The Homeostasis Of The Immune System

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02 Nov 2017

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ABSTRACT

Regulatory T cells (Tregs) play a pivotal role in the homeostasis of the immune system and in the modulation of the immune response and have emerged as key players in the development and maintenance of peripheral immune tolerance. Broadly speaking, CD4+ T cells possessing the ability to suppress immune responses can be divided into two types: naturally occurring (nTreg) and inducible (iTreg) or adaptive regulatory cells. Naturally occurring thymic-derived CD4+CD25+ regulatory T cells are a T cell subset with immunosuppressive properties that constitutes 5–10% of the total peripheral CD4+T cells. While regulatory T cells in normal conditions regulate ongoing immune responses and prevent autoimmunity, imbalanced function or number of these cells, either enhanced or decreased, might lead to tumour development and autoimmunity respectively. These cells thus play a major role in autoimmune diseases, transplantation tolerance, infectious diseases, allergic disease and tumour immunity. These natural properties make regulatory T cells attractive tools for novel immunotherapeutic approaches. The in vivo manipulation or depletion of Tregs may help devise effective immunotherapy for patients with cancer, autoimmunity, graft versus host disease, infectious diseases and allergic diseases. It is crucial to understand regulatory T cell biology before attempting therapies, including (i) the injection of expanded regulatory T cells to cure autoimmune disease or prevent graft-versus-host disease or (ii) the depletion or inhibition of regulatory T cells in cancer therapy. Recent findings in murine models and studies in humans have opened new avenues to study Treg biology and their therapeutic potential. This overview attempts to provide a framework for integrating these concepts of basic and translational research.

Natl Med J India 2012;25:000–0

INTRODUCTION

Regulatory T cells (Tregs) play a critical role in modulation of the immune response and have emerged as key players in the development and maintenance of peripheral immune tolerance. The purpose of this review is to present the current perspective on key developments in the biology and function, role and application of Tregs relevant to a variety of clinical conditions and avenues for their therapeutic modulation.

ORIGIN AND DISCOVERY

As self-reactive T and B cells can be demonstrated in the peripheral blood of the host (as a result of breakdown of clonal deletion process or central tolerance) and the self-reactive cells can under certain circumstances, be activated to cause tissue damage in autoimmunity, Gershon in 1971 suggested that there was probably a second regulatory mechanism and that a subset of suppressor T cells should exist in the host to maintain tolerance to self-antigens in the periphery.1 Due to conflicting results, the concept of T cell suppression fell into disrepute and research waned till the late 1980s. It took more than two decades to overcome the lack of evidence for phenotypic or functional markers of so called suppressor T cells, as suggested by Gershon, and identify a subset of CD4+ T cells co-expressing CD25 that downregulate the activation and expansion of self-reactive T cells.

There was resurgence of interest in this field with the seminal observations of the Sakaguchi group in the mid-1990s in what is now termed Tregs.2 They demonstrated that a minor population of CD4+ T cells that co-expresssed the CD25 antigen (the IL-2R α chain) function as Tregs in the normal adult and the fact that CD4+CD25+ T cells exhibit an enormously suppressive activity and prevent autoimmunity in a murine model. This and other data collectively suggested the existence of a thymically produced suppressive T cell population, which was responsible for the establishment and maintenance of peripheral self-tolerance.

The understanding of Treg cell biology took a leap forward in 2003 following the delineation of the functional and developmental role played by the transcription repressor—the X-chromosome-encoded forkhead-winged-helix transcription factor, FoxP3, as the key player in the biology of CD4+CD25+ Treg cells and as a specific lineage marker and master regulator of Treg cells.3,4 FoxP3 protein remains the best and the most specific marker of Treg cells todate. Subsequent studies in the mouse showed that FoxP3-deficient animals lack Treg cells, whereas over expression of the FoxP3 protein leads to profound immune suppression. Young males with the IPEX syndrome (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) or a mutant mouse strain, scurfy, both succumb to similar autoimmune and inflammatory diseases due to uncontrolled activation of CD4+ T cells.5 Both IPEX patients and scurfy mice have mutations in a common gene, FoxP3.6

Subsets of regulatory T cells

Currently, various subsets of Tregs have been identified based on their expression of cell surface markers, production of cytokines and mechanisms of action (Table I, Figs 1 and 2). They are broadly divided into 2 subsets, naturally occurring (nTreg) and induced (iTreg), based on their ontogeny and mode of action (Figs 1 and 3).7

In brief, Treg cells classified on the basis of origin, generation and mechanism of action, appear to have complementary and overlapping functions in the control of immune response. In peripheral T cell immunoregulation, the nTreg cell subset can work in synchrony with iTreg cells to control the activation and function of adaptive immune response. nTreg suppression is primarily cytokine-independent while iTreg is primarily cytokine-dependent. nTregs which are generated in the thymus, are characterized by the constitutive expression of intracellular cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor family related gene (GITR) and transcription factor forkheadbox P3 (FoxP3),8 while antigen-induced or adaptive iTregs, CD4+ Tr1 and Th3 cells, which are induced in the periphery upon encountering cognate antigens are mainly identified by expression of immunosuppressive cytokines interleukin-10 (IL-10) and/or transforming growth factor-β (TGF-β), respectively.9–12

