Báo cáo khoa học: Tec family kinases: Itk signaling and the development of NKT ab and cd T cells potx

MINIREVIEW
Tec family kinases: Itk signaling and the development of
NKT ab and cd T cells
Qian Qi
1,2
, Arun Kumar Kannan
1,2,3
and Avery August
1,2
1 Department of Veterinary & Biomedical Sciences, Center for Molecular Immunology & Infectious Disease, The Pennsylvania State
University, University Park, PA, USA
2 Department of Microbiology & Immunology, Cornell University, Ithaca, NY, USA
3 Immunology & Infectious Disease Graduate Program, The Pennsylvania State University, University Park, PA, USA
Introduction
Interleukin-2-inducible T-cell kinase (Itk) is a member
of the Tec family of nonreceptor protein tyrosine
kinases which includes Rlk and Tec, and is important
for effective signaling through the T-cell receptor
(TCR) [1,2]. There are additional Tec family kinases
that signal from other receptors and have essential
functions in other cell types, and these are reviewed in
the accompanying minireviews [3]. In the absence
of Itk, there are severe defects in activation of key
signaling components including phospholipase C
(PLC)c, which results in reduced influx of Ca
2+
, and
defective activation of extracellular signal-regulated
kinase ⁄ mitogen-activated protein kinase (ERK ⁄
MAPK), with resultant reduction in the activation of
the transcription factors nuclear factor for activated

T cells (NFAT), nuclear factor kappa-light chain
enhancer of activated B cells (NFjB) and activator
protein-1 [4]. A number of studies have examined the
Keywords
development; ERK; Id3; Interleukin-4; PLC;
PLZF; SLP-76; signaling; T-bet; T cell
receptor
Correspondence
A. August, Department of Microbiology &
Immunology, C5 171 VMC, Cornell
University, Ithaca, NY 14853-6401, USA
Fax: +1 607 253 3384
Tel: +1 607 253 3400
E-mail:
Note
Q. Qi and A. K. Kannan contributed equally
to this work
(Received 31 August 2010, revised 28
October 2010, accepted 25 February 2011)
doi:10.1111/j.1742-4658.2011.08074.x
The Tec family tyrosine kinase interleukin-2 inducible T-cell kinase (Itk) is
predominantly expressed in T cells and has been shown to be critical for
the development, function and differentiation of conventional ab T cells.
However, less is known about its role in nonconventional T cells such as
NKT and cd T cells. In this minireview, we discuss evidence for a role for
Itk in the development of invariant NKT ab cells, as well as a smaller pop-
ulation NKT-like cd T cells. We discuss how these cells take what could be
the same signaling pathway regulated by Itk, and interpret it to give differ-
ent outcomes with regards to development and function.
Abbreviations

DN, double negative; ERK, extracellular signal-regulated kinase; Id3, inhibitor of DNA binding 3; IFN, interferon; IL, interleukin; i NKT, invariant
natural killer T cells; Itk, interlukin-2 inducible T-cell kinase; MAPK, mitogen-activated protein kinase; NFAT, nuclear factor for activated
T cells; NFjB, nuclear factor kappa-light chain enhancer of activated B cells; NK, natural killer cells; PLC, phospholipase C; PLZF,
promyelocytic leukemia zinc finger protein; SAP, signaling lymphocyte activating molecule-associated protein; SLP-76, Src homology
2-domain containing leukocyte protein of 76 kDa; TCR, T-cell receptor.
1970 FEBS Journal 278 (2011) 1970–1979 ª 2011 The Authors Journal compilation ª 2011 FEBS
role of Itk in T-cell development. In the absence of Itk,
there is a partial block in the development of ab T cells
and a reduced ratio of CD4 to CD8 single positive thy-
mocytes in both the thymus and periphery [5]. In addi-
tion, the absence of Itk was also found to affect positive
and negative selection of thymocytes using TCR trans-
genic mouse models, suggesting that Itk regulates the
strength of the signal emanating from the TCR during
T-cell selection [5–7]. Furthermore, combined deletion
of Itk and Rlk leads to a further reduction in the TCR
signal strength, resulting in the conversion of negative
to positive selection and a rescue of T-cell numbers in
T cell receptor transgenic mice [6].
More recently, Itk-deficient mice were reported to
have reduced development of naı
¨
ve or conventional
CD4
+
and CD8
+
T cells, and normal or increased
development of CD4
+

and CD8
+
T cells, which have
an activated memory-cell-like phenotype ([8–12],
reviewed in [13] and [14]). These cells have increased
expression of CD44 and CD122, have preformed mes-
sage for interferon (IFN)- c, and are able to rapidly
produce cytokines upon stimulation [8–12]. These cells,
also referred to as innate memory phenotype T cells or
nonconventional T cells, may develop via an indepen-
dent pathway, dependent on expression of major histo-
compatability complex molecules on bone-marrow-
derived cells. These nonconventional or innate memory
phenotype T cells share characteristics with invariant
natural killer T cells (iNKT) and cd T cells, including
the ability to rapidly produce cytokines, as well as
alternative modes of development. The data that are
accumulating suggest that the role of Itk in the devel-
opment and function of iNKT cells and cd T cells
seems to be quite different from conventional ab
T cells. This minireview focuses on this aspect of Itk,
its role in the development and function of iNKT and
NKT-like cd T cells.
Itk and i NKT cell development
NKT cells are a subset of innate T cells characterized by
their expression of the NK1.1 marker along with the ab
TCR. Although these cells carry an antigen-specific
TCR, they are characterized by their shared functions
with natural killer (NK) cells. Thus NKT cells can
directly kill target cells in an antigen-nonspecific fash-

ion, but can also respond to stimulation via their TCR
in an antigen-specific fashion. Like NK cells, they have
the ability to rapidly produce large amounts of cyto-
kines upon stimulation by ligands that interact with
either their NK receptors or their TCRs. These cells
share portions of their developmental program with
conventional T cells. iNKT are a subset of NKT cells
that largely express an invariant ab TCR. Both iNKT
and conventional T cells develop in the thymus from
T-cell progenitors derived from bone marrow, and
progress through the CD4
+
CD8
+
double-positive thy-
mocytes stage. However, iNKT cells diverge during
positive selection and, in sharp contrast to conventional
T cells, are selected to express a restricted ab TCR rep-
ertoire characterized by a semiinvariant TCR chain
formed through VDJ recombination. Although the pro-
cess is stochastic, a majority of NKT cells carry a TCR
composed of Va14–Ja18 segments, combined with either
Vb8.2 or Vb7. These cells recognize glycolipids, proto-
typically a-galactosyl ceramide (although a number of
other ligands have been identified), in the context of the
nonclassical major histocompatibility complex molecule
CD1d [15]. Because of current technical difficulties in
the isolation and analysis of other NKT cell subsets and
their comparatively lower numbers, iNKT cells repre-
sent the most widely studied NKT cell lineage.

