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

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
+

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.

⁄ 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

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

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

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

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

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

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-

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-

This work was supported by National Institutes of Health
Grants AI51626, AI065566, and AI073955 (to A.A.).
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