Journal of Atherosclerosis and Thrombosis Vol.19, No.

6 503

Review

Regulatory T Cells in Atherogenesis

Naoto Sasaki, Tomoya Yamashita, Masafumi Takeda and Ken-ichi Hirata

Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan

Atherosclerosis is believed to be an inflammatory condition of the arterial wall. It has become appar-
ent that various types of cells of innate and adaptive immunity participate in atherogenesis. T cells
are of particular interest because they mediate pathogenic immune responses involved in the accelera-
tion of atherosclerosis. Recent studies from several independent groups indicated that subsets of reg-
ulatory T cells (Tregs) actively mediate immunologic tolerance and inhibit atherosclerosis develop-
ment or progression through the down-regulation of effector T-cell responses. It is likely that there is
an imbalance between pathogenic effector T cells and Tregs under atherosclerotic conditions. Recent
evidence suggests that in addition to the thymus, gut-associated lymphoid tissues are the main sites
for the generation of several subsets of peripherally inducible Tregs. This indicates that intervention
in the gut environment to promote an endogenous regulatory immune response may serve as a possi-
ble therapeutic approach to suppress atherosclerotic diseases. In this review, we discuss not only the
possible role of Tregs in the prevention of atherosclerosis, but also promising strategies to prevent or
cure atherosclerotic diseases by promoting an endogenous regulatory immune response, particularly
by oral immune modulation.

J Atheroscler Thromb, 2012; 19:503-515.

Key words; Atherosclerosis, Immune system, Inflammation, Regulatory T cells, Oral immune modulation

cyte adhesion molecules in endothelial cells. Through

Introduction
Atherosclerosis is an inflammatory condition of
the arterial wall, which is regulated by cells of innate
and adaptive immunity 1-4). This causes unstable
plaques and thrombotic occlusion of the artery, lead-
ing to severe clinical events, including acute coronary
syndrome (ACS) and stroke, which account for
approximately a quarter of deaths in Japan.
Both animal and human studies have shown that
hypercholesterolemia is associated with the accumula-
tion and retention of low-density lipoprotein (LDL)
in the arterial intima, which is considered to be an ini-
tial inflammatory response in the artery wall in ath-
erogenesis. LDL is prone to oxidative and enzymatic
modifications, leading to the up-regulation of leuko-
Address for correspondence: Naoto Sasaki, Division of
Cardiovascular Medicine, Department of Internal Medicine,
Kobe University Graduate School of Medicine, 7-5-1,
Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan
E-mail: nao10@mrh.biglobe.ne.jp
Received: June 26, 2011
Accepted for publication: December 7, 2011
the activated endothelium, monocytes enter the sub-
endothelial space or intima and differentiate into mac-
rophages. These processes are critical for the initiation
of atherosclerosis both in humans and animals. Modi-
fied LDL particles are taken up by scavenger receptors
on macrophages, which results in intracellular choles-
terol accumulation and transformation of monocytes
into foam cells. These differentiated macrophages also
express toll-like receptors, which bind lipopolysaccha-
ride, oxidized LDL (oxLDL), heat-shock protein
(HSP) 65/60, and other ligands, inducing their activa-
tion and leading to the production of pro-inflamma-
tory molecules. Numerous studies of human athero-
sclerotic lesions have provided important clues to the
pathogenesis of atherosclerosis, including immune
cells. Because of the limited elucidation of the molec-
ular mechanisms of atherogenesis in human studies,
two strains of genetically altered mice, apolipoprotein
E-deficient (ApoE /) and low-density lipoprotein
receptor-deficient (LDLR /) mice, have been used for
many studies.
Atherosclerotic lesions in human and ApoE / or
504 Sasaki et al .
Figure 1

IFN-: Proatherogenic
Th1

Antigen presentation

Nave APC
APC CD4+ Th2 Treg
T cell
IL-4: Proatherogenic?
IL-5, 10: Anti-atherogenic

Th17

IL-17: Proatherogenic?

Fig. 1. Role of T Lymphocytes in Atherogenic and Protective Immune Responses in Ath-

erogenesis.
After antigen presentation by antigen-presenting cells (APCs) such as macrophages or dendritic
cells to nave T cells, adaptive T-cell-mediated immune responses occur in the lymphoid organs
and possibly also in atherosclerotic lesions. Depending on stimulation by several cytokines or regu-
lation by transcription factors, nave CD4 T cells can differentiate into T helper type 1 (Th1), T
helper type 2 (Th2), and T helper type 17 (Th17) lineage. Regulatory T cells (Tregs) suppress
polyclonal T cell activation and the differentiation of nave T cells into Th1, Th2, and Th17 lin-
eage and down-regulate APC function.

