Regulatory T cells in cancer immunotherapy

TL;DR: It is hoped that combination of Treg-cell targeting with the activation of tumor-specific effector T cells will make the current cancer immunotherapy more effective.

Abstract: FOXP3-expressing regulatory T (Treg) cells, which suppress aberrant immune response against self-antigens, also suppress anti-tumor immune response. Infiltration of a large number of Treg cells into tumor tissues is often associated with poor prognosis. There is accumulating evidence that the removal of Treg cells is able to evoke and enhance anti-tumor immune response. However, systemic depletion of Treg cells may concurrently elicit deleterious autoimmunity. One strategy for evoking effective tumor immunity without autoimmunity is to specifically target terminally differentiated effector Treg cells rather than all FOXP3+ T cells, because effector Treg cells are the predominant cell type in tumor tissues. Various cell surface molecules, including chemokine receptors such as CCR4, that are specifically expressed by effector Treg cells can be the candidates for depleting effector Treg cells by specific cell-depleting monoclonal antibodies. In addition, other immunological characteristics of effector Treg cells, such as their high expression of CTLA-4, active proliferation, and apoptosis-prone tendency, can be exploited to control specifically their functions. For example, anti-CTLA-4 antibody may kill effector Treg cells or attenuate their suppressive activity. It is hoped that combination of Treg-cell targeting (e.g., by reducing Treg cells or attenuating their suppressive activity in tumor tissues) with the activation of tumor-specific effector T cells (e.g., by cancer vaccine or immune checkpoint blockade) will make the current cancer immunotherapy more effective.

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REVIEW
Regulatory T cells in cancer immunotherapy
Atsushi Tanaka
1, 2
, Shimon Sakaguchi
1
1
Laboratory of Experimental Immunology, WPI Immunology Frontier Research Center, Osaka University, Suita 565-0871, Japan;
2
Department of Frontier Research in Tumor Immunology, Graduate School of Medicine, Osaka University, Suita 565-0871, Japan
FOXP3-expressing regulatory T (Treg) cells, which suppress aberrant immune response against self-antigens, also
suppress anti-tumor immune response. Inltration of a large number of Treg cells into tumor tissues is often associ-
ated with poor prognosis. There is accumulating evidence that the removal of Treg cells is able to evoke and enhance
anti-tumor immune response. However, systemic depletion of Treg cells may concurrently elicit deleterious autoim-
munity. One strategy for evoking effective tumor immunity without autoimmunity is to specically target terminally
differentiated effector Treg cells rather than all FOXP3
+
T cells, because effector Treg cells are the predominant cell
type in tumor tissues. Various cell surface molecules, including chemokine receptors such as CCR4, that are speci-
cally expressed by effector Treg cells can be the candidates for depleting effector Treg cells by specic cell-depleting
monoclonal antibodies. In addition, other immunological characteristics of effector Treg cells, such as their high ex-
pression of CTLA-4, active proliferation, and apoptosis-prone tendency, can be exploited to control specically their
functions. For example, anti-CTLA-4 antibody may kill effector Treg cells or attenuate their suppressive activity. It
is hoped that combination of Treg-cell targeting (e.g., by reducing Treg cells or attenuating their suppressive activity
in tumor tissues) with the activation of tumor-specic effector T cells (e.g., by cancer vaccine or immune checkpoint
blockade) will make the current cancer immunotherapy more effective.
Keywords: Treg; CTLA-4; cancer; immunotherapy
Cell Research (2017) 27:109-118. doi:10.1038/cr.2016.151; published online 20 December 2016
Correspondence: Shimon Sakaguchi
E-mail: shimon@ifrec.osaka-u.ac.jp
Introduction
A number of studies have shown that self-antigen or
tumor antigen-specic CD4
+
and CD8
+
T cells are present
in healthy individuals [1-4]. How such self- or tumor-re-
active T cells are controlled in healthy or tumor-bearing
individuals remains to be determined. Mechanisms for
the maintenance of immunological self-tolerance (i.e.,
unresponsiveness to self-antigens) not only prevent au-
toimmunity but also hamper effective tumor immunity
because many tumor antigens recognized by autologous
lymphocytes are normal self-antigens or quasi-self-anti-
gens with genetic mutations. This is one reason why it is
difcult to elicit strong tumor immunity in cancer-bear-
ing patients by cancer vaccine alone [5]. It also suggests
that effective tumor immunity can be evoked by breach-
ing a certain mechanism(s) of immunological self-toler-
ance systemically or locally in tumor tissues.
