Regulatory T cells: friend or foe in immunity to infection?
Kingston H.G. Mills
Immune Regulation Research Group, Department of Biochemistry,
Trinity College, Dublin 2, Ireland
Email:kingston.mills@tcd.ie
Preface
Homeostasis in the immune system is dependant on a balance between responses that control
infection and tumours and the reciprocal responses that prevent inflammation and
autoimmune diseases. It is now recognized that regulatory T (Tr) cells play a critical role in
suppressing immune responses to self-antigen and preventing autoimmune diseases. Evidence
is also emerging that Tr cells control immune responses to bacteria, viruses, parasites and
fungi. This article explores the possibilities that Tr cells can be both beneficial to the host in
limiting the immunopathology associated with anti-pathogen immune responses, and to the
pathogen through subversion of protective immune responses of the host.
Introduction
Protection against infection is fundamental to survival of all animals and is mediated
by the immune system, which has evolved innate and adaptive mechanisms to deal with
invading microbes. The effector mechanisms employed by the host to control infection
include the production of inflammatory cytokines and chemokines, recruitment of
inflammatory cells to the site of infection and activation of cytotoxic T lymphocytes (CTL)
and natural killer (NK) cells, which lyse host cells infected with the pathogen (FIG. 1). While
this helps to eliminate or slow the spread of the organisms, if not tightly controlled, this
response can result in severe inflammation and collateral tissue damage1. A further potential
for damage arises because the cells and molecules of the immune system that respond to
pathogen antigens can also respond to self-antigens and if uncontrolled this can result in
autoimmune disease2. Inflammation and the immune response to pathogens is regulated by a
variety of host suppressor mechanisms, including the production of anti-inflammatory
cytokines by cells of the innate immune system in response to conserved pathogen-derived
products3,4. However, recent evidence suggests that the adaptive immune system may also
help to control infection-induced immunopathology through the generation of antigen-specific
Tr cells (Fig. 1). Therefore Tr cells may play a protective role in immunity to infection.
It is also possible that pathogens may exploit Tr cells to subvert the protective immune
responses of the host. Although the infections caused by many pathogens are self-limiting in
immunocompetent hosts, other pathogens can persist and cause chronic infections. In
infections, such as those caused by HIV, hepatitis C (HCV) virus and many parasites, the
pathogen persists because the appropriate immune response for its elimination either fails to
develop or is suppressed. Furthermore, there is evidence that the incidence of allergy/asthma
and autoimmune diseases are lower in individuals infected with helminth parasites or exposed
to microbial products as children5,6. It appears that many, if not all, pathogens that cause
persistent or chronic infections have evolved strategies to subvert host protective immune
responses. These strategies include evasion of humoral and cellular immunity by antigenic
variation, interference with antigen processing or presentation and subversion of phagocytosis
and killing by cells of the innate immune system7. However, relevant to this discussion, a
common immune subversion strategy employed by many pathogens is to enhance the
production of anti-inflammatory or immunosuppressive responses, which normally function
to control or terminate protective effector immune responses of the host. This can be achieved
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through the production of molecules with homology to human cytokines, such as viral
Interleukin-10 (IL-10), direct induction of immunosuppressive cytokines, such as IL-10 and
transforming growth factor (TGF)-, by innate cells in response to pathogen-derived
molecules3,8 or indirectly, through the generation of Tr cells.
An understanding of the role of Tr cells in immune homeostasis is far from complete
and there are a number of important unanswered questions. How do regulatory mechanisms
control the development of autoimmunity, while at the same time permitting the same type of
immune responses to mediate protection against infection? What is the advantage to the host
of inducing microbe-activated Tr cells that suppress immune responses that facilitate
pathogen elimination from the host? However, our knowledge has increased in the last few
years and a number of studies, primarily in murine models of infection with bacteria, viruses,
parasites and fungi have demonstrated that Tr cells specific for pathogen antigens are induced
during infection. Furthermore, studies involving depletion or transfer of CD4+CD25+
regulatory T cells have provided evidence that natural Tr cells can influence the immune
response to pathogens and infectious disease outcome. This article reviews the recent
evidence for pathogen-specific Tr cells and their role in infection, focusing on the protective
role of Tr cells in immunity to pathogens, as a means of limiting infection-induced
immunopthology, as well as the exploitation of Tr cells by pathogens as a novel immune
subversion mechanism to prolong their survival in the host.
Tr cell biology
Functional subtypes of natural and inducible Tr cells. It is now firmly established that there
are both natural (or constitutive) and inducible (or adaptive) populations of Tr cells (FIG. 2),
which may have complementary and overlapping functions in the control of immune
responses. However, the lineage relationship, if any, between these subtypes remains to be
defined. The lack of definitive cell surface marker for either population has compromised
advances in the field and has lead to some confusion as to the precise nature of the cells under
study in different laboratories. It appears that the natural self-antigen reactive CD4+CD25+ Tr
cells develop in the thymus and emerge into peripheral tissues where they suppress the
activation of other self-reactive T cells9,10. By contrast, IL-10 or TGF- secreting Tr cells,
termed Tr1 or Th3 cells respectively, are generated from naïve T cells in the periphery after
encounter with antigen and under the direction of dendritic cells (DC) whose activation status
is distinct from those that promote the differentiation of T helper 1 (Th1) or Th2 cells. In
addition to these well defined populations of CD4+ Tr cells, there is also evidence for an
immunosuppressive function of CD8+ Tr cells that secrete either IL-10 or TGF-11,12.
Furthermore, antigen-activated CD8  T cells prevent insulin-dependent diabetes in mice13
and IL-10 and TGF- producing  Tr cells suppress anti-tumour CTL and natural killer (NK)
activity14. In addition, NKT cells that co-express NK cell and T cell markers secrete
regulatory cytokines, including IL-1015. Therefore, NKT and  T cells may also be
categorized as Tr cells.