LDLR / mice have both CD4 and CD8 T cells,
and the former population is predominant 5-7). After
antigen presentation by antigen-presenting cells
(APCs) such as macrophages or dendritic cells (DCs)
to nave T cells, adaptive T-cell-mediated immune
responses occur. CD4 T-cell clones derived from ath-
erosclerotic plaques, which are restricted by major his-
tocompatibility complex (MHC) class molecules,
have been shown to recognize oxLDL, HSP-60, and
other antigens from microorganisms such as Chla-
mydia pneumonia, which are supposed to be candi-
date self-antigens for atherogenesis 3, 8). Although there
are many CD8 T cells in atherosclerotic lesions,
which recognize viral antigens presented on MHC
class molecules, little is known about their roles in
atherogenesis. It is now well known that in addition to
macrophage activation, CD4 T-cell-mediated patho-
genic immune responses play an important role in
atherogenesis in humans and mice 3, 9). Depending on
stimulation by several cytokines or regulation by spe-
cific transcription factors, nave CD4 T cells can dif-
ferentiate into T helper type 1 (Th1), T helper type 2
(Th2), and T helper type 17 (Th17) lineage (Fig. 1).
Th1 cells are the major CD4 T cells in mouse and
human atherosclerotic plaques and are shown to pro-
mote atherosclerotic disease by producing inflamma-
tory cytokines such as interferon-10, 11). The role of
Th2-mediated immune responses in atherosclerosis
remains controversial depending on the produced
cytokines or animal models used and needs further
investigations 12, 13). Th17 cells, which have been
recently identified, play a highly pathogenic role by
producing inflammatory cytokine interleukin (IL)-
17 14, 15). There are several isoforms of IL-17, including
IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-
17F 16). Th17 cells are known to produce IL-17A and
IL-17F, and the former is reported to play the most
critical role in mediating autoimmune diseases. Exper-
imental evidence suggests that pathogenic immune
responses caused by this subset massively contribute to
the progression of several autoimmune diseases in
mice. Several studies suggest a proatherogenic role for
Th17 cells in hyperlipidemic mice 17-20), whereas exper-
imental data from another group demonstrated a pro-
tective role of IL-17 in atherosclerosis 21). A recent
study used a genetic deletion of IL-17A and showed
that IL-17A is involved in systemic and vascular
inflammation, but did not alter plaque burden in
experimental atherosclerosis 22). Thus, the role of Th17
cells in atherosclerotic diseases is still controversial.
Accumulating evidence suggests that several sub-
sets of regulatory T cells (Tregs), which have been
shown to maintain immunological unresponsiveness
to self-antigens and suppress excessive immune
 Regulatory T Cells in Atherogenesis 505

A
Gut-associated
Thymus lymphoid tissue

Lymphoid organs or Treg

atherosclerotic plaque CD25
Cell contact-dependent
(CTLA-4-CD80/86 pathway)
TGF- Foxp3

Suppression

Effector
APC
T cell

Effetor T cells become anergic

or die through apoptosis

Atherosclerosis

B
Gut-associated lymphoid tissue

Lymphoid organs or
atherosclerotic plaque
LAP

LAP+
Tr1
Treg TGF-
IL-10

Macrophage Effector Macrophage Effector

T cell T cell

Atherosclerosis

Fig. 2. Several Subsets of Tregs May Play a Protective Role in Atherogenesis: a

Hypothesis.
Several subsets of Tregs can differentiate from nave T cells in gut-associated lymphoid
tissues, as well as in the thymus. A, CD25Foxp3 Tregs may inhibit APC function,
possibly in a cell-contact dependent manner such as down-regulation of CD80/86
expression on APCs by CTLA-4 or through the production of TGF-, which may lead
to anergy or apoptosis of effector T cells, suppression of their responses, and attenua-
tion of atherosclerosis development. APC, antigen-presenting cell; Foxp3, forkhead
box P3. B, Peripherally generated Tregs such as Tr1 cells and CD4LAP Tregs down-
regulate effector T cell and macrophage function through the production of IL-10 or
TGF-, respectively, leading to reduction of atherosclerosis. LAP, latency-associated
peptide.