Phenotype or marker molecules of Regulatory T cells

Treg cells were originally defined on the basis of constitutive expression of surface CD4 antigen and surface CD25 antigen (IL-2 receptor α-chain) at high density.13–15 Early studies lacked the incorporation of FoxP3 which has been recognized as a master regulation and lineage-specification factor for Tregs. More recently, studies have shown that reciprocal expression of the IL-7 receptor (CD127) on FoxP3+ Treg cells is a more specific way to quantify Treg cells.16,17 nTreg cells constitute approximately 5%–10% of the peripheral CD4+ T cell population in normal naive mice and healthy humans18 and are characterized by the constitutive expression of CD25 (IL-2 receptor α-chain) and low expression levels of CD45RB.

The CD4+CD25+ phenotype for Tregs has been insufficient to define them as CD25, is not T cell restricted and cannot be used to distinguish Treg cells from effector T cells (Teff). While the murine CD4+CD25+ population is highly enriched in Tregs, in humans CD25+ cells contain both Treg and Teff populations. To obtain enriched Treg cell with little Teff contamination, it is necessary to gate on the CD4+CD25high population that has regulatory activity. This population accounts for only 1%–3% of human CD4+CD25+ T cells. The CD4+CD25+CD127low population contains approximately 80% of the FoxP3+ cells and is significantly larger than the CD4+CD25high population. Overall, the available data indicate that FoxP3 identifies a broader regulatory T cell population than that defined by CD4+CD25+ or CD4+CD25+CD127low expression alone. Definition of Treg cells by combining CD127 and FoxP3 has the advantage of including not only Treg cells expressing high levels of CD25 but also Treg cells with low CD25 expression and excluding at the same time activated conventional T cells.19 While a strong correlation between FoxP3 and CD25 expression in the resting CD4+ T cell population has been reported, low levels of FoxP3 are detectable in CD25−CD4+ T cells.19,20 Thus, it seems that FoxP3 expression too, in humans, might not be confined to Treg cells.

Other cell-surface markers associated with Treg cell phenotype and function are CTLA-4 (or CD152), GITR, CD62 ligand (CD62L), transforming growth factor β1 (TGF-β1), IL-10, lymphocyte activation gene-3 (LAG-3), integrin αEβ7 (CD103), neuropilin-1 (Nrp1) and OX40 (CD134).6,10

Function

T cell mediated suppression by Tregs is a key mechanism for preserving self-tolerance, not only to autoantigens but also to foreign antigens in mice and humans, indicating that Tregs play a pivotal role in the homeostasis of the immune system. Tregs have thus emerged as key players in the induction and maintenance of immunological tolerance. Regulatory T cells provide protection from autoimmune disease, graft-versus-host disease, transplant rejection and overwhelming tissue destruction during infections. Conversely, high Treg numbers enable cancer cells to evade the host immune response.21,22

REGULATORY T CELLS IN HUMAN DISEASE

Tumour immunity

Cancer develops as a part of ‘self’ and many tumour-associated antigens are self-antigens recognized by autologous T-cells. Therefore, the mechanisms that maintain immunological tolerance, central or peripheral, to self-antigens may inhibit the generation of effective tumour immunity. Naturally occurring Treg cells impede immune surveillance against autologous tumour cells and may inhibit the generation, activation, proliferation and effector function of tumour-infiltrating T-cells.

Expansion of CD4+CD25+ Treg cells within the tumour microenvironment, draining lymph nodes and peripheral blood has so far been accepted as a hallmark of cancer. In the context of cancer, the importance of Tregs lies in the fact that increased numbers may favour tumour development or growth and influence the course of the disease. Augmented Treg cell frequencies have been linked to tumour stage, prognosis and survival.

Non-specific depletion of CD4+ T cells can lead to the induction of efficient antitumour immunity. Onizuka et al. in 1999 were the first to suggest that Tregs played an important role in inhibiting tumor immunity. They showed that reduction of CD4+CD25+ Treg cells by anti-CD25 monoclonal antibody treatment resulted in rejection of immunogenic tumours in none or less responding animals.23

Various murine tumour models exploring the suppressive function of Tregs in tumours such as fibrosarcoma, melanoma, colon carcinoma, pancreatic cancer and prostate dysplasia have been addressed by different workers.24 The relatively early induction of Treg cells in tumour development implies that their presence would precede the time of diagnosis in most patients. Treg cells are attracted to the site of the tumour and one of the possible mechanisms is shown in Fig 4. The in vivo manipulation or depletion of Treg cells may help devise effective immunotherapy for cancer patients and represent a novel way of provoking tumour immunity. However, further work is needed to understand how enrichment of Treg cells occurs in cancer patients and if accumulation or preferential induction of tumour-specific Tregs plays a role during tumour progression.