Among the various mechanisms of immunological
self-tolerance, immune suppression by endogenous Fox-
p3
+
CD25
+
CD4
+
Treg cells is essential and indispensable
as illustrated by spontaneous autoimmune disease devel-
opment when Treg cells are rendered decient. For ex-
ample, mutations of the gene encoding the Treg-specic
transcription factor Foxp3 impair Treg cell development
and cause a fatal multi-organ autoimmune disease called
immune dysregulation, polyendocrinopathy, enteropathy,
and X-linked (IPEX) syndrome [6]. Depletion of Fox-
p3
+
CD25
+
CD4
+
Treg cells by a variety of methods is also
able to cause similar autoimmune diseases in otherwise
normal rodents [7].
On the other hand, it is now well substantiated that a
large number of Treg cells infiltrate into tumor tissues
of various cancers and their abundant presence is often
associated with poor clinical prognosis. Experimentally,
the role of Treg cells in tumor immunity was rst demon-
strated by an attempt to determine a common basis be-
tween tumor immunity and autoimmunity [8]. Removal
of Treg cells using cell-depleting anti-CD25 antibody,
either by in vivo antibody administration to mice or
Cell Research (2017) 27:109-118.
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SPRINGER NATURE | Cell Research | Vol 27 No 1 | January 2017
transfer of cell suspension depleted in vitro of CD25
+
Treg cells into histocompatible T-cell-deficient mice,
effectively eradicated a variety of inoculated syngeneic
tumors [8, 9]. The mice showed an increase of tumor-in-
ltrating CD8
+
T cells with strong tumor-specic killing
activity, and upon re-challenge with the same tumor
cells, exhibited more rapid rejection than the primary
rejection, indicating the establishment of tumor-specic
immunity [8, 10]. These studies have thus demonstrated
that the removal of Treg cells is able to evoke effective
anti-tumor immunity by abrogating immunological un-
responsiveness to syngeneic tumors, albeit it may also
cause autoimmunity, especially if Treg cells are depleted
systemically.
In this review, we discuss molecular basis of Treg
functions and their behavior in tumor tissues, and strat-
egies to target Treg cells, in particular their subsets, in
order to evoke effective anti-tumor immunity in humans,
without eliciting deleterious autoimmunity.
Treg cell function in relation to tumor immunity
T-cell receptor repertoire of Treg cells
The T-cell receptor (TCR) repertoire of Treg cells
is broad and skewed to a certain extent to recognizing
self-antigens. That is, in the course of T-cell selection in
the thymus, a developing Treg cell exhibits a higher TCR
afnity than a conventional T (Tconv) cell for the MHC/
self-peptide ligand that selects both [11]. Assuming that
TCR recognition of peptides is cross-reactive (and de-
generate) and a particular TCR is able to recognize a mil-
lion different peptides of 10 amino acid length [12, 13],
the TCR repertoire of Treg cells as well as Tconv cells
is broad and able to recognize a wide spectrum of self
and non-self antigens including quasi-self-tumor anti-
gens. Given the antigen-primed state of endogenous Treg
cells (as illustrated by higher level expression of T-cell
accessory molecules such as LFA-1), it is reasonable to
assume that Treg cells recognizing a particular self- or
tumor antigen are more easily activated than naive Tconv
cells recognizing the same antigen, ensuring Treg-medi-
ated dominant tolerance [14].
Treg-mediated suppression mechanisms
Treg cells are able to control not only T cells but also
B cells, NK cells, dendritic cells (DCs), and macro-
phages via humoral and cell-cell contact mechanisms [6].
A variety of molecules are involved in Treg-mediated
suppression mechanisms, including CTLA-4 (cytotoxic
T-lymphocyte-associated protein 4), IL-2, IL-10, TGF-β,
IL-35, GITR (glucocorticoid-induced TNF receptor),
LAG3 (lymphocyte-activation gene 3), granzyme B, ade-
nosine, and cAMP [6] (Figure 1 and Table 1). Given that
ectopic Foxp3 expression in Tconv cells is able to confer
Treg-like suppressive activity, the molecule(s) mediating
a core suppressive mechanism may well be controlled
by Foxp3. In addition, among various mechanisms of
Treg-dependent suppression, those important for main-
taining self-tolerance (i.e., the suppression mechanisms
whose impairment causes autoimmune disease) have the
most impact on tumor immunity. On these assumptions,
there are only a few molecules whose expression is con-
trolled by Foxp3 directly or indirectly and whose de-
ciency abrogates Treg-suppressive function and causes
severe autoimmune diseases. The candidates include IL-
2, IL-2 receptor subunits, and CTLA-4. Foxp3 indeed
controls the expression of these molecules and decien-
cies of IL-2, CD25 (IL-2 receptor α-chain), CD122 (IL-
2 receptor β-chain), or CTLA-4 produce similar autoim-
mune diseases as observed in Foxp3 deciency [6].
