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2012 40: 186 originally published online 5 January 2012Toxicol PatholRichard A. Peterson

Regulatory T-Cells: Diverse Phenotypes Integral to Immune Homeostasis and Suppression

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Regulatory T-Cells: Diverse Phenotypes Integral toImmune Homeostasis and Suppression

RICHARD A. PETERSON

GlaxoSmithKline, Research Triangle Park, North Carolina, USA

ABSTRACT

Regulatory T-cells (TREG) are diverse populations of lymphocytes that regulate the adaptive immune response in higher vertebrates. TREG delete

autoreactive T-cells, induce tolerance, and dampen inflammation. TREG cell deficiency in humans (i.e., IPEX [Immunodysregulation, Polyendocri-

nopathy and Enteropathy, X-linked syndrome]) and animal models (e.g., ‘‘Scurfy’’ mouse) is associated with multisystemic autoimmune disease.

TREG in humans and laboratory animal species are similar in type and regulatory function. A molecular marker of and the cell lineage specification

factor for TREG is FOXP3, a forkhead box transcription factor. CD4þ TREG are either natural (nTREG), which are thymus-derived

CD4þCD25þFOXP3þ T-cells, or inducible (i.e., Tr1 cells that secrete IL-10, Th3 cells that secrete TGF-b and IL-10, and Foxp3þ Treg). The proin-

flammatory Th17 subset has been a major focus of research. TH17 CD4þ effector T-cells secrete IL-17, IL-21, and IL-22 in autoimmune and inflam-

matory disease, and are dynamically balanced with TREG cell development. Other lymphocyte subsets with regulatory function include: inducible

CD8þ TREG, CD3þCD4�CD8� TREG (double-negative), CD4þVa14þ (NKTREG), and gd T-cells. TREG have four regulatory modes of action: secre-

tion of inhibitory cytokines (e.g., IL-10 and TGF-b), granzyme-perforin-induced apoptosis of effector lymphocytes, depriving effector T-cells of

cytokines leading to apoptosis, or inhibition of dendritic cell function. The role of TREG in mucosal sites, inflammation/infection, pregnancy, and

cancer as well as a review of TREG as a modulatory target in drug development will be covered.

Keywords: immunopathology; regulatory T-cells; lymphocytes; flow cytometry; Scurfy mouse; FOXP3; IPEX.

INTRODUCTION TO REGULATORY T-CELLS

This article will review the scientific literature and provide an

overview of Regulatory T-cell (TREG) characterization, function,

role in the immune response, at mucosal sites, during pregnancy

and in the tumor microenvironment, and as a target for modulation

in drug development. There has been enhanced focus on the TREG

in the field of immunology, which translates into a prominent

increase in the number of citations over the past decade (Figure 1).

TREG are diverse populations of lymphocytes that regulate

the adaptive immune response in higher vertebrates. TREG

delete autoreactive T-cells, induce tolerance, and dampen

inflammation (Akdis 2009; Ozdemir et al. 2009; Dittel 2008;

Orihara et al. 2008; Sakaguchi et al. 2008; Simpson 2008;

Bluestone and Tang 2005; Coutinho et al. 2005).

Over the past 40 years, the field of immunology has slowly

evolved, becoming much more complex than was initially

hypothesized in the 1960s. Between 1966 and 1968, several

groups identified B-lymphocytes as the source of the antibody

response and T-lymphocytes as critical in the delayed-type

hypersensitivity (DTH) response (Claman et al. 1966; Mosier

1967; Mitchell et al. 1968). In 1971, Gershon and Kondo iden-

tified negative interference on positively acting lymphocytes

during inflammation and referred to it as ‘‘infectious immuno-

logical tolerance’’ (Gershon and Kondo 1971). This was the

first indication that a ‘‘suppressive’’ process was active during

inflammation. The ‘‘suppressor T-cell’’ hypothesis was put

forth by two groups in the later years of the 1970s (Vadas et

al. 1976; Okumura et al. 1977). A series of negative results

from experiments conducted by several groups led to a loss

of confidence in the ‘‘suppressor T-cell’’ hypothesis, rejection

of the hypothesis by the field of immunology, and more

recently reacceptance (Kapp and Bucy 2008; Germain 2008;

Lehner 2008). The rejection of the ‘‘suppressor T-cell’’ hypoth-

esis did not explain away the presence of a dampening of

immune responses commonly seen in the inflammatory milieu.

In 1989, Mosmann and Coffman identified the T-helper (TH)-1

and (TH)-2 CD4þ T-cell subsets based upon the profiles of

cytokines secreted after activation. TH1 cells primarily secrete

IFN-g, IL-15, and TNF-a, while TH2 cells secrete IL-4, IL-5,

IL-6, and IL-13. Powrie et al. showed in severe combined

immunodeficient (SCID) mice that subsets of CD4þ T-cells

The author declared no potential conflicts of interests with respect to the

authorship and/or publication of this article. The author received no financial

support for the research and/or authorship of this article.

Address correspondence to: Richard A. Peterson II, DVM, PhD, DACVP,

Safety Assessment, 9.3009.2D, GlaxoSmithKline, 5 Moore Drive, Research

Triangle Park, NC 27709; phone: (919) 315-2450; fax: (919) 483-6858;

e-mail: [email protected].

Abbreviations: A2A, adenosine receptor; APC, antigen presenting cell; BREG,

regulatory B-cell; CD, cluster of differentiation; CMV, cytomegalovirus; CTLA-

4, cytotoxic T-lymphocyte antigen-4; DN, double negative; DTH, delayed-type

hypersensitivity; EAE, experimental autoimmune encephalomyelitis; FIV, feline

immunodeficiency virus; FLG-2, fibrinogen-like protein-2; FOXP3, forkhead box

protein-3; GVHD, graft-versus-host disease; HCV, hepatitis C virus; HIV, human

immunodeficiency virus; IBD, inflammatory bowel disease; IFNg, interferon

gamma; IL, interleukin; IPEX, immunodysregulation polyendocrinopathy entero-

pathy X-linked syndrome; LAG3, lymphocyte-activating gene-3; MHC, major

histocompatability complex; miR, microRNA; MoAb, monoclonal antibody;

mTOR, mammalian target of rapamycin; NK cell, natural killer cell; NKTREG,

natural killer regulatory T-cell; SCID, severe combined immunodeficient; TCR,

T-cell receptor; TGF-b, transforming growth factor beta; TH0, T-helper 0 cell;

TH1, T-helper 1 cell; TH2, T-helper 2 cell; TH3, T-helper 3 cell; TH17, T-helper

17 cell; TREG, regulatory T-cell; Tr1, type 1 regulatory T-cells.