Solid cancers

Numerous investigators have reported the important role played by Tregs in the immune response and prognosis of solid tumours.6,24 Increased percentages of Tregs were first reported in tumour-infiltrating lymphocytes in non-small-cell lung cancer and ovarian cancer.25 Following this initial observation, further studies concluded that Tregs are increased not only in the tumour microenvironment of patients with invasive breast or pancreatic cancers but also in the peripheral blood, suggesting that the increase of Treg cells is a generalized phenomenon.26 In malignant melanoma, patients have a 2-fold increased frequency of Treg cells in metastatic lymph nodes compared with tumour-free nodes.27 In patients with gastric carcinoma, the frequency of peripheral Tregs is inversely correlated with their prognosis.28 Curative resections significantly reduced the Treg numbers, which increased again in patients with a relapse after a curative resection.29 It has been demonstrated that Tregs suppress tumour-specific T cell mediated immunity in ovarian cancer, contributing to tumour growth and accumulate during progression and are associated with decreased survival.30 Subsequently, Treg frequencies were also found to be increased in squamous cell carcinoma of the head and neck,31 hepatocellular carcinoma32 and mesotheliomas.33 Hence, increased Treg cells appears to be a common theme in most human solid tumours. Moreover, there seems to be a stage-dependent increase of Treg cells with frequencies of Tregs probably correlated to overall survival. These findings provide compelling evidence of the correlation between tumour growth and Treg cell frequencies.

Haematological malignancies

In comparison to the interest in Treg numbers in solid tumours, developments in the role of Tregs in haematological malignancies took a while to occur and are relatively recent.6,24 A variety of methods to study Tregs in haematological malignancies have been devised, the most predominant of these being the use of flow cytometry.

The first study in this field demonstrated large populations of both IL-10-secreting Tr1 and CD4+CD25+ Treg cells in infiltrating lymphocytes and peripheral blood mononuclear cells in Hodgkin lymphoma.34 In patients with B-cell chronic lymphocytic leukaemia, a stage-dependent increase of CD4+CD25highFoxP3+CTLA4+GITR+Tregs with frequencies reducing after fludarabine therapy has been established.35 However, patients with decreased Tregs are prone to autoimmune disease and increased frequency of autoimmune haemolytic anaemia and thrombocytopenia is linked to fludarabine therapy.

Similar studies have reported an expanded pool of Tregs in B-cell non-Hodgkin lymphomas,36,37 acute myeloid leukaemia (AML)38,39 and chronic myeloid leukaemia.40 In a similar vein, increased frequencies of CD4+CD25highFoxP3+ Tregs have also been shown in multiple myeloma as well as its premalignant precursor monoclonal gammopathy of undetermined significance.41 There is also data to suggest that peripheral expansion of Tregs occurs frequently in high-risk myelodysplastic syndrome as well as at disease progression.42 Taken together, these data establish the concept of increased Treg cells in haematological malignancies. However, some of the early studies need to be validated by using more specific markers such as FoxP3 as well as more sophisticated and standardized functional assays.

Transplantation tolerance

The finding that depletion of CD4+CD25+ Tregs from normal mice enhances graft rejection suggests that these cells are involved in transplantation tolerance. Vice versa, an increase of the absolute Treg cell number may lead to significant prolongation of graft survival.43–45 Several studies indicate that Tregs not only play a role in solid organ transplantation but seem also to inhibit graft versus host disease (GVHD) after stem cell transplantation (SCT). Murine studies have established an important role for Tregs in the suppression of GVHD without compromising a graft versus leukaemia (GVL) effect and clinical studies of the feasibility and clinical consequences of adoptive transfer of this cellular population after allogeneic SCT are ongoing. Strategies that augment a GVL effect without increasing the risk of GVHD are required to improve the outcome after allogeneic SCT.

In humans, prospective studies have shown that Treg cell frequencies were significantly lower in patients with acute GVHD (aGVHD) than those who did not have aGVHD or who underwent autologous SCT.46 Moreover, there was an inverse linear correlation between Treg frequencies and severity of aGVHD. Similarly, patients with chronic GVHD (cGVHD) had a lower frequency of Tregs as compared to those who had no cGVHD.47 Increased Treg content in the graft was found to be associated with less aGVHD in alloSCT after myeloablative conditioning regimens in some,48,49 but not all studies.50 The reason for these apparent disparate results is not known. The Stanford group has demonstrated that Tregs suppress early expansion of alloreactive donor T cells and CD25 expression, decreasing GVHD but without abrogating the GVL effect.51

