kinase Itk in T-Cell Signaling
ab TCR Thymic
Conventional Itk-dependent
DP stromal
ab T
MHC cell (Fewer in Itk -/- mice)
SLAM receptors
DP ab TCR
DP HC ab NKT
CD1d
SLAM receptors
DN ab TCR Itk-inhibited
Innate-like
DP HC
ab T (More in Itk -/- mice)
MHC
SLAM receptors
gd TCR
DN ? gd NKT Itk-independent
gd TCR
DN ? Conventional
gd T
Figure 4. Regulation of T-cell lineages by Itk. In the thymus, progenitor T cells (CD42CD82CD32; DN) develop
into gd T cells and ab T cells. Cells developing in the ab T-cell lineage progress through an intermediate
CD4þCD8þ (DP) stage prior to their maturation into mature conventional ab T cells, CD1d-restricted ab
NKT cells and other subsets of innate-like ab T cells. Conventional ab T cells, which are selected on classical
MHC molecules expressed on thymic stromal cells, and ab NKT cells, which are selected on the MHC class
IB molecule, CD1d, expressed on hematopoietic cells (HC) and dependent on SLAM receptor signaling, are
greatly reduced in the absence of Itk. In contrast, innate-like ab T cells, which are also selected by
recognition of MHC molecules on HC and require SLAM receptor signaling, and “innate-like” gd T cells
(gd NKT) are substantially increased in number in Itk -/- mice. A third subset of cells, illustrated by
conventional gd T cells, appear unaffected by the presence versus the absence of Itk. Currently, the cell–cell
interactions required for gd T-cell development are unknown.
Schaeffer et al. 2000; Lucas et al. 2002). Further upon activation (Atherly et al. 2006; Broussard
examination of these CD8 SP populations sug- et al. 2006; Dubois et al. 2006; Hu et al. 2007).
gested that these cells do not resemble conven- These phenotypes are characteristic of memory
tional naive CD8 SP thymocytes. Instead, cells in the periphery—however, developmental
virtually all of the CD8 SP thymocytes in these studies and fetal thymic organ cultures provided
mice showed markers seen on memory or acti- evidence that Itk-deficient CD8 cells developed
vated T cells, including CD44 and CD122 (the these characteristics within the thymus (Atherly
IL-2Rb chain), and rapidly expressed cytokines et al. 2006; Broussard et al. 2006; Dubois et al.
Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002287 13Downloaded from http://cshperspectives.cshlp.org/ on July 26, 2021 - Published by Cold Spring Harbor Laboratory Press
A.H. Andreotti et al.
2006). Although genetic crosses showed a re- is not clear. Reciprocal transfers of Itk -/-b2m-/-
quirement for MHC Class I for their develop- bone marrow provided evidence that the devel-
ment, transfers of bone marrow from Itk -/- or opment of these innate phenotypes required
Rlk -/-Itk -/- mice into irradiated b2m-deficient selection on hematopoietic cells (Horai et al.
mice, which lack MHC-Class I on their radio- 2007). One possibility is that poor selection of
resistant thymic stroma, showed that Itk -/- and conventional thymocytes could permit selec-
Rlk -/-Itk -/- CD8þ thymocytes could be selected tion or expansion of cells selected on hema-
on hematopoietically derived cells (Broussard topoietic cells. However, transfer of WT bone
et al. 2006). Although these observations are marrow into irradiated b2m-deficient hosts
atypical for conventional thymocytes, these fea- prevents selection of conventional CD8þ T cells
tures are all reminiscent of NKT cells and other and yet does not give rise to a large number of
related innate T-cell lineages; thus, many innate innate CD8 cells, suggesting that development
T-cell lineages have been shown to be selected of these cells is normally suppressed. Given
on double-positive thymocytes, to express acti- the rapid production of cytokines showed by
vation or memory cell markers, and to rapidly these cells, it is likely that the generation of these
produce cytokines even in the thymus (Berg cells may be under tight control.
2007). Like NKT cells, Itk -/- and Rlk -/-Itk -/- Several theories could account for the devel-
CD8þ cells also expressed NK cell markers and opment of this population in Itk -/- mice. Itk
high levels of the transcription factor Eosmeso- could play a negative role in signaling pathways
dermin, and were dependent on IL-15 (Atherly required for the development of these lineages,
et al. 2006; Dubois et al. 2006). Moreover, like such those downstream of SLAM family recep-
NKT cells, development of these CD8 cells was tors. Alternatively, these cells might normally
dependent on SAP, a small adaptor molecule be deleted in wild-type animals—negative
that is required for signaling from the SLAM selection can occur on hematopoietic cells,
family of receptors that are expressed on hema- which express costimulatory molecules, such
topoietic cells, but not on the thymic epithe- as CD28, thought to be important in this proc-
lium (Horai et al. 2007). Together these ess (Punt et al. 1994). Alternatively, defective
characteristics suggested that Itk-deficiency led TCR signaling may make these cells more
to the preferential selection of an innate CD8 susceptible to signals from costimulatory mole-
cell population on hematopoietic cells within cules, such as SLAM family members, or cyto-
the thymus (Fig. 4). kines that may be required either for selection
Although this phenotype is primarily seen or development of these phenotypes (Atherly
in CD8 cells, there is also a small population of et al. 2006; Dubois et al. 2006; Horai et al.
