Chapter 3 - Regulatory T Cells in Cancer

https://doi.org/10.1016/S0065-230X(10)07003-XGet rights and content

Abstract

At the present time, regulatory T cells (Tregs) are an integral part of immunology but the route from discovery of “suppressive” lymphocytes in the 1980s to the current established concept of Tregs almost 20 years later has been a rollercoaster ride. Tregs are essential for maintaining self-tolerance as defects in their compartment lead to severe autoimmune diseases. This vitally important function exists alongside the detrimental effects on tumor immunosurveillance and antitumor immunity. Beginning with the identification of CD4+CD25+ Tregs in 1995, the list of Treg subsets, suppressive mechanisms, and knowledge about their various origins is steadily growing. Increase in Tregs within tumors and circulation of cancer patients, observed in early studies, implied their involvement in pathogenesis and disease progression. Several mechanisms, ranging from proliferation to specific trafficking networks, have been identified to account for their systemic and/or local accumulation. Since various immunotherapeutic approaches are being utilized for cancer therapy, various strategies to overcome the antagonistic effects exerted by Tregs are being currently explored. An overview on the biology of Tregs present in cancer patients, their clinical impact, and methods for modulating them is given in this review. Despite the extensive studies on Tregs in cancer many questions still remain unanswered. Even the paradigm that Tregs generally are disadvantageous for the control of malignancies is now under scrutiny. Insight into the specific role of Tregs in different types of neoplasias is the key for targeting them in a way that is beneficial for the clinical outcome.

Introduction

The current view on immunology can arguably be thought to begin with the discovery that adaptive immunity is composed of two major types of lymphocytes; the B (bone marrow-derived) and T (thymus-derived) cells (Miller, 1961, Mosier, 1967). Almost concurrently, anecdotal observations were already extending the role of T cells, beyond functioning as effectors and positive regulators, to suppressors of immunological responses. Pioneering studies by Gershon and Kondo in the early 1970s demonstrated for the first time that lymphocytes can suppress T cell responses in an antigen-specific manner (Gershon and Kondo, 1970) and that transfer of antigen-experienced T cells into naïve mice can lead to an antigen-specific tolerance by attenuating T cell activity (Gershon and Kondo, 1971). With great foresight, this cell population was named “suppressor cells” and fit perfectly into the dogma of homeostatic immunoregulation. It was hypothesized that by sustaining quantitatively and qualitatively optimal responses, the immune system facilitated an efficient elimination of pathogens and simultaneously prevented autoimmunity (Penhale et al., 1973). Based on the observations that T cells from tumor bearing hosts were endowed with immunosuppressive capacities preventing the rejection of even highly immunogenic tumors by immunocompetent hosts, potential interconnections between “suppressor cells” and malignancies were presumed (Berendt & North, 1980, Fujimoto et al., 1975). Despite the great significance of these findings, a growing skepticism led to a major loss of momentum and interest for almost 20 years. The main reasons for this were the failure to unequivocally define these cells together with a number of key misleading publications on MHC regions postulated as characteristic for “suppressor cells;” in particular, the illusory I-J locus as well as T-T suppressor hybridomas not transcribing T cell receptor (TCR) genes (Moller, 1988, Simpson, 2008).

Finally, in 1995 Sakaguchi and colleagues initiated the renaissance of the “suppressive cells” (Sakaguchi et al., 1995). In very elegant experiments they showed that transfer of thymic CD25-depleted T cells induced autoimmune diseases in athymic nude mice, while addition of a small proportion of CD4+CD25+ T cells was sufficient to maintain tolerance. Accordingly, the CD25 molecule was the first promising candidate for a phenotypic definition of “suppressive cells” that were named as thymus-derived naturally occurring regulatory T cells (nTregs). CD25 is the α-chain of the high-affinity receptor for interleukin-2 (IL-2R). Although nTregs do not produce IL-2 (Allan et al., 2005) they are vitally dependent on IL-2 production by their environment. This is markedly illustrated by the development of a lethal lymphoproliferative disease in mice deficient for IL-2 or the IL-2Rβ, which resulted in dysregulated T cell activation and severe alterations within the nTreg compartment (Suzuki et al., 1995). The constitutive expression of the IL-2R on nTregs may reflect this dependence on external IL-2. Several models to date have explored how IL-2 signaling contributes to suppressive function, thymic development, and homeostasis of Tregs (Bayer et al., 2005, Furtado et al., 2002, Setoguchi et al., 2005). Interestingly, IL-2 is one of the primary cytokines secreted by effector T cells upon stimulation (Sojka et al., 2004), and drives proliferation and clonal expansion of T cells (Morgan et al., 1976). In parallel, IL-2 appears to be crucial for mechanisms involved in the termination of T cell responses, thereby forming a sophisticated negative feedback circuit.