186

Toxicologic Pathology, 40: 186-204, 2012

Copyright # 2012 by The Author(s)

ISSN: 0192-6233 print / 1533-1601 online

DOI: 10.1177/0192623311430693

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induce and protect from intestinal inflammation (Powrie et al.

1994). This once again defined a population of lymphocytes

with immunosuppressive actions, which was identified as a

population expressing CD45RBlow while the inducing cells

were CD45RBhigh. In 1995, Sakaguchi et al. found that immu-

nologic self-tolerance was maintained by activated CD4þ T-

cells expressing CD25 (IL-2R alpha chain). Depletion of this

CD4þCD25þ population led to development of autoimmune dis-

ease (Sakaguchi et al. 1995). Two groups, Hori et al. and Fontenot

et al., identified a CD4þCD25þFOXP3þ cell population that was

found to be thymically derived, natural TREG cells (Hori et al.

2003; Fontenot et al. 2003, 2005). FOXP3 has been shown to

be critical for regulatory action of most subtypes of TREG.

TYPES OF REGULATORY T-CELLS (TREG, NATURAL,

AND ADAPTIVE/INDUCED) CD4þCD25þ TREG

Natural TREG are thymus-derived CD4þCD25þFOXP3þ T-

cells (Figure 2). They arise in the thymus during early stages of

human fetal development (gestational week 14) (Cupedo et al.

2005) and are resistant to thymic deletion (Lim et al. 2006).

nTREG differentiate from thymocytes that express TCRs with

an increased affinity for self-peptide-MHC complexes (Lim

et al. 2006; Maggi et al. 2005; Schwartz 2005). nTREG can sup-

press the following cell types: CD4þ T-cells, dendritic cells,

CD8þ T-cells, NKT cells, NK cells, monocytes/macrophages,

B-cells, mast cells, basophils, eosinophils, and osteoblasts

(Shevach et al. 2009; Lim et al. 2006). FOXP3þ expression

is necessary for thymocytes to commit to the TREG lineage

(Lim et al. 2006). Resting nTREG are CD45RAþFOXP3low,

while activated nTREG are CD45ROþFOXP3high (Beissert

et al. 2006; Lim et al. 2006). Natural TREG also express the

following markers: T cell activation/differentiation markers

(CD45RO [memory phenotype; activated], CD45RB [resting],

CD25 [both]), adhesion molecules (CD62L [both], CD44

[both] and Integrin a4b7 [resting]), cytotoxic T lymphocyte-

associated protein-4 (CTLA-4; both), co-stimulatory molecule

CD28 (both), chemokine receptors (CCR7 [both], CXCR4

[both], CCR9 [resting]), glucocorticoid-induced TNFR-

related protein (GITR, both), OX40 (CD134, both), and folate

receptor-4 (FR4 in rodents, both) (Lim et al. 2006). IL-2 is essen-

tial for nTREG development, function, and homeostasis (i.e.,

CD25 is the a-chain of the high affinity IL2R) (Malek et al.

2008). Severe autoimmunity is seen in IL-2-, IL2Ra-, and IL-

2Rb-deficient mice, which also lack a normal number of TREG

cells (Malek et al. 2008). The nTREG profile in mammals is highly

conserved. Nonhuman primates have the same nTREG protein

expression and functional profile as that of humans including two

FOXP3 isoforms (Roncarolo and Battaglia 2007; Bloom 2006),

while in rodents there are only subtle differences primarily in the

type of granzyme expressed (i.e., B versus A) and only a single

FOXP3 isoform (Roncarolo and Battaglia 2007).

ADAPTIVE/INDUCED TREG

The two most common types of adaptive or induced TREG

are the T regulatory type 1 cells (Tr1) and TH3 TREG cells (Fig-

ure 2). Tr1 cells secrete IL-10 and also have an IL-10-

dependent induction process (Fujio et al. 2010; Beissert et al.

2006; Roncarolo et al. 2006). They are capable of secreting

high levels of IL-10 and TGF-b in the human and mouse and

also secrete low levels of IL-2, IL-5, and IFN-g (Fujio et al.

2010; Roncarolo et al. 2006). An important growth factor for

Tr1 cells is IL-15, which can support Tr1 cell proliferation even

without TCR activation (Fujio et al. 2010; Roncarolo et al.

2006). They are CD4þ, anergic, and proliferate poorly upon

antigen-specific activation which is likely due to the autocrine

production of IL-10 leading to suppression of proliferation

(Fujio et al. 2010; Roncarolo et al. 2006). The mechanism of

suppressive effects with Tr1 cells is soluble factor-based (i.e.,

IL-10), and the suppressive effects of Tr1 cells are negated

by anti-IL-10 neutralizing antibody in vitro (Fujio et al.

2010; Roncarolo et al. 2006). There is no specific marker for

Tr1 cells, although repressor of GATA-3 (ROG) shows poten-

tial utility (Fujio et al. 2010; Roncarolo et al. 2006), but it is not

specific for this cell population.

TH3 cells secrete TGF-b and IL-10 and express FOXP3

(Beissert et al. 2006). They are induced from naıve CD4þ

T-cells by TGF-b and have an important role in oral tolerance

to non-self antigens and negating autoimmune reactions (Wan

and Flavell 2007). TH3 cells secrete TGF-b, which has immu-

nosuppressive effects acting through a soluble-factor mechan-

ism. TH3 cells have a reciprocal relationship with TH17 cells

(Korn et al. 2009; Nistala and Wedderburn 2009). TH17 cells

are highly proinflammatory and are thought to be important in

autoimmune disease (Abraham and Cho 2009; Crome et al.

2009; Korn et al. 2009; Nistala and Wedderburn 2009;

Romagnani et al. 2009); this will be discussed in detail later

in the article. There has been no specific surface marker

identified for TH3 cells, although expression of FOXP3 is

induced in TH3 cells (i.e., cannot differentiate from nTREG)

(Korn et al. 2009).

