Hindawi Publishing Corporation
Clinical and Developmental Immunology
Volume 2011, Article ID 294968, 9 pages
MechanismsThat Regulate PeripheralImmuneResponsesto
School of Health Sciences, University of Notre Dame Australia, 19 Mouat Street, Fremantle, WA 6959, Australia
Correspondence should be addressed to Gerard F. Hoyne, email@example.com
Received 16 January 2011; Accepted 16 February 2011
Academic Editor: Aziz Alami Chentoufi
Copyright © 2011 Gerard F. Hoyne. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The immune system must balance the need to maintain a diverse repertoire of lymphocytes to be able to fight infection with the
need to maintain tolerance to self-proteins. The immune system places strict regulation over the ability of T cells to produce the
major T cell growth factor interleukin 2 as this cytokine can influence a variety of immune outcomes. T cells require the delivery
of two signals, one through the antigen receptor and a second through the costimulatory receptor CD28. The immune system uses
a variety of E3 ubiquitin ligases to target signaling proteins that function downstream of the TCR and CD28 receptors. Mutations
in these E3 ligases can lead to a breakdown in immune tolerance and development of autoimmunity. This paper will examine the
role of a range of E3 ubiquitin ligases and signaling pathways that influence the development of T-cell effector responses and the
development of organ-specific autoimmune diseases such as type 1 diabetes.
The immune system has evolved to protect the body from
infectious pathogens through both innate and adaptive
immune responses. The adaptive immune response is built
upon a diverse repertoire of antigen-specific T and B lym-
phocytes that learn to distinguish between self- from non-
self- antigens during their differentiation in the thymus and
bone marrow, respectively. Central tolerance is established
in the thymus by the elimination of autoreactive thymocytes
that display a TCR with high affinity for self-peptide/MHC
complexes . Despite the relative efficiency of clonal
deletion not all tissue-specific antigens are expressed in the
thymus and thus a small proportion of autoreactive T cells
can escape thymic deletion, complete their maturation and
enter the peripheral circulation. The immune system has
multiple checkpoints in place to limit the activation and
last decade has led to an improved understanding of some
of these checkpoints involved in peripheral regulation of the
Autoimmunity arises following a failure in either central
or peripheral tolerance mechanisms. In the periphery the
immune system has a range of mechanisms available that
control the fate of autoreactive T cells, including immune
privilege, immune ignorance, activation-induced cell death,
clonal anergy, and immune suppression-mediated by regula-
tory T (Treg) cells [3–5]. Autoimmune diseases are classified
as organ-specific or systemic depending on the source of the
autoantigens. Type 1 diabetes (T1D) is an example of an
in tolerance in both CD4+ and CD8+ T cells and B cells
that express antigen receptors specific for proteins derived
from the islets of Langerhans in the pancreas. Some of the
key target autoantigens in T1D include insulin, GAD65,
and insulin adenoma 2 (IA2) protein. Systemic autoimmune
diseases are typified by systemic lupus erythematosus (SLE)
or lupus and are due to the generation of high-affinity anti-
self-antibodies specific for ubiquitous cellular proteins or
Type 1 diabetes arises as a result of a breakdown of
destruction of the pancreatic beta cells and loss of insulin
secretion that is mediated by CD4+ and CD8+ T cells .
Patients can produce anti-insulin antibodies indicating that
T1D reflects a generalized breakdown in immunological
tolerance that allows islet-reactive CD4+ Th cells to provide
help to autoreactive B cells.