Although CD25 was sufficient to characterize and further analyze a relatively homogeneous population of nTregs in mice, the same approach was rather challenging in humans. The reason is the limited specificity provided by CD25, whose intrinsic expression at varying levels can be noted in approximately 30% of the T cells and is further upregulated on effector T cells upon stimulation (Baecher-Allan et al., 2004). Unlike mice kept under pathogen-free conditions, humans are continually exposed to immunogenic stimuli resulting in T cell activation and potential CD25 upregulation. In pathological conditions associated with ongoing inflammation this problem is even more pronounced. Consequently, studying Tregs in autoimmune and malignant diseases is complicated further. It may even be speculated that past studies describing CD25+ Tregs as functionally defective may have been influenced by contamination of activated CD25+ effector T cells (Dejaco et al., 2006). In the steady effort to define Tregs more accurately, it was demonstrated that up to 5% of human peripheral CD4+ T cells that express CD25 at high levels are endowed with strong immunosuppressive capacities. This observation narrowed the phenotype of human Tregs further down to CD4+CD25high T cells (Baecher-Allan et al., 2001). Due to the lack of a standardized methodological cut off point for CD25high expression, comparability between clinical studies remained difficult and elevated levels of CD25 expression on effector T cells under conditions of severe inflammatory activity could not be excluded (Han et al., 2008, Seddiki et al., 2006).

Efforts to identify the genetic defects responsible for the severe autoimmune disorders in patients with the IPEX (Immunodysregulation, Polyendocrinopathy, Enteropathy, X-linked) syndrome led to the discovery of germline mutations resulting in a FOXP3 gene deletion on the X-chromosome (Bennett et al., 2001, Chatila et al., 2000). The FOXP3 gene encodes for a transcription factor (TF) of the forkhead-box/winged-helix family. Extensive studies in mice and humans revealed the critical importance of the FOXP3 TF as a master regulator of nTreg development and function. Late double-positive lymphocytes that already express FOXP3 at early thymic developmental stages appear to be destined for the nTreg lineage (Tai et al., 2005, Zhou et al., 2009). Ectopic expression of FOXP3 by retroviral gene transfer in CD4+CD25 T cells has been shown in vitro and in vivo to result in phenotypic and functional suppressive cells demonstrating the plasticity of lymphocytes and the pivotal role of FOXP3 for nTregs (Fontenot et al., 2003, Hori et al., 2003). Concordant to the CD25 expression-based characterization of Tregs, the majority of CD4+FOXP3+ T cells were found to be CD25high (Baecher-Allan et al., 2004, Roncador et al., 2005). FOXP3 dimerizes with the nuclear factor of activated T cells (NF-AT) leading to suppression of IL-2, IL-4, and interferon-γ (IFN-γ) expression, while inducing CD25, cytotoxic T lymphocyte antigen 4 (CTLA-4), and gluco-corticoid-induced TNF receptor family-related gene/protein (GITR) (Lopes et al., 2007, Wu et al., 2006). Like CD25, both CTLA-4 and GITR are also upregulated on effector T cells upon activation (Ermann & Fathman, 2003, Roncador et al., 2005, Tai et al., 2005). Although FOXP3 is presently considered the most reliable (intracellular) phenotypic marker for nTregs, major concerns arose when it became evident that FOXP3 expression could be transiently induced in CD4+ and CD8+ effector T cells upon stimulation, albeit at lower levels (Gavin et al., 2006, Roncador et al., 2005, Roncarolo & Gregori, 2008, Walker et al., 2003, Ziegler, 2007). Consequently, Zou and colleagues suggested a combination of FOXP3 and intracellular cytokine staining, especially for IL-2, IFN-γ, and tumor necrosis factor-α (TNF-α), as an accurate tool to identify nTregs based on the fact that activated FOXP3+ conventional T cells express these polyfunctional cytokines in contrast to nTregs (Kryczek et al., 2009). A promising approach to overcome these impediments can be initiated at the epigenetic level. A major criterion for the lineage commitment of nTregs is the sustained, stable expression of FOXP3 as compared to the transient expression found in FOXP3+ effector T cells. A static gene expression can be achieved stably through remodeling of the chromatin structure by epigenetic modifications like DNA methylation. In fact a specific methylation pattern, particularly a demethylated DNA sequence within the FOXP3 locus, associated with stable FOXP3 expression upon in vitro expansion, was identified as nTreg-specific and defined as a Treg-specific demethylated region (Baron et al., 2007). This methodology has recently been further optimized allowing enumeration of nTregs in clinical samples such as peripheral blood (PB) and tissues (Wieczorek et al., 2009). Furthermore, two studies have demonstrated that expression of the IL-7R α-chain (CD127) is a useful marker for discriminating between activated conventional T cells and nTregs (Liu et al., 2006b, Seddiki et al., 2006). Suppressive CD4+ T cells are negative or weakly positive for CD127, which inversely correlates with the FOXP3 expression, regardless of the CD25 levels. Consequently, the following proposed phenotype of CD4+CD25+CD127low/negFOXP3+ T cells corresponds to the majority of nTregs. Importantly, this phenotype allows a more homogeneous purification of viable CD4+CD25+CD127low/neg nTregs.