Unlike conventional T cells, iNKT cells are selected
by CD1d expression on immature double-positive
thymocytes [15,16]. Efficient selection of iNKT cells
also depends on TCR signaling in response to cognate
antigen in the context of CD1d. Indeed, a number of
signaling molecules that lie downstream of the TCR
can affect the development of iNKT cells (for review
see [15,17]). iNKT cells pass through at least four
stages of maturation based on their surface phenotype
and expression of cytokines (Fig. 1) (reviewed in [17]).
The earliest characterized iNKT cell progenitor is
CD24
+
⁄ NK1.1
)
⁄ CD44
)
(stage 0), and these progeni-
tors can respond to interleukin (IL)-7. These cells then
downregulate the expression of CD24 as they progress
through to stage 1 (CD24
)
⁄ NK1.1
)
⁄ CD44
)
). As the
cells progress to stage 2 they upregulate CD44
(CD24
)

⁄ NK1.1
)
⁄ CD44
+
). At stages 1 and 2, these
cells undergo extensive proliferation thus expanding
the positively selected iNKT cell pool. Stage 3 marks
the final maturation that can occur in either the thy-
mus or the periphery. At this stage, most cells are
CD44
hi
⁄ NK1.1
+
and can secrete large amounts IFN-c
and IL-4 [17]. These fully mature iNKT express high
levels of the IL-15 receptor CD122 and their homeo-
stasis is regulated by IL-15. The final maturation step,
most clearly defined by the upregulation of NK1.1, is
an important checkpoint to ensure normal numbers
and frequency of iNKT cells in the periphery. This
maturation step is also clinically relevant, as it has
been implicated in ontogeny of autoimmunity induc-
tion in nonobese diabetic (NOD) mice [18,19].
The Tec family kinases that are expressed in conven-
tional T cells are also expressed in iNKT cells. Itk is
the most abundantly expressed, followed by Txk ⁄ Rlk
Q. Qi et al. Itk and i NKT and cd T cells
FEBS Journal 278 (2011) 1970–1979 ª 2011 The Authors Journal compilation ª 2011 FEBS 1971
(referred to as Txk) then Tec. All are upregulated in
the mature NK1.1

+
fraction when compared with
NK1.1
)
cells in the thymus, although the expression
levels are similar in the fractions in the periphery [20].
In the absence of Itk, there are reduced numbers of
iNKT cells in the thymus and periphery [21]. More
detailed analysis of the development of Itk-null
i NKT cells revealed that they upregulate CD44, but
fail to upregulate CD69, CD122 and NK1.1, thus fail-
ing to progress to stage 3. The absence of Txk along
with Itk results in a more severe block at the stage
2 ⁄ stage 3 transition point, suggesting that Txk may
play some compensatory role in this developmental
pathway [20]. In the absence of Itk, the splenic iNKT-
cell population is increased in the CD44
lo
⁄ NK1.1
)
⁄
CD69
)
population [20,22], which is exaggerated in the
Itk ⁄ Txk double-knockout mice [20].
In the absence of Itk, there are also increased levels
of apoptosis in peripheral iNKT cells [20]. This is sug-
gested to correlate with decreased expression of the
IL-15 receptor beta chain, CD122, which affects the
IL-15 responsiveness of these cells in the periphery.

CD122 expression is regulated in part by the transcrip-
tional factor T-bet, the absence of which also results in
a block on iNKT cell development [23,24]. Indeed,
T-bet expression is reduced in Itk-deficient iNKT cells,
and Itk may regulate the expression of T-bet in these
cells, thus regulating iNKT cell development [20].
Role of Itk in i NKT cell function
Analysis of the remaining Itk-null i NKT cells for cyto-
kine production revealed that although these cells
possess preformed mRNA for IL-4, IL-5, IL-13 and
IFN-c, they lack the capacity to translate and secrete
these cytokines upon antigenic stimulation both
in vitro and in vivo [20,22,25]. By contrast, bypassing
the TCR with the addition of 4b-phorbol 12-myristate
13-acetate and the calcium ionophore ionomycin can
rescue cytokine secretion, indicating that TCR signals
are defective for cytokine secretion in these cells in the
absence of Itk. Thus although iNKT cell development
is reduced in the absence of Itk, cells that can make it
through this pathway are functionally able to make
and secrete cytokines if full TCR signals are applied.
Thus the absence of Itk does not affect the capacity of
these cells to generate preformed cytokine mRNA and
become poised for cytokine secretion [20,22].
In conventional T cells, particularly Th1 cells, IFN-c
is predominantly regulated by T-bet, whereas IL-4 is
Fig. 1. Involvement of Itk in the development of i NKT and NKT-like cd T cells. During T-cell development in the thymus, cd T cells separate
from ab T cells during the CD4
)
CD8