2Clinical and Developmental Immunology
It was originally thought that clonal anergy and regula-
tory T cells were entirely distinct mechanisms that are used
to control peripheral immune responses. However, studies
in recent years have uncovered a remarkable overlap with
the mechanisms of clonal anergy and also the generation
of regulatory T cells in the periphery. In particular the
discussion will examine the group of proteins known as
ubiquitin ligases and how these proteins have emerged as
an important class of negative regulators of the immune
response not only in animals but also in humans. In addition
I will examine how signaling through the AKT/mammalian
target of rapamycin (mTOR) pathway has important roles
in balancing the choice between immunity and suppres-
Naive T cells remain in a quiescent state as they circulate
through secondary lymphoid tissues, the blood and lymph,
and in the absence of an antigenic signal rely on survival
signals transmitted via growth factor receptors (e.g., IL-7R,
IL-2 because they recruit a number of different nuclear
repressor proteins to the Il2 gene locus (e.g., Ikaros, p50 NF-
κB dimers, Blimp1, Tob, and Smad proteins) that mediate
epigenetic modification to repress the Il2 gene transcription
T-cell activation is dependent on the delivery of two
separate signals. Signal one is mediated through the TCR
and signal two through the costimulatory receptor CD28 in
response to binding of its ligands CD80/CD86 which leads
to phosphorylation of a number of intracellular signaling
pathways including phospholipase γ-1, protein kinase-θ,
MAPK, JNK, PI3K, and Iκ-B kinase (IKK) that leads to
the recruitment of transcription factors (e.g., NFAT, AP-
1, and NF-κB) critical to the transcription of the Il2 gene
. IL-2 is a multifunctional cytokine that plays a role
in T-cell mitogenesis stimulating growth of activated T
cells via paracrine and autocrine signalling through the
IL-2 receptor (IL-2R) . It can promote Th1 and Th2
cell differentiation; it is required for the generation of
CD8+ T cell memory responses and plays a crucial role
in the maintenance and homeostasis of Treg cells in the
periphery. The delivery of a costimulatory signal during
T cell activation leads to derepression of the Il2 gene,
and Il2 mRNA transcripts become stabilized and translated
into protein to drive T cell mitogenesis. Given the role
that IL-2 has in coordinating the proliferative response
of effector T cells, it is not surprising that the immune
system has placed Il2 gene transcription under tight regu-
lation in an effort to restrict the inappropriate activation
of this gene in na¨ ıve T cells in vivo and to limit the
potential for a breakdown in immune tolerance and for
autoimmunity. Therefore the secretion of IL-2 following T-
cell activation represents an important checkpoint that can
determine the outcome between immunity and tolerance
Ligation of TCR on T cells in the absence of CD28 costim-
ulation leads to the development of clonal anergy which is
and the IKK pathways and results in reduced activity of
the nuclear factors AP-1 and NF-κB and deficient IL-2 gene
cells . The original studies performed on mouse Th1 cell
clones in vitro showed that ligation of TCR in the absence
of CD28 costimulation leads to a hyporesponsive state and
a failure to secrete IL-2 leading to an abortive proliferative
response . It is also possible to induce anergy in na¨ ıve
CD4+ and CD8+ T cells in vivo that is identified by a failure
of these cells to secrete IL-2 and inflammatory cytokines,
for example, TNF-α and IFN-γ upon restimulation with
antigen. Around the same time, human CD4+ T cells were
also shown to be subject to clonal anergy in vitro through
cognate presentation of peptide between MHCII expressing
T cells, (i.e., T-T presentation in the absence of APCs)
. The induction of anergy can also be induced by
artificially elevating calcium levels which leads to activation
of calcineurin and NFAT activity in the cells [17, 18]. Finally
delivery of soluble antigens via the intravenous or mucosal
routes is particularly conducive to tolerance induction that
reflects many aspects of anergy [19–23].
Clonal anergy is an active process that requires new
protein synthesis and is associated with an anergic gene
expression profile that is characterized by increased expres-
sion of a number of E3 ubiquitin ligases including Cbl-
b, Itch, and Grail . Recent studies have also identified
that the induction of anergy is associated with induction
of numerous negative regulators of TCR signaling including
diacylglycerol kinase, caspase3, Traf6, Ikaros, Egr2, Egr3, and
CREM (cyclic AMP response element modulator) [18, 24–
29]. Early studies showed that anergy was associated with
defective Ras/MAPK activation and AP1 transactivation, but
the causal relationship of Ras activation to anergy induction
was difficult to prove [30, 31]. More recently gene expression
profiling of anergic cells revealed that inhibitors of RasGRP1
were increased [18, 32] and diacylglycerol kinase (DGK) is
required for the induction of T-cell anergy and is induced in
response to NFAT. DGK functions by inhibiting Ras activa-
tion by diminishing the second messenger diacylglycerol that
would activate RasGRP1 [32, 33].
4.Regulation of T-CellActivationby
Multiple E3 ubiquitin ligases are required for regulation of
lymphocyte development and activation and immune toler-
ance [34, 35]. Ubiquitination is a highly conserved process
where proteins become tagged with ubiquitin, and this can
target them for degradation by the proteasome. Ubiquitin is
added by the sequential activity of three enzymes: E1 is an
activating enzyme, E2 is a conjugating enzyme, and E3 is a
Proteins can either be subject to monoubiquitination or
polyubiquitination (Table 1) [36, 37].