Recent studies have reported that the CpG island associated with the promoter of the FoxP3 gene is hypermethylated in CD4+CD25+ cells and that administration of DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (AZA) up-regulates expression of FoxP3, resulting in Treg expansion in vitro.52,53 In murine transplantation models administration of AZA after transplantation results in expansion of Tregs and a reduction in the incidence of aGVHD without apparent abrogation of a GVL effect.54

Autoimmunity

Sakaguchi et al. first reported in 1995 that the transfer of CD4+CD25- T cells into athymic nude mice induces various organ-specific autoimmune diseases, whereas the cotransfer of a small number of CD4+C25+ T cells could completely prevent the onset of autoimmunity.2

The IPEX syndrome alluded to previously made it apparent that a deficiency of nTregs caused by mutations in the FoxP3 gene resulted in autoimmune lesions in multiple tissues that manifest early in life.6 Apart from this mutation, humans expressing a defective form of the transcription factor AIRE (autoimmune regulator) develop multiorgan autoimmune disease.55 Mutations of the AIRE gene are seen in APECED, an autosomal monogenetic disease characterized by autoimmune polyendocrinopathies, mucocutaneous candidiasis and ectodermal dystrophies. It has been assumed that mutations within the AIRE gene may lead to insufficient negative selection of self-reactive T cell clones and defective generation of thymus-derived Treg cells.6

Decreased numbers and functional activity of CD4+CD25+ FoxP3+ T cells (Tregs) are associated with impaired immune homeostasis and an increased susceptibility to development of various autoimmune diseases.56 Several studies in experimental animal as well as human autoimmune disorders, such as multiple sclerosis, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), type 1 diabetes, psoriasis, myasthenia gravis, Kawasaki disease, autoimmune polyglandular syndrome type II and autoimmune lymphoproliferative syndrome57 have reported a decrease in circulating CD4+CD25+ Tregs compared to healthy individuals.

It is likely that most autoimmune diseases have a common mechanism. A question which needs to be answered is whether the onset of autoimmune diseases is determined by a dysfunction/deficiency of naturally occurring Tregs by an imbalance between self-reactive T cells and natural Tregs. CD4+CD25+ Tregs from active RA patients are both phenotypically and functionally abnormal. Increased number of Tregs has been detected in the synovial fluid of patients with active rheumatic disease, suggesting a migration of Tregs from the peripheral blood to the site of inflammation. Moreover, Tregs of patients with RA showed a diminished in vitro activity to suppress the production of interferon- g (IFN- g) and tumour necrosis factor-α (TNF-α) by the CD4+CD25- effector T cells which may contribute to the ongoing inflammation.58 Several cytokines can influence the activity and number of Tregs, negatively or positively, in physiological and pathological conditions. IL-6 appears critical in the inhibition of differentiation of Foxp3+ Tregs and the suppressive activity of Tregs.59 IL-6 is also found in high levels in SF from the joints of patients with active RA which renders responder T cells resistant to CD4+CD25+ Treg suppression.60

TNF has been described to inhibit the suppressive function of both naturally occurring Tregs and TGF-β-induced Tregs. TNF-mediated inhibition of suppressive function is related to a decrease in FoxP3 mRNA and protein expression by the Tregs. Notably, CD4+CD25high Tregs isolated from patients with active RA expressed reduced levels of FoxP3 mRNA and protein. Treatment with anti-TNF antibody (infliximab) increased FoxP3 mRNA and protein expression by CD4+CD25high Tregs and restored their suppressive function.61 CD4+CD25high Tregs isolated from patients with active SLE expressed reduced levels of FoxP3 mRNA and protein and poorly suppressed the proliferation and cytokine secretion of CD4+ effector T cells in vitro. In vitro activation of CD4+CD25high Tregs from these patients restored their suppressive function. Overproduction of TNF may also contribute to the defective Treg function in patients with active SLE.62

An autoimmune pathophysiology has also been postulated for several haematological disorders. In vitro studies in aplastic anaemia (AA), pure red cell aplasia, autoimmune haemolytic anaemia (AIHA) and primary immune thrombocytopenia (ITP) support the view that the disease mechanism may in part be mediated by self-reactive T cells.6 In a murine model of AIHA, depletion of CD4+CD25+ Treg cells resulted in increased incidence of AIHA in C57/B16 mice.63 In patients with ITP, naturally occurring CD4+CD25+ Tregs are both functionally impaired and reduced in number. It has been found that the number of CD4+CD25+ T cells was significantly lower in ITP patients in the severe phase. However, the number of those cells increased in the patients at the complete remission phase, especially after a splenectomy.64

A study undertaken to investigate role of Tregs in preventing immune thrombocytopenia in a murine model revealed that Treg-deficient mice prepared by inoculation of Treg-depleted CD4+CD25– T cells isolated from BALB/c mice into syngeneic nude mice intravenously spontaneously developed thrombocytopenia. Simultaneous transfer of Tregs completely prevented the onset of thrombocytopenia, but Treg transfer after the onset of thrombocytopenia had no apparent effect. These results indicate that Tregs play a critical role in preventing murine autoantibody-mediated thrombocytopenia by engaging cytotoxic T lymphocyte-associated antigen 4.65