FIGURE 1.—There has been a steady increase in the number of regula-

tory T-cell (TREG) citations in the scientific literature from the mid-

1990s to the present (from Scopus). The key milestones in TREG

research are highlighted on the horizontal axis.

Vol. 40, No. 2, 2012 REGULATORY T-CELLS 187



OTHER T-CELLS WITH REGULATORY FUNCTION

Other T-cell populations that have been shown to have reg-

ulatory function include inducible CD8þ TREG cells,

CD8þCD28� TREG cells, and CD3þCD4�CD8� TREG

(double-negative [DN]). CD8þ TREG (Kapp and Bucy 2008;

Beissert et al. 2006; Nakamura et al. 2003; Ke and Kapp

1996; Jiang et al. 1992) are CD8þ T-cells that have regulatory

activity on CD4þ TH1 cells. This TREG population inhibits

priming of CD8þ T-cells, CD4þ T-cells, and antibody

responses against oral antigens. They are induced by manipula-

tion of co-stimulatory molecule interactions (i.e., CD137 and

CD40) primarily, although antigens introduced into immune

privileged sites (i.e., anterior chamber of eye leading to anterior

chamber-associated autoimmune deviation [ACAID]) elicit

CD8þ TREG due to high levels of TGF-b and IL-10 at this site

(Biros 2008; Niederkorn 2008). They have also been shown to

arise spontaneously in Experimental Allergic Encephalitis

(EAE) and murine Ovalbumin-induced oral tolerance models

and in tumor-bearing hosts due to IL-10 secretion by APCs

in the neoplasm. They are well characterized in rodents, but

their importance in humans is uncertain. CD8þCD28- TREG

(Cortesini et al. 2001; Chang et al. 2002) are FOXP3� and are

induced by inhibitory immunoglobulin-like transcript (ILT)-3

and ILT-4 receptor expression on dendritic cells and down-

regulation of CD80 and CD86. They interfere with the

CD28/B7 pathway with allogeneic antigens and CD154/

CD40 pathway with xenogeneic antigens. CD8þCD28� TREG

regulate immune responses by direct cell-to-cell interactions

with dendritic cells; therefore they are directly tolerogenic.

This cell population has been described in rodents and humans.

CD3þCD4�CD8� (DN) TREG are TCRabþ, CD4�, and CD8�

(therefore DN). They promote the development of tolerance

and regulate autoimmunity (Strober et al. 1996). This cell pop-

ulation has been defined and characterized in heart xenotrans-

plantation experiments (Chen et al. 2003, 2005; Zhang et al.

2006; Ma et al. 2008). DN TREG are poorly suppressive in naıve

mice but are actively suppressive in recipients of heart xeno-

grafts co-transferred with donor DN Tregs. They have been

shown to prevent heart xenograft rejection (i.e., transplant tol-

erance). It has been shown that antigen presenting cells (APC)

from tolerant mice had down-regulation of MHCII, CD40, and

B7 molecules, and likely were in an immature state. The

FIGURE 2.—TREG are either formed naturally by thymic differentiation (nTREG) or are induced in the periphery (iTREG) from naıve T-cells (TH0).

Types of iTREG include TH3, Tr1, which are CD4þCD25þFOXP3þ, and CD4þCD25-FOXP3þ iTREG. The main effector CD4þ subsets are TH1,

TH2, and TH17. The cytokines that are important in inducing these cells from TH0 cells and the cytokines that these cells secrete are listed.

188 PETERSON TOXICOLOGIC PATHOLOGY



significance of DN TREG in nontransplant scenarios is therefore

uncertain and more data is needed.

CD4þVa14þ (NKTREG)

Natural killer T-cells coexpress NK receptors (i.e., NK1.1 or

CD161) as well as a conserved T-cell receptor (TCR) molecule

(Beissert et al. 2006), Va14. Va14 pairs with Vb8 in the mouse

and Vb11 in the human and are restricted by CD1d, a conserved

MHCI-type molecule that presents glycolipids. NKTREG cells

are generated in the thymus and can be CD4þ, CD8þ, DN, or

double positive (DP). In mice, DN NKTREG are the dominant

phenotype (Zeng et al. 1999); while in humans, CD4þNKTREG

are most efficient (Jiang et al. 2005). They have been studied pri-

marily in rodent xenogeneic transplantation models (Ikehara et

al. 2000). They secrete IL-4, IL-10, TGF-b (paracrine), and

induce cytotoxicity by cell-to-cell contact, and the targets of

NKTREG cells are T-cells and APCs (Ikehara et al. 2000; Zeng

et al. 1999). The significance of NKTREG cells in immune regu-

lation outside of xenotransplantation is uncertain.

gd T-CELLS (MUCOSAL/INTRAEPITHELIAL)

Gamma delta (gd) T-cells, which express TCRgd and have a

mucosal distribution, are primarily suppressive and are associ-

ated with mucosal tolerance (Kapp and Ke 1997; Ke et al.

1997; Mowat 1994), but can also function to regulate autoim-

munity (Weiner et al. 1994), elicit ACAID in the eye (Biros

2008), and function in tumor immunity (Seo et al. 1999). These

cells do not recognize peptides associated with MHC receptors

(Kronenberg 1994); alternatively they recognize cellular pro-

teins including heat shock proteins (Munk et al. 1989) and non-

classic MHC molecules like CD1 (Brossay et al. 1998) and Qa-

1 (Vidovic et al. 1989). gd T-cells that infiltrate tumors have

FIGURE 3.—TREG can cause suppression of effector T-cells and dendritic cells through secretion of soluble factors including IL-10, TGF-b,

fibrinogen-like protein-2 (FLG-2), granzyme A or B and perforin, and adenosine. The figure shows the signaling pathways and the suppressive

effects on effector T-cells and dendritic cells.




cytokine profiles that are similar to Tr1 cells (i.e., IL-10 and

TGF-b) and inhibit the immune response against neoplasia

(Kapp et al. 2004; Seo and Tokura 1999; Seo et al. 1999; Kapp

and Ke 1997; Ke et al. 1997). Therefore, gd T-cells are a minor

population of mucosal and intratumoral T-cells with regulatory

activity and major roles in mucosal tolerance and tumor

immunity.