Clinical and Developmental Immunology3
Table 1: E3 Ligases that play an important role in preventing autoimmunity.
NameReferences Type of E3 ligase
Ubiquitinates PLC-γ1 and PKC-θ downstream of TCR signalling. Also binds to and
ubiquitinates P85 subunit of PI3K to inhibit CD28-mediated triggering of PI3K.
Critical role in T-cell anergy.
Important role in Th cell differentiation. Binds to a number of substrates including
Notch, c-Flip, Smad2, p63, and p73. Ubiquitinates Bcl-10, JunB, Cbl-b, and PKC-θ
and targets them for degradation. Binds to and is regulated by the E3 ligase Ndfip1.
Binds to and ubiquitinates a number of targets that targets them for degradation
including JunB, Cbl-b, c-Cbl, Notch, PTEN, PLC-γ1, and Bcl-10.
Associates with Nedd4 and is a negative regulator of Itch. Other targets include p63,
p73, and c-Flip.
Selectively inhibits RhoGTPase activity but does not affect the Ras/MAPK kinase
pathway. Inhibits IL-2 production and T-cell proliferation.
Binds to and ubiquitinates NEMO targeting it for degradation. Leads to
upregulation of NF-κB signalling in response to TCR signaling.
Binds to RNA-protein complexes and targets themfor degradation. Important role
in posttranscriptional regulation of target gene expression. Targets include ICOS,
CXCR5, PDCD1, CCL5, Il21, and CD100.
Cbl-b [40–45] RING type
Itch [46–50] HECT type
Nedd4 HECT type
Ndfip1[52–54] HECT type
Grail [55–57]RING type
Traf6[26, 58] RING type
Roquin [59–61] RING type
Cbl-b and its close paralogue, c-Cbl, are RING (really
expression of activated receptors with receptor tyrosine
kinase activity (e.g., the EGF receptor) through endocytosis
and targeting them for ubiquitin-mediated degradation.
C-Cbl is required for TCR modulation in thymocytes,
while the two proteins appear to function redundantly for
promoting TCR downregulation in peripheral T cells. Cblb
is induced during the induction of anergy [17, 18, 38], and
its expression is regulated by the early growth response genes
(Egr) 2 and Egr3 . However Cblb has a nonredundant
role in the induction and maintenance of peripheral T-cell
anergy. Cblb-deficient mice are susceptible to autoimmune
diseases, and T cells display hyperproliferation in response
to TCR signaling and produce IL-2 in the absence of a
costimulatory signal. The Komeda diabetes prone (KDP)
rat strain develops spontaneous islet-specific autoimmunity
that is caused by a loss of function mutation in Cblb, but
the emergence of spontaneous disease requires additional
susceptibility factors, notably a diabetes-susceptible MHC
haplotype . The autoimmune regulator (Aire) gene is a
transcription factor that controls the expression of tissue-
specific self-antigens within the thymus. In particular it is
crucial for clonal deletion of self-reactive lymphocytes [40–
42]. Aire deficiency in both mice and humans can trigger
spontaneous autoimmune disease such as type 1 diabetes
with variable latency [40, 43], and it was recently revealed
that Cblb acts as critical failsafe mechanism to help restrain
autoreactive T cells in the periphery as a result of an AIRE
deficiency . Combined mutations in Aire and Cblb lead
to a lethal autoimmunity with destruction of the exocrine
pancreas and multiorgan inflammation within 5 weeks of
life, whereas on their own neither gene mutation could
trigger the lethal autoimmunity .
Similarly, we find that islet-specific autoimmunity is only
caused by Cblb deficiency in mice when combined with a
TCR transgene that makes high numbers of islet-reactive
CD4 cells in their thymus and expresses the neoself-antigen
hen egg lysozyme on pancreatic beta cells . The Cblb
deficiency does not affect negative selection of autoreactive T
thymus . However, the islet reactive T cells from Clbb−/−
TCR x insHel double transgenic mice proliferated strongly
and produced cytokines following restimulation with self-
antigen in vitro indicating a breakdown in T cell anergy
compared to wild-type cells from TCR x insHel which were
unresponsive to the Hel stimulation. We also observed that
the Cblb deficiency reduced the formation of inducible Tregs
in response to TGF-β signaling, and these cells were less
suppressive compared to wild-type iTregs . This will be
discussed further below.