In yet another study, it was reported that thrombopoietic agents in patients with ITP have profound effects in restoring immune tolerance and improving regulatory T cell activity.66

Two studies have shown that Tregs are significantly lower at presentation in almost all patients with AA than in controls.67,68 Activated and resting Tregs were reduced in AA. Moreover, a functional impairment of Tregs was observed.68 Treg defects are also now implicated in autoimmune marrow failure.67

A study strongly suggests that the deficiency of Tregs might play an important role in the immunopathophysiology of autoimmune neutropenia in childhood.69

In an elegant study, it has been shown that abnormalities in Treg cells may contribute to the pathogenesis of autoimmune complications associated with Wiscott-Aldrich syndrome.70

Figure 1

CD4+CD25+ T cells in patients with aplastic anemia and in healthy control subjects. (A) Peripheral blood mononuclear cells from patients with aplastic anemia and healthy control subjects were stained with anti-CD4 and anti-CD25 antibodies followed by intracellular anti-FOXP3 antibodies. The upper gate on the dot plots (gate a) represents CD4+CD25hi+ T cells and the lower gate (gate b) the CD4+CD25+ T cells. FOXP3 expression was gated on CD4+CD25hi+ T cell population (gate a). Representative dot plots are shown from a healthy control subject, a patient with aplastic anemia, and another patient before and after immunosuppressive treatment. Tx, treatment. (B,C) Relative numbers of CD4+CD25hi+ T and CD4+CD25hi+ FOXP3+ T cells from all healthy control subjects and patients with aplastic anemia examined are shown. All patients were examined before receiving any immunosuppressive treatment. Differences between patients and control subjects were statistically significant (P < .001). (D) CD4+CD25+ T cells' nuclear extracts from healthy volunteers and patients with aplastic anemia were analyzed for FOXP3 expression. All patients examined had significantly decreased FOXP3 protein levels compared with healthy donors (P < .001). All patients' samples were analyzed side-by-side with healthy donors' samples. (E) Cytoplasmic extracts from CD4+CD25+ T cells from patients and healthy donors were analyzed by immunoblot for NFAT1 expression. All patients examined showed diminished or undetectable NFAT1 protein levels compared with healthy control subjects (P < .001). All patients' samples were run side-by-side with control subjects' samples. There were no differences in TLR2 expression between patients and control subjects. (F) RNA from CD4+CD25+ T cells from patients with aplastic anemia and control subjects was analyzed for FOXP3 gene expression in quantitative polymerase chain reaction experiments. Patients with aplastic anemia showed decreased FOXP3 mRNA/actin copies compared with healthy control subjects (P = .03). Horizontal lines represent mean values. (G-I) The densitometric intensities of immunoblot results from all the subjects studied for FOXP3, NFAT1, and TLR2 expression are collectively presented. Horizontal lines represent mean values. Results are plus or minus standard error of the mean (± SEM).

Deficient CD4+ CD25+ FOXP3+ T regulatory cells in acquired aplastic anemia

Blood. Blood;110(5):1603-1606.

Infectious diseases

Treg cells have a role to play in infectious diseases by blunting overactive and damaging immune responses to pathogens. Another but not necessarily exclusive role of Tregs might be to moderate the degree of inflammation and damage in a tissue in the course of any early infection.

In infectious diseases, however, the situation is ambivalent. Sometimes, Tregs are useful and at other times not.

During infection, Tregs appear to play a pivotal role in preventing immunopathology and limiting collateral damage to the host caused by immune responses to the pathogen. However, Tregs can also be exploited by the pathogen to subvert protective immune responses and thereby, prolong their survival in the host. This is a particular feature of pathogens that causes chronic infection, including many parasites, viruses and bacteria. In many of these infections, a failure to clear the infection can be attributed directly to potent Treg responses, and depletion or inactivation of Tregs in mouse models can enhance pathogen clearance.

Tregs are intricately involved in the immune response to many parasitic infections. During malarial infection increased numbers of CD4+CD25+FoxP3+ T cells have been found in both human and murine malaria infection. Higher Tregs numbers are associated with increased parasite load and development of human infection caused by P. falciparum.71

The CD4+ regulatory T cell population has also been found to be significantly higher in several filarial infections. The human filarial parasite has the inherent ability to stimulate Treg activity, providing further support for the hypothesis that these cells play an important role in the infective process. Adult parasites also induce FoxP3 expression. A study indicated that, in the mouse at least, filarial parasites expand the frequency and activity of FoxP3-expressing T cells at the site of infection, and that these cells have functional in vitro suppressive capacity indicative of Tregs.72