responses 23), inhibit atherosclerosis development or kines in atherosclerosis, IL-10 and transforming
progression through the down-regulation of activated growth factor (TGF)-, play a key role in the suppres-
T-cell responses (Fig. 2A and 2B) 24-27). It has been sive function or differentiation of several subsets of
demonstrated that two major anti-inflammatory cyto- Tregs, suggesting that the protective effects of these
506 Sasaki et al .
cytokines in atherogenesis are partly due to the modu-
lation of Treg functions 28, 29). Deficiency of Treg func-
tion or development has been shown to be a primary
cause of some autoimmune diseases in humans and
animals 30-32). Similarly, the balance between effector T
cells and Tregs may be important for the control of
atherosclerotic diseases 28). In consideration of this, we
believe that promotion of an endogenous regulatory
immune response has therapeutic potential to suppress
atherosclerotic disease.
In this review, we discuss the current knowledge
of Treg generation and function, the proposed spe-
cific roles of Tregs in atherosclerosis (summarized in
Table 1), and the strategies to prevent or cure athero-
sclerotic diseases by promoting an endogenous regula-
tory immune response, particularly by oral immune
modulation.
Generation and Function of Regulatory T Cells
Sakaguchi et al. first reported that naturally aris-
ing Tregs constitutively express high levels of CD25
(IL-2 receptor -chain) molecule, and that depletion
of the CD4CD25 T cell population, which consti-
tutes 5-10% of peripheral CD4 T cells in normal
nave mice and humans, elicits autoimmunity similar
to its human counterparts 30). Firm evidence has shown
that Tregs, which are produced by normal thymus,
play a crucial role in dominant suppression of patho-
genic immune responses and maintenance of self-tol-
erance and immune homeostasis 23). In addition, natu-
ral Tregs specifically express transcription factor Foxp3
(forkhead box P3), which is essential for their devel-
opment and function and is currently the most reli-
able molecular marker for them 33-35). Mutations of
human gene FOXP3 cause the genetic disease IPEX
(immune dysregulation, polyendocrinopathy, enterop-
athy, X-linked syndrome), in which autoimmune dis-
eases such as type 1 diabetes, allergy and inflammatory
bowel disease can be observed at high frequency 32).
Foxp3 natural Tregs suppress the proliferation of tis-
sue-specific autoimmune T cells and their differentia-
tion into Th1, Th2, and Th17 lineage in vivo. More-
over, they inhibit polyclonal T cell activation and the
function of other types of lymphoid cells, including B
cells, macrophages and DCs (Fig. 1) 36).
Several lines of evidence have suggested that
Treg-mediated suppression includes multiple mecha-
nisms, such as secretion of immunosuppressive soluble
factors (IL-10, IL-35, TGF-, granzyme, and perfo-
rin); deprivation of IL-2 by Tregs, which is necessary
for the maintenance of responder T cells; cell-contact-
dependent suppression, and functional modification
or killing of APCs, which may operate synergistically
and in a complementary manner 36-38).
Recent evidence has demonstrated that Foxp3
Tregs can differentiate from nave T cells in the
periphery under certain conditions, as well as in the
thymus 39, 40). Peripherally generated Tregs are called
adaptive or inducible Tregs (iTreg) and are reported to
have similar immunological properties with thymus-
derived Tregs. In addition to Foxp3 iTregs, several
kinds of iTregs, including T regulatory type 1 (Tr1) or
T helper type 3 (Th3) cells, have been reported. Tr1
cells without Foxp3 expression were demonstrated in
Peyers patches of mice fed a low dose of -lactoglobu-
lin and produced immunomodulatory cytokine
IL-10 41). Th3 cells are induced in mucosal tissues dur-
ing oral tolerance and produce high amounts of
TGF- in an antigen-specific manner 41, 42). Also, it is
reported that some Th3 cells are TGF--induced
Foxp3 iTregs. Recently, CD4LAP (latency-associ-
ated peptide) Treg has been identified as a new subset
of Tregs that suppresses autoimmune diseases in
mice 43-47). This Treg population is linked to inflamma-
tory bowel disease in mice 48). Recent studies from
other groups and our group have consistently shown
that oral or nasal anti-CD3 antibody is biologically
active and induces CD4LAP Tregs that suppress
experimental autoimmune encephalitis (EAE) 43), auto-
immune diabetes 44), lupus 45, 46), collagen-induced
arthritis 47), type 2 diabetes 49), and atherosclerosis 27) in
a TGF--dependent fashion (Fig. 2B).
Protective Roles of Treg Cells in Experimental
Atherosclerosis
Based on the role of the immunoregulatory func-
tions of Tregs described above, it can be speculated
that antigen-specific Tregs can be produced in
response to a number of altered self antigens including
LDL, oxidized epitopes on apoptotic cells and HSP
65/60 to dampen inflammation within the atheroscle-
rotic plaques 8, 50). However, at the same time, much
more activated effector T cells, which have been pre-
sented such self-antigens by APCs in the plaques, can
be seen in atherosclerotic lesions and regional lymph
nodes. Under atherosclerotic conditions, there might
be an imbalance between effector T cell and Treg
responses both in the atherosclerotic lesions and in
systemic lymphoid organs 28). Tregs control pathologi-
cal immune responses in an antigen-specific manner.
Importantly, once they are activated after antigen pre-
sentation by APCs, they exert suppressive function
regardless of their antigen specificity 23); therefore, in
addition to antigen-specific Tregs, polyclonal activated
Tregs may be also important for the suppression of
pathogenic immune responses in atherosclerosis,
 Regulatory T Cells in Atherogenesis 507

Table 1. Current Knowledge of Possible Roles of Tregs in Atherogenesis

Description References
/ / /
What Is Known ? Treg deficiency due to reconstitution with CD80 /CD86 or CD28 bone marrow in
LDLR / mice is associated with the significant increase of atherosclerotic lesions. 24
/ / /
Transfer of CD28 splenocytes (Treg deficiency) to ApoE Rag-2 mice with lymphocyte
deficiency accelerates atherosclerosis development compared with the transfer of wild-type 24
splenocytes.
Adoptive transfer of natural CD4CD25 Tregs reduces atherosclerosis development in
ApoE / mice. 26