A NON-T LYMPHOCYTE SUBSET WITH REGULATORY FUNCTION

Another cell type exhibiting regulatory activity is the regu-

latory B-cell (BREG) which has been recently reviewed by Li et

al. (2011). BREG cells are beyond the scope of this review, but

are mentioned here briefly to showcase the diversity of lym-

phocytes with regulatory function in the immune system and

for completeness-sake. BREG cells have been shown in various

animal disease models, including collagen-induced arthritis

(Trentham et al. 1977), experimental autoimmune encephalitis

(EAE) (Wolf et al. 1996), nonobese diabetes mellitus (Tian et

al. 2001), inflammatory bowel disease (Mizoguchi et al. 2000),

and systemic lupus erythematosus (Lenert et al. 2005), and

function primarily to regulate T-cells, B-cells, and NKT cells.

BREG cells act by soluble factor-based means by secreting IL-

10 (Reigert and Bar-Or 2008), TGF-b (Takenoshita et al. 2002)

and producing anti-inflammatory antibodies (Diamond et al.

2003). Cell-cell interactions are an important mode of BREG

regulatory function and include CD1d binding (Yanaba et al.

2008) by CD1dhiCD5þ cells and CD40/CD40 L interactions

(Quezada et al. 2004). More research into specific roles for

BREG is needed to more completely define this cell type.

TREG BASICS

TREG cells function at sites of inflammation in close spatial

proximity to effector T-cells, thus allowing direct interactions

between the two cell types (Askenasy et al. 2008). TREG cells

FIGURE 4.—TREG can cause suppression of effector T-cells and dendritic cells through cell contact-based mechanisms, including galectin-1 bind-

ing, CTLA-4 binding to CD80/86, lymphocyte activation gene-3 (LAG-3) binding to MHCII molecules, and neuropilin-1 binding. The figure

shows the ligands, receptors, and the suppressive effects on effector T-cells and dendritic cells.




are attracted by inflammatory signals, although they are less

chemotactic than effector T-cells (Siegmund et al. 2005).

When arriving at the site of inflammation, TREG down-

regulate chemotactic receptors and adhesive interactions to

arrest further migration (Siegmund et al. 2005). nTREG are

mitotically inactive under basal conditions (Kuniyasu et al.

2000). Activated TREG-mediated immune suppression is

antigen-nonspecific (i.e., not MHCII-restricted) (Bienvenu et

al. 2005), although induction of suppressor function in TREG

requires antigen-specific stimulation (i.e., via TCR) leading

to tissue-specific suppressive effects (‘‘bystander’’ suppres-

sion) (Shevach 2009; Weiner 1997). TREG have also been asso-

ciated with regulating lymphopenia-induced lymphocyte

proliferation, which is a normal homeostatic event that occurs

after lymphopenia of numerous causes including those of a

physiologic and pathologic nature. Lymphopenia-induced lym-

phocyte proliferation does not increase the number of naıve

lymphocytes, but instead the number of memory T-cells

increase, which might result in potential self-reactive clones

and subsequent autoimmunity. TREG have an important role

in regulating this process (Datta and Sarvetnick 2009).

EFFECTS OF ACUTE AND CHRONIC STRESS ON HUMAN TREG

beta 1-adrenergic and glucocorticoid a receptors are overex-

pressed in TREG versus effector T-cells (Freier et al. 2010; Ata-

nackovic et al. 2006). These receptors likely mediate TREG

responses to stress (Freier et al. 2010; Atanackovic et al.

2006). TREG numbers have been shown to decrease with acute

physiologic stress (Freier et al. 2010; Atanackovic et al. 2006;

Atanackovic et al. 2002). Based on these findings, chronic

stress could exacerbate autoimmune disease and other inflam-

matory conditions (Freier et al. 2010).

MODES OF ACTION OF TREG REGULATION

TREG have primary effect on T-cells and/or dendritic cells

by three main regulatory modes of action: (1) soluble factors

(Figure 3), (2) cell-to-cell contact (Figure 4) and (3) competi-

tion for growth factors (local effect) (Figure 5). Soluble factors

include IL-10 and TGF-b with direct suppressive effects on

effector T-cells (Joetham et al. 2007; Annacker et al. 2003),

fibrinogen-like protein-2 (FLG-2) with apoptotic effects on

effector T-cells and prevention of maturation of dendritic cells

(Shalev et al. 2009), granzyme A/B and perforin apoptotic

effects on effector T-cells (Grossman et al. 2004), and produc-

tion of adenosine by CD39/73 cleavage of ATP, which causes

cell cycle arrest in effector T-cells and prevention of matura-

tion and decreased antigen presenting capability in dendritic

cells by binding to the A2A receptor on these cell types (Deaglio

FIGURE 5.—TREG can cause suppression of effector T-cells by providing competition for growth factors (primarily IL-2), which is essential for

their survival in the periphery. TREG express multiple copies of IL-2R/CD25, which bind up local IL-2 competing with effector T-cells for

IL-2 stimulation. The figure shows the ligands, receptors, and the suppressive effects on effector T-cells.

FIGURE 6.—FOXP3 and CD3 dual immunofluorescence on tissue from

an area of inflammation. Note the green nuclear labeling (FOXP3þ) and

the red cytoplasmic labelling (CD3þ). The image shows a group of T-

cells with several FOXP3þ TREG cells in the field. 400�magnification.




et al. 2007). Galectin-1 binding to effector T-cells and dendritic

cells resulting in cell cycle arrest and/or apoptosis (Garin et al.

2007), CTLA4 and CD80/86 binding to dendritic cells causing

decreased costimulation and decreased antigen presentation

(Read et al. 2000), lymphocyte activating gene-3 (LAG3 or

CD223, a CD4 homolog) binding to MHCII molecules on den-

dritic cells leading to prevention of maturation and a reduction in

antigen presentation capability (Workman and Vignali 2004),

and neuropilin-1 binding to dendritic cells resulting in decreased

antigen presentation (Sarris et al. 2008) are examples of cell

contact-based mediated effects of TREG. TREG can also deny

IL-2 to other inflammatory cells by binding the cytokine and act-

ing as a ‘‘sink’’ leading to effector T-cell apoptosis (Pandiyan et

al. 2007). Therefore, TREG have a variety of mechanisms that

can be used to suppress an immune response and are able to use

all or a subset of these mechanisms simultaneously.