Natural CD25+ Tr cells were first defined in 1995 by Sakaguchi and colleagues, who
showed that lymphoid cell populations from which CD4+ T cells expressing the IL-2
receptor (CD25) had been removed caused spontaneous development of various T cell
mediated autoimmune diseases when transferred into athymic nude mice16. Furthermore, reconstitution with CD4+CD25+ T cells prevented the development of autoimmunity. This
discovery, along with the work of Powrie and others on a CD45Blow population, challenged
traditional theories on clonal deletion as the sole mechanism of self tolerance and provided
convincing evidence that autoantigen-reactive T cells that cause autoimmune diseases are
controlled through active suppression by natural Tr cells17. The CD4+CD25+ Tr cells, which
constitute 5-10% of peripheral T cells, are continuously produced in the thymus as a
functionally mature population of T cells that includes cells with immunosuppresive activity.
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However, CD25 is not a definitive marker of natural Tr cells; CD25 is an activation marker
for T cells and therefore is expressed on effector Th1 and Th2 cells as well as suppressor T
cells, and suppressive function has also been documented in CD25- T cells. These
observations led to attempts to find alternative markers for Tr cells. Putative Tr cellassociated markers include surface expression of CD45RBlow, CD38, CD62L, CD103,
glucocorticoid-induced TNF receptor (GITR) or expression of the transcriptional repressor
FoxP317-19. The latter appears to be the most promising marker of natural Tr cells and recent
studies have shown that transfection with FoxP3 confers regulatory activity on CD25- T
cells20. T cell receptor (TCR) engagement appears to be necessary for optimal suppressive
activity and it has been assumed that circulating CD4+CD25+ Tr cells are activated following
recognition of self-antigens in vivo9, however evidence of natural Tr cell antigen-specificity is
still limited.
A unique cytokine production profile, rather than surface makers, has been used to
define at least two populations of inducible Tr cells. Although it had been recognized for
some time that T cells with suppressor or anergic activity could be generated in vivo in certain
situations, for example in oral tolerance induction21,22 or during infection with certain
pathogens, such as rabies virus23, Brugia malayi24 and Mycobacteria tuberculosis25, it was not
until the mid 1990s that nomenclature was applied to these cells. Weiner and colleagues
demonstrated that the induction of oral tolerance and the prevention of Th1-mediated
autoimmune diseases by feeding self-antigens was associated with the generation of TGF-secreting T cells in the gut26. These T cells, which were distinct from Th2 cells in that they
secreted high levels of TGF- and varying amounts of IL-4 and IL-10, were named Th3 cells.
In 1997 Groux and colleagues demonstrated that repeated in vitro antigen stimulation of T
cells isolated from ovalbumin specific TCR transgenic mice in the presence of IL-10 resulted
in the expansion of a population of Tr cells that produced high levels of IL-10 and were
capable of suppressing Th1 responses and Th1 mediated autoimmune diseases and called
these cells Tr1 cells27. More recently it has been demonstrated that antigen-specific Tr1 cells
can be generated in vivo during certain infections and that IL-10 may be a differentiation
factor rather than growth factor for Tr1 cells28. Since Th2 cells secrete the
immunosuppressive or anti-inflammatory cytokines IL-10 and IL-4, these cells may also have
regulatory as well as effector function, but are distinguished from Th3 and Tr1 cells through
the production of high levels of IL-4 and relatively lower levels of IL-10 and lack of TGF-
Targets of suppressor activity.
Immunity to intracellular pathogens is mediated by CD4+ Th1 cells and CD8+ CTL, whereas
immunity to extracellular pathogens is mediated by antibody and Th2 cells. Innate immune
responses also play a protective role early in infection and instruct the adaptive immune
response (FIG. 1). Each of these effector mechanisms can be suppressed by natural and
inducible Tr cells. It has been demonstrated that Tr1 cells or CD4+CD25+ Tr cells can
suppress proliferation and cytokine production by naïve CD4+CD25- T cells or antigenspecific Th1 or Th2 cells in vitro28-33. There is more limited evidence that Tr cells can
suppress pathogen-specific T cells in vivo, including suppression of IFN- production by Th1
cells in responses to B. pertussis by Tr1 cells28 and by CD25- T cells in response to
Leishmania major by CD25+ T cells29. More recently it has been demonstrated that CD25+ T
cells can suppress activation of CD8+ T cells in vitro34 and secondary CD8+ T cell responses
to Listeria monoctytogenes35 and herpes simplex virus (HSV)36 in vivo. Finally, there is
evidence that Tr cells can suppress the innate cell infiltration and activation that leads to
inflammatory pathology induced with Helicobacter hepaticus in the colon37. Therefore, the
targets of suppressor activity by Tr cells cells are immune responses that confer protection
against infection with microbes, but also responses that can cause collateral damage to host
tissue during infection.
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Mechanism of suppression. The suppressive function of natural and inducible Tr cells on
effector T cells is currently a subject of some debate but in different model systems has been
shown to be mediated either through secretion of immunosuppressive cytokines or by cell-tocell contact (FIG. 3). Many studies have demonstrated that suppression mediated by Tr1 or
Th3 cells can be reversed using anti-IL-10 or anti-TGF- antibodies. IL-10 inhibits TNF-
and IL-12 production, whereas TGF-1 inhibits Th1 responses through its effect on
expression of the transcription factor T-bet and IL-12R38-40. It has been reported that TGF-1
production by Tr cells induces IL-10 secretion in Th1 cells by Smad4-induced activation of
the IL-10 promoter41. This suggests that there may be interdependent as well distinct roles for
IL-10 and TGF- in the immunosuppressive function of inducible Tr cells. Cytokine mediated
suppression may also operate at the level of the antigen presenting cell, since IL-10 and Tr
cells can inhibit MHC class II and co-stimulatory molecule expression on DC40,42.