Peripherally generated Tregs such as Tr1 cells play a protective role in the development of
atherosclerosis in ApoE / mice through the production of IL-10. 61
/
Oral tolerance induction to oxidized LDL or heat-shock protein-60 in LDLR mice
attenuates atherosclerosis in both early and advanced stages, in association with increased
numbers of CD25Foxp3 Tregs in the MLNs and spleen, and increased expression of Treg- 62, 64
associated molecules in atherosclerotic lesions.
Intravenously administered FcR-non-binding CD3-specific antibody induces Foxp3 Tregs
producing TGF- and inhibits atherosclerosis development and progression in LDLR / mice. 73

Oral anti-CD3 antibody treatment induces CD4LAP Tregs and CD4CD25Foxp3 Tregs
in the MLNs and spleen and inhibits atherosclerotic plaque formation in ApoE / mice 27
through a TGF--dependent mechanism.
What Is Unknown ? Previous studies do not provide direct evidence of the atheroprotective role of endogenous
Foxp3 Tregs, and further studies with more sophisticated methods will be needed to clarify
this issue.
Although several mechanisms of Treg-mediated immune suppression have been proposed, the
core mechanism of Treg-mediated regulation in atherosclerosis remains unclear.
It remains unclear whether, in addition to polyclonal activated Tregs, antigen-specific Tregs are
important for the suppression of pathogenic immune responses in atherosclerosis, because we
still do not know which antigen is the most critical for disease initiation and progression.
Although recent studies suggest that Tregs migrate to atherosclerotic lesions, where antigen
presentation may occur as well as in lymphoid organs, the actual roles of intraplaque Tregs in
atherogenesis have not been clarified.
In the clinical setting, it is unknown whether impaired function or reduced numbers of Tregs
may contribute to the progression of atherosclerotic diseases in humans.
Foxp3, forkhead box P3; MLNs, mesenteric lymph nodes.