)
DN thymocyte stage, although the exact separation point is unclear. Itk and SLP-76 regulate the develop-
ment of NKT like Vc1.1
+
⁄ Vd6.2 ⁄ 3
+
cd T cells, likely due to its ability to mediate TCR signal strength, regulating MAPK signaling, thus affecting
the expression of PLZF and Id3. i NKT cells arise from CD4
+
CD8
+
double-positive thymocyte precursors through positive selection and develop
through a series of developmental stages that ultimately become mature i NKT cells. Itk and Txk are involved in the final maturation of
i NKT cells, which may be through regulating of the same pathway of TCR signal strength affecting the expression of transcription factors
T-bet and PLZF, which is interpreted differently by developing i NKT cells. Dashed arrows indicate hypothesized or indirect interactions.
Itk and i NKT and cd T cells Q. Qi et al.
1972 FEBS Journal 278 (2011) 1970–1979 ª 2011 The Authors Journal compilation ª 2011 FEBS
regulated by the transcription factor GATA-3 [26].
However, in the absence of Itk, iNKT cells that do
develop express IFN-c mRNA, despite the reduced
expression of T-bet (although GATA-3 expression is
normal) [20,22]. These findings suggest that IFN-c
expression in iNKT cells may have less dependence on
T-bet. Recently, the transcription factor promyelocytic
leukemia zinc finger protein (PLZF), has been sug-
gested to be a major regulator of i NKT cell develop-
ment and function, with PLZF primarily expressed in
iNKT cells and other nonconventional T cells [27].
PLZF belongs to the BTB-zinc finger family of tran-
scription factors and in the absence of PLZF, the num-

bers of mature iNKT cells is greatly reduced [28]. The
homeostasis of these cells is also affected as a majority
of iNKT cells in PLZF-null mice accumulate to lymph
nodes, whereas the majority of iNKT cells in wild-type
mice are found in the liver [28]. These PLZF-null
iNKT cells also lack preformed mRNA for cytokines
and are defective in cytokine production following
TCR stimulation [28]. Whether Itk regulates expression
and ⁄ or function of PLZF is unclear at this time,
although there are some interesting findings along
these lines as discussed below.
Signaling by Itk in i NKT cells
Itk plays a critical role in the increase in intracellular
calcium in T cells, in part by interacting with Src
homology 2-domain containing leukocyte protein of
76 kDa (SLP-76) and regulating tyrosine phosphoryla-
tion and activation of PLC-c1 (Fig. 2) [1,2]. SLP-76 is
also critical for the development of iNKT cells, partic-
ularly the Itk binding site Y145 [29]. Thus the absence
of the Itk signaling pathway results in reduced NFAT
activation and expression of NFAT-regulated genes
[30]. NFATc1 ⁄ NFAT2 is selectively upregulated after
TCR stimulation in CD4
+
iNKT cells and this can
lead to a substantial increase in IL-4 production by
these cells [31]. This NFAT activation may be due to
Fig. 2. Signaling pathway leading to i NKT and NKT-like cd T cells regulated by Itk. Depiction of the signaling pathway used by the TCR and
modulated by Itk that results in the development of i NKT ab and cd T cells. Note that in the case of the TCR, these pathways seem to be
shared (negative regulation of PLZF, positive regulation of ERK), but lead to different developmental outcomes. Other pathways depicted

such as PKC-h, CARMA–MALT–Bcl10 and NFjB that are critical for the development of i NKT cells are depicted for comparison. Dashed
lines indicate proposed but indirect interactions.
Q. Qi et al. Itk and i NKT and cd T cells
FEBS Journal 278 (2011) 1970–1979 ª 2011 The Authors Journal compilation ª 2011 FEBS 1973
activation of the calcium calcineurin–NFAT–Erg2 axis
[32]. Although, further experiments are needed, the
well-documented regulation of the Ca
2+
response,
NFATc1 activation by Itk following TCR ligation in
conventional T cells could be conserved in iNKT cells
[1]. If conserved, this Itk-regulated signaling pathway
leading to NFAT activation could be severely compro-
mised, resulting in the observed deficiencies. Indeed, in
conventional T cells, Itk is important for the induction
of the transcription factor Egr2 (as well as Egr1 and -3),
which lies downstream of NFAT [33]. Egr2 is uniquely
critical for the development of iNKT cells, with the
block observed at a similar stage to that observed in the
absence of Itk in mice lacking this factor [20,32]. i NKT
cell development and function in NFAT-deficient mice
have not yet been analyzed, although the calcium path-
way and calcineurin is critical for the development of
these cells [32,34].
As discussed above, Itk-deficient iNKT cells express
low levels of T-bet, and Itk may regulate the expres-
sion of this critical transcription factor. Indeed, it has
been suggested that Itk regulates T-bet levels in
iNKT cells in the thymus, and that thymic egress
favors those cells that express T-bet and CD122 [20].

In addition, iNKT cell development is dependent on
signaling lymphocyte-activating molecule (SLAM),
SLAM-associated protein (SAP), Src family kinase
Fyn, PKCh, Bcl-10 and NFjB [17]. NFjB and PKCh
are dispensable for selection of conventional T cells
but critical for iNKT cell development [17]. Itk has
been shown to modulate the localization of PKCh,as
well as the activation of NFjB, and it is likely that
the signaling pathway that Itk regulates is conserved
in both conventional and iNKT cells [35,36].
Recent reports suggest that there may be some func-
tional interaction between Itk and PLZF. PLZF is
selectively upregulated in the CD4
+
CD44
hi
memory
phenotype T cells that are found in the absence of Itk,
although none of the CD8
+
subsets expressed PLZF.
These CD4
+
CD44
hi
cells have features of innate mem-
ory phenotype cells discussed above [27]. In addition,
the absence of PLZF leads to a block iNKT cell devel-
opment at the initial stages of maturation [28], and
both mice deficient in Itk and those that express a

transgene for PLZF have a developmental block in
stage 2 of iNKT cell maturation, all of which results in
a severe reduction in the number and frequency of
mature and functional iNKT cells. These seemingly
opposing results might be explained by the tight regu-
lation of PLZF expression during iNKT cell matura-
tion. Cells in stage 1 express high levels of PLZF, and
expression is decreased as the cells progress to stage 2,
with levels are greatly reduced during final maturation
to stage 3 [28]. Thus, the phenotype of PLZF-null mice
could be due to the reliance of iNKT cells on this tran-
scriptional regulator during the early stages of their
development. The constitutive expression of PLZF in
PLZF transgenic mice may lead to defective regulation
of iNKT cell development and function.
The findings reported to date favor a view that Itk
regulates TCR signals which, dependent on the T-cell
type, will have differential outcomes. Itk signals are
important for the development of naı
¨
ve or conven-
tional T cells, and are less important or not important
for the development of nonconventional of innate
memory phenotype T cells. By contrast, Itk is critical
for the development of the iNKT-cell population. It
remains to be seen if the molecular signals regulated
by Itk are conserved in all of these T cell types. We
next discuss another type of T cell whose development
is dependent on Itk below, a small subset of cd T cell
that have properties of NKT cells and express the