The suppressive mechanisms of the CD4+CD25+ Tr cells are not clear, but there is
evidence that cell-to-cell contact is required and expression of the inhibitory co-stimulatory
molecule CTLA-4 may be involved43. However there is also conflicting evidence on roles for
IL-10 and secreted or surface bound TGF-29,43,44. It has also been suggested that Tr cells
may inhibit pathogenic effector T cell responses by competing for shared resources within a
normal immune system45. Thus, while the mechanisms of suppression by Tr1 and Th3 cells
appear to be primarily cytokine mediated, CD4+CD25+ Tr cells may use multiple and as yet
unidentified mechanisms to mediate suppression.
Pathogen-specific Tr cells
Cell depletion and transfer, as well as cytokine knockout or inhibition experiments have
provided considerable indirect evidence of a role for inducible (TABLE 1) and natural
(TABLE 2) Tr cells in infection. However, there are still a limited number of reports
demonstrating specificity of Tr for pathogen antigens.
Tr1/Th3cells: Although many studies have demonstrated that pathogens, especially those that
cause chronic infections or are associated with immunosuppression, induce the regulatory
cytokines, IL-10 and TGF-, the source of these cytokines has not always been defined. In
some cases it has been demonstrated that innate cells, usually macrophages or more rarely
DC, are the source and in others it has been shown to come from T cells8. However the
distinction between Th2 and Tr cell-derived IL-10 or TGF- has not always been made. The
definitive demonstration of antigen-specific Tr cells is dependant on the generation of
antigen-specific T cell clones or on careful ex vivo intracellular cytokine staining of antigenstimulated T cells, showing high IL-10, no IL-4 and low (human) or no (mouse) IFN-
production.
The first definitive reports of inducible antigen-specific Tr1-type clones generated
during infection were made in mice infected with B. pertussis28 and humans infected with
HCV46 or the nemotade parasite, Onchocerca volvulus47,48. The B. pertussis study showed
direct evidence of suppression of Th1 cells by Tr1 clones specific for bacterial antigens and
the latter reports showed indirect evidence, through enhanced IFN- production in the
presence of anti-IL-10 antibodies. The studies with B. pertussis28 and on virus-specific CD8+
Tr cells in chronic HCV infection49 suggest that antigen-specific Tr are recruited to the
mucosal site of infection. More recently, antigen-specific Tr1-type cells have been
demonstrated in a number of other chronic infections, including Epstein-Barr virus (EBV)50,
M. tuberculosis25,51,52, HIV53 and during infection with murine leukemia virus, a murine
model for AIDS54. It has also been demonstrated that IL-10 producing Tr are induced in vitro
by DCs stimulated with phosphatidylserine from Schistosoma mansoni55. Although the
problems of cultivating and cloning antigen-specific Tr cells in vitro have hampered advances
4
in this area, it is tempting to speculate that Tr cells are induced during infection with most if
not all pathogens, especially those that cause persistent or chronic infections.
Natural Tr cells: Most studies on CD4+CD25+ Tr cells in infection have demonstrated a role
for these cells in controlling anti-pathogen immunity, but few studies have demonstrated
specificity for pathogen antigens (TABLE 2). CD4+CD25+ Tr cells specific for pathogenderived antigens have been demonstrated to accumulate at the site of infection in the dermis
soon after infection with L. major and suppress IFN- production and the ability of effector T
cells to eliminate the parasite from the host29. CD4+CD45RBlow Tr cells from H. hepaticus
infected but not uninfected mice prevent the development of intestinal inflammation triggered
in RAG-/- mice by transfer of CD4+ T cells from IL-10-/- mice56. The observation that
CD4+CD45RBlow Tr cells from H. hepaticus infected wild type mice inhibit IFN- production
by T cells from IL-10 deficient mice and produce IL-10 after exposure to H. hepaticus
antigens in vitro argues that these Tr cells, rather than being endogenous, represent a memory
population resulting from previous exposure to bacterial antigen.
Host protective role of Tr cells in infection
There is convincing evidence of a protective role of Tr cells against autoimmune diseases and
also in allograft rejection and allergy, where they suppress potentially pathogenic immune
responses mediated by effector Th1, Th2 cells or CTLs2,16,27,57-59 . Since the latter responses
also play key roles in protection against pathogens, it might appear counterintuitive that Tr
could have a protective role in infection. However in many infectious diseases immune
responses to the pathogen can result in collateral damage to host tissues and
immunoregulatory mechanisms, including the induction of Tr cells, are essential to control
this immunopathology.
Viruses. IL-10 secreting CD4+ and CD8+ Tr cells have been demonstrated in HCV infection
and there is indirect evidence that both populations may inhibit HCV-specific T cells in
chronically infected individuals46,49,60. However it has been suggested that HCV-specific
CTLs that home to the liver produce IL-10 and help to reduce liver inflammation49.
Furthermore, HCV infected patients with reduced numbers of CD4+CD25+ T cells often
develop an autoimmune syndrome, called mixed cryoglobulinemia, characterized by B cell
proliferation and autoantibody production61. Thus, while Tr cells may prevent viral clearance
they also prevent immunopathology and development of autoimmunity.