where we still do not know which antigen is the most
critical for disease initiation and progression.
Recently, Ait-Oufella H et al. demonstrated that
endogenous CD4CD25 natural Tregs play a protec-
tive role in atherogenesis in mice 24). It is known that
the generation and homeostasis of Tregs needs interac-
tions between the costimulatory molecules CD80/
CD86 and CD28 51). The authors investigated the
effect of CD80/CD86 or CD28 deficiency on the
development of atherosclerosis in LDLR / mice using
the irradiation/bone marrow transplantation model.
They found that Treg deficiency due to reconstitution
with CD80 //CD86 / or CD28 / bone marrow
was associated with a significant increase of atheroscle-
rotic lesions. Moreover, they also demonstrated that
transfer of CD28 / splenocytes to ApoE /Rag-2 /
mice, which are deficient in lymphocytes, accelerated
atherosclerosis development compared with the trans-
fer of wild-type splenocytes. Another group supports
this idea, showing that adoptive transfer of natural
CD4CD25 Tregs reduces atherosclerosis develop-
ment in ApoE / mice 26). Collectively, these data sug-
gest a crucial role for endogenous Tregs in the control
of atherosclerosis (Fig. 2A). However, these in vivo
studies described above do not provide direct evidence
for the atheroprotective role of Tregs, because Ait-
508 Sasaki et al .
Oufella H et al. used a bone marrow transplantation
model with CD80 //CD86 / or CD28 / bone
marrow, where deficiency of CD80/CD86 or CD28
could affect the function of not only Tregs but also
other effector T cells 51). In addition, Tregs are reported
to have constitutively high expression of CD25,
whereas this molecule is upregulated upon activation
of effector T cells 52), suggesting the possibility that the
previous studies described above could not evaluate
the exact effects of Treg deficiency on the development
of atherosclerosis. It is now well known that natural
Tregs specifically express Foxp3, which is a master reg-
ulator of Treg development and function and therefore
is currently the most reliable molecular marker for
natural Tregs 23, 33). The question of whether Foxp3
Tregs play a critical role in the suppression of athero-
genesis will be directly addressed by analyzing bacte-
rial artificial chromosome transgenic mice expressing a
diphtheria toxin receptor (DTR) under the control of
the foxp3 gene locus 53) or foxp3DTR knock-in mice 54)
allowing selective and efficient depletion of Foxp3
Tregs by diphtheria toxin injection.
Tregs may play a protective role against vascular
dysfunction not only in hypercholesterolemia but also
in hypertension. A recent study demonstrated that
adoptive transfer of Tregs had a protective effect on
the cardiac damage caused by angiotensin , a major
mediator of hypertension and cardiac damage,
although it had no impact on angiotensin -induced
arterial hypertension 55). Notably, recent accumulating
evidence clearly suggested that the pathogenic T cell
response contributes to hypertensive disease and vas-
cular dysfunction in mouse models 56). Thus, further
studies are needed to identify the role for Tregs in the
prevention of hypertension and consequent reduction
of atherosclerosis.
Strategies for Enhancing Treg-Mediated Immune
Regulation in Atherosclerosis
Recent studies have established that T-cell
costimulatory or coinhibitory pathways may contrib-
ute to nave T cell activation and enhancement of
effector and memory T cell responses or negative regu-
lation of T-cell responses, respectively. It has now
become evident that these pathways play crucial roles
not only in modulating nave and effector T cell func-
tion but also in Treg generation and function,
although the precise roles of these pathways in Treg
function remain to be determined 51). A recent study
documented that inducible costimulatory molecule
(ICOS), which is a CD28 family member involved in
the costimulatory pathway, plays a pivotal role in the
regulation of atherosclerosis through the modulation
of Treg responses 25). One of the important coinhibi-
tory molecules is cytotoxic T lymphocyte-associated
protein 4 (CTLA-4), which is a CD28 family member
expressed only on activated T cells including Foxp3
Tregs. B7 family molecules, such as B7-1 (CD80) and
B7-2 (CD86), which are mainly expressed on macro-
phages and DCs, bind to CTLA-4, possibly resulting
in suppression of T-cell activation, although the pre-
cise molecular mechanisms for this negative regulation
are still not well-defined 57). Interestingly, CTLA-4 is
induced on conventional T cells only after activation,
whereas Foxp3 Tregs constitutively express this mole-
cule. Moreover, CTLA-4-dependent suppression is
supposed to be a key mechanism for Treg-mediated
suppression 58, 59). One mechanism for CTLA-4-depen-
dent suppression is reported to be down-modulation
of APC function by reducing their CD80 or CD86
expression 58, 59). Taken together, CTLA-4 is considered
to be a possible therapeutic target for atherosclerotic
disease via promotion of Treg suppressor function, as
well as down-regulation of effector T cell function.
Thus, blockade of costimulatory pathways or enhance-
ment of coinhibitory pathways could be effective
treatments for atherosclerotic disease through modula-
tion of Treg function as well as effector T cell func-
tion 60); however, it is possible that blockade of the
costimulatory pathway may impair both effector T
cell and Treg function. The effects of the modulation
of costimulatory or coinhibitory pathways on each T
cell subset should be clearly defined before this
approach is applied to clinical use.
In addition to natural Foxp3 Tregs produced in