Tregs are also involved in immunoregulatory mechanisms in trypanosomiasis, toxoplasmosis and schistosomiasis. Visceral leishmaniasis (VL) represents a parasitic disease that has been shown not to induce expansion of natural Tregs. In a study among patients with Kala-azar, frequencies of FoxP3+ cells in patient with VL before and after treatment did not increase, neither were they elevated when compared to endemic controls. It was therefore concluded that active VL is not associated with increased frequencies of peripheral FoxP3 Tregs or accumulation at the site of infection. While active VL does not induce expansion of Treg, it has been shown in animal models that Treg is directly responsible for its reactivation.73

Several studies have reported involvement of Tregs in viral diseases as Tregs might affect the magnitude of the immune response and therefore the outcome of viral clearance. After depletion of Tregs by anti-CD25 antibody in Herpes simplex virus infected mice, increased CD4+ T cell responses, enhanced CD8+ proliferative and cytotoxic T lymphocyte (CTL) responses, and increased mucosal antibody levels were reported as compared to nondepleted animals. In addition, viral clearance occurred more rapidly in Treg-depleted mice. In humans with chronic hepatitis B (HBV) and hepatitis C virus (HCV) infection, an increase in peripheral CD4+CD25+ Tregs, as compared to healthy individuals, has been described. Moreover, these Tregs are able to suppress HCV-specific CD8+ T cell immune responses. Besides increased levels of Tregs in patients, IL-10 producing Tr1 cells could also be isolated and cloned from patients with chronic HCV infection, but not from patients who cleared the infection. Following the demonstration of the role of Tregs in suppressing antiviral immune responses, several in vitro studies showed that depletion of Tregs from peripheral blood of patients with viral infection results in increased T cell responses to HBV, HCV, cytomegalovirus (CMV), and human immunodeficiency virus (HIV).10 While the presented results are clear for HBV, HCV and CMV infections, the influence of Tregs during HIV infection might be more complex.

Substantial research has been done on the role of regulatory T cells (Tregs) in HIV infection. There is usually an increased frequency of Tregs in lymphoid tissues of HIV-infected persons. In HIV infection, though most studies demonstrate Treg expansion as compared with healthy controls,74 in one study, Tregs were depleted.75 To date, it is still not clear whether Tregs are detrimental to HIV infection because they suppress HIV-specific responses, or if they are beneficial because they decrease immune activation associated with HIV infection.76

Mycobacterium tuberculosis (Mtb) is a good example of a bacterium that causes chronic infection, where Tregs have been implicated in pathogen persistence and protection against immune-mediated pathology. Tregs are enhanced in patients with active disease, where they impair immune responses, especially IFN-g production by protective Th1 cells. Tregs are prominent in tubercular granulomas and pleural effusions. FoxP3+ Tregs are significantly higher in the peripheral blood of patients with active compared with latent tuberculosis (TB). Tregs may also render patients with latent TB susceptible to reactivation. Ex vivo depletion of Tregs in human peripheral blood mononuclear cells from patients with active disease enhances IFN-g production in response to Mtb antigens.77

Allergic diseases

Allergy, atopic or non-atopic, is an altered immune reactivity to harmless environmental allergens to which one has previously been exposed. Allergic response to an allergen is subject to a complex regulatory control, which includes Tregs. Experiments in mice models and humans have clearly shown a deficiency of Tregs in allergic diseases such as asthma, allergic rhinitis, eczema and atopic dermatitis.78 Different regulatory cell types have been implicated in the prevention of atopic sensitization in mouse models and humans. In addition to naturally occurring Tregs, adaptive Tregs, induced in response to foreign antigens, have been demonstrated. The cytokines most commonly implicated in Treg-mediated suppression of allergic asthma are TGF-β and IL-10.79

A decreased expression of FoxP3 has been reported in allergic diseases. In humans, along with typical disease manifestations such as elevated IgE levels, eczema, insulin-dependent diabetes mellitus, intestinal inflammation, asthma and other recurrent respiratory disorders have also been associated with IPEX in which there is a mutation of the FoxP3 gene. Although eczema is a common feature of IPEX-deficiency, there is less documentation of asthma in IPEX patients. The most likely reason for this is that there is inherently significant uncertainty in the diagnosis of asthma in young children.80

According to the ‘hygiene hypothesis’ which was postulated to explain the increased prevalence of allergic diseases in developed countries in recent years, early childhood exposures to pathogen-associated products inversely correlates with the incidence of allergic diseases in adulthood. With the rapid progress of research in the field of Tregs in recent years, it appears that microbial infections are conducive to the development of these cells which are then able to exercise their immunosuppressive functions to dampen unwarranted immune responses against foreign or self-antigens.80

REGULATORY T CELLS AS POTENTIAL IMMUNOTHERAPY

There are a variety of circumstances where manipulation of Treg cells could prove beneficial. Studies using mouse experimental models have provided compelling evidence that Tregs can induce transplantation tolerance, prevent GVHD, cause regression of tumours and control allergy and autoimmunity.