More direct evidence of a protective role for Tr in preventing immunopathology has
come from studies in mouse models of viral infection. Theiler’s virus infection of mice
induces a demyelinating disease mediated by CD4+ T cells, and transfer of virus-specific
CD8+ Tr cells has been shown to prevent inflammation and the pathogenic effects of the
CD4+ T cells12. In footpad infection of mice with HSV, removal of CD25+ T cells enhances
the virus-specific CD8+ T cell response and enhances viral clearance36. However, Th1
responses and the severity of T cell mediated lesions in the cornea of HSV-infected mice were
more severe if mice were depleted of CD25+ T cells before infection62. CD4+CD25+ Tr cells
therefore appear to reduce the severity of immune-mediated inflammatory lesions by
preventing the induction of pathogenic CD4+ T cells and limiting the migration of these cells
to inflammatory sites in the tissues. Therefore in chronic viral infections Tr cells may play a
beneficial role to the host by maintaining a balance between efficient effectors and memory
responses, but with a low level inflammation that causes minimal damage to the host.
Bacteria. Indirect evidence of a protective role for IL-10-producing Tr cells in host defence
against bacteria-induced immune mediated pathology has come from studies demonstrating
that disease severity is exacerbated in IL-10 deficient mice. IL-10-/- mice succumb to primary
and secondary infection with L. monocytogenes; the number of cells in the inflammatory
5
infiltrate, the inflammatory cytokine production in the brain and the severity of the brain
lesions is enhanced in the knockout mice63. Peritonitis and mortality from E. coli infection is
enhanced in IL-10-/- mice, despite accelerated clearance of the bacteria in knockout compared
with wild-type mice64. The colonization of the gastric mucosa by H. pylori is reduced in IL10-/- mice, but the severity of chronic active gastritis is significantly higher than in the
wildtype mice65. Similarly, IL-10-/- mice infected with H. hepaticus develop severe
inflammation associated with IL-12 production and Th1 responses66. The IL-10 that helps to
limit inflammation during bacterial infection may be in part derived from innate cells.
However, it has been shown that the induction of IL-10 by macrophage and DC in response to
certain pathogen-derived molecules, facilitates the induction of Tr1 cells, thus amplifying the
effect of innate IL-1028. Indeed, studies with Toll-like receptor (TLR)-4 defective mice have
suggested that both innate and Tr1 cell-derived IL-10 may help to limit inflammatory
pathology in the lungs induced by B. pertussis infection. B. pertussis stimulates IL-10
production from DC and macrophages and generates Tr1 cells in the respiratory tract of
infected mice. However, TLR-4 defective mice have reduced IL-10 production from DC and
macrophages and do not generate Tr1 cells when infected with B. pertussis67. These mice
have significantly greater cellular infiltrate, lung damage and higher bacterial load than
normal mice, suggesting that the induction of Tr1 cells helps to limit inflammatory pathology
and thereby enhance pathogen elimination by preventing damage to the ciliated epithelia cells
required for removal of the bacteria from the lungs.
Direct evidence for the role of CD4+CD25+ Tr cells in the prevention of intestinal
inflammation has come from the demonstration that H. hepaticus infection induces a
population of CD4+CD45RBlow Tr cells that inhibit the development of colitis in IL-10deficient mice56. Removal of CD25+ T cells from the lymph node cells used to reconstitute
athymic mice prior to infection with H. pylori reduced the bacterial load but enhanced
gastritis68. Furthermore, transfer of CD4+CD25+ Tr cells from normal mice prevented H.
hepaticus triggered intestinal inflammation in RAG2-/- mice by IL-10 and TGF- dependent
mechanisms37. The Tr cells did not affect bacterial colonization in the gut, rather the
protective effect of the Tr cells appeared to be mediated by suppressing T cell dependant and
innate inflammatory responses, including recruitment of neutrophils and macrophages and
activation of NK cells in the intestine37.
Fungi: Pneumocystis carnii causes pneumonia in immunocompromised individuals. In a
mouse model, transfer of CD4+CD25- T cells to RAG2-/- mice infected with P. carnii reduced
the pathogen burden, but these mice developed severe lung inflammation and a fatal wasting
disease. Co-transfer of CD4+CD25+ prevented lung inflammation and the development of
disease induced by CD4-CD25- T cells, but enhanced the pathogen load69. A similar situation
has been reported for Candida albicans infection. Th1 cells mediate protection against C.
albicans and in the absence of CD4+CD25+ T cells, which are not induced in CD86 or CD28
deficient mice or where IL-10 signalling is deficient, the fungal growth is reduced, but
inflammatory pathology is enhanced70. Furthermore, transfer of IL-10 and TGF- secreting
CD4+CD25+ T cells decreased inflammation in CD86 deficient mice. In a separate study, an
absence of TLR-2 was associated with impaired IL-10 production, reduced CD4+CD25+ Tr
cells and enhanced inflammatory infiltrate, but lower pathogen burden in C. albicans infected
mice71. Thus, while Tr cells may compromise fungal clearance, they may also be beneficial to
the host, limiting infection-induced pathology.
Parasites. In human malaria, polymorphisms in the TNF- promoter have been associated
with disease severity; among children with severe malaria those with the TNF-308A allele
had lower plasma levels of IL-10 than TNF72. Furthermore, higher ratios of IL-10 to TNF-
in children with mild malaria is suggestive of a role for IL-10 in controlling the excessive
6
inflammatory activities of TNF-73. Although these studies have not yet provided direct
evidence that IL-10 is produced by parasite-specific Tr cells, it has been suggested that Tr
cells may contribute to the anti-inflammatory cytokine pool that controls TNF-mediated
inflammation in malaria1. These observations are complemented by studies in mice which
have shown that infection with P. chabaudi chabaudi is more severe in IL-10-/- mice, which is
associated with enhanced inflammation, including increased TNF-, IFN- and IL-12
production; treatment with anti-TNF- enhances survival74. Furthermore, treatment of
infected mice with a neutralizing anti-TGF- antibody exacerbated infection with P. berghei
and P. chabaudi chabaudi and treatment with recombinant TGF- slowed the rate of parasite
growth and enhanced survival75. CD4+CD25+ cells and CD8+ T cells from malaria infected
mice secreted high levels of TGF- in response to parasite antigens in vitro76, suggesting that
antigen-specific CD8 Tr cells may help to control malaria-induced inflammation.
