Functional control of regulatory T cells and cancer immunotherapy

Seminars in Cancer Biology 16 (2006) 106–114

Review

Functional control of regulatory T cells and cancer immunotherapy Rong-Fu Wang ∗ The Center for Cell and Gene Therapy and Department of Immunology, Baylor College of Medicine, ALKEK Building, N1120, Houston, TX 77030, USA

Abstract Regulatory T (Treg) cells induce immune tolerance by suppressing host immune responses against self- or non-self-antigens. Hence, they not only play critical roles in preventing autoimmune diseases, but also may have detrimental effects on vaccines directed to cancer and infectious diseases. Understanding the antigen specificity and functional control of Treg cells will be crucial to the development of effective cancer immunotherapy. This review will discuss different subsets of Treg cells, the factors that contribute to Treg cell generation and suppressive function, and the ability of signaling through Toll-like receptor 8 to reverse the suppressive function of Treg cells. Importantly, this TLR pathway does not depend on interaction with dendritic cells, but operates independently in Treg cells, relying on TLR8 (with MyD88 as its sole receptor-proximal adaptor) to transduce signals generated by TLR8 ligands. Linking TLR signaling to the functional control of Treg cells opens intriguing opportunities to shift the balance between CD4+ T-helper and Treg cells, in ways that may improve the outcome of cancer immunotherapy. © 2005 Elsevier Ltd. All rights reserved. Keywords: Immune suppression; Tumor Immunology; Cancer vaccines; Regulatory T cells

Contents 1. 2.

3. 4. 5.

6. 7.

8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subsets of Treg cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Naturally occurring CD4+ CD25+ Treg cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Antigen-induced Tr-1 and Th3 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Antigen-specific CD4+ Treg cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. CD8+ Treg cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. TCR ␥␦+ Treg cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tumor cells induce CD4+ Treg cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antigen specificity of CD4+ Treg cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors that affect the generation of antigen-specific Treg cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Modes of antigen presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Peptide-binding affinity and antigen expression levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. DC and costimulatory molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of CD4+ Treg cell-mediated immune suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Linking of TLR signaling to the functional control of Treg cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Triggering of TLRs on DCs shifts the balance between Treg and effector T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Human TLR 8 directly reverses the suppressive function of Treg cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

106 107 107 107 107 107 107 107 108 108 108 108 109 109 109 110 110 110 111 111 111

1. Introduction ∗

Tel.: +1 713 798 1244; fax: +1 713 798 1263. E-mail address: [email protected].

1044-579X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcancer.2005.11.004

T cells play an essential role in the immunosurveillance and destruction of cancer cells [1], but this knowledge has not

R.-F. Wang / Seminars in Cancer Biology 16 (2006) 106–114

been translated into clinically effective immunotherapies. Identification of antigens from melanomas and other tumors that are recognized by T cells set the stage for developing more effective, antigen-specific cancer immunotherapy [2–5]. While vaccination with peptides or dendritic cells (DCs) pulsed with antigenic peptides derived from various melanoma antigens has clear potential in cancer therapy, ongoing clinical trials have not produced any compelling evidence of therapeutic benefit [6,7]. Part of the difficulty appears to lie in the presence of CD4+ regulatory T (Treg) cells at tumor sites. Indeed, the results of recent studies imply that Treg cells may significantly suppress immune responses, leading to immune tolerance of tumor cells. Thus, the ability to control the suppressive function of Treg cells will be crucial to the development of effective immunotherapy of cancer patients. 2. Subsets of Treg cells Naturally occurring CD4+ CD25+ Treg cells have been extensively studied in mice and humans, and several additional subsets of Treg cells have been identified and characterized as well, leading to the current view of Treg cell heterogeneity, in which subsets of these cells are defined by distinct suppressive mechanisms. 2.1. Naturally occurring CD4+ CD25+ Treg cells This subset represents a small fraction (5–6%) of the overall CD4+ T cell population and is derived from thymus without specific antigen stimulation. These cells express a high level of GITR and Foxp3 molecules and mediate immune suppression through a cell–cell contact mechanism [8–13]. 2.2. Antigen-induced Tr-1 and Th3 cells These Treg cells are induced in peripheral tissues by MHC/peptide stimulation, secrete a large amount of IL-10 and/or TGF-␤, and suppress immune responses through a cytokine-dependent mechanism [14–16]. 2.3. Antigen-specific