Tumour immunity

The suppressive Tregs present within the tumour microenvironment and draining lymph nodes may help mediate immune evasion. Therefore, in cancers, the general objective has been to blunt or negate the activity of Tregs so that anti-tumour effector T cell function can proceed more effectively. Targeting Treg cells therefore provides an attractive immunotherapeutic strategy to potentially influence the suppressed immune response in tumour patients thereby altering and supporting conventional anti-tumour therapy. However, such strategies have to be undertaken cautiously as this leads to modification of immune homeostasis which may predispose to the development of autoimmune diseases.

The Sakaguchi group and others have described tumour models in mice where suppression of Tregs with anti-CD25 or anti-GITR monoclonal antibody appears to achieve this antitumour effect.2 In a study in a murine model, an agonistic anti-GITR antibody targeting Treg cells has been successfully applied in tumour-bearing mice.81

A specific approach for therapeutic targeting of regulatory T cells may be to attempt to remove Tregs by targeting the CD25 receptor using an IL-2 diphtheria toxin conjugate. Pilot studies in cancer patients indicate that this achieves Treg cell reduction in the peripheral blood and has led in some cases to clinical improvement. Studies suggest that the effectiveness of a cancer vaccine would be augmented by this synergistic approach. The depletion of CD25+ T cells with anti-CD25 monoclonal antibody before dendritic cell-based vaccination dramatically improved survival in mice compared with those receiving vaccination alone. A recombinant IL-2 diphtheria toxin conjugate DAB(389)IL-2 (denileukin diftitox) has been used in a study to selectively eliminate CD25-expressing Treg cells from the peripheral blood mononuclear cells of patients with cancer.82 This strategy significantly reduced the number of Tregs present in the peripheral blood of metastatic renal cell carcinoma patients. In yet another study, analysis of circulating Tregs in patients with metastatic prostate cancer vaccinated with PSA-TRICOM vaccine showed a decrease in Treg suppressive function in post- versus pre-vaccination scenario and its possible association with improved overall survival.83

There are various potential ways by which the frequencies or suppressive functions of Tregs can be down-regulated. Depletion of Tregs with anti-CD25 agents has been the most common approach. Pharmacological means to deplete Tregs include reagents such as denileukin diftitox (anti-CD25 Ab),84 low-dose cyclophosphamide, fludarabine, ipilimumab (anti-CTLA-4, a fully human monoclonal antibody),85 anti-GITR mAb, anti-OX40, anti-TGF, anti-toll-like receptor and 1-methyl-d-tryptophan (D-1MT).11 Denileukin diftitox (Ontak) has been approved by the US Food and Drug Administration (FDA) to treat cutaneous T cell leukemia/lymphoma for the past 3 years with no reported increased incidence in autoimmune disorders. Additional reports confirm that denileukin diftitox depletes the number of Tregs and the suppression mediated by the CD4+CD25+ T cell population and improves immunity in individuals with renal cell carcinoma and melanoma.86

Ipilimumab has been approved for the treatment of unresectable or metastatic melanoma, while anti-TGF is being used in trials in myelofibrosis, mesothelioma and systemic sclerosis, anti-OX40 in trials in prostate cancer and asthma and D-1MT in trials in refractory solid tumours.11 A phase I/II trial evaluating ipilimumab in patients with follicular lymphoma is currently ongoing.85 Low-dose cyclophosphamide has been successfully tested in murine cancer models to cause immunologically mediated regression of immunogenic lymphomas. Cyclophosphamide depletes Tregs and boosts efficacy of a dendritic cell vaccine in mouse models for melanoma or colon carcinoma. This immunostimulatory agent, thus, might be successfully integrated into chemoimmunotherapy.11

The mammalian target of rapamycin (mTOR) inhibitor everolimus in addition to inhibiting immune responses enhances Treg conversion by several distinct pathways. The converted Tregs can be further expanded by injection of IL-2. The combined use of everolimus and IL-2/IL-2αβ complexes in vivo makes it feasible to achieve highly effective antigen-driven conversion of naive T cells into Treg and their expansion in vivo and thereby the described protocols constitute important tools to achieve immunological tolerance by Treg vaccination.87 Vaccination against FoxP3 improved tumour immunity in a model for renal cell carcinoma. CpG treatment lowers FoxP3+ T cells in lymph nodes of melanoma patients.88

While there are still limitations of currently available Treg-depletion strategies, their employment has already begun to show a translational impact. However, further studies to extend these findings are warranted.