The characterization of “suppressive cells” based on CD25 expression heralded a new era of Treg research. More than 10 years later this process is still ongoing and has definitely gained momentum. One of the research areas with the strongest interest in Treg biology has traditionally been cancer research. The biology of human Tregs and their various subtypes, their complex role in cancer and translational approaches in modern cancer therapy are discussed in subsequent sections.

Section snippets

Regulatory T Cell Subsets

Several studies have demonstrated that nTregs are primarily formed by high-avidity selection of CD4 single-positive thymocytes through major histocompatibility complex (MHC) class II-dependent TCR interactions (Apostolou et al., 2002, Bensinger et al., 2001, Fontenot et al., 2005b, Jordan et al., 2001, Larkin et al., 2008, Modigliani et al., 1996, Sakaguchi, 2001). However, other contributory mechanisms like selective survival rather than induced differentiation (van Santen et al., 2004) or the

Mechanisms Mediating the Suppressive Function

In the past decade extensive studies have been performed to further explore the underlying cellular and molecular mechanisms of Treg-mediated immunomodulation (summarized in Fig. 1), which has led to significant improvement in our understanding.

Proliferation and cytokine production of conventional T cells can be inhibited upon TCR activation of Tregs (Takahashi et al., 1998, Thornton & Shevach, 1998). This process is cell-to-cell contact dependent and leads to an inhibition of IL-2 production.

Regulatory T Cells in Cancer

The role of the immune system in cancerogenesis and tumor progression has been the subject of much controversy since the 1950s when Burnet and Thomas formulated their concept of “tumor immunosurveillance”; a process through which the immune system recognizes and (ideally) eliminates self-cells that have undergone malignant transformation (Burnet, 1957). Numerous observations in clinical and experimental settings have fortified this concept that was further advanced by the model of “immune

Compartmental Redistribution

Increasing evidence confirms the hypothesis that Tregs selectively migrate to the site where regulation is required (Fig. 2A). This system, relying on interactions between chemokines/chemokine-receptors and integrins/integrin-receptors (Wei et al., 2006), is often usurped by tumors. Curiel and colleagues were the first to show in ovarian cancer a CCL22-orchestrated migration of CCR4-expressing Tregs toward tumor tissue and malignant ascites (Curiel et al., 2004). In addition to tumor cells,

Antigen Specificity of Tregs in Cancer

As nTregs, like conventional T cells are educated in the thymus, possess somatically rearranged TCRs and recognize self-Ags they should in theory be able to recognize tumor-associated antigens (TAAs). Mouse studies show that antigen-specific Tregs may be more suppressive compared to nonspecific Tregs. Wang and colleagues were the first ones to generate Treg clones specific for the LAGE1 cancer testis antigen from TILs of melanoma patients. These Treg clones required antigen-specific activation