CD4 and NK1.1 markers.
Itk and NKT cd T-cell development
Compared with ab T cells, the cd T-cell population is
minor, comprising  5–10% of the total T cells in the
blood and lymphoid organs. Although the cell num-
bers are low in the periphery, cd T cells are more
abundant in the skin and reproductive tract (as
reviewed previously [37]). In this section, we discuss
the role of Itk in the development of peripheral cd
T cells, although Itk also plays a role in the develop-
ment of skin cd T cells [38].
The cd T-cell population contains many distinct sub-
sets which reside in different tissues, including the sec-
ondary lymphoid organs and the epithelial layers of
tissue such as the skin, intestinal epithelium and lung.
The different subsets of cd T cells express distinct cd
TCRs and develop at different times in the thymus.
Skin cd T cells, also called skin-resident intraepithelial
T lymphocytes, uniquely express Vc3 ⁄ Vd1, arise from
fetal thymic precursor at around day 13, and become
mature and migrate to the skin before birth in mice
[39]. Vc4
+
cd T cells are generated later than Vc3
+
cd
T cells in the fetal thymus and migrate to epithelial
layers of reproductive tract, lung and tongue [39,40].
By contrast, cd T cells in the secondary lymphoid
organs are only produced in the adult thymus, and

they predominantly express Vc2 and Vc1.1 along with
diverse Vd chains [41–45]. Populations of cd T cells
that can uniquely secrete specific cytokines, including
IL-17 or IL-4 have been described previously [45,46].
The IL-4 secreting population has been described as
having properties of NKT cells [47].
Itk and i NKT and cd T cells Q. Qi et al.
1974 FEBS Journal 278 (2011) 1970–1979 ª 2011 The Authors Journal compilation ª 2011 FEBS
An increasing number of studies suggest that TCR
signaling strength determines T-cell lineage commit-
ment, with stronger signals favoring cd T-cell develop-
ment, whereas weaker signals favor ab T-cell
development [48–50]. Although Itk may modulate TCR
signal strength, and one might expect that reduced sig-
nal strength received by Itk-null developing T cells
would lead to reduced cd T-cell development, previous
analysis of these cells in Itk-null mice suggested that this
population is not affected [5]. However, we and others
have recently reported that in the absence of Itk, the
percentage and numbers of cd T cells in the adult thy-
mus and secondary lymphoid organs is dramatically
increased [51,52]. Further analysis showed that this
increase is mainly due to the accumulation of a
Vc1.1 ⁄ Vd6.3 subset of cd T cells, which express high
levels of CD4 and NK1.1 [51,52]. This Vc1.1 ⁄ Vd6.3 sub-
set of cd T cells are the same cd T-cell population previ-
ously shown to secrete IL-4 and exhibit properties of
NKT cells [47,53]. It has been suggested that these
NKT cell-like Vc1.1 ⁄ Vd6.3 cd T cells may receive stron-
ger TCR signals than other cd T-cell subsets, which in

wild-type animals could lead to negative selection
during development [53]. Because Itk may act as an
amplifier in the TCR signaling, Itk deficiency may affect
the SLP-76 signaling complex and dampen the TCR-
mediated Ca
2+
influx and activation of PLCc1, weaken-
ing downstream signals, such as ERK ⁄ MAPK, NFAT
and activator protein-1 [54]. Thus the Itk deficiency
may decrease TCR signal strength and allow some
Vc1.1 ⁄ Vd6.3 cd T cells to survive negative selection.
SLP-76 is an adaptor protein that interacts with,
and is important for the activation of Itk and other
signaling proteins during TCR signaling [55–57]. It is
therefore of considerable interest that transgenic mice
expressing two SLP-76 mutants including one carry-
ing a mutant of the Itk binding site (Y145F, Y112-
128F) also exhibit significantly increased numbers of
Vc1.1 ⁄ Vd6.3 cd T cells [29,58]. Thymocytes expressing
these SLP-76 mutants have defects in TCR mediated
PLC-c1 activation, Ca
2+
influx and Erk activation,
demonstrating that TCR signal strength is weakened
in these T cells [29]. These data suggest that Itk regu-
lates the development of Vc1.1 ⁄ Vd6.3 cd T cells
through altered TCR signaling strength via SLP-76.
As discussed above, SAP is important in iNKT cell
development. SAP deficiency in these SLP-76 trans-
genic mice results in normalization (i.e. reduced

numbers compared to the SLP-76 mutants) of the
altered numbers of Vc1.1 ⁄ Vd6.3 cd T cells, suggesting
that SAP is also involved in the developmental pathway
of these Vc1.1 ⁄ Vd6.3 cd T cells [58]. Because SLP-76
and Itk interact during TCR signaling (via Y145), it is
possible that SAP also modulates the pathway regu-
lated by Itk in the development of Vc1.1 ⁄ Vd6.3 cd
T cells. Inhibitor of DNA binding 3 (Id3) is an E-pro-
tein inhibitor that is downstream of MAPK signaling
pathway. Similar to Itk-null mice, mice lacking Id3
have alterations in cd T-cell development, and also
show increased numbers of Vc1.1 ⁄ Vd6.3 cd T cells
[58–61]. PLZF, shown to regulate iNKT cell develop-
ment, has also been shown to regulate the development
and function of this Vc1.1 ⁄ Vd6.2 ⁄ 3 cd T-cell popula-
tion [58]. Itk may thus modulate TCR signals that
regulate expression of PLZF and Id3, thus affecting
development of this unique cd T-cell population.
Because the microenvironments in the fetal thymus
and adult thymus are different, the production of dis-
tinct subsets of cd T cells during different stages of
ontogeny suggests that they have distinct developmen-
tal mechanisms. We have also found that that mice
lacking Itk have significantly reduced numbers of
another unique population of cd T cells that carry the
Vc3 ⁄ Vd1 cd TCR and home to the skin, skin-resident
intraepithelial T lymphocytes, suggesting that Itk is
important for their development [38,62]. Further analy-
sis indicates that Itk regulates the migration and hom-
ing, but not maturation and homeostasis, of these cd