CD4+

Treg cells

Like those naturally occurring counterpart, antigen-specific CD4+ Treg cells express a high level of GITR and Foxp3, and suppress immune responses through a cell-contact-dependent mechanism once they are activated after exposure to a specific antigen [17,18]. Although the origin of CD4+ Treg cells remains largely obscure, they may arise from antigen experienced CD4+ CD25− T cells in the suppressive cytokine milieu of tumor sites [19,20] or after interaction with naturally occurring CD4+ CD25+ T cells, as previously suggested [21,22]. Recent studies in both humans and animal systems demonstrate the conversion of peripheral na¨ıve CD4+ CD25− T cells to CD4+ CD25+ Treg cells by activation or TGF-␤ stimulation of the bulk T cell population [23,24]. Results obtained in transgenic models show that the extent of self-peptide stimulation affects the peripheral generation and expansion of CD4+ CD25+ Treg cells, and thus

107

directs the selection and accumulation of such cells in sites where the self-peptide is expressed [25–27]. Different subsets of DCs can induce both Th1 and Th2 effector cells depending on the dose of antigen: high antigen doses induce Th1 cell development, whereas low antigen doses induce Th2 cell development [28]. Chronic stimulation with low doses of antigen favors the generation of naturally occurring CD4+ CD25+ Treg cells in vivo [29] and antigen-specific Tr1 cells both in vivo and in vitro [30–32]. 2.4. CD8+ Treg cells Not all Treg cells are CD4+ T cells. Indeed, CD8+ Treg cells have been identified that mediate immune suppression in an antigen-dependent manner [33–35]. Such cells suppress antigenactivated CD4+ T cells in a TCR-specific manner restricted by the MHC class Ib molecule Qa-1 [36,37]. CD8+ CD28− Treg cells have also been reported to suppress antigen-presenting cells, including DCs that present the same peptide/MHC complexes to which the CD8+ Treg cells have been primed [35]. In contrast to naturally occurring CD4+ CD25+ Treg cells, the CD8+ Treg cells are generated or induced only after antigen priming [34,35,38]. Whether CD8+ Treg cells can be generated by tumor cells or tumor-infiltrating DCs, and suppress immune cells (CD4+ , CD8+ effector cells and DCs) at tumor sites, remains unclear. 2.5. TCR γδ+ Treg cells TCR-␥␦+ T cells represent a small population of T cells consisting of ␥ and ␦ TCR chains with limited TCR usage. They are distinct from ␣␤ T cells [39,40], and may function as professional antigen-presenting cells (APC) [41] and regulatory cells [42]. Although ␥␦+ Treg cells are enriched in peripheral tissues, including skin, gut and solid tumors [43], their presence at tumor sites has yet to be demonstrated. 3. Tumor cells induce CD4+ Treg cells Tumor-specific CD4+ Treg cells presented in tumor sites may play a significant role in the suppression of antitumor immunity. North and co-workers [44], in a series of experiments with immunogenic murine tumors, demonstrated the complete regression of large established tumors after adoptive transfer of tumor-reactive T cells from immune donors, but only when the tumors were growing in T-cell deficient mice. Such antitumor immunity could be inhibited by an infusion of splenic T cells from T-cell intact, tumor-bearing donors that had lost their concomitant immunity [44]. Combined treatment with tumorreactive T cells and cyclophosphamide can cause established tumors to completely regress in immune-competent mice [45]. Thus, elimination of CD4+ suppressor T cells by any of several different strategies enhanced antitumor immunity [46–49]. The role of CD4+ Treg cells in antitumor immunity was recently reevaluated in a poorly immunogenic murine tumor model [50]. The existence of CD4+ T cell-mediated immune suppression has also been demonstrated in human cancers [51,52]. CD4+ CD25+

108

R.-F. Wang / Seminars in Cancer Biology 16 (2006) 106–114

T cells were identified as Treg cells, and depletion of such T cells resulted in various autoimmune diseases [53]. Interestingly, the removal of CD4+ CD25+ T cells by an anti-CD25 antibody in animal models enhanced antitumor responses [8,54,55]. Recently, increased proportions of CD4+ CD25+ Treg cells in the total CD4+ T cell populations were detected in several different patients with different types of cancers, including lung, breast and ovarian tumors [56–58], as well as metastatic melanoma lymph nodes [59], were recently reported. In recent studies, we demonstrated the presence of antigen-specific CD4+ Treg cells at tumor sites [17,18] showing that Treg cells suppressed the proliferation of na¨ıve CD4+ T cells and inhibited IL2 secretion by CD4+ effector cells upon activation by tumor-specific ligands.

enhanced antitumor immunity or induce Treg cells for immune suppression, depending on many factors that determine the fate of T cells after antigen exposure.

4. Antigen specificity of CD4+ Treg cells

It appears that antigenic ligands that preferentially stimulate CD4+ Treg cells tend to be those antigens that are processed and presented by whole tumor cells, but not by DCs pulsed with the same tumor cell lysates. One possible explanation is that MHC class II-positive tumor cells are capable of presenting antigens to CD4+ T cells with secondary signaling through B7-CD28 interaction because the majority of tumor cells do not express B7 molecules. Further studies are required to determine whether the way in which antigenic ligands are presented influences the fate of T cells to become Th or Treg cells.