TREG MARKERS (IMMUNOHISTOCHEMISTRY, FLOW CYTOMETRY,

INTRAVITAL MICROSCOPY, AND MIRNA ANALYSIS)

Immunofluorescent and chromogenic IHC and flow cyto-

metry (de Boer et al. 2007; Roncador et al. 2005) for the

following markers has been shown to be useful in characteriz-

ing TREG in tissue sections and isolated cells: FOXP3 (Figures

6, 7, 8, and 9), CD25 (alpha chain of IL2R) (Figures 7, 8, and

9), and CD62L (L-Selectin) (Figures 6 and 7). Intercellular

interactions of TREG cells and effector T-cells have been visua-

lized in rodent models using intravital microscopy (Tang and

Krummel 2006). Differences in micro-RNA (miRNA) in

human TREG versus effector T-cells have been shown (Hezova

et al. 2010). There was a significant increase in miR-146a and

decreases in miR-20b, 31, 99a, 100, 125b, 151, 335, and 365 in

TREG when compared with effector T-cells. The pattern of

increased expression of miR-146a in combination with

decreases in the other miRNAs is a possible marker of TREG.

FOXP3: FUNCTION AND BIOLOGY

FOXP3, a forkhead box transcription factor, is a molecular

marker and cell lineage specification factor for TREG (Fontenot

et al. 2003; Hori et al. 2003). FOXP3 is critical for the thymic

differentiation of ab TCRþ thymocytes into nTREG (Sakaguchi

et al. 2008; Fontenot et al. 2003; Hori et al. 2003). The FOXP3

gene product is referred to as scurfin and functions by

FIGURE 7.—Flow cytometry was performed on blood from male and female CD-IGS rats to evaluate the percentage of

CD4þCD25þCD62LþFOXP3þ (nTREG and iTREG) cells. The image highlights the gating strategy and steps used in this evaluation.




regulating a set of genes required for (1) the suppressor activity

of TREG, proliferative, and metabolic fitness of TREG and (2)

repressing alternative T cell differentiation pathways (Brun-

kow et al. 2001). It has been shown that a higher level of

expression of FOXP3 is sufficient for suppressive activity of

non-TREG T-cells in rodents (Sakaguchi et al. 2008; Lourenco

and La Cava 2011). Several pathways are important in the

induction of FOXP3 expression including TGF-b, IL-2, reti-

noic acid, 17-b-estradiol, and signaling through the

sphingosine-1-phosphate receptor 1 (S1PR1) (Figure 10).

TGF-b is essential for induction of FOXP3 expression in naıve

T-cells (i.e., iTREG including TH3 cells), but in the human it

does not necessarily confer regulatory function (Lourenco and

La Cava 2011).

FOXP3 DEFICIENCY

The ‘‘Scurfy’’ Mouse

In mice (B.Cg-Foxp3sf), a frameshift mutation in the FOXP3

gene results in a truncated protein lacking the forkhead domain

and is responsible for the ‘‘Scurfy’’ phenotype (Clark et al.

1999; Ochs et al. 2002; Patel 2001). Scurfy is an X-linked

recessive mutation in the mouse that is lethal in hemizygous

males 16 to 25 days after birth (Ochs et al. 2002; Godfrey

et al. 1991). Scurfy mice have an overproliferation of CD4þ

T-lymphocytes and other cell types due to uncontrolled

cytokine secretion with extensive multiorgan infiltration due

to lack of functional TREG (Ochs et al. 2002; Godfrey et al.

1991). Lymphadenopathy, parenchymal organ infiltration, and

subcutaneous inflammatory cell infiltrates (e.g., ear pinnae) are

prominent (Figures 11 and 12) (Godfrey et al. 1991). The phe-

notype of Scurfy mice is similar to mouse models that lack

CTLA-4 or TGF-b, and in humans with immunodysregulation,

polyendocrinopathy and enteropathy, X-linked syndrome

(IPEX) (Clark et al. 1999; Ochs et al. 2002; Patel 2001).

IPEX in Humans

IPEX is a rare human multisystemic autoimmune disease

(X-linked: females are carriers, males have the disease) that

is linked to the dysfunction of the transcriptional activator

FOXP3 which results in dysfunction of regulatory T-cells with

subsequent autoimmunity (Ochs et al. 2002). Mutations in the

forkhead domain of FOXP3 disrupt FOXP3 protein: DNA

interactions (Ochs et al. 2002; Patel 2001). The disorder man-

ifests with enteropathy, food allergy, massive lymphoprolifera-

tion, psoriasiform or eczematous dermatitis, nail dystrophy,

autoimmune endocrinopathies (primarily insulin-dependent

diabetes mellitus and autoimmune thyroid disease), Coombs-

positive anemia, autoimmune thrombocytopenia, autoimmune

neutropenia, tubular nephropathy, and autoimmune skin condi-

tions such as alopecia universalis and bullous pemphigoid

(Ochs et al. 2002). Treatment for IPEX includes immunosup-

pressive agents (e.g., cyclosporin A, FK506) alone or in com-

bination with steroids (Ochs et al. 2002). There has been

limited success in treating the syndrome with bone marrow

transplantation (Ochs et al. 2002).

FIGURE 8.—The graphs of flow cytometry data using markers useful in

characterizing TREG show the percentage of CD3þCD4þ cells,

CD62Lþ cells in the CD3þCD4þpopulation, CD25þcells in the

CD3þCD4þ population, and FOXP3þ cells in the CD3þCD4þ popu-

lation cell subsets in male and female rats. There was no definitive dif-

ference in cell percentages between males and females in this

evaluation.

FIGURE 9.—The graphs of flow cytometry data show the percentages of

FOXP3þ cells in the CD4þCD25þ population and CD25þ cells in the

CD4þFOXP3þ population. The majority (approximately 80%) of

CD4þCD25þ cells are also FOXP3þ, which would include nTREG and

TH3 cells. Only a minority (approximately 30%) of CD4þFOXP3þ

cells are also CD25þ, which means approximately 70% of the

CD4þFOXP3þ population likely represents an iTREG cell population.




TH17 CELLS AND TREG: A RECIPROCAL BALANCE

TH17 cells are a subpopulation of effector T cells that are

very pro-inflammatory and implicated in allergic/autoimmune

disease (Abraham and Cho 2009; Crome et al. 2009; Korn et al.