IL-10 producing CD4+CD25+ T cells are induced in mice during infection with S.
mansoni and these Tr cells (as well as innate IL-10) help to protect the host from the
hepatocyte damage induced by the parasites egg’s and to prevent death from the infection
immune-mediated pathology77. Similarly, depletion of CD4+CD25+ T cells enhanced the
parasite burden, severity of colon lesions and colitis in L. major infected SCID mice
adoptively transferred with spleen cells78. Although infection of IL-10-/- mice with L. major is
associated with enhanced parasite-specific immune responses and pathogen clearance from
the host, this sterilizing cure results in a loss of memory and therefore resistance to reinfection by the same parasite29. Therefore Tr cells appear to control the immune responses
sufficiently to contain but not eradiate the infection, thereby suppressing potentially
pathogenic T cell effector responses, but allowing the maintenance of T cell memory.
Tr cells in pathogen immune subversion
Although beneficial to the host through preventing immunopthology and enabling memory
development, Tr cells can also be beneficial to the pathogen, enabling it to establish a chronic
infection. Many pathogens have evolved strategies that facilitate their persistence, largely
through their ability to evade or subvert the host immune response. One strategy is to induce a
state of immunosuppression, either through direct interference with host immune effector
mechanisms or through the production of immunosuppressive cytokines. Many viruses
produce antagonists of pro-inflammatory cytokines or their receptors, or molecules that are
homologous to IL-10 or TGF-, or stimulate the production of anti-inflammatory cytokines
from macrophages or other innate immune cells3,8. It has recently being recognized that this
can also be extended to the induction of T cells with suppressor activity, including natural and
inducible Tr cells.
Viruses. The majority of patients infected with HCV remain persistently infected despite the
induction of HCV-specific antibody and T cell responses. Many chronically infected patients
remain disease free for decades; others go on to develop cirrhosis of the liver and in certain
cases, heptocarcinoma. It has recently been demonstrated that patients with chronic HCV
infection have circulating HCV-specific CD4+ Tr1 cells46 and CD8+ Tr cells49. These Tr cells
appear to be capable of inhibiting protective anti-viral immunity, since addition of a
neutralizing anti-IL-10 antibody significantly enhanced HCV-specific IFN- production by T
cells in vitro60. Furthermore, there is a higher frequency of CD4+CD25+ Tr cells in patients
with chronic infections compared with those that have cleared the infection79. These
CD4+CD25+ Tr cells were capable of suppressing HCV-specific CTL responses, suggesting
that natural Tr cells may also contribute to chronic infection by suppressing protective
immune responses. This is consistent with findings in HSV-infected mice, where CD4+CD25+
Tr cells suppress virus-specific CD8+ T cell responses and delay viral clearance36
7
Retroviruses, such as HIV, usually persist for the lifetime of the infected host and
escape immunity by antigen variation. The immunodeficiency syndrome in the later stages of
AIDS is a direct reflection of a reduction in the number of CD4+ T cells. However, even
before the numbers of CD4+ T cells start to decline, immune responses to HIV and unrelated
pathogens are suppressed. One explanation for this is the switch from Th1- to Th2-dominated
responses that has been observed with disease progression80. Alternatively, activation of Tr
cells that inhibit Th1 and CTL responses in vivo may explain the immunosuppression during
retroviral infection prior to depletion of CD4+ T cells. Individuals with progressive or active
HIV replication have a high frequency of IL-10-producing CD4+ T cells; these cells comprise
those that produce IL-10 constitutively and those that produce it upon gag-stimulation53.
Furthermore, CD4+CD25+ T cells from HIV infected individual suppress proliferation and
cytokine production by CD8+ and CD4+ T cells in response to HIV antigens81 and depletion of
CD4+CD25+ Tr cells from peripheral blood mononuclear cells (PBMC) enhances the
frequency of CD8+ and CD4+ T cells secreting IFN-- in response to HIV81,82 and
cytomegalovirus82 antigens. In addition, HIV antigens induce TGF- secreting CD8+ Tr cells
which inhibit IFN- secretion by CD8+ T cells specific for vaccinia virus. Thus, Tr cells
specific for HIV antigens may contribute to general immunosuppresion during retrovial
infection11 and this conclusion is supported by studies in animal models of retroviral
infection. Infection of cats with feline immunodeficiency virus (FIV) is associated with the
activation of CD4+CD25+CTLA4+ Tr cells that inhibit proliferation and IL-2 production by
CD4+CD25- T cells from normal cats31. Furthermore, ablation of IL-10 secreting Tr cells in
mice prevented progression of murine AIDS, an immunodeficiency syndrome induced by
murine leukaemia virus54. Persistent infection of mice with the murine retrovirus, Friend
retrovirus, is associated with a decreased ability to develop anti-tumour immune responses83.
IL-10 producing CD4+ T cells from mice persistently infected with Friend virus suppress IFN production by CD8+ T cells84. Similarly, in humans infected with EBV, Tr1 cells are
induced against the LMP1 protein and these cells inhibit Th1 responses to other EBV
proteins, which may facilitate viral persistence and promote the induction of associated
tumours50. These findings suggest that in some cases, virus-specific Tr cells not only prevent
pathogen elimination, but may promote a generalized state of immune suppression in vivo
such that the individual is more susceptible to secondary infections with other pathogens or
has reduced resistance to tumours.