the thymus, it is reported that peripherally generated
Tregs such as Tr1 and Th3 cells play a protective role
in the development of atherosclerosis in mice through
the production of IL-10 or TGF-, respectively
(Fig. 2B) 61, 62). Mucosal tolerance induction has been
shown to inhibit various autoimmune diseases in
mice, partly through the induction of several types of
adaptive Tregs 63); therefore, this implies that mucosal
tolerance induction might be an effective way to treat
atherosclerosis. For example, it was reported that oral
tolerance induction to oxLDL or HSP-60 in LDLR /
mice attenuated atherosclerosis in both early and
advanced stages, in association with increased num-
bers of CD25Foxp3 Tregs, increased production of
TGF- or IL-10 in the mesenteric lymph nodes
(MLNs) and spleen, and increased expression of Treg-
associated molecules in atherosclerotic lesions 62, 64).
Recent evidence suggests that modulation of
Treg-mediated immune responses, mainly via increas-
ing their number, represents a novel therapeutic
approach against atherosclerosis. Most previous stud-
 Regulatory T Cells in Atherogenesis
Table 2. Blood Treg Count in Patients with Coronary Artery Disease
Reference Treg definition
high
Mor et al. 2006 CD4 CD25
Ammirati et al. 2010 CD4CD25 high CD127 low
SA, stable angina; MI, myocardial infarction; UA, unstable angina.
ies have focused on searching for methods to increase
the number of Tregs, whereas strategies for enhancing
their suppressive function have not been extensively
examined so far. As described before, in the fields of
immunology, mechanisms of Treg-mediated suppres-
sion have been actively investigated and have attracted
much attention 36-38). Although several mechanisms
have been proposed, it remains unclear to what extent
each mechanism contributes to suppression. It is pos-
sible that the dominant mechanism may vary depend-
ing on the situation or subset of Tregs. It is tempting
to investigate the core mechanism of Treg-mediated
immune suppression in atherosclerosis if it exists.
Interestingly, a recent in vitro study using a co-culture
system demonstrated that human CD25Foxp3
Tregs may inhibit proinflammatory activation in
human umbilical vein endothelial cells via the cell-
contact-dependent pathway or production of soluble
factors such as IL-10 and TGF-65). The same group
also reported that co-culture with mouse Tregs mark-
edly suppressed macrophage foam-cell formation
through the same pathways 66). Since it is well known
that endothelial dysfunction and macrophage foam-cell
formation play key roles in the initiation and progres-
sion of various forms of atherosclerotic diseases, these
results support the idea that Tregs are closely involved
in the protection against atherosclerosis. The elucida-
tion of core Treg-mediated suppressive mechanisms will
contribute to the development of more effective thera-
peutic tools for restricting atherosclerosis based on the
modulation of Treg-mediated immune regulation.
Role of Tregs in Clinical Atherosclerotic Disease
It has now become evident that Tregs play protec-
tive roles in atherogenesis in several experimental mouse
models. Before translating this idea into the clinical set-
ting, to ascertain whether impaired function or reduced
numbers of Tregs may contribute to the progression of
atherosclerotic diseases in humans, extensive clinical
studies are required. Mor A et al. demonstrated that
patients with ACS exhibited a reduced number and
compromised functional properties of CD25 Tregs 67).
In contrast, there is a recent clinical report showing that
Treg levels were significantly increased in the periphery
of ST-elevation ACS patients, whereas their numbers
509
SA MI UA
not changed decreased not determined
not changed increased decreased
were significantly decreased in non-ST-elevation ACS
patients (Table 2) 68). This study also demonstrated
that circulating Treg levels correlated neither with the
intima-media thickness of the common carotid artery
nor with its progression, indicating that peripheral
Treg levels may not be a useful marker for evaluation
of the severity of atherosclerosis. Thus, the role of
Tregs in atherosclerotic diseases, including ACS,
remains unclear and needs further clarification.
Assuming that Tregs migrate into regional lymph
nodes, where self-antigens are presented, are activated
by DCs, and also migrate into inflamed tissues to
dampen local inflammation, it is possible that Treg
numbers in the peripheral blood may not reflect their
systemic status. Where can Tregs be found and work
in the body under physiological and pathological con-
ditions ? A recent report demonstrated that Tregs in
the circulation infiltrate the periphery, traffic to drain-
ing lymph nodes, and then recirculate back to the
inflamed tissues to suppress pathogenic immune
responses 69). Therefore, the dynamics of Treg trafficking
and localization under atherosclerotic condition should
be examined to establish the clinical importance of
Tregs for the prevention of atherosclerotic diseases.
CD4LAP Tregs and Oral Anti-CD3 Antibody
Therapy in Autoimmunity
Administration of FcR-non-binding anti-CD3
monoclonal antibody has been shown to induce long-
lasting immune tolerance through the induction of
TGF--producing CD4CD25 Tregs and to be an
effective treatment for autoimmune diabetes in mice
and humans 70, 71), and acute transplant rejection in
humans 72). Notably, it has been shown that intrave-
nously administered FcR-non-binding CD3-specific
antibody induces a regulatory immune response and
inhibits atherosclerosis development and progres-
sion 73). Interestingly, as described before, recent stud-