Itch is a HECT (homologous to the E6-associated
protein carboxy terminus) type E3 ubiquitin ligase and is
upregulated following induction of anergy [17, 46]. Itch has
an N-terminal protein kinase C-related domain, four WW
domains, and a C-terminal HECT domain. The Itchy mouse
strain was identified as the naturally occurring non-agouti-
lethal 18H mutation that leads to a loss of function mutation
in the Itch gene . Itch can promote the ubiquitination of
multiple proteins including Bcl-10, JunB, Cbl-b, and PKC-
θ [17, 48, 49]. Itch-deficient mice develop a spontaneous
and lethal systemic proinflammatory disease consistent with
a failure of peripheral tolerance. The disease is associated
with an expansion of Th2-type T cells that trigger a chronic
pulmonary interstitial inflammation with elevated levels
of IgE antibodies . PLC-γ1 and PKC-θ are induced
by calcium/calcineurin signaling, and Itch targets both
attenuate TCR signaling . Reduced levels of PLC-γ1 and
PKC-θ shorten the longevity of the immunological synapse
and thus reduce the interactions between T cells and APCs.
In addition, Itch also targets Jun B for degradation which is
required for the formation of the AP1 transcription factor
4Clinical and Developmental Immunology
Neural precursor cell-expressed developmentally down-
regulated 4 (Nedd4) is a HECT E3 ligase that is also
expressed in T cells and can target multiple proteins for
ubiquitination and degradation including JunB, Cbl-b, c-
Cbl, Notch, PTEN, PLC-γ1, and Bcl10 which indicates that
there may be a functional overlap between Itch and Nedd4
in T cells. Using fetal liver (FL) chimeras, Yang et al. 
showed that reconstitution of irradiated mice with Nedd4-
deficient FL cells led to normal T-cell development but these
cells proliferated poorly following stimulation in vitro and
produced less cytokines and the recipient animals did not
develop any sign of overt autoimmunity. The defective T-
cell activation was independent of JunB but instead could
be attributed to a failure to ubiquitinate and degrade Cblb.
Therefore, Nedd4 is required to degrade Cbl-b in order for
T-cell activation to proceed .
Ndfip1 was originally identified through its association
with the HECT E3 ligase Nedd4 and was later found to be
a negative regulator of Itch [52, 53]. Ndfip1−/−develops a
severe inflammatory disease that manifests clinically similar
to that observed in Itch−/−mice. The Ndfip1-deficient
mice develop severe skin inflammation with weight loss,
splenomegaly, hepatomegaly, and premature death. The
similarity between the disease phenotypes of Itch−/−and
Ndfip1−/−mice may relate to the target proteins regulated
by Itch, for example, Jun B which is upregulated and can
promote Th2 differentiation [48, 54]. However, other targets
of Ndfip1 include p63, p73, and c-Flip, but it is not known if
dysregulation of any of these targets was responsible for the
Grail is a RING-type E3 ubiquitin ligase that was
found to be induced following the induction of T-cell
anergy in CD4+ T cells . Ectopic expression of Grail
in CD4+ T cells inhibits IL-2 production and proliferation
following stimulation antigen pulsed APCs . In addition
over-expression of Grail in T cells can selectively inhibit
RhoGTPase activity but does not affect Ras activation and
MAPK signalling . This suggests that Grail has a separate
regulatory function in controlling TCR signaling that is
independent of the Cblb E3 ligase. Grail-deficient mice were
resistant to immune tolerance induction, and they were
more susceptible to autoimmune diseases . Similar to
the Cblb−/−mice, na¨ ıve T cells from Grail-deficient animals
display a hyperproliferative response and increased cytokine
production in the absence of a costimulation. In addition,
loss of Grail function Tregs displays reduced suppressive
Traf6 is an E3 ligase with an N-terminal RING finger
domain, and one of its targets includes the NF-κB essential
modifier (NEMO) which is a member of the IκB kinase com-
plex that functions downstream of TCR signalling. T-cell-
specific deletion of Traf6 leads to a multiorgan inflammatory
disease with splenomegaly and lymphadenopathy. The Traf6
deficient cells display Th2 polarization and the mice produce
deficient T cells hyperproliferate in response to anti-CD3
only in the absence of CD28 costimulation, and this was
related to increased phosphorylation of PI3K p85 and AKT
with E3 ligase activity. The Sanroque mouse strain was
identified through an ENU mutagenesis screen for regulators
of systemic autoimmunity. The Rch31san/sanmice carry a
point mutation in the conserved Roq domain generating a
hypomorphic allele that gives rise to a lethal spontaneous
systemic lupus-like disease . The Rch31san/sanmice have
spontaneous germinal centre formation, increased numbers
of T follicular helper cells (Tfh), increased serum levels of
double stranded DNA antibodies, and elevated serum Ig
levels. Tfh cells of the Rch31san/sanmice constitutively express
high levels of inducible costimulator molecule (ICOS) which
can promote spontaneous germinal centre responses .