Most studies have focused on the role of Treg cells in the prevention of various organ-specific autoimmune diseases [53,60]. Because of the importance of CD4+ Treg cells in modulating host immune responses, their generation and maintenance have long been thought to require the presence of target antigen or tissues [61–64]. However, the natural target antigens recognized by these cells remain mainly unknown [15,19,65]. Our current knowledge of the antigen specificity of CD4+ Treg cells has come largely from studies with antigen-specific TCR transgenic animals [19,27,66–68]. Expression of the TCR variable beta chain is as diverse in CD4+ Treg cells as in effector T cells [69]. A recent study demonstrated that a large proportion of naturally occurring CD4+ CD25+ Treg cells recognize MHC class II-bound peptides from the peripheral tissues more efficiently than do CD25− T cells [70]. Thus, bulk CD4+ CD25+ T cell populations display very diverse specificities for autoantigens (tissue-specific self-antigens) and tumor antigens, making it more difficult to identify the physiological ligands recognized by antigen-specific CD4+ Treg cells. In contrast to the antigenspecific CD4+ Treg cells in autoimmune diseases, which often have compromised function [71], tumor-infiltrating CD4+ T cells provide an enriched source for establishing tumor-specific CD4+ Treg cells. Identification of LAGE1 and ARTC1 as natural ligands for CD4+ Treg cell clones established from cancer patients provides compelling evidence that antigen-specific CD4+ Treg cells are present at tumor sites and mediate antigenspecific and local immune suppression of antitumor immunity [17]. Both LAGE1 and ARTC1 are dominantly expressed in cancer and normal testis, but not in other normal tissues tested, therefore providing the specificity for CD4+ Treg cells. Interestingly, NY-ESO-1, a LAGE1 homolog, is one of the most immunogenic tumor antigens identified to date. Unlike NYESO-1, it appears that LAGE1 preferentially stimulates CD4+ Treg cells rather than CD4+ Th cells because all T cell clones tested possessed the properties associated with CD4+ Treg cells. Consistent with this notion, immunization of mice with Dna J-like 2, an autoantigen identified by recombinant expression cloning (SEREX), elicited suppressive immune response mediated by CD4+ CD25+ Treg cells [72,73]. Thus, tumor antigens or autoantigens may stimulate a CD4+ Th cell response for

5. Factors that affect the generation of antigen-specific Treg cells Foxp3 has recently been identified as a Treg cell lineagespecific factor that controls the development of Treg cells [74,75]. Other factors that can influence the generation and maintenance of Treg cells include TCR signal strength, costimulatory molecules and cytokines [76,77]. 5.1. Modes of antigen presentation

5.2. Peptide-binding affinity and antigen expression levels Recently, we generated a panel of EBV-encoded nuclear antigen 1 (EBNA1)-specific CD4+ T-cell lines and clones that recognize naturally processed EBNA1-P607–619 and -P561–573 peptides in the context of HLA-DQ2 and HLA-DR11, -DR12 and -DR13 molecules, respectively [78]. Phenotypic and functional analyses of these CD4+ T cells revealed that they represent EBNA1-specific CD4+ T helper as well as Treg cells. CD4+ Treg cells do not secrete IL-10 and TGF-␤ cytokines but do express CD25, GITR and Foxp3, and are capable of suppressing the proliferation of na¨ıve CD4+ and CD8+ T cells stimulated with mitogenic anti-CD3 antibody. The suppressive activity of these CD4+ Treg cells is mediated by cell–cell contact or, in part, by a cytokine dependent mechanism. Importantly, these Treg cells suppress IL-2 secretion by CD4+ effector T cells specific for either EBNA1 or a melanoma antigen [78], suggesting that they induce immune suppression. The fact that both CD4+ helper and regulatory T cells recognizing the same EBNA1 peptide can be generated raises an important issue regarding peptidebased immunotherapy against EBV-associated malignancies. Although antigen-specific CD4+ helper T cells can be induced by peptide-based vaccines, the simultaneous expansion of Treg cells with the same peptide specificity may ultimately inhibit effector (CD4+ and CD8+ ) T-cell responses against tumors. These observations suggest that the success of peptide-based vaccines against cancer and other diseases may likely depend upon our ability to identify antigens/peptides that preferentially activate helper T cells, and to devise strategies for regulating the balance between CD4+ helper and regulatory T cells.