2009 and 2007; Nistala and Wedderburn 2009; Romagnani et

al. 2009). TH17 cells secrete IL-17, IL-22, IL-21 and IL-17F

(Korn et al. 2009, 2007; Veldhoen et al. 2006; Bettelli et al.

2006; Mangan et al. 2006). TGF-b is critical in induced/adap-

tive TREG cell (i.e., TH3 TREG) differentiation as well as devel-

opment of TH17 effector cells (Wan and Flavell 2007) (Figure

13). Naıve T-cells develop into a ‘‘transitional’’ cell-type that

expresses FOXP3 (TH3 TREG cell marker) and ROR-gt and

RORa (TH17 effector cell markers) in the presence of TGF-b(Figure 14) (Nistala et al. 2009; Korn et al. 2007; Veldhoen

et al. 2006; Bettelli et al. 2006; Mangan et al. 2006). Further

exposure to TGF-b or IL-6/IL-21 in the inflammatory milieu

leads to TH3 cell differentiation or TH17 effector cell differen-

tiation (Zhou et al. 2008; Korn et al. 2007; Veldhoen et al.

2006; Bettelli et al. 2006; Mangan et al. 2006). Therefore, a

reciprocal balance between TH17 cells and TH3 TREG cells

exists (Bettelli et al. 2006) where IL-6 and IL-21 inhibit TH3

cell differentiation to the benefit of TH17 cells (Korn et al.

2009, 2007) and TGF-b elicits TH3 cell differentiation to the

disadvantage of TH17 cells (Zhou et al. 2008).

ROLE OF TREG IN MUCOSAL SITES

Oral tolerance is an important mechanism in the mucosal

immune system whereby oral administration of a specific anti-

gen induces unresponsiveness of systemic immune responses

with the same or different antigens (Chen et al. 2007; Weiner

et al. 1994). TGF-b is critical for the induction of oral tolerance

(Chen et al. 2007; Weiner et al. 1994). Low-dose of antigen

cells leads to ‘‘dampening,’’ a paracrine effect, of the immune

response due to TGF-b derived from nTREG, TH3, and Tr1 cells.

High-dose of an antigen is associated with mucosal T-cell

anergy and apoptosis (Chen et al. 2007; Weiner 1997; Weiner

FIGURE 10.—The figure highlights several pathways of induction of FOXP3 expression including the ligands, receptors, and/or intermediary steps

in induction of gene expression. A schematic of the FOXP3 gene shows the promoters and important transcription factors that bind to the pro-

moters. There are 11 coding exons in both the mouse and human FOXP3 genes.




et al. 1994). At mucosal sites, TREG are responsible for

‘‘bystander’’ suppression, where TGF-b produced at mucosal

sites during development of oral tolerance regulates immune

responses on T-cells, B-cells, NK cells, macrophages, and den-

dritic cells in an antigen nonspecific manner (Weiner 1997).

Animal models of inflammatory bowel disease (IBD) have been

used to study TREG and mucosal tolerance (Blumberg 2009).

Researchers have adoptively transferred CD4þCD45RBhigh

T-cells into SCID or Rag-1 mice (Powrie et al. 1994) or have

relied on the 2, 4,6-trinitrobenzene sulfonic acid (TNBS)-induced

colitis rodent model (Te Velde et al. 2006). A negative effect of

TREG in mucosal immunity is the secretion of TGF-b from TREG

in the face of IL-6 and IL-21 in the inflammatory milieu. TGF-bcan lead to differentiation of TH17 cells and subsequent inhibition

of TREG differentiation, resulting in more severe inflammation

(Korn et al. 2009, 2007). TREG have a critical role in tolerance and

‘‘bystander’’ suppression of mucosal sites with resident gdT-cells.

ROLE OF TREG IN INFLAMMATORY/INFECTIOUS DISEASE

Both natural and induced TREG are important in suppressing

inflammation in infectious disease (Figure 15; Abraham and

Medhitzov 2011; Liu et al. 2010; Jager and Kuchroo 2010;

Scholzen et al. 2009; de Boer et al. 2007; Belkaid and Rouse

2005). nTREG are activated by microbial infections and prod-

ucts of the resulting inflammatory milieu. TREG express toll-

like receptors 4, 5, 7, and 8, allowing the cells to respond to

bacterial products such as lipopolysaccharide leading to

increased TREG survival and proliferation (Caramahlo et al.

2003). Probiotics have been shown to elicit activation of TREG,

which then suppress TH1-mediated colitis in the TNBS mouse

colitis model (Di Giacinto et al. 2005). Microbes are also able

to manipulate antigen-presenting cells leading to priming/acti-

vation of TREG and elicit secretion of chemokines and cyto-

kines that favor induction, priming, recruitment, and survival

of TREG, thus using TREG for their own benefit (Cabrera et al.

2004; Hesse et al. 2004; Caramahlo et al. 2003; Belkaid et al.

2002). These microbial organisms include virus (HIV [Aandahl

et al. 2004], HCV [Cabrera et al. 2004], CMV [Aandahl et al.

2004], FIV [Vahlenkamp et al. 2004], Herpes simplex virus

[Suvas et al. 2004]), bacteria (Helicobacter spp. [Lundgren et

al. 2003; Kullberg et al. 2002] and Listeria monocytogenes

[Kursar et al. 2002]), fungi (Candida albicans [Montagnoli et

al. 2002] and Pneumocystis carinii [Hori et al. 2002]), and pro-

tozoal (Leishmania major [Belkaid et al. 2002], Schistosoma

mansoni [Hesse et al. 2004] and Plasmodium spp. [Scholzen

et al. 2009]). TREG can become infected with viral pathogens

(e.g., HIV and FIV), and it is uncertain if they remain suppres-

sive when infected (Joshi et al. 2004; Oswald-Richter et al.

2004). Animal infection models and infected humans show

accumulation of TREG at the site of infection with leishmania-

sis, herpes simplex virus, and schistosomiasis (Hesse et al.