Transplantation

Reinforcement of the function of Treg cells in transplantation may mediate graft survival and this has set the stage for the potential therapeutic application of Tregs in transplant patients. In rodents, Treg depletion from the donor graft accelerates GVHD lethality, suggesting a role of endogenous donor Treg-mediated suppression during the GVHD response. Studies in murine models have shown that inducible Treg cells were much less effective at preventing GVHD than naturally occurring Tregs. However, recent studies in a human xenogenic GVHD model reveal comparable suppression by in vitro-expanded, polyclonally activated iTregs and ex vivo expanded nTregs.89

Within 8 years of the first demonstration of the efficacy of Tregs in suppressing GVHD in mouse models, three clinical trials evaluating the safety and efficacy of Tregs in treating GVHD have been reported, all demonstrating promising potentially safe and reliable efficacy profiles.90–92 In these preliminary studies in humans, Tregs obtained from adult donors or umbilical cord blood was administrated after transplantation to prevent GVHD.91,92

The first-in-man trial involved two patients.90 The first patient had chronic GVHD 2 years after transplantation. After receiving 0.1x106/kg fluorescence-activated cell sorting (FACS) purified ex vivo expanded Tregs from the donor, the symptoms subsided and the patient was successfully withdrawn from immunosuppression. The second patient had acute disease that progressed despite three infusions with an accumulative dose of 3x106/kg expanded donor Tregs. A larger scale phase I trial has recently been concluded in which it was shown that ex vivo-expanded Treg infusions from a third party could be used as supplemental GVHD prophylaxis after double umbilical cord blood transplantation.91 Twenty-three patients with advanced haematological malignancy were enrolled and treated with two units of umbilical cord blood as source of stem cells and effector T cells. Tregs were isolated using anti-CD25 immunomagnetic bead selection from third-party cord blood samples that had 4–6 HLA match with the recipient. Tregs infused with the graft were detectable in the peripheral blood up to day 14 after transplantation. During the 1-year period after Treg infusion, the investigators observed no dose-limiting toxicities or increase in adverse events when compared with historical controls. Incidences of severe acute GVHD were significantly reduced in patients who received Treg therapy.

In the third trial, freshly isolated anti-CD25 immunomagnetic bead-enriched donor Tregs without ex vivo expansion were infused 4 days before the infusion of CD34+ cells and conventional T cells from the same donors in 28 patients with high-risk haematological malignancies undergoing haploidentical transplantation.92 No adjunct immunosuppression was given after transplant. Patients demonstrated accelerated immune reconstitution, reduced CMV reactivation, and a lower incidence of tumor relapse and GVHD when compared with historical controls. Only 2 (7%) patients developed aGVHD. These encouraging early experiences support further investigation of the efficacy of Treg therapy in controlling GVHD and applying Treg therapy in other disease settings.45

There are numerous ongoing trials in AML which are testing whether Treg manipulation in AML can decrease GVHD. Strategies that augment a GVL effect without increasing the risk of GVHD are required to improve the outcome after allogeneic SCT.54 There is also interest to consider wider application of Treg therapy in other disease settings such as solid organ transplantation. However, several aspects of Treg biology that have particular relevance to organ transplantation remain to be fully elucidated.

An immunosuppressive drug, rapamycin, has been used to selectively isolate and expand CD4+CD25high Foxp+ Tregs. This provides additional justification for their clinical use in future cell therapy-based trials as an adjunct therapy with Tregs.93

The main obstacle for clinical application of human CD4+CD25+ Treg cells is their low number in peripheral blood. The numbers of nTreg that can be isolated from periphery are far too small to be clinically effective. Clinical trials with ex vivo-expanded human FoxP3+ Treg cells isolated from peripheral blood are currently under question mainly because purified CD25+ T cells may contain contaminant non-Treg cells.

Autoimmunity

In autoimmunity, the problem is often a failure of one or more regulatory cells types to function effectively. Here the challenge is to find ways to boost their activity. The practical problem here is to use Tregs that can interfere with ongoing autoimmune disease and cause its reversal or even resolution. Research in animal models has demonstrated that Tregs can be used to treat many autoimmune diseases such as type 1 diabetes, inflammatory bowel disease, systemic lupus erythematosus, multiple sclerosis, RA and autoimmune gastritis.

FACS-based isolation can provide high-yield CD4+CD25+CD127low Tregs for ex vivo expansion of highly pure Tregs and this protocol has been approved by the US FDA for a phase I safety trial in type 1 diabetic patients (NCT01210664).94 CD3-specific antibodies have been used successfully in patients with autoimmune diseases, particularly Type 1 diabetes. The in vivo induction of Tregs using, for example, anti-CD3 antibodies, or autoantigenic peptides currently appears closer to clinical application.

Recent studies demonstrate that natural Tregs are unstable and dysfunctional in inflammatory conditions. The stability and function of induced Tregs (iTregs) in established autoimmune diseases, their advantage as therapeutics under these conditions and proper generation and manipulation of iTregs used for cellular therapy is yet another area of active investigation which is being explored. Immunomodulation with manipulation of Tregs may become a part of treatment of autoimmune disorders in the future.



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