A cardinal feature of Treg cells is that they consti-
tutively express high-affinity IL-2 receptor composed of
CD25, CD122, and CD132 (common γ-chain) at a high lev-
el, but scarcely produce IL-2. Treg cells therefore highly de-
pend on exogenous IL-2, which is mainly produced by acti-
vated Tconv cells, for their survival and proliferation. Foxp3
binds to and attenuate AML1 and NFAT, two transcription
factors necessary for IL-2 production, thus repressesing
IL-2 production [15, 16]. In fact, neutralization of circu-
lating IL-2 by administration of anti-IL-2 antibody can
compromise Treg function and survival, causing severe
autoimmunity as produced by Treg deficiency [6]. On
the other hand, the constitutive expression of high-af-
nity IL-2 receptor combined with self-insufciency in
IL-2 production determines that Treg cells absorb IL-2
from their surroundings, thus limiting the amount of IL-2
available for Tconv cells and consequently suppressing
the activation and proliferation of the latter [17]. Accord-
ingly, addition of exogenous IL-2 abrogates Treg-sup-
pressive function in vitro [14, 18]. Therefore, IL-2 and
IL-2 receptor can be key targets for controlling Treg cell
survival and suppressive function [19].
CTLA-4 is a highly potent co-inhibitory molecule
expressed constitutively by Treg cells and by Tconv cells
after activation. In mice, Treg-specic deletion of CTLA-
4 elicits systemic hyper-proliferation of Tconv cells and
fatal autoimmune diseases affecting multiple organs, in-
cluding severe myocarditis [20]. Recently, heterozygous
CTLA-4 mutations in humans were identied in patients
with multiple autoimmune symptoms accompanied by
impaired suppressive function of Treg cells [21, 22]. As
CTLA-4 has much higher affinity than CD28 for their
common ligands CD80 and CD86, CTLA-4 expressed by
Treg cells may physically outcompete CD28 on Tconv

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111
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Figure 1 Treg suppression mechanisms. Treg cells, which scarcely produce IL-2, deprive IL-2 from the surrounding via
their high afnity IL-2 receptor, making it unavailable for responder T cells. They also constitutively express CTLA-4, which
down-modulates CD80/CD86 expression by antigen-presenting cells (APCs), thus depriving co-stimulatory signal to respond-
er T cells. Treg cells also produce immune-suppressive cytokines such as IL-10, which also down-modulates APC functions.
Under this deprivation of co-stimulatory signal, responder T cells with high-afnity TCRs for the presented antigen die by
apoptosis, those with intermediate afnity TCRs are rendered anergic, and those with low-afnity TCRs stay dormant. This
IL-2/IL-2 receptor-dependent and CTLA-4-dependent mechanism forms a core basis of Treg-mediated suppression in various
tissues including cancer.
Table 1 Key mechanisms of suppression by Treg cells
Molecule(s) Mechanism of suppression
IL-2 receptor/IL-2 Constitutive expression of high afnity IL-2 receptor α chain (CD25) and dependency on
exogenous IL-2 by Treg cells together limit the amount of IL-2 available to Tconv cells,
thereby hindering the activation and proliferation of the latter
CTLA-4 Constitutively expressed CTLA-4 on Treg cells preferentially binds to and downregulates
CD80/CD86 co-stimulatory molecules on antigen-presenting cells, depriving Tconv cells of
the co-stimulatory signal
IL-10 and other immune-suppressive Treg cells produce immune-suppressive cytokines, such as IL-10 and TGF-β; form extracellular
cytokines and substances adenosine from ATP by CD39 and CD73; and can also mediate direct killing of Tconv or antigen-
presenting cells by secreting granzymes
cells for binding to CD80/CD86 on antigen-presenting
cells, thereby inhibiting co-stimulation of Tconv cells
[23, 24]. Furthermore, CTLA-4 on Treg cells down-mod-
ulates expression of CD80 and CD86 on DCs, thereby
hindering the activation of Tconv cells at this level [20,
25, 26]. Thus, Treg expression of CTLA-4 is essential for
Treg-mediated immune suppression.