Roquin regulates ICOS expression levels by binding to the 3?
untranslated region of the Icos mRNA, promoting its degra-
dation . In the sensitized TCR x insHel model whereby
the HEL neoself-antigen is expressed on the pancreatic islets
, the expression of a high frequency of islet reactive T
cells expressing the HEL-specific TCR, these Rch31san/sanTCR
x insHel mice have rapidly develop type 1 diabetes and
generate anti-Hel islet-specific autoantibodies . This is
an unusual feature of autoimmunity in this animal model
as Cblb−/−TCR x insHel mice do not make anti-Hel islet
autoantibodies despite developing type 1 diabetes at high
When the Roquin mutation was combined with the Aire
deficiency in mice, it was predicted that this combination
would lead to rapid autoimmune disease given the severity
of systemic autoimmunity observed in the Rch31san/sanmice.
However, the Aire−/−Rch31san/sanmice displayed normal
survival rates and remained overtly healthy with no clinical
signs of autoimmunity for over 140 days. In contrast,
Aire−/−Cblb−/−mice died with a mean survival age of 25d
with severe weight loss, and surprisingly the immunological
destruction was restricted to the exocrine tissue of the pan-
creas . As a result, the mice did not become diabetic as
the islet tissue was unaffected by the autoimmune response.
These studies highlight that Cblb appears to have a unique
role in restricting the activation of autoreactive T cells in
the periphery that is especially crucial when Aire-mediated
clonal deletion is compromised in the thymus.
There has been some significant progress made in the
peripheral immune system. These checkpoints are crucial to
prevent Il2 gene transcription and inappropriate activation
of dampening the TCR signal that is controlled by multiple
E3 ligases. The specificity of each of these ligases is crucial
to control immune responses under different environmental
conditions. The outcome of responses may be influenced
by the range of cytokines or even different subsets of APCs
present within different tissues.
5.Cblb and Itch inRegulatory
Regulatory T cells that express the forkhead/winged helix
transcription factor Foxp3 play a critical role in vivo in
Clinical and Developmental Immunology5
suppressing immune responses. Mutations in Foxp3 in mice
(scurfy) and humans with immunodysregulation, polyen-
docrinopathy, enteropathy, and X-linked syndrome (IPEX)
both lead to overwhelming multiorgan autoimmunity, dia-
betes, and early death [63–65]. Numerous studies have
shown that Foxp3 is a master regulator of Treg development
and function [66–68]. Overexpression of Foxp3 in na¨ ıve T
cells can convert them to a Treg phenotype that enables
them to suppress the response of other T cells either in
vitro or in vivo [66–68]. Foxp3 is a transcriptional repressor
that is recruited to the Il2 and Ifnγ loci in T cells and
modulates histone acetylation . It is now apparent that
there are two types of Tregs in the immune system. The
natural Tregs (nTregs) are selected in the thymus during
CD4+ T-cell differentiation; these cells represent ∼5–10% of
the peripheral CD4+ T cell pool and play crucial roles in
regulating T-cell responses to self-antigens . The second
subset is known as inducible Tregs whereby na¨ ıve CD4+
CD25− T cells cultured in the presence of TGF-β (IL-10 and
IL-2) can switch on Foxp3 and differentiate as a Treg cell
[70–73]. These iTregs function in an equivalent manner in
being able to suppress responses of other T cells whether in
vitro or in vivo. The mechanism of suppression by Tregs is
mediated by secretion of inhibitory cytokines (e.g., IL10, IL-
35, or TGF-β) and/or cell-cell contact .