ies have revealed that oral or nasal anti-CD3 antibody
with or without the Fc portion is biologically active
and induces CD4LAP Tregs that suppress several
autoimmune diseases in mice, and that this therapy
would not be expected to have side effects regardless
of the presence of Fc portion 43-47). Based on the results
provided by animal experiments, murine anti-CD3
510 Sasaki et al .
monoclonal antibody (OKT3) was orally administered
to healthy human subjects 74). Oral OKT3 administra-
tion exerted many favorable immunologic effects, such
as enhanced T cell proliferation, suppressed Th1 and
Th17 responses, increased TGF- and IL-10 expres-
sion, and decreased IL-23 and IL-6 expression in DCs.
The findings from this report demonstrated that oral
anti-CD3 monoclonal antibody is safe and biologi-
cally active not only in mice but also in humans.
Based on the efficacy and safety of this antibody treat-
ment in several mouse studies and human healthy vol-
unteers, a clinical study using oral OKT3 has been
performed for the treatment of human autoimmune
diseases, including nonalcoholic steatohepatitis and
type 2 diabetes, and showed beneficial effects in these
patients 75).
Given this background, we hypothesized that
the induction of CD4LAP Tregs by oral anti-CD3
antibody treatment would inhibit atherosclerosis in
ApoE / mice 27).
Oral Immune Modulation by Anti-CD3 Antibody
Could Be Used as a Promising Therapeutic Approach
for Atherosclerosis
We fed 6-week-old male ApoE / mice 5 g
hamster anti-CD3-specific antibody or 5 g hamster
IgG by gastric intubation once a day for 5 consecutive
days. Two days after the last feeding of anti-CD3 anti-
body, we found a significant increase in LAP Tregs
and Foxp3 Tregs in the CD4 T cell population in
MLNs of anti-CD3-treated mice. We next analyzed
the cytokine and chemokine production profile from
spleen lymphocytes on day 7 after treatment, which
showed suppressed Th1 and Th2 immune responses
in anti-CD3-treated mice. We also observed that
TGF- is the only cytokine with anti-inflammatory
properties that is enhanced by anti-CD3 antibody
treatment. Six-week-old ApoE / mice on a standard
diet were given anti-CD3 antibody or control hamster
IgG orally on 5 consecutive days, and atherosclerosis
was assessed at 16 weeks of age. Oral administration
of anti-CD3 antibody significantly reduced athero-
sclerotic lesion formation and the accumulation of
macrophages and CD4 T cells in plaques compared
with controls. Neutralization of TGF- in vivo using
anti-TGF- neutralizing antibody abrogated the pre-
ventive effect of oral anti-CD3 antibody, suggesting
that TGF- plays a crucial role in the inhibition of
atherosclerosis development by oral anti-CD3 anti-
body. It is likely that CD4LAP Tregs and CD4
Foxp3 Tregs induced by oral anti-CD3 antibody
reach mesenteric lymph nodes and other lymphoid
organs such as the spleen through the bloodstream,
where they may suppress pathogenic immune responses
through the production of TGF-, leading to the
reduction of atherosclerotic plaque formation and
inflammatory cell recruitment into plaque (Fig. 3).
Recent studies suggest that antigen presentation
may occur within atherosclerotic plaque in addition to
the lymphoid organs, and that Tregs migrate to ath-
erosclerotic lesions to suppress local immune responses,
although the actual roles of intraplaque Tregs in ath-
erogenesis have not been clarified yet 9, 28). By immu-
nohistochemical study using anti-Foxp3 antibody, we
found an increase in the number of natural Tregs
within the plaque of anti-CD3-treated mice. These
data indicate that oral anti-CD3 antibody treatment
could inhibit the migration of effector T cells into the
lesion selectively and relatively increase the proportion
of natural Tregs in atherosclerotic plaque that might
suppress pathogenic T cell immune responses or mac-
rophage activation as well as in the lymphoid organs.
It is reported that Tregs express several kinds of hom-
ing receptors whose expression plays a role in control-
ling their migration into inflamed tissues or infectious
sites 76). Although we did not examine the expression
patterns of homing receptors in Tregs after oral anti-
CD3 antibody administration, it is possible that some
alterations of such receptor expression may contribute
to Treg accumulation in atherosclerotic plaque. If Treg
migration into atherosclerotic lesions is important to
suppress local immune reactions, manipulation of Treg
trafficking and localization by modulating their hom-
ing receptors could be useful to treat atherosclerosis.
It has been reported that immunosuppressive
effects by intravenous FcR-binding anti-CD3 anti-
body may involve mechanisms such as the depletion
of pathogenic T cells from the bloodstream or lym-
phoid organs and modulation of the T-cell receptor on
T cells 72). Different from the case of intravenous FcR-
binding anti-CD3 antibody administration, we did
not observe T-cell receptor down-modulation or
depletion of T cells; however, comparing parenteral
FcR-non-binding anti-CD3 antibody with oral FcR-
binding or non-binding anti-CD3 antibody, there
might be some common mechanisms underlying the
suppression of autoimmunity, because both appear to
work mainly by inducing TGF--producing Tregs
rather than eliminating pathogenic T cells. One possi-
bility to understand this similarity is that after oral
administration, FcR-binding anti-CD3 antibody
might lose its Fc portion in the gut and consequently
produce F(ab')2 (FcR-non-binding anti-CD3 anti-
body), a small amount of which might enter the
bloodstream, leading to the induction of Tregs.
TGF- has a broad spectrum of functions such
 Regulatory T Cells in Atherogenesis 511