Bacteria. A proportion of individuals infected with M. tuberculosis do not have positive skin
test responses to purified protein derivative (PPD) and this absence of delayed type
hypersensitivity (DTH) responses to mycobacterial antigens is associated with a poorer
clinical outcome. T cells from patients with positive PPD skin tests proliferated and secreted
IFN- and IL-10 in response to PDD, whereas T cell from non-responding patients produced
IL-10, but not IFN-25,51. Furthermore, anti-IL-10 antibodies enhanced PPD-specific IFN-
production by T cells from non-responding patients25. This suggests that Tr1 cells that
suppress Th1 responses to PPD mediate T cell suppression in tuberculosis patients. In
addition, recent studies in IL-10 transgenic mice demonstrate that reactivation of chronic
Mycobacterium tuberculosis infection and suppression of protective Th1 responses is strongly
influenced by the expression of IL-10 during the latent phase of infection85. Furthermore, cell
mediated immunity to M. bovis BCG is enhanced in IL-10-/- mice and the knockout mice
eliminate the bacteria faster than wildtype mice86. Collectively these finding suggest that IL10-producing cells, probably Tr1 cells as well as innate cells, contribute to the chronic state of
mycobacterial infections.
Similarly in Yersinia enterocolitica infection, the V antigen stimulates IL-10
production from macrophages, which suppresses production of the protective cytokine, TNF, and IL-10-/- mice are highly resistant to Yersinia infection87. Although Yersinia-specific Tr
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cells have not yet been documented, it is likely that the bacteria-triggered IL-10 production
from macrophages facilitats suppression of protective immunity, either directly or through the
induction of Tr cells. This conclusion is supported by studies with B. pertussis, where two
virulence factors Filamentous haemagglutinin (FHA) and CyaA, which stimulate IL-10
production from macrophages and DC, direct the induction of Tr1 cells in vivo28,88.
Suppression of local Th1 responses early in infection with B. pertussis89 appears to result
from the induction of Tr cells, which are detectable in the lung during acute infection 28, even
before the appearance of Th1 cells (P. McGuirk and K. Mills unpublished). Furthermore cotransfer of B. pertussis-specific Tr1 clones with Th1 cells from convalescent mice to naïve
mice prior to infection with B. pertussis suppressed Th1 responses and exacerbated
infection28. Therefore it appears that persistence of infection with certain bacteria may be
related to the induction of IL-10-producing CD4+ Tr1 cells and that this may be a strategy
used by the bacteria to evade host immune responses. Indirect evidence of a suppressive role
for CD4+ Tr cells in the development of memory CD8+ T cells has also been provided from
studies with Listeria monocytogenes in mice, where removal of CD4+ T cells enhances the
generation of CD8+ T cell responses and protection against infection induced by
immunization with killed Listeria90.
Parasites. Infection with malaria parasites is persistent and is associated with suppressed
immune responses to the parasite and to un-related antigens. In a murine model of malaria,
depletion of CD4+CD25+ Tr cells protected mice against lethal infection with Plasmodium
yoelii91 and reduced the parasite burden in naive and immunized mice infected with P.
berghei92. CD25+ cell depletion also reversed the defect in the proliferative response to
parasitized red blood cells91. Furthermore, treatment of mice with anti-TGF- and anti-IL-10
reduced parasitemia and enhanced survival of P. yoelii infected mice76. Similarly, Infection of
IL-10-/- mice with L. major results in more rapid clearance of the infection93. Although
susceptibility to Leishmania infection in BALB/c mice has been associated with IL-4
production and Th2 polarization, recent evidence suggests that IL-10 production by Tr cells
may play an important role in persistence of L. major infection in mice94. IL-10-/- mice clear
L. major infection more rapidly than wild-type mice. Furthermore, CD4+CD25+ Tr cells
specific for leishmania antigens have been shown to accumulate at the site of infection in the
dermis soon after infection. These Tr cells, suppress the ability of effector T cells to eliminate
the parasite from the host29. Therefore, pathogens have evolved strategies for persistence
through subversion of protective immune responses via activation of anti-inflammatory
cytokine production from innate cells and through activation of natural and inducible Tr cells.
Pathogen-activated dendritic cells induce Tr cells During infection, the differentiation of
naive T cells into distinct effector CD4+ T cells subtypes is controlled by DCs and regulatory
cytokines produced by innate cells (FIG. 4). Following binding of conserved pathogenderived molecules to pathogen recognition receptors (PRR), such as TLRs, on the surface of
immature DC at the site of infection, the DC matures and migrates to the lymph node where it
presents antigen to naive T cells. The differentiation of naïve T cells into Th1 cells is
promoted by the production of IL-12, IL-23 and IL-27 and Th2 cells by IL-4 and IL-6.
Although there is some evidence that immature DC may selectively activate Tr cells 95, it
appears that T cells induced with immature DC are anergic rather than regulatory and that the
DC that direct the induction of Tr have an intermediate phenotype, including enhanced MHC
class II expression and CD86, but low levels of CD40 and ICAM-132,88,96. This is supported
by studies, which have demonstrated that DC lacking surface expression of CD40 suppressed
a primed immune response and induced IL-10 secreting CD4+ Tr cells96. Furthermore,
ICAM-1–LFA-1 interaction is thought to promote the induction of Th1 cells independently of
IL-12. Thus DC, in which CD40 and ICAM-1 expression is suppressed but CD80 and CD86
enhanced, may promote the induction of Tr1, while blocking Th1 differentiation8.
9
The cytokine environment that promotes the differentiation of Tr cells is also distinct
from that which drives Th1 and Th2 cells. Pathogen molecules that inhibit IL-12 and enhance
IL-10 production have been shown to promote the induction of Tr1 cells in vitro and in vivo8.
FHA from B. pertussis activates IL-10 and inhibits IL-12 production from DC and
macrophages and FHA-stimulated DC promote the expansion of IL-10 secreting T cells from
naive T cells in vitro28. Furthermore, incubation of FHA-stimulated DC with anti-IL-10
antibody prevents the induction of Tr1 cells, suggesting that IL-10 is a differentiation factor
for Tr1 cells. Cholera toxin (CT) and B. pertussis CyaA also inhibit IL-12 production and
CD40 expression on DC and synergise with TLR ligands in activating IL-10 production from
DC and macrophages32,88. DC activated by these pathogen-derived molecules induce Tr1 and
Th2 cells.