TGF-β has long been known to exert anti-inflammatory
effects on the immune system. Deletion of TGF-β leads to
abnormal T cell responses and rapid autoimmunity [72, 74–
76]. It also plays an important role in Treg generation and
maintenance [72, 73] and together with IL-2 is important
for the thymic development of nTregs . TGF-β is also
a potent inhibitor of T-cell proliferative response. However,
in recent years there has been a significant advance made
in understanding the plasticity of CD4+ Th cell lineage
commitment. The Th1-Th2 paradigm stood unchallenged
for nearly 20 years but in the last 5 years several new
Th cell subsets have been defined. Activation of na¨ ıve T
cells in the presence of TGF-β + IL-6 can induce RORγτ
expression, and this promotes the differentiation of Th17
cells which play critical roles in bacterial immunity as
well as being implicated in autoimmune diseases .
Investigating how TGF-β influences the differentiation of Th
cell subsets with completely distinct effector functions (i.e.,
Treg (suppression) and Th17 cell autoimmunity) has been
a major focus for immunologists. It is now clear that when T
cells encounter TGF-β alone this can be sufficient to induce
Foxp3 expression and allow iTreg development to proceed
[70–73]. In contrast, the presence of IL-6 antagonizes Foxp3
expression by a direct effect on AKT activity  (discussed
TGF-β signalling by the TGF-βIIR leads to the phospho-
rylation of Smad2/3 which forms a complex with Smad4
and this complex is translocated to the nucleus to regulate
transcription of target genes [80, 81]. One of the targets is
Smad7 which is an inhibitory Smad used to attenuate TGF-
β signalling by competing with Smad 2/3 for binding to
the receptor [80, 81]. The intron 1 of the Foxp3 gene in T
cells contains two NFATs and a Smad3 binding sites that
are located upstream of the Stat5 and CREB binding sites
. Na¨ ıve CD4+ CD25− T cells from Cblb−/−and Itch−/−
mice show poor induction of Foxp3 expression, and the
iTregs induced are functionally less suppressive in coculture
experiments with wild-type na¨ ıve T cells [45, 46, 83, 84].
To understand the molecular basis of Itch regulation of
Foxp3 expression in iTreg cells studies by Liu and colleagues
identified that TGF-β-induced early gene product (TIEG1 or
KLF10) is induced in response to TGF-β stimulation .
Itch can bind to and ubiquitinate TIEG1 leading to both
mono- and polyubiquitinated forms. Itch and TIEG1 can
bind to the of Foxp3 promoter leading to its transactivation.
Forced expression of TIEG1 in wild-type T cells can induce
robust Foxp3 expression, and cell proliferation was inhibited
in the presence of TGF-β . In contrast, overexpression of
and these cells were resistant to TGF-β inhibition in vitro.
they were also resistant to the inhibitory effects of TGF-
β in vitro, they failed to induce Foxp3 expression in the
presence of TGF-β in vitro, and they were less effective
in mediating suppression of growth of wild-type na¨ ıve
T effector cells. Finally, replacing TIEG1 in TIEG1−/−T
cells through retroviral transduction could restore Foxp3
expression, and these cells could mediate immune sup-
pression . Taken together these results have identified
an important pathway by which Itch and TIEG1 controls
iTreg cell development by regulating Foxp3 expression. This
is likely to have important implications in the control of
autoimmune responses to tissue-specific antigens in the
6.Regulation of T-CellActivationby
The AKT-mTOR pathway has emerged as a central check-
point that controls the fate of multiple cell types in the
immune system [85–87]. Signaling downstream of the TCR
and growth factor receptors (e.g., IL-2R) leads to activation
of PI3 kinase (PI3K), the serine threonine kinase AKT, and
mTOR which in turn leads to increased activity of the
cyclin-dependent kinase (e.g., CDK2) that allows cells to
traverse the G1 cell cycle restriction check point to stimulate
proliferation and avoid the induction of anergy. The AKT-
cells to integrate environmental cues such as nutrient and
amino acid availability, senses energy stores, and directs
cellular proliferation . The AKT-mTOR pathway stim-
ulates a switch in cellular metabolism from catabolic to
anabolic pathways in activated T cells leading to an increase
in glycolysis and the expression of nutrient transporters .