skin-resident intraepithelial T lymphocytes. Thus Itk
plays distinct roles in the development of different cd
T-cell subsets.
Role of Itk in NKT-cell-like function of
Vc1.1/Vd6.3 cd T cells
It has long been observed that naı
¨
ve Itk-null mice have
high levels of serum IgE despite the observed defects
in Th2 cytokine secretion from conventional and
iNKT cells. IgE production is highly dependent on
IL-4, and the CD4
+
NKT-like Vc1.1 ⁄ Vd6.3 cd T cells
can rapidly secrete IL-4 in vitro and in vivo [53,58,63].
Several published studies suggest that IL-4-secreting cd
T cells contribute to helping B cells class switch to pro-
duce IgE. Mice lacking ab T cells have normal B-cell
phenotypes, germinal center formation and production
of antibodies, particularly IgG1 and IgE, which was
suggested to be due to the IL-4 production by cd T cells
[64,65]. Human cd T cells can also induce class switch-
ing in B cells to produce IgE [66]. In an allergic asthma
model, mice lacking cd T cells had decreased production
of IgE, which were rescued by adding IL-4, suggesting
that cd T cells are important for IL-4 production and
help the production of IgE and IgG1 [67].
The CD4
+
NKT-like cd T cells observed in the Itk-

null mice largely carry the Vc1.1 ⁄ Vd6.2 ⁄ 3 TCR, and
stimulation of these cells purified from either wild-type
Q. Qi et al. Itk and i NKT and cd T cells
FEBS Journal 278 (2011) 1970–1979 ª 2011 The Authors Journal compilation ª 2011 FEBS 1975
or Itk-null mice via the TCR induces large amounts of
IL-4 [51,52]. The finding that these IL-4 producing
CD4
+
NKT-cell-like cd T cells accumulate in Itk-null
mice suggests that these cells may be responsible for
this paradoxical finding. Indeed, removing cd T cells
from the Itk-null mice results in significantly reduced
serum IgE [51,52], and transfer of these cells along
with wild-type B cells induced class switch and IgE
production in RAG-null mice [52]. These Itk-null
NKT-like cd T cells express CD40L and OX40,
costimulatory molecules that provide B-cell help, in
response to anti-TCR d stimulation [51]. Interestingly,
LAT mutant mice (Y175 ⁄ 195⁄ 235F), which have no
ab T cells but accumulate high numbers of cd T cells
in peripheral lymphoid organs, have high levels of
serum IgE and IgG1, suggesting that LAT may also
play a role in the development of similar if not the
same population of NKT-like cd T cells that secrete
IL-4 and can induce B-cell class switch [68].
One signal, many outcomes
The combination of studies on conventional ab T-cell
development, iNKT cell and cd T-cell development,
including NKT-like cd T cells, suggests that signaling
pathways regulated by Itk may be interpreted quite dif-

ferently dependent on the cell type. Itk is critical for
effective development of conventional ab T cells, the
major T-cell population that participates in the immune
response [1,13]. Similarly, signals regulated by Itk are
important for effective development of iNKT cells, but
interestingly not their primed state with preformed
cytokine message, although Itk is required for cytokine
secretion. By contrast, Itk seems to play a negative reg-
ulatory role in the development of NKT-like cd T cells
(carrying the Vc1.1 ⁄ Vd6.2 ⁄ 3 TCR), and does not affect
their ability to secrete IL-4, but does affect the ability
of other cd T-cell populations to secrete IFN-c.
What can we surmise from these findings about the
signals regulated by Itk downstream of the TCR dur-
ing the development of these various subsets of T cells?
Based on the studies to date, it is clear that the cal-
cium pathway and SLP-76 are critical mediators of
Itk. SLP-76, and particularly the Itk-binding site
within SLP-76, is critical for the development of
iNKT cells and plays a role in restraining the develop-
ment of NKT-like Vc1.1 ⁄ Vd6.2 ⁄ 3 cd T cells, perhaps
due to negative selection. Similarly, the Ras ⁄ Erk ⁄
MAPK pathway, also downstream of Itk, was previ-
ously described as not being critical for the develop-
ment of iNKT or cd T cells [69]. However, given the
more recent studies, a re-examination of the role of
Ras in the development of these cells seems to be war-
ranted. With regards to transcriptional targets of the
Itk pathway, although the spotlight has been on
NFAT, other factors are coming into focus, in particu-

lar, Egr family members (Egr1, -2 and -3), Id3 and
PLZF. However, these pathways have different effects
on iNKT cells versus NKT-like Vc1.1 ⁄ Vd6.2 ⁄ 3 cd
T cells. In iNKT cells, the pathway is required,
whereas in NKT-like Vc1.1 ⁄ Vd6.2 ⁄ 3 cd T cells, the
pathway restrains. Given that both cell types can
secrete IL-4, it is likely that the production of this
cytokine, and the T-cell types that can produce it, need
to be tightly controlled. Like iNKT cells, NKT-like
Vc1.1 ⁄ Vd6.2 ⁄ 3 cd
T cells seem to have a conserved
ligand. Although yet to be identified, this ligand (or
related ligands), may be involved in negative selection
of these cells, likely via the Itk–SLP-76–ERK ⁄ MAPK
module. Manipulating these pathways may result in
differential manipulation of these cells and thus the
immune response. Future experiments determining
whether the same signaling pathway is used differen-
tially by these different T-cell populations will be very
informative. Nevertheless, these findings have impor-
tant implications for the potential use of Itk inhibitors
in various inflammatory diseases [2].
We have recently reported that iNKT cell develop-
ment can be partially rescued by a kinase deleted
mutant of Itk, suggesting that kinase activity is only
partially required for the development of these cells.
This partial rescue correlated with rescued expression
of CD122 and T-bet, and suppression of Eomeso-
dermin [70].
Acknowledgements