Cancer Vaccines and Regulatory T Cells

Abundant evidence exists that clinical responses to cancer vaccines are influenced by the disease stage at the time of vaccination. Tumor burden and Treg levels typically tend to go hand-in-hand. For example, patients with advanced melanoma have significantly higher circulating Tregs than those with minimal residual disease (Nicholaou et al., 2009). Tregs may be induced or expanded by cancer vaccines as illustrated in studies with melanoma patients, where immunological and clinical responses

Targeting Regulatory T Cells in Cancer Therapy

Taken together Tregs regardless origin, impede tumor surveillance and appear in many cases to be directly linked to the disease pathogenesis. In studies dating back to the 1980s performed by Robert North and colleagues, Treg depletion was shown to be an elegant approach for increasing immune reactivity against cancer. Especially to date, where various forms of immunotherapies find their way into cancer treatment it appears inevitable to counteract the suppressive effects of Tregs. Nevertheless,

Concluding Remarks

Tregs efficiently suppress innate and adaptive immunity. Despite the extensive research that has been carried out, many aspects of Treg biology in cancer remain to be explored. Vast majority of preclinical and clinical studies have linked the presence of Tregs to an increased risk for development as well as progression of cancer. This paradigm is currently under scrutiny as it has been convincingly shown that Tregs can act in a beneficial fashion in inflammatory driven malignancies, explaining

Acknowledgments

This work was supported by grants from the Swedish Cancer Society, the Cancer Society of Stockholm, the Swedish Medical Research Council, an ALF-Project grant from the Stockholm City Council, and the German Research Foundation (DFG).

References (348)

  • N.G. Chakraborty et al.

    Regulatory T-cell response and tumor vaccine-induced cytotoxic T lymphocytes in human melanoma

    Hum. Immunol.

    (2004)
  • T.A. Chatila

    Role of regulatory T cells in human diseases

    J. Allergy Clin. Immunol.

    (2005)
  • C. Chauveau et al.

    Heme oxygenase-1 expression inhibits dendritic cell maturation and proinflammatory function but conserves IL-10 expression

    Blood

    (2005)
  • D.J. Chung et al.

    Indoleamine 2,3-dioxygenase-expressing mature human monocyte-derived dendritic cells expand potent autologous regulatory T cells

    Blood

    (2009)
  • L. Cosmi et al.

    Human CD8+CD25+ thymocytes share phenotypic and functional features with CD4+CD25+ regulatory thymocytes

    Blood

    (2003)
  • L. Cosmi et al.

    Th2 cells are less susceptible than Th1 cells to the suppressive activity of CD25+ regulatory thymocytes because of their responsiveness to different cytokines

    Blood

    (2004)
  • M.A. Curotto de Lafaille et al.

    Natural and adaptive foxp3+ regulatory T cells: more of the same or a division of labor?

    Immunity

    (2009)
  • M.A. Curotto de Lafaille et al.

    Adaptive Foxp3+ regulatory T cell-dependent and -independent control of allergic inflammation

    Immunity

    (2008)
  • A. Curti et al.

    Modulation of tryptophan catabolism by human leukemic cells results in the conversion of CD25- into CD25+ T regulatory cells

    Blood

    (2007)
  • S.E. Erdman et al.

    CD4+ CD25+ regulatory T lymphocytes inhibit microbially induced colon cancer in Rag2-deficient mice

    Am. J. Pathol.

    (2003)
  • G. Filaci et al.

    CD8+ T suppressor cells are back to the game: are they players in autoimmunity?

    Autoimmun. Rev.

    (2002)
  • J.D. Fontenot et al.

    Regulatory T cell lineage specification by the forkhead transcription factor foxp3

    Immunity

    (2005)
  • F. Foss et al.

    A phase-1 trial of bexarotene and denileukin diftitox in patients with relapsed or refractory cutaneous T-cell lymphoma

    Blood

    (2005)
  • J. Fu et al.

    Increased regulatory T cells correlate with CD8 T-cell impairment and poor survival in hepatocellular carcinoma patients

    Gastroenterology

    (2007)
  • W.Z. Ai et al.

    Follicular lymphoma B cells induce the conversion of conventional CD4+ T cells to T-regulatory cells

    Int. J. Cancer

    (2009)
  • Y. Akasaki et al.