Lactobacillus paracase inhibits proliferation and Th1 and Th2 cytokine production by
allo-reactive T cells, but enhances IL-10 and TGF- production, suggesting that the bacteria
promoted the induction of Tr cells97 Candida hyphae induce IL-4 and IL-10 secretion by DC
and hyphae pulsed DC expand CD4+CD25+ T cells in vivo; furthermore anti-IL-10 receptor
antibody prevented expansion of CD4+CD25+ T cells in candida infected mice70. It appears
however that the source of innate IL-10 that promotes the induction of Tr1 cells is not
confined to DC. The NS4 protein from HCV stimulates IL-10 production from monocytes
(but not DC) which in turn activate DC to induce Tr1 cells at the expense of Th1 cells60. EBV
infection is associated with the induction of Tr1-type cells specific for the LMP-1 protein.
EBV produces a viral homologue of mammalian IL-10 (vIL-10), which is expressed, together
with the latent protein LMP1, during the lytic cycle together with LMP-1. Since LMP1specifc IL-10 secreting T cells are induced in EBV infected individuals, vIL-10 may help to
promote the differentiation of these Tr1 cells in vivo50. Therefore, the production of
immunoregulatry cytokines, IL-10 and TGF-, and possible IFN-, by innate cells in
response to certain pathogen derived products, together with IL-12 suppression and selective
activation of co-stimulatory molecule expression on DC may have a major influence on the
induction of Tr cells during infection. Finally, it has also been suggested that Tr cells may
respond directly to physiological or pathogenic ligand interaction with TLR-498 or CD4699
expressed on the surface of T cells.
Conclusions and prospects for therapies
The study of Tr cells in the context of infection has demonstrated that these cells form an
essential component of the host immune protective armoury through their ability to limit
immunopathology and allow the development of immunological memory. However Tr cells
can also be detrimental to the host as they can be exploited by pathogens to facilitate their
persistence by suppressing anti-pathogen protective immune responses. This review has
provided evidence from different studies that Tr cells can be both beneficial and detrimental
to the host in response to the same pathogen. An explanation for these apparently
contradictory findings may lie in the balance between regulatory and effector cells in different
individuals, disease settings and experimental systems. The different outcomes of infection,
persistence, resolution with excessive collateral damage, or resolution with limited
immunopatholgy and development of memory may be influenced by the ratio of Tr to effector
T cells (FIG. 5). This hypothesis is supported by a recent report demonstrating that reinfection of mice with L. major at a secondary site enhanced the number of Tr cells, resulting
in disease reactivation at the primary site of infection; the equilibrium between effector and
regulatory T cells controlled the efficiency of recall immune responses and disease reactivation100.
These studies have also helped to increase our understanding of the role of Tr cells in
immune homeostasis and how they may be manipulated in the treatment of human diseases.
In a normal healthy individual the immune system must be capable of preventing the
development of autoimmune diseases by suppressing immune responses to self-antigens. It
10
must also be able to mount immune responses that control infections to a range of pathogenic
organisms. Immune homeostasis is achieved through a careful balance between effector and
suppressor responses, possibly through an appropriate frequency of Th1, Th2 and CTL versus
natural and induced Tr cells (FIG. 5). The development of autoimmunity and allergy may in
part arise from a deficit in Tr cells, whereas development of cancer and chronic infections
may be associated with an excess of Tr cells. Therefore manipulation of this balance has
opened up new approaches to therapy for a range of human diseases.
The expansion of Tr cells using strategies traditionally associated with induction of
tolerance, has had some success in reducing symptoms of autoimmune diseases in animal
models26,101. However, this needs further development before appropriate approaches can be
routinely applied to humans. Studies in tumour models have shown that alteration in the ratio
of Tr versus Th1/CTL cells can affect tumour survival; removal of CD4+CD25+ T cells
enhances anti-tumour immunity102, while therapy with Th1/CTl promoting versus Tr1
promoting pathogen-derived molecules results in reduction versus enhancement of tumour
survival (A. Jarnicki, J. Lysaght, S. Todryk and K. Mills, unpublished). In murine infectious
disease models, there is some evidence that removal of CD4+CD25+ Tr cells can help to
resolve infection36,91. However the application of this approach to humans will not be
straightforward or without risk and there are many unanswered questions. First, will it be
possible to deplete CD4+CD25+ cells in vivo using monoclonal antibodies? Second, will
transient removal of CD4+CD25+ cells lead to resolution of infection and if not, will more
persistent removal of CD4+CD25+ cells open up risks of developing autoimmunity?
Inhibition of pathogen-induced Tr1/Th3 cells or the cytokines they secrete is an alternative
approach for the treatment of chronic infections. Studies with PBMC from patients infected
with HCV60 or with M. tuberculosis25 have demonstrated that antigen-specific IFN--secretion
can be enhanced in vitro following addition of anti-IL-10. Targeting IL-10 and TGF- has
the advantage of inhibiting the innate cytokines that promote Tr cells as well as the products
of Tr cells that mediate suppression. However this is also not without risk as antiinflammatory cytokines may enhance T cells that mediate pathogen clearance, but if
uncontrolled, the same T cells can contribute to immunopathology. Therefore the key to
success with immunotherapeutic approaches will be to elicit the correct balance of
effector/pathogenic and regulatory T cells.
Acknowledgements
Kingston Mills is supported by Science Foundation Ireland, The Irish Health Research Board,
and Enterprise Ireland. I am grateful to Peter McGuirk and Ed Lavelle for helpful discussions.