Treg cells require TCR stimulation to exert suppres-
sive activity; without antigen stimulation, they fail to
suppress immune response [14]. TCR stimulation of Treg

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cells further upregulates CTLA-4 and other accessory
molecules, particularly adhesion molecules such as LFA-
1, whose deficiency compromises suppressive activity.
When TCR afnity for a stimulating antigen is the same
between Treg cells and Tconv cells, Treg cells can be ac-
tivated to exert suppression at a much lower antigen con-
centration than Tconv cells [14]. This indicates that con-
stitutively high expression of LFA-1 and other accessory
molecules prior to TCR stimulation may contribute to
setting a lower threshold for TCR-induced activation of
Treg cells [27]. On the other hand, another key feature of
Treg cells is their hypoproliferation upon TCR stimula-
tion in vitro; albeit a fraction of Treg cells can proliferate
actively in response to TCR stimulation in vivo [14, 28-
29]. An excessive amount of IL-2 or agonistic anti-CD28
antibody is able to abrogate the in vitro hypo-responsive-
ness. Ectopic expression of Foxp3 in CD4
+
T cells also
converts them into a state of hypo-responsiveness upon
TCR stimulation [30]. It has also been recently demon-
strated that Treg cells, especially Ki-67
+
Treg cells, are
highly dependent on tonic TCR signaling for their pro-
liferation [31, 32], and ablation of TCR signaling results
in caspase-mediated apoptosis of these cells in mice. It
is also of note that Foxp3-dependent repression of the
expression of proximal TCR signaling molecules (e.g.,
ZAP-70) in Treg cells may contribute to their unique sen-
sitivity to TCR stimulation. For example, whereas TCR
stimulation upregulates the expression of ZAP-70 in
Tconv cells, it downregulates ZAP-70 expression in Treg
cells [33]. Foxp3 binds to the promoter region of ZAP-70
gene [33-35] and retroviral expression of Foxp3 in Tconv
cells reduces their ZAP-70 expression [33]. It remains to
be determined how TCR signaling attenuation at the lev-
el of ZAP-70 may contribute to Treg-specic functions.
It might affect Treg cell selection in the thymus to skew
the TCR repertoire toward higher self-reactivity [11].
As another possibility, the TCR signal attenuation might
rescue Treg cells from activation-induced cell death upon
antigen exposure, enabling them to survive better than
Tconv cells and exert dominant control over the latter at
tumor sites.
In summary, constitutively high expression of CD25
and CTLA-4, dependency on exogenous IL-2, and TCR
stimulation have essential roles in producing and shaping
Treg functions, especially Treg-mediated suppression.
Indeed, adapting this triad is able to convert Tconv cells
into Treg-like suppressive T cells effective in vivo and
in vitro, indicating these three events are minimally re-
quired for constructing Treg-suppressive activity [26].
These molecular processes are also good targets to con-
trol Treg function and development in the context of tu-
mor immunity.
Treg migration to nonlymphoid tissues
Treg cells are widely distributed in both lymphoid
and nonlymphoid tissues, including tumors, where they
affect immune responses and inflammation. Treg cells
in nonlymphoid tissues are predominantly effector Treg
cells (CD44
hi
CD62L
lo
) and are highly proliferative [36].
In mice, migration of Treg cells to nonlymphoid tissues,
such as the skin and the lungs, requires chemokine re-
ceptor CCR4 (C-C chemokine receptor type 4) [37]. Treg
cells decient in CCR4 expression fail to migrate, and
this causes severe inammatory diseases in the skin and
the lungs.
Cell fate and function of Treg-suppressed Tconv
cells
Until recently, the fate of responder T cells after they
are suppressed by Treg cells was poorly understood (e.g.,
if they die by apoptosis, become anergic, or stay dormant
during the period of suppression). A recent in vitro study
has now shown that the presence of a large number of
Treg cells can render self-antigen-/tumor antigen-specic
human CD8
+
T cells (e.g., recognizing Melan-A antigen)
anergic [38]. These anergic CD8
+
T cells are hyporespon-
sive to antigen stimulation (i.e., hypoproliferative and
producing very little IL-2 and other cytokines), express
co-inhibitory molecules such as CTLA-4, and are pheno-
typically naive (i.e., CD45RA
high
and CCR7
+
). Induction
of anergy depends on the intensity of Treg suppression
(determined by the number and suppressive activity of
the Treg cells) and TCR afnity of the responder T cells.