Recent studies have revealed that AKT/mTOR signaling is
T cells, the differentiation of CD4+ Th cell lineages (e.g.,
Th1, Th2 Th17) , CD8+ T cell memory [90, 91], as well
as the differentiation of Treg cells in the periphery  and
lymphocyte trafficking . AKT can in turn activate mTOR
and induce the phosphorylation of the Foxo transcription
factors (e.g., Foxo 1 and Foxo 3a) which leads to their
6Clinical and Developmental Immunology
nuclear export and subsequent degradation . Treatment
of T cells with the mTOR inhibitor rapamycin could block
mTOR activity and induce T cell anergy even when T cells
received a TCR and costimulatory signal . The induction
of anergy requires the complete inhibition of mTOR activity,
and there is heightened activation of the calcineurin/NFAT
pathway . The AKT-mTOR pathway acts downstream
of several energy sensing pathways in eukaryotic cells, that
can modulate expression of genes that are involved in the
maintenance of anergy, or indirectly mediates degradation of
the gene products .
7.mTOR inTreg Development andFunction
As discussed above, activation of na¨ ıve T cells in the
presence of TGF-β readily induces expression of Foxp3. This
such as Cbl-b and PTEN [94, 95]. Likewise, constitutive
AKT activity in T cells can inhibit Foxp3 expression .
Delgoffe et al.  recently showed that deletion of mTOR
leads to increased expression of Foxp3 in T cells and that
mTOR deficient CD4+ T cells are hypersensitive to TGF-
β treatment and switch on Foxp3. The effect on Foxp3
expression appears to be related to mTORC2 activity and
not mTORC1. The Foxo transcription factors, Foxo1 and
Foxo3a, are nuclear proteins that function downstream
of AKT/mTOR signaling, and their activity is regulated
by AKT-mediated phosphorylation that inactivates them
and they are exported from the nucleus. One of the
targets of the E3 ligase Cbl-b is the p85 subunit of
PI3K. Active PI3K is required for activation of AKT and
so in anergic cells, which express higher levels of Cbl-
b, can inactivate PI3K signaling leading to inhibition of
mTOR/AKT signalling. Two recent studies have identified
that regulation of the Foxo1 and Foxo3a transcription factors
is essential for TGF-β-mediated iTreg cell differentiation
In the absence of Cblb the Foxo1 and Foxo3 proteins
become phosphorylated indicating heightened levels of
PI3K-AKT signaling, and this leads to impaired expression
of Foxp3 in Cblb−/−T cells when cultured in the presence
of TGF-β [83, 96]. The loss of Cblb does not impact on
nTreg differentiation in the thymus and does not influence
cell numbers in the periphery. Ouyang et al. found that in
the absence of both Foxo1 and Foxo3a T cells were unable to
induce Foxp3 expression and when cultured in the presence
double knockout mice also showed increased expression of
proinflammatory genes (e.g., IL-17 and IFN-γ) which are
normally absent from iTreg cells. In addition, many of the
is surprising is that the PI3K/AKT/mTOR/Foxo pathway
appears to be more active in the iTreg cells and not the
nTreg cells that develop in the thymus. From the recent
studies it appears that AKT/mTOR signaling only affects
the induction of Foxp3 expression and it does not have any
influence on the maintenance of Foxp3 expression in iTreg
The development of T1D is caused by the breakdown of
immune tolerance to islet-specific antigens in the pancreas.
At present we do not understand how the breakdown
in immune tolerance to islet-specific antigen occurs in
most individuals. The last decade has seen some important
developments in the identification of key genes that play
an essential role in the regulation of peripheral immune
responses as well as the further clarification of autoimmune
checkpoints that try to limit organ-specific autoimmune
diseases such as T1D. The recent discovery regarding the
plasticity of CD4+ Th cell development in vivo has led to a
greater insight into the process of disease pathology. Under-
standing the molecular regulation between inflammatory T
cell subsets and Treg cells offers the potential for therapeutic
intervention in diseases such as T1D.
Clearly the immune system places an important effort
in controlling the activation of na¨ ıve T cells as it strives
to prevent inappropriate T effector cell activation and
differentiation. As highlighted in this paper the family of E3
ubiquitin ligases play a critical role in the immune system
and dysregulation of any of one of these genes can have
by disrupting processes such as clonal anergy, Treg differen-
tiation, and generation of autoantibody responses. The next
should begin to uncover immune checkpoints that have not
understanding of the disease process of T1D in humans that
may eventually lead to the “holy grail” where we can cure
people of this debilitating and life-threatening disease.
This work was supported by project grants from the
JuvenileDiabetes ResearchFoundation, 4-2006-1025 and the
Diabetes Australia Research Trust project grant.
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