2004; Suvas et al. 2004; Belkaid et al. 2002), in the peripheral

blood with HIV and HCV (Oswald-Richter et al. 2004) and in

lymphoid organs of HIV patients (Andersson et al. 2005). A

good example of the role of TREG in infection would be Plas-

modium spp. that causes malaria in human patients and rodent

models and is associated with increased numbers of TREG (i.e.,

natural and induced), which have a direct association with the

parasitic load in the host. TREG accumulate in areas of inflam-

mation induced by the organisms and inhibit TH1 responses

(i.e., both protective and pathologic) primarily by the suppres-

sive effects of IL-10 and TGF-b, and ultimately result in either

clearance of the organism and resolution or clinical cases of

malaria depending on the level of TREG cell induction and the

organism’s manipulation of this lymphocyte population

(Scholzen et al. 2009). TREG has also been shown to be a factor

FIGURE 11.—The ‘‘Scurfy’’ mouse has a frameshift mutation in the

forkhead box transcription factor, FOXP3. This results in early lethal-

ity due to unregulated inflammation (parenchymal organs, subcutis,

and lymphoid organs are the main sites of inflammatory cell accumu-

lation) due to the lack of functional TREG cells. Note the gross pathol-

ogy image of a Scurfy mouse with marked hepato- and splenomegaly

and evidence of subcutaneous lesions at the tail base.

FIGURE 12.—Note the prominent mononuclear inflammatory cell accu-

mulation in the dermis and upper subcutis from a ‘‘Scurfy’’ mouse.

Hematoxylin and eosin, 200� magnification.




in the development of silica-induced pulmonary fibrosis in a

mouse model. Depletion of TREG leads to the maintenance of

a TH1 response and subsequent attenuation of fibrosis versus

the shift to a TH2 response that normally predisposes to devel-

opment of fibrosis in this model (Liu et al. 2010). The balance

between effector T-cells and TREG is critical in determining the

immune response to pathogens (Figure 16).

ROLE OF TREG IN ORGAN TRANSPLANTATION

TREG have been shown to have a pivotal role in transplant

tolerance leading to graft acceptance and prevention of rejec-

tion in xenotransplantation (Muller et al. 2009; Zhang et al.

2009; Le and Chao 2007; Yong et al. 2007). Adoptive transfer

of TREG into mice has been shown to inhibit, delay, and prevent

development of graft versus host disease (GVHD) (Taylor et al.

2002; Cohen et al. 2002) as well as to prevent allograft (i.e.,

pancreatic islet cell transplant) rejection (Battaglia et al.

2006; Gregori et al. 2001). Post-transplant immunosuppressive

drugs have been shown to have variable effects on TREG.

Mycophenolate mofetil increases TREG numbers and induces

transplant tolerance when given with vitamin D (Han et al.

2005; Huang et al. 2003; Gregori et al. 2001), calcineurin inhi-

bitors (e.g., cyclosporine, tacrolimus, sirolimus) decrease TREG

and abrogate transplant tolerance (Shoji et al. 2005; Larsen et

al. 1996), and Rapamycin and methylprednisolone do not seem

to have effects on TREG or transplant tolerance (Blaha et al.

2003).

ROLE OF TREG IN PREGNANCY

Natural TREG, CD8þ TREG, TH3 cells, and Tr1 cells have

been shown to be important in immune tolerance during preg-

nancy (Guerin et al. 2009; Trowsdale and Betz 2006; Aluvihare

FIGURE 13.—There is a reciprocal relationship between TH17 (proinflammatory effector T-cell subset that are associated with autoimmune dis-

ease) and TH3 cells (iTREG that are CD4þCD25þFOXP3þ), which is elicited during differentiation of naıve TH0 cells, but final differentiation

depends on the cytokine milieu. TGF-b is critical in differentiation of both cell types but depends on the cytokine milieu. The schematic shows the

cytokine pathways in the differentiation of TH17 and TH3 cells from TH0 effector cells.




et al. 2004). IL-2/STAT5 signaling and 17-b-estradiol (E2)

during pregnancy cause induction of FOXP3 and increased

TREG numbers (i.e., humans and animal models) by induction

of FOXP3þ iTREG and TREG cell expansion (Fainboim and

Arruvito 2011). Fetal alloantigen (Zhao et al. 2007) and placen-

tal trophoblast antigens such as heat shock protein-60, carci-

noembryonic antigen, CD274 (i.e., ligand for programmed

cell death-1 receptor), and human leukocyte antigen-G

(Taglauer et al. 2008; Yang et al. 2006; Shao et al. 2005;

LeMaoult et al. 2004) are also important in TREG cell induc-

tion/activation and expansion during early pregnancy. Paternal

alloantigens and TGF-b in seminal fluid can also induce/acti-

vate TREG (Robertson et al. 2009; Robertson and Sharkey

2001). During the course of pregnancy, there are increased

numbers of CD4þCD25þ cells in the circulation in early preg-

nancy, reaching an apex during the second trimester and declin-

ing until the postpartum period where they reach a number that

is nearly at the prepregnancy level (Heikkinen et al. 2004).

TREG are significantly decreased in deciduas from spontaneous

vaginal deliveries when compared with Caesarean section,

indicating a potential role of declining TREG in fetal delivery

(Xhao et al. 2007; Sindram-Trujillo et al. 2004). The ovary nor-

mally contains a population of thymic-derived nTREG (Samy et

al. 2005), and the testis has been shown to have a locally

induced iTREG population that are specifically targeted to testi-

cular antigens and contributes to testicular immune privilege

(Nasr et al. 2005). TREG have been shown to be necessary for

immune tolerance of the conceptus, oocytes, and spermatozoa

(Trowsdale and Betz 2006). There are increased numbers of

TREG in the blood, decidual tissue, and lymph nodes draining

the uterus (Tilburgs et al. 2008; Thuere et al. 2007). TREG cell

depletion and/or decreased function have been shown to lead to

loss of pregnancy in rodent models (Aluvihare et al. 2004) and

in patients who have miscarried (Yang et al. 2008; Sasaki et al.

2004). Infertility, miscarriage, and pre-eclampsia have been

linked with decreased TREG number and/or function (Guerin

et al. 2009; Tilburgs et al. 2008; Thuere et al. 2007; Trowsdale

and Betz 2006; Aluvihare et al. 2004).

FIGURE 14.—This schematic focuses on the differentiation pathways of TH0 to TH17 and TH3 cells showing a transitional state (FOXP3þ, ROR-gtþ

and RORaþ cell) where addition of certain cytokines/factors drives differentiation to TH3 cells (FOXP3þ) leading to suppression or TH17 cells

(ROR-gtþ and RORaþ) resulting in inflammation/autoimmune disease.