CD4 cells that show these properties in Itk -/- 2007). Further analyses will provide clues to
mice. These CD44hiCD4þ cells are also depend- the regulation of these interesting lineages that
ent on SAP and express PLZF, a transcription appear to be at the boundary of innate and adap-
factor responsible for the innate characteristics tive immune responses (Berg 2007).
of NKT cells (PLS and LJB, unpublished data
and Broussard et al. 2006; Hu and August
Itk Signaling Regulates ab and gd NKT-Cell
2008; Raberger et al. 2008). Although the select-
Maturation
ing cell population for the innate type T cells in
Itk -/- mice is yet undetermined, the large num- In addition to defects in the development
ber of CD8 cells with this characteristic (and and selection of conventional naı̈ve CD4þ and
relatively low numbers of CD4 cells) suggests CD8þ T-cells, abTCRþ NKT-cell maturation
that it is likely to be DP thymocytes, which is also impaired in the absence of Itk and Rlk
express MHC Class I but not Class II, and are (Gadue and Stein 2002; Au-Yeung and Fowell
the selecting cells for NKT cells (Bendelac 1995). 2007; Felices and Berg 2008; and Fig. 5). In these
Why loss of Itk leads to the preferential Tec kinase-deficient mice, ab NKT cell num-
development of CD8 cells with this phenotype bers are substantially reduced; furthermore, of
14 Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002287Downloaded from http://cshperspectives.cshlp.org/ on July 26, 2021 - Published by Cold Spring Harbor Laboratory Press
Tec Kinase Itk in T-Cell Signaling
the cells that remain, few represent the most switching to IgE. As with the majority of gd
mature ab NKT subset characterized by high T cells, the ligand recognized by the Vg1.1/
expression of NK1.1 and CD122, and down- Vd6.3þ TCR is unknown, nor is it known
regulation of CD4. Itk -/- and Itk -/-Rlk -/- NKT whether activation of these cells requires signal-
cells also show poor responses to TCR stimula- ing through the gd TCR.
tion, producing little to no IL-4 and IFNg These findings raise several interesting
following in vivo activation. Thus, similar to issues regarding the development and signaling
conventional ab T cells, ab NKT cells require properties of gd T cells. In contrast to ab T cells,
Tec kinases for their development and function. gd T cells are not reduced in the absence of Itk;
In stark contrast to the requirements for further, the gd NKT cell population is, in fact,
abTCRþ T cells, the development of gd T cells greatly expanded. These data indicate that the
does not require Itk signaling (Fig. 4). The signals regulating gd T-cell development do
major gd T-cell populations that arise in the not require Itk, a situation comparable to that
fetus and adult are largely unchanged in Itk -/- seen for innate CD8þ ab T cells. The expansion
mice (Felices et al. 2009; C. Yin and LJB, unpub. of both the gd NKT and the innate CD8þ pop-
observ.). Indeed, overall gḋT cell numbers in ulation in Itk -/- mice may reflect the fact that the
Itk -/- mice are significantly increased compared majority of these cells normally undergo nega-
to wild-type mice, because of dramatic increases tive selection, but that their deletion is impaired
in one particular gd T-cell subset, the gd NKT in the absence of Itk.
cells (Felices et al. 2009; Qi et al. 2009). This A second interesting issue is the apparently
subset expresses the Vg1.1/Vd6.3 TCR; how- robust TCR signaling in Itk -/- gd T cells. ab T
ever, in all other regards, these gd T cells share cells lacking Itk have substantially reduced
characteristics of CD1d-specific ab NKT cells responses to TCR signaling, particularly in their
rather than traits associated with most adult- ability to synthesize effector cytokines. Itk -/- gd
derived gd T cells (Vicari et al. 1996; Azuara T cells, however, produce copious amounts of
et al. 1997; Lees et al. 2001). For instance, the cytokines in response to gd TCR stimulation.
majority of gd NKT cells express CD4, NK1.1, These data suggest that the biochemical path-
and the signature NKT cell transcription factor, ways downstream of the gd TCR are likely
PLZF (Felices et al. 2009; Kreslavsky et al. 2009). distinct from those in ab T cells. Consistent
As a result, gd NKT cells, like their ab NKT cell with this idea, mice carrying a mutant version
counterparts, produce both IFNg and TH2 of the adapter protein LAT also have few ab T
cytokines. In addition, these cells preferentially cells but greatly increased numbers of gd T cells
home to the liver, rather than to lymphoid that overproduce TH2 cytokines; further, these
organs such as the spleen (Lees et al. 2001; Feli- mice share the hyper-IgE syndrome seen in
ces et al. 2009). Itk -/- mice (Nunez-Cruz et al. 2003). This LAT
Surprisingly, this expanded population of mutant is lacking a critical tyrosine in the cyto-
gd NKT cells is responsible for a spontaneous plasmic tail that is essential for efficient TCR
hyper-IgE syndrome observed in Itk -/- mice signaling in ab T cells, reinforcing the notion
(Felices et al. 2009; Qi et al. 2009). When that aḃ and gd TCR signaling have distinct
Itk -/- mice are crossed to mice lacking gd T cells, biochemical requirements.
the elevated serum IgE and germinal center
hyperplasia seen in Itk -/- mice disappear. Itk -/-
ITK AND HUMAN DISEASE
splenic gd NKT cells constitutively express
ICOS, and readily upregulate CD40-ligand Although mutations affecting Btk were identi-
and OX40 following gd TCR stimulation in fied as the cause of X-linked Agammaglobuline-
vitro (Felices et al. 2009). These findings suggest mia 17 years ago (Rawlings et al. 1993; Thomas
that Itk -/- gd NKT cells are being activated et al. 1993; Tsukada et al. 1993; Vetrie et al.
in vivo, allowing them to provide T cell help 1993), alterations in Itk have only recently
to B cells and secrete cytokines that promote been associated with human disorders. These
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