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Figure Legends
Figure 1. Immunity to infection. Innate immune effector cells, including macrophages,
dendritic cells (DC) neutrophils, and NK cells, together with various protein components of
the complement system, provide the first line of defence against invading microbes. Binding
of conserved pathogen-derived molecules to pathogen recognition receptors (PRRs) on
macrophages and DC activate the production of pro-inflammatory cytokines and chemokines,
which help to attract other effector cells to the site of infection. Pathogen-activated DC
present antigen to T cells and promote the differentiation of naïve T cells into various
subtypes of effector CD4+ and CD8+ T cells. CD4+ Th1 cells secrete IFN-, which activates
the anti-microbial activity of macrophages and helps B cell production of IgG2a antibodies,
whereas Th2 cells provide help for B cell production of IgG1, IgA and IgE. CD8+ T cells lyse
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host cells infected with viruses and intracellular bacteria and parasites. Many of these
responses can cause host tissue damage, for example, excessive inflammation from
uncontrolled pro-inflammatory cytokines and chemokine production by innate cells and Th1
cells, eosinophilia and allergic reactions from uncontrolled Th2 responses and killing of host
cells by CD8+ CTL and NK cells. In the normal individuals, regulatory T (Tr) cells (natural Tr
circulating in the periphery and those induced by infection), help to control these effector
functions and associated damage to host tissues.
Figure 2. Natural and inducible Tr cells. Natural Tr cells express the cell surface marker
CD25 and the transcriptional repressor FoxP3. These cells mature and migrate from the
thymus and represent 5-10% of peripheral T cells in a normal mouse. Other populations of
antigen-specific Tr cells can be induced from naïve CD4+CD25- or CD8+CD25- in the
periphery under the influence of semi-mature dendritic cells, IL-10, TGF- and possibly IFN. The inducible populations of Tr cells include distinct subtypes of CD4+ cells; Tr1 cells
which secrete high levels of IL-10, no IL-4 and low or no IFN- and Th3 cells which secrete
high levels of TGF-. Although CD8+ T cells are normally associated with CTL function and
IFN- production, these cells or a subtype of these cells can secrete IL-10 and have been
called CD8+ Tr cells.
Figure 3. Targets of Tr cells and mechanisms of suppression. CD4+CD25+FoxP3+ natural
Tr cells inhibit proliferation of CD25- T cells. The mechanism of suppression appears to be
multifactorial, and includes cell-to-cell contact. CD4+CD25+ T cells express CTLA-4, which
interacts with CD80/CD86 on the APC and this interaction delivers a negative signal for T
cell activation. There is also some evidence that secreted or surface-bound TGF- or secreted
IL-10 may play a role in suppression by natural Tr cells. NK T cells and inducible populations
of Tr cells, which include Tr1, Th3 and CD8+Tr cells secrete IL-10 and/or TGF-. These
immunosuppressive cytokines inhibit proliferation and cytokine production by effector T
cells, including Th1, Th2 and CD8+ CTL, either directly or through their inhibitory influence
on maturation and activation of DC or other APC.
Figure 4. Role of pathogen-derived molecules in promoting the induction of Tr versus
Th1/Th2 cells. Pathogens produces a range of conserved molecules, which interact with
pathogen recognition receptors (PRR) on innate cells, including macrophages and DC. The
majority of TLR ligands, including LPS and CpG motifs and viral RNA, activate IL-12 and
IL-27 production and DC maturation (upregulated surface expression of CD80, CD86 and
CD40), leading to Th1 cell induction. Distinct families of pathogen-derived molecules,
including filamentous haemagglutinin (FHA) and adenylate cyclase toxin (CyaA) from B.
pertussis, cholera toxin (CT), hepatitis C virus non-structural protein 4 (HCV-NS4),
Schistosome-specific phosphatidylserine (PS) interact with PRR, including CD11b/CD18,
GM1 or TLR2 on DC, stimulate IL-10 and inhibit IL-12 production by macrophages and DC
and activate DC into a semi-mature or intermediate phenotype, which promotes the induction
of Tr1/Th3 cells. Finally products of Helminth parasites, yeast hyphae, CT and CyaA, and
E.coli heat labile enterotoxin (LT), which stimulate IL-4 or IL-6 production from DC or other
innate cells, promote the induction of Th2 cells. IL-10 and TGF- produced by Tr cells and
from innate cells inhibit the activation of Th1 cells, which mediate immunity to intracellular
pathogens and Th2 cells, which mediate immunity to extracellular pathogens. However, these
immunosuppressive cytokines also prevent innate inflammatory responses and autoimmunity
and allergy, which is mediated by pathogenic Th1 and Th2 cells respectively.
Figure 5. Protective immunity versus immunopathology is dependant on a balance
between regulatory and effector T cells. Proposed model where certain pathogens in
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different individuals can either: A) Stimulate potent pathogen-specific Tr cell responses,
through selective induction of innate IL-10, which together with natural CD25+ Tr cells, can
inhibit the generation and function of effector T cells and prevent clearance of the microbes,
an immune evasion strategy by many pathogens that cause chronic infections. B) Induce Th1biased or Th2-biased immune responses in certain individuals, by activating innate IL-12 or
IL-4 production respectively. In cases where Tr cells are limiting, either through a defect in
natural CD25+ Tr cells or limited induction of Tr1/Th3 cells by the pathogen, these effector
cells can mediate clearance of the microbe. However in the absence of control by an
appropriate complement of Tr cells, theses effector T cells can result in pathological damage
to host tissue. C) Induce balanced number of regulatory and effector T cells, possibly by
stimulating both IL-10 and IL-12/IL-4 production by innate cells, thus allowing effector T cell
mediated clearance of the microbe, with Tr cells controlling the inflammatory responses, thus
limiting damage to host tissue.
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