R.-F. Wang / Seminars in Cancer Biology 16 (2006) 106–114

The expression level or dose of a particular antigen may be an important factor in determining whether CD4+ T cells are deleted, or become Treg or Th cells. When TCR (TS1) transgenic mice expressing a T cell receptor (TCR) that is specific for the murine I-Ed -restricted determinant from HA (site 1) were crossed with different HA transgene mouse lines (HA12, HA28, PevHA and ␤-myo-HA) expressing different levels of HA antigens under control of various promoters, the variable HA expression led to deletion of the corresponding HA-specific T cells or to development of HA-specific CD4+ CD25+ Treg cells [25,79]. Of particular interest was that about 50% of S1 (specific for HA site 1 peptide) T cells in TS1xHA28 as well as TS1xPevHA double Tg mice possessed a CD4+ CD25+ Treg phenotype [25,79]. Similar findings have been described in a separate study with Ova-specific TCR Tg (DO.11.10) mice (in a BALB/c genetic background), which expressed a TCR specific Ova323–339 epitope. When DO.11.10 mice were crossed with two Ova (RIP-Ovahigh and RIP-Ovalow ) Tg mice, 37% of the T cells in DO.11xRIP-Ovahigh double Tg mice developed IL-10 producing CD4+ CD25+ Treg cells, while the percentage of T cells in DO.11xRIP-Ovalow double Tg mice remained unchanged [80]. These observations demonstrate that the levels of antigen expression dictates whether CD4+ T cells are deleted or become antigen-specific CD4+ Treg or Th cells. Further studies are required to determine the role of antigen expression levels in deciding the fate of CD4+ Treg cells in the thymus and peripheral tissues. Identification of more tumor-specific or tissue-specific ligands for these CD4+ Treg cells will help to clarify the intrinsic properties of CD4+ Treg cells that foster their preferential stimulation at tumor sites.

109

late in the peripheral tissues, and actively monitor their environment by endocytosis and macropinocytosis of pathogens (microbes and viruses) or antigens. It is generally accepted that DCs present antigens to T cells through MHC/peptide-TCR engagement (the first signal), and activate T cells through B7CD28 interaction (the second signal). However, distinct developmental stages of DCs, costimulatory molecules and cytokines dictate the consequences of immune responses: immunity versus tolerance [90,91]. Immature DCs tend to induce immune tolerance rather than immunity by either deleting reactive T cells or inducing regulatory T cells [90,92,93]. Targeting of antigens to a specific subset of immature DCs promotes the induction of antigeninduced Tr1 cells [31,94], while mature antigen-bearing DCs are capable of stimulating and expanding naturally occurring CD4+ CD25+ Treg cells both in vitro and in vivo [95–99]. These studies suggest that in addition to eliciting potent effector T cell responses, DCs have the ability to induce and expand CD4+ Treg cells [100]. CD28-B7 costimulatory interactions, which are required as a second signal for the activation of T cells, may also play an important role in Treg development, as illustrated by the greatly reduced Treg cell population in both CD28−/− and B7-1/B7-2 double knockout mice [101–103]. The B7 molecule is required for conversion of na¨ıve T cells to Treg cells in vivo [104]. Constitutively expressed B7 costimulator function to suppress T cell activation and maintain self-tolerance, principally by sustaining a population of regulatory T cells [105]. 6. Mechanisms of CD4+ Treg cell-mediated immune suppression

5.3. Cytokines IL-2 signaling is critical for the development of Treg cells, as demonstrated by greatly reduced numbers of CD4+ Treg cells in IL-2-deficient mice [81] or in normal mice treated with neutralized antibody against circulating IL-2 [82]. IL-2 neutralization inhibits physiological proliferation of peripheral CD25+ CD4+ Treg cells, thus triggering the early onset of diabetes in diabetesprone nonobese diabetic mice and producing a wide spectrum of T cell-mediated autoimmune diseases [82]. IL-10 and TGF-␤ have been implicated in the induction of antigen-specific Tr1 cells in vitro and in vivo [24,83–85], while IL-6 and unidentified soluble factors secreted by DCs have been reported to render effector cells refractory to suppression by CD4+ Treg cells, rather than turn off the suppressive function of Treg cells [86]. IL-2, TGF-␤ and IL-10 also influence the ability of naturally occurring and antigen-induced CD4+ CD25+ Treg cells to convert CD4+ CD25− cells to suppressive Treg cells [87]. TGF-␤ plays an important role in the induction and maintenance of Foxp3 expression, suppressive function and homeostasis in peripheral CD4+ CD25+ Treg cells [75,88,89]. 5.4. DC and costimulatory molecules Dendritic cells are pivotally positioned at the interface of innate and adaptive immunity. Immature DCs reside and circu-

It has been clearly demonstrated that CD4+ Treg cells require antigen-specific activation or polyclonal TCR stimulation to exert their suppressive function. Once activated, they can suppress CD4+ and CD8+ T cells in an antigen-nonspecific manner. Several mechanisms have been proposed to explain how CD4+ Treg cells inhibit CD4+ effector T cells [19,106]. Some investigators suggest that IL-10 and/or TGF-␤ are directly involved in T-cell-mediated suppression, while others contend that cell–cell contact is required for suppression [19,84]. These discrepancies may reflect the use of