This work was supported by National Institutes of Health
Grants AI51626, AI065566, and AI073955 (to A.A.).
References
1 Readinger J, Mueller K, Venegas A, Horai R &
Schwartzberg P (2009) Tec kinases regulate
T-lymphocyte development and function: new insights
into the roles of Itk and Rlk ⁄ Txk. Immunol Rev 228,
93–114.
2 Sahu N & August A (2009) ITK inhibitors in inflamma-
tion and immune-mediated disorders. Curr Top Med
Chem 9, 690–703.
3 Ellmeier W, Abramova A & Schebesta A (2011) Tec
family kinases: regulation of FceRI-mediated mast-cell
activation. FEBS J 278, 1990–2000.
4 Schwartzberg P, Finkelstein L & Readinger J (2005)
Tec-family kinases: regulators of T-helper-cell differenti-
ation. Nat Rev Immunol 5, 284–295.
Itk and i NKT and cd T cells Q. Qi et al.
1976 FEBS Journal 278 (2011) 1970–1979 ª 2011 The Authors Journal compilation ª 2011 FEBS
5 Liao X & Littman D (1995) Altered T cell receptor sig-
naling and disrupted T cell development in mice lacking
Itk. Immunity 3, 757–769.
6 Schaeffer E, Broussard C, Debnath J, Anderson S,
McVicar D & Schwartzberg P (2000) Tec family kinases
modulate thresholds for thymocyte development and
selection. J Exp Med 192, 987–1000.
7 Schaeffer E et al. (1999) Requirement for Tec kinases
Rlk and Itk in T cell receptor signaling and immunity.
Science 284, 638–641.
8 Broussard C, Fleischacker C, Horai R, Chetana M,

Venegas AM, Sharp LL, Hedrick SM, Fowlkes BJ &
Schwartzberg PL (2006) Altered development of CD8
+
T cell lineages in mice deficient for the tec kinases Itk
and Rlk. Immunity 25, 93–104.
9 Horai R, Mueller K, Handon R, Cannons J, Anderson
S, Kirby M & Schwartzberg P (2007) Requirements for
selection of conventional and innate T lymphocyte lin-
eages. Immunity 27, 775–785.
10 Hu J & August A (2008) Naive and innate memory
phenotype CD4
+
T cells have different requirements
for active Itk for their development. J Immunol 180,
6544–6552.
11 Hu J, Sahu N, Walsh E & August A (2007) Memory
phenotype CD8
+
T cells with innate function selectively
develop in the absence of active Itk. Eur J Immunol 37,
2892–2899.
12 Atherly L, Lucas J, Felices M, Yin C, Reiner S & Berg
L (2006) The Tec family tyrosine kinases Itk and Rlk
regulate the development of conventional CD8
+
T cells.
Immunity 25, 79–91.
13 Berg LJ (2007) Signalling through TEC kinases regu-
lates conventional versus innate CD8(+) T-cell develop-
ment. Nat Rev Immunol 7, 479–485.

14 Gomez-Rodriguez J, Kraus Z & Schwartzberg P(2010)
Tec family kinases Itk and Rlk ⁄ Txk in T lymphocytes:
cross-regulation of cytokine production and T-cell fates.
FEBS J 278, 1980–1989.
15 Bendelac A, Savage P & Teyton L (2007) The biology
of NKT cells. Annu Rev Immunol 25, 297–336.
16 Wei D, Lee H, Park S, Beaudoin L, Teyton L, Lehuen
A & Bendelac A (2005) Expansion and long-range dif-
ferentiation of the NKT cell lineage in mice expressing
CD1d exclusively on cortical thymocytes. J Exp Med
202, 239–248.
17 Godfrey D, Stankovic S & Baxter A (2010) Raising the
NKT cell family. Nat Immunol 11, 197–206.
18 Hammond K, Poulton L, Palmisano L, Silveira P,
Godfrey D & Baxter A (1998) T cell receptor
(TCR)CD4CD8 (NKT) thymocytes prevent insulin-
dependent diabetes mellitus in nonobese diabetic
(NOD) ⁄ Lt mice by the influence of interleukin (IL)-4
and ⁄ or IL-10. J Exp Med 187, 1047–1056.
19 Lehuen A, Lantz O, Beaudoin L, Laloux V, Carnaud C,
Bendelac A, Bach J & Monteiro R (1998) Overexpres-
sion of natural killer T cells protects V14–J281
transgenic nonobese diabetic mice against diabetes.
J Exp Med 188 , 1831–1839.
20 Felices M & Berg L (2008) The Tec kinases Itk and Rlk
regulate NKT cell maturation, cytokine production,
and survival. J Immunol 180, 3007–3018.
21 Gadue P & Stein P (2002) NK T cell precursors exhibit
differential cytokine regulation and require Itk for
efficient maturation. J Immunol 169, 2397–2406.