Upon Treg-mediated down-modulation of CD80/CD86
expression by antigen-presenting cells, as discussed
above, T cells with high afnity TCR for the stimulating
antigen die by apoptosis, whereas T cells with interme-
diate afnity TCR are rendered anergic, and T cells with
low-affinity TCR stay dormant. Thus, Treg cells can
induce long-term self-tolerance, and hinder effective tu-
mor immunity, by determining the fate of self-reactive or
tumor-reactive T cells (Figure 1).
It is of note that a sizable fraction of self-antigen-spe-
cific CD8
+
T cells (e.g., those specific for Melan-A as
detected by peptide/MHC tetramer staining) present
in the peripheral blood of normal individuals are CT-
LA-4-expressing but naive in phenotype (i.e., CCR7
+
and CD45RA
high
) and functionally anergic. In addition,
CTLA-4-negative naive CD8
+
T cells positive for the
same tetramer staining can be activated to proliferate in
response to antigen stimulation. It appears that the latter
population of non-anergic naive T cells give rise to effec-
tor T cells to cause autoimmune disease or mediate effec-
tive tumor immunity when Treg-mediated suppression is

Atsushi Tanaka and Shimon Sakaguchi
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breached, as discussed below.
Functional/phenotypic heterogeneity of human
FOXP3
+
T cells
There is accumulating evidence that FOXP3
+
T cells
in humans are heterogeneous in phenotype and func-
tion, consisting of suppressive and non-suppressive
subpopulations. For example, naive CD4
+
T cells tran-
siently express FOXP3 at a low level upon in vitro TCR
stimulation; yet they are not measurably suppressive.
Attempts to delineate suppressive and non-suppressive
FOXP3
+
CD4
+
T cells present in the peripheral blood
of healthy individuals have shown that FOXP3
+
CD4
+
T cells can be divided into three subpopulations based
on expression levels of FOXP3 (or CD25) and the cell
surface molecule CD45RA [39] (Figure 2). They are:
(i) FOXP3
lo
CD45RA
+
CD25
lo
cells (Fraction I or Fr. I),
designated as naive or resting Treg cells; (ii) FOXP3
hi
C-
D45RA
–
CD25
hi
cells (Fr. II), designated as effector or
activated Treg cells, which are terminally differentiated
from Fr. I cells upon antigen stimulation and highly sup-
pressive; and (iii) FOXP3
lo
CD45RA
–
CD25
lo
non-Treg
cells (Fr. III), which do not possess suppressive activity
but can secrete pro-inammatory cytokines. Furthermore,
adjacent populations of highly suppressive Fr. II and
non-suppressive Fr. III can be better differentiated by the
expression of CD15s (sialyl Lewis x), a sugar antigen, on
suppressive Treg cells, at least for those in the peripheral
blood [40]. This classication of FOXP3
+
CD4
+
T cells is
instrumental in dening suppressive or non-suppressive
FOXP3
+
subpopulations, delineating developmental stag-
es of Treg cells, and assessing their adaptive processes in
physiological and pathological immune responses.
Tumor-inltrating Treg cells
Accumulating studies have demonstrated that a large
number of Treg cells inltrate into various types of tu-
mors in humans and mice. In humans, tumors in the head
and neck [41], breast [42], lung [43], liver [44], gastroin-
testinal tract [45, 46], pancreas [47], and ovary [48, 49]
have been shown to harbor a large number of tumor-in-
filtrating Treg cells [50]. Importantly, decreased ratios
of tumor-inltrating CD8
+
T cells to FOXP3
+
Treg cells
were shown to correlate with poor prognosis, especially
Figure 2 Functional classication of human FOXP3
+
CD4
+
T-cell subpopulations in tumor tissue. Human FOXP3
+
T cells in the
peripheral blood and lymph nodes are composed of heterogeneous subpopulations containing suppressive Treg cells (naive
and effector Treg cells) and activated non-Treg cells without suppression function. These subpopulations are designated as
Fraction (Fr.) I, II, and III for naive Treg (nTreg), effector Treg (eTreg), and non-Treg cells, respectively. CD25 surface mark-
er can be used in the place of FOXP3 because of their correlative expression in humans. Majority of cancers are inltrated
predominantly by effector Treg cells, whereas certain cancers are inltrated by both effector Treg cells and non-Treg cells.
Tumor-inltrating effector Treg cells predominantly express various cell surface molecules including CTLA-4, CCR4, and PD-1.