Figure 3

Anti-CD3 Ab Lymphoid organs or

atherosclerotic plaque
APC

Positive feedback effect

LAP+ Macrophage
Treg

LAP+ TGF-
Treg
Blood Foxp3+
Intestine Treg Effector
T cell

Foxp3+
Treg

APC

Atherosclerotic plaque

Fig. 3. Possible Mechanisms of Atherosclerosis Reduction by Oral Administration

of Anti-CD3 Antibody.
Oral anti-CD3 antibody induces CD4LAP and CD4Foxp3 Tregs, possibly in gut-
associated lymphoid tissues, which migrate into other lymphoid organs, subsequently lead-
ing to suppression of pathogenic effector immune responses in a TGF--dependent man-
ner. Up-regulated TGF- secretion in the gut from CD4LAP Tregs could lead to a fur-
ther increase in the number of not only CD4LAP Tregs but also CD4Foxp3 Tregs, as
well as enhancement of their suppressive function. In addition to the lymphoid organs,
Tregs may migrate to atherosclerotic plaques and dampen local immune responses, result-
ing in the attenuation of atherosclerotic lesion formation.

as immune suppressive or promoting properties
depending on the situation. TGF- has been attract-
ing much attention as a potent anti-atherosclerotic
cytokine 77). TGF-, which is produced by many types
of cells in atherosclerotic plaques, suppresses patho-
genic immune responses such as the recruitment of
inflammatory cells into plaques or foam cell forma-
tion, and increases collagen biosynthesis 78, 79). To
investigate the intrinsic function of TGF- in T cells,
several groups have used transgenic approaches to
block TGF- signaling in T cells by expressing domi-
nant negative TGF- receptors, and found that ath-
erosclerosis-prone mice such as ApoE / mice or
LDLR / mice with specific deletion of TGF- signal-
ing in T cells show markedly accelerated plaque for-
mation and inflammatory cell infiltration, associated
with the enhancement of both Th1 and Th2 immune
responses 80, 81). These studies demonstrate that TGF-
signaling in T cells is indispensable for the regulation
of atherosclerotic disease progression. In consideration
of this, our data suggest that increased TGF- pro-
duction from T cells, possibly from CD4LAP Tregs,
may be a key mechanism for the preventive effect of
oral anti-CD3 antibody on atherosclerosis through the
down-regulation of both Th1 and Th2 immune
responses.
Intriguingly, in addition to an increased number
of LAP Tregs, we found a significant increase in
Foxp3 Tregs in MLNs of anti-CD3-treated mice.
This induction of Foxp3 Tregs was not observed in
previous studies by other groups 43, 44). Recent studies
suggest that differentiation of CD4Foxp3 Tregs
from nave T cells may be facilitated by dendritic cells
in gut-associated lymphoid tissue in the presence of
TGF-39, 40), and that retinoic acid produced by den-
dritic cells plays an important role in their differentia-
tion 82-84). In consideration of this, it is highly possible
that up-regulated TGF- secretion in the gut from
anti-CD3-induced CD4LAP Tregs could lead to the
induction of Foxp3 Tregs. Interestingly, several recent
lines of evidence indicate a reciprocal relationship
between Tregs and Th17 cells 85, 86). It has been
reported that TGF- stimulation leads to the differen-
tiation of nave T cells to Th17 cells or CD4Foxp3
Tregs in mice in the presence or absence of IL-6,
respectively 87). Our data indicate a shift from Th17
512 Sasaki et al .
cells to CD4Foxp3 Tregs in oral anti-CD3-treated
mice, which might partially affect the athero-protec-
tive effects of oral anti-CD3, although whether Th17
cells accelerates atherosclerosis as well as other autoim-
mune diseases remains unclear so far.
Mucosal tolerance induction has been shown to
inhibit various autoimmune diseases in mice and one
of the most important mechanisms for this efficacy is
believed to be the induction of various Tregs 63). In this
context, low doses of oral antigen loading may play a
critical role in increasing the number of self-antigen-
specific Tregs. Similarly, autoimmune diseases have
been shown to be suppressed by only low doses of oral
anti-CD3 antibody, not by high doses, associated with
an increase in LAP Tregs, although there is no evi-
dence of antigen specificity with oral anti-CD3 anti-
body 43). Ochi et al. proposed the idea that, as observed
in mucosal tolerance induction, low doses of oral anti-
CD3 antibody administration may result in the induc-
tion of Tregs by delivering a weak signal to T cells 88).
Based on the idea that the immunological effects of
oral anti-CD3 antibody do not seem to be antigen
specific, this could be applied for the treatment of a
wide range of autoimmunity diseases, including ath-
erosclerosis, in which many candidate antigens can
elicit pathogenic immune responses. Although further
elucidation of the cellular and molecular mechanisms
underlying the induction of various Tregs by oral anti-
CD3 antibody administration is needed, we believe
that modulation of the Treg response via the intestinal
approach may represent an attractive strategy for the
treatment and prevention of atherosclerosis.
Concluding Remarks
It has now become evident that several types of
Tregs are essential for the regulation of pathogenic T
cell immune responses in atherogenesis, at least in
mice. Enhancement of an endogenous regulatory
immune response could be a novel therapeutic
approach against atherosclerotic disease; however, fur-
ther clinical studies are required to examine whether
this could also apply to humans. We are interested in
reagents, antibodies or novel methods that selectively
affect Treg function or development but have less
effect on effector T cell function. In consideration of
the clinical applications, this seems to be quite impor-
tant because therapies that might affect both Treg and
effector T cell function could activate excessive
immune responses, as observed in intravenous injec-
tion of anti-CD3 antibody or super-agonist anti-
CD28 antibody 89). As another approach to enhance
endogenous regulatory immune responses, cell-based
therapy such as transfer of ex vivo expanded Tregs into
patients with autoimmune disease or graft-versus-host
disease after bone marrow transplantation is currently
under investigation. If cellular therapy is successful for
the treatment of such inflammatory diseases, this
could be a possible therapeutic approach to treat ath-
erosclerosis in humans; however, several key issues
need to be addressed before their clinical use. Such cell
therapy may come into clinical use if very pure Tregs
can be separated and kept stable after expansion upon
antigenic stimulation. Importantly, we should be
aware of severe adverse effects, which have not been
observed in animal experiments, before applying new
methods to promote regulatory immune responses in
the clinical setting.
Acknowledgments
We thank Drs Shimon Sakaguchi and Tomoyuki
Yamaguchi (Laboratory of Experimental Immunology,
WPI Immunology Frontier Research Center, Osaka
University) for valuable discussions.
Sources of Funding
This work was supported by a Grant-in Aid for
Scientific Research in Japan and Research Grants from
Jinsenkai Medical Foundation, Kanae Medical Foun-
dation, Suzuken Memorial Foundation, Uehara Medi-
cal Foundation, Medical Research Fund of Hyogo
Medical Association, Cardiovascular Research Fund,
Hyogo Science and Technology Association, and
Takeda Science Foundation.
Disclosures
None.
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