22 Au-Yeung B & Fowell D (2007) A key role for Itk in
both IFN gamma and IL-4 production by NKT cells.
J Immunol 179, 111–119.
23 Townsend M, Weinmann A, Matsuda J, Salomon R,
Farnham P, Biron C, Gapin L & Glimcher L (2004)
T-bet regulates the terminal maturation and
homeostasis of NK and Valpha14i NKT cells. Immunity
20, 477–494.
24 Matsuda J, Zhang Q, Ndonye R, Richardson S, Howell
A & Gapin L (2006) T-bet concomitantly controls
migration, survival, and effector functions during the
development of Valpha14i NKT cells. Blood 107, 2797–
2805.
25 Stetson D, Mohrs M, Reinhardt R, Baron J, Wang Z,
Gapin L, Kronenberg M & Locksley R (2003) Constitu-
tive cytokine mRNAs mark natural killer (NK) and
NK T cells poised for rapid effector function. J Exp
Med 198, 1069–1076.
26 Zhu J, Yamane H & Paul W (2010) Differentiation of
effector CD4 T cell populations. Annu Rev Immunol 28,
445–489.
27 Raberger J et al. (2008) The transcriptional regulator
PLZF induces the development of CD44 high memory
phenotype T cells. Proc Natl Acad Sci USA 105, 17919–
17924. Epub 2008 Nov 12.
28 Kovalovsky D, Uche OU, Eladad S, Hobbs RM, Yi W,
Alonzo E, Chua K, Eidson M, Kim HJ, Im JS et al.
(2008) The BTB-zinc finger transcriptional regulator
PLZF controls the development of invariant natural
killer T cell effector functions. Nat Immunol 9

,
1055–1064.
29 Jordan M, Smith J, Burns J, Austin J, Nichols K,
Aschenbrenner A & Koretzky G (2008) Complementation
in trans of altered thymocyte development in mice express-
ing mutant forms of the adaptor molecule SLP76. Immu-
nity 28, 359–369.
30 Fowell D, Shinkai K, Liao X, Beebe A, Coffman R,
Littman D & Locksley R (1999) Impaired NFATc
translocation and failure of Th2 development in Itk
deficient CD4 T cells. Immunity 11, 399–409.
31 Wang Z, Kusam S, Munugalavadla V, Kapur R, Brut-
kiewicz R & Dent A (2006) Regulation of Th2 cytokine
expression in NKT cells: unconventional use of Stat6,
GATA-3, and NFAT2. J Immunol 176, 880–888.
32 Lazarevic V, Zullo A, Schweitzer M, Staton T, Gallo E,
Crabtree G & Glimcher L (2009) The gene encoding early
Q. Qi et al. Itk and i NKT and cd T cells
FEBS Journal 278 (2011) 1970–1979 ª 2011 The Authors Journal compilation ª 2011 FEBS 1977
growth response 2, a target of the transcription factor
NFAT, is required for the development and maturation
of natural killer T cells. Nat Immunol 10, 306–313.
33 Miller AT & Berg LJ (2002) Defective Fas ligand
expression and activation-induced cell death in the
absence of IL-2-inducible T cell kinase. J Immunol 168,
2163–2172.
34 Oh-hora M (2009) Calcium signaling in the develop-
ment and function of T-lineage cells. Immunol Rev 231,
210–224.
35 Thuille N, Lutz-Nicoladoni C, Letschka T, Hermann-

Kleiter N, Heit I & Baier G (2009) PKCtheta and Itk
functionally interact during primary mouse CD3
+
T cell activation. Immunol Lett 126, 54–59.
36 Schaeffer E, Yap G, Lewi C, Czar M, McVicar D,
Cheever A, Sher A & Schwartzberg P (2001) Mutation
of Tec family kinases alters T helper cell differentiation.
Nat Immunol 2, 1183–1188.
37 Xiong N & Raulet D (2007) Development and selection
of gammadelta T cells. Immunol Rev 215, 15–31.
38 Xia M, Qi Q, Jin Y, Wiest D, August A & Xiong N
(2010) Differential roles of IL-2-inducible T cell kinase-
mediated TCR signals in tissue-specific localization and
maintenance of skin intraepithelial T cells. J Immunol
184, 6807–6814.
39 Jameson J, Witherden D & Havran WL (2003) T-cell
effector mechanisms: gammadelta and CD1d-restricted
subsets. Curr Opin Immunol 15, 349–353.
40 Bonneville M, Ito K, Krecko EG, Itohara S, Kappes D,
Ishida I, Kanagawa O, Janeway CA, Murphy DB &
Tonegawa S (1989) Recognition of a self major histocom-
patibility complex TL region product by gamma delta
T-cell receptors. Proc Natl Acad Sci USA 86, 5928–5932.
41 Heilig JS & Tonegawa S (1986) Diversity of murine
gamma genes and expression in fetal and adult T lym-
phocytes. Nature 322, 836–840.
42 Korman AJ, Marusic-Galesic S, Spencer D, Kruisbeek
AM & Raulet DH (1988) Predominant variable region
gene usage by gamma ⁄ delta T cell receptor-bearing cells
in the adult thymus. J Exp Med 168, 1021–1040.

43 Takagaki Y, Nakanishi N, Ishida I, Kanagawa O &
Tonegawa S (1989) T cell receptor-gamma and -delta
genes preferentially utilized by adult thymocytes for the
surface expression. J Immunol 142, 2112–2121.
44 Pereira P, Hermitte V, Lembezat MP, Boucontet L,
Azuara V & Grigoriadou K (2000) Developmentally
regulated and lineage-specific rearrangement of T cell
receptor Valpha ⁄ delta gene segments. Eur J Immunol
30, 1988–1997.
45 Jensen KD, Su X, Shin S, Li L, Youssef S, Yamasaki
S, Steinman L, Saito T, Locksley RM, Davis MM et al.
(2008) Thymic selection determines gammadelta T cell
effector fate: antigen-naive cells make interleukin-17
and antigen-experienced cells make interferon gamma.
Immunity 29, 90–100.
46 Gerber D, Azuara V, Levraud J, Huang S, Lembezat
M & Pereira P (1999) IL-4-producing gamma delta
T cells that express a very restricted TCR repertoire are
preferentially localized in liver and spleen. J Immunol
163, 3076–3082.
47 Azuara V, Levraud J, Lembezat M & Pereira P (1997)
A novel subset of adult gamma delta thymocytes that
secretes a distinct pattern of cytokines and expresses a
very restricted T cell receptor repertoire. Eur J Immunol
27, 544–553.
48 Hayes SM, Li L & Love PE (2005) TCR signal strength
influences alphabeta ⁄ gammadelta lineage fate. Immunity
22, 583–593.
49 Haks MC, Lefebvre JM, Lauritsen JP, Carleton M,
Rhodes M, Miyazaki T, Kappes DJ & Wiest DL (2005)