    Induction of a CD4+ T regulatory type 1 response by cyclooxygenase-2-overexpressing glioma

    J. Immunol.

    (2004)
  • S.E. Allan et al.

    The role of 2 FOXP3 isoforms in the generation of human CD4+ Tregs

    J. Clin. Invest.

    (2005)
  • T. Alvaro et al.

    Outcome in Hodgkin's lymphoma can be predicted from the presence of accompanying cytotoxic and regulatory T cells

    Clin. Cancer Res.

    (2005)
  • M.H. Andersen et al.

    Identification of heme oxygenase-1-specific regulatory CD8+ T cells in cancer patients

    J. Clin. Invest.

    (2009)
  • G. Angelini et al.

    Antigen-presenting dendritic cells provide the reducing extracellular microenvironment required for T lymphocyte activation

    Proc. Natl. Acad. Sci. USA

    (2002)
  • O. Annacker et al.

    CD25+ CD4+ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10

    J. Immunol.

    (2001)
  • S.M. Ansell et al.

    Phase I study of ipilimumab, an anti-CTLA-4 monoclonal antibody, in patients with relapsed and refractory B-cell non-Hodgkin lymphoma

    Clin. Cancer Res.

    (2009)
  • I. Apostolou et al.

    In vivo instruction of suppressor commitment in naive T cells

    J. Exp. Med.

    (2004)
  • I. Apostolou et al.

    Origin of regulatory T cells with known specificity for antigen

    Nat. Immunol.

    (2002)
  • P. Attia et al.

    Inability of a fusion protein of IL-2 and diphtheria toxin (Denileukin Diftitox, DAB389IL-2, ONTAK) to eliminate regulatory T lymphocytes in patients with melanoma

    J. Immunother.

    (2005)
  • T. Azuma et al.

    Human CD4+ CD25+ regulatory T cells suppress NKT cell functions

    Cancer Res.

    (2003)
  • B. Baban et al.

    IDO activates regulatory T cells and blocks their conversion into Th17-like T cells

    J. Immunol.

    (2009)
  • C. Badoual et al.

    Prognostic value of tumor-infiltrating CD4+ T-cell subpopulations in head and neck cancers

    Clin. Cancer Res.

    (2006)
  • C. Baecher-Allan et al.

    CD4+CD25high regulatory cells in human peripheral blood

    J. Immunol.

    (2001)
  • E. Bardel et al.

    Human CD4+ CD25+ Foxp3+ regulatory T cells do not constitutively express IL-35

    J. Immunol.

    (2008)
  • U. Baron et al.

    DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3(+) conventional T cells

    Eur. J. Immunol.

    (2007)
  • G.J. Bates et al.

    Quantification of regulatory T cells enables the identification of high-risk breast cancer patients and those at risk of late relapse

    J. Clin. Oncol.

    (2006)
  • M. Battaglia et al.

    Induction of transplantation tolerance via regulatory T cells

    Inflamm. Allergy Drug Targets

    (2006)
  • M. Battaglia et al.

    Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients

    J. Immunol.

    (2006)
  • A.L. Bayer et al.

    Essential role for interleukin-2 for CD4(+)CD25(+) T regulatory cell development during the neonatal period

    J. Exp. Med.

    (2005)
  • C.L. Bennett et al.

    The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3

    Nat. Genet.

    (2001)
  • S.J. Bensinger et al.

    Major histocompatibility complex class II-positive cortical epithelium mediates the selection of CD4(+)25(+) immunoregulatory T cells

    J. Exp. Med.

    (2001)
  • M.J. Berendt et al.

    T-cell-mediated suppression of anti-tumor immunity. An explanation for progressive growth of an immunogenic tumor

    J. Exp. Med.

    (1980)
  • C. Bergmann et al.

    Expansion of human T regulatory type 1 cells in the microenvironment of cyclooxygenase 2 overexpressing head and neck squamous cell carcinoma

    Cancer Res.

    (2007)
  • E. Bettelli et al.

    Foxp3 interacts with nuclear factor of activated T cells and NF-kappa B to repress cytokine gene expression and effector functions of T helper cells

    Proc. Natl. Acad. Sci. USA

    (2005)
  • Cited by (0)

    View full text