ROLE OF TREG IN CANCER

TREG have been shown to dampen the cytotoxic immune

response (CD8þ/NK cell) as well as suppressing overall level

of host immune response to the neoplasm in cancer. Research-

ers have looked at numbers and location of TREG and the effects

on cancer patient outcome (Menetrier-Caux et al. 2009). They

showed that TREG are present in a large panel of solid tumors in

humans. TREG in pulmonary, pancreatic, gastric, hepatic, and

ovarian carcinomas have been correlated with a negative out-

come; while in B-cell lymphoma, head and neck cancer and

colonic carcinoma TREG have been associated with a positive

patient outcome (Menetrier-Caux et al. 2009). TREG had no

effect on survival with prostatic, renal, and squamous cell car-

cinomas (Menetrier-Caux et al. 2009). In breast cancer, TREG in

peri-tumoral lymphoid aggregates were correlated with a

negative outcome, while intra-tumoral TREG were associated

with a positive outcome. (Menetrier-Caux et al. 2009) The

results of vaccine-induced cancer T-cell mediated immu-

notherapy (i.e., cancer vaccine therapy) have proven to be dis-

appointing (Welters et al. 2008) due to the vaccine’s inability to

induce effector T-cells, TREG already present in the tumor, and/

or induction of TREG by the vaccine itself. Based on these find-

ings, the success of cancer vaccine therapy will depend on the

balance between TREG and effector cells at the start of therapy

(Welters et al. 2008). Neoplastic TREG cells in Sezary Syn-

drome (leukemic variant of cutaneous T-cell lymphoma in the

human) express variant splices of FOXP3. The neoplastic cells

with variant FOXP3 are able to function as TREG and work to

dampen the host immune response against the neoplastic

T-lymphocytes (Krejsgaard et al. 2008).

FIGURE 15.—During an infection with a pathogenic organism complex networks develop where nTREG undergo antigen-specific expansion and Tr1

and iTREG cells undergo pathogen-specific differentiation in the presence of tolerogenic cytokines (IL-10 and TGF-b). All three types of TREG

cells then work to suppress effector T-cells in the inflammatory milieu by soluble factors, cell-to-cell contact, or competing for IL-2. nTREG can

also induce infectious tolerance in dendritic cells. Together these result in suppression of immune responses to pathogens. Pathogens can also use

TREG for their own benefit to evade the immune response.




TREG AS A MODULATORY TARGET IN DRUG DEVELOPMENT

Pharmacological agents have been developed that control

TREG number and/or function (Ohkura et al. 2011; Nizar et

al. 2010; Thorburn and Hansbro 2010; Nandakumar et al.

2009; Tao et al. 2007; Yang et al. 2006). Molecules that

increase TREG number include rTGF-b, FTY720 (Fingolimod,

a S1P1 modulator), retinoic acids, histone deacetylase inhibi-

tors, and probiotics. TREG suppressive function is augmented

with the following agents: recombinant IL-2, Rapamycin

(mTOR pathway inhibitor) in type-1-diabetes mellitus and

renal transplantation, and histone deacetylase inhibitors. His-

tone deacetylase inhibitors that are also known as chromatin

modifying agents (e.g., HDAC9) lead to increased TREG num-

ber and function by a combination of FOXP3 protein acetyla-

tion, TREG chromatin remodeling, and promotion of TREG

development (Tao et al. 2007). While agents that block the

TGF-b and/or CTLA-4 signaling pathways decrease TREG

number and/or function. Aromatase inhibitors (e.g., Letrozole)

used for estrogen modulation in cancer chemotherapy regimens

and recombinant IL-2 lead to decreased TREG numbers.

Biopharmaceutical agents have been developed to modulate

TREG (Khan et al. 2011; Ohkura et al. 2011; De Serres et al.

2011; Weber et al. 2009) including Tremelimumab (CTLA-4

modulation) in advanced melanoma which leads to increased

effector T-cell numbers with no change in TREG number,

CD25 modulation by Daclizumab in multiple sclerosis (MS)

and advanced breast cancer, Denileukin diftitox as a cancer

vaccine, LMB-2 and RFT5-SMPY-dgA in advanced melanoma

decrease TREG number, anti-GITR MoAb (DTA1) which leads

to decreased TREG number, agonist anti-OX40 MoAb (OX86)

and anti-FR4 MoAb which decrease TREG number, and Alem-

tuzumab (anti-CD52 MoAb) which increases TREG number.

Adoptive transfer of TREG has been evaluated in clinical

trials of patients that have received human stem cell transplants

to induce transplant tolerance, patients with GVHD, and type 1

diabetes mellitus patients to ameliorate autoimmune disease

with a good patient safety profile and showed that the proce-

dure is feasible, yet efficacy still needs to be shown convin-

cingly (Riley et al. 2009; Trzonkowski et al. 2009).

CONCLUSIONS

TREG have both positive and negative functions in the

immune system. Positive aspects of TREG include suppression

of immune responses including autoimmunity, maintaining

immune homeostasis, as well as tolerance in xenotransplanta-

tion, at mucosal sites, and during pregnancy. Negative aspects

FIGURE 16.—The balance between effector cells and TREG cells in the inflammatory milieu dictate the course of and ultimate resolution or per-

sistence of the inflammatory response to an infectious agent. There are advantages and disadvantages for both the host and microbe if there is an

imbalance in the numbers/function of effector cells and TREG.




of TREG can include manipulation by some infectious agents to

avoid the host’s immune response and in cancer TREG can sup-

press the body’s cytotoxic immune response to neoplastic cells.

The balance between TREG and effector cells determines the

fate of an immune response. The fact that the immune system

has so many sources of regulatory cells which overlap function-

ally but have slightly different induction/activation pathways

underscores the importance of TREG in host survival.

ACKNOWLEDGMENTS

I would like to acknowledge the following researchers:

Virginia Godfrey, DVM, PhD, DACVP, from the University

of North Carolina at Chapel Hill for her kind donation

of the ‘‘Scurfy’’ mouse images; Caroline Genell from the

GlaxoSmithKline, Safety Assessment, Immunotoxicology

Group for the rat TREG flow cytometry data; and John

Spaull of the GlaxoSmithKline MCT Cell Biology Group

for the FOXP3 immunofluorescence images.

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