Attenuation of gammadeltaTCR signaling efficiently
diverts thymocytes to the alphabeta lineage. Immunity
22, 595–606.
50 Kreslavsky T, Garbe AI, Krueger A & von Boehmer H
(2008) T cell receptor-instructed alphabeta versus gam-
madelta lineage commitment revealed by single-cell
analysis. J Exp Med 205, 1173–1186.
51 Felices M, Yin CC, Kosaka Y, Kang J & Berg LJ
(2009) Tec kinase Itk in gammadeltaT cells is pivotal
for controlling IgE production in vivo. Proc Natl Acad
Sci USA 106, 8308–8313.
52 Qi Q, Xia M, Hu J, Hicks E, Iyer A, Xiong N &
August A (2009) Enhanced development of CD4
+
gam-
madelta T cells in the absence of Itk results in elevated
IgE production. Blood 114, 564–571.
53 Kreslavsky T et al. (2009) TCR-inducible PLZF tran-
scription factor required for innate phenotype of a sub-
set of gammadelta T cells with restricted TCR diversity.
Proc Natl Acad Sci USA 106, 12453–12458.
54 Berg LJ, Finkelstein LD, Lucas JA & Schwartzberg PL
(2005) Tec family kinases in T lymphocyte development
and function. Annu Rev Immunol 23, 549–600.
55 Bunnell SC, Diehn M, Yaffe MB, Findell PR, Cantley
LC & Berg LJ (2000) Biochemical interactions integrat-
ing Itk with the T cell receptor-initiated signaling
cascade. J Biol Chem 275, 2219–2230.
56 Qi Q & August A (2007) Keeping the (kinase) party
going: SLP-76 and ITK dance to the beat. Sci STKE

2007, pe39.
57 Bogin Y, Ainey C, Beach D & Yablonski D (2007)
SLP-76 mediates and maintains activation of the Tec
family kinase ITK via the T cell antigen receptor-
induced association between SLP-76 and ITK. Proc
Natl Acad Sci USA 104 , 6638–6643.
58 Alonzo ES, Gottschalk RA, Das J, Egawa T,
Hobbs RM, Pandolfi PP, Pereira P, Nichols KE,
Koretzky GA, Jordan MS et al. (2010) Development of
promyelocytic zinc finger and ThPOK-expressing innate
gamma delta T cells is controlled by strength of TCR
signaling and Id3. J Immunol 184, 1268–1279.
Itk and i NKT and cd T cells Q. Qi et al.
1978 FEBS Journal 278 (2011) 1970–1979 ª 2011 The Authors Journal compilation ª 2011 FEBS
59 Verykokakis M, Boos M, Bendelac A, Adams E, Pere-
ira P & Kee B (2010) Inhibitor of DNA binding 3 limits
development of murine slam-associated adaptor pro-
tein-dependent ‘innate’ gammadelta T cells. PLoS ONE
5, e9303.
60 Lauritsen JP, Wong GW, Lee SY, Lefebvre JM,
Ciofani M, Rhodes M, Kappes DJ, Zu´ n
˜
iga-Pflu
¨
cker JC
& Wiest DL (2009) Marked induction of the helix-loop-
helix protein Id3 promotes the gammadelta T cell fate
and renders their functional maturation Notch
independent. Immunity 31, 565–575.
61 Ueda-Hayakawa I, Mahlios J & Zhuang Y (2009) Id3

restricts the developmental potential of gamma delta
lineage during thymopoiesis. J Immunol 182, 5306–
5316.
62 Xiong N, Kang C & Raulet D (2004) Positive selection
of dendritic epidermal gammadelta T cell precursors in
the fetal thymus determines expression of skin-homing
receptors. Immunity 21, 121–131.
63 Wen L, Barber DF, Pao W, Wong FS, Owen MJ &
Hayday A (1998) Primary gamma delta cell clones can
be defined phenotypically and functionally as Th1 ⁄ Th2
cells and illustrate the association of CD4 with Th2
differentiation. J Immunol 160, 1965–1974.
64 Wen L et al. (1994) Immunoglobulin synthesis and
generalized autoimmunity in mice congenitally
deficient in alpha beta(+) T cells. Nature 369,
654–658.
65 Wen L et al. (1996) Germinal center formation, immu-
noglobulin class switching, and autoantibody produc-
tion driven by ‘non alpha ⁄ beta’ T cells. J Exp Med 183,
2271–2282.
66 Horner AA, Jabara H, Ramesh N & Geha RS (1995)
gamma ⁄ delta T lymphocytes express CD40 ligand and
induce isotype switching in B lymphocytes. J Exp Med
181, 1239–1244.
67 Zuany-Amorim C, Ruffie C, Haile S, Vargaftig BB,
Pereira P & Pretolani M (1998) Requirement for gam-
madelta T cells in allergic airway inflammation. Science
280, 1265–1267.
68 Nun
˜

ez-Cruz S, Aguado E, Richelme S, Chetaille B,
Mura AM, Richelme M, Pouyet L, Jouvin-Marche E,
Xerri L, Malissen B et al. (2003) LAT regulates
gammadelta T cell homeostasis and differentiation. Nat
Immunol 4, 999–1008.
69 Alberola-Ila J, Hogquist K, Swan K, Bevan M &
Perlmutter R (1996) Positive and negative selection
invoke distinct signaling pathways. J Exp Med 184, 9–18.
70 Qi Q, Xia M, Bai Y, Yu S, Cantorna M & August A
(2011) Interleukin-2-inducible T cell kinase (Itk)
network edge dependence for the maturation of iNKT
cell. J Biol Chem 286, 138–146.
Q. Qi et al. Itk and i NKT and cd T cells
FEBS Journal 278 (2011) 1970–1979 ª 2011 The Authors Journal compilation ª 2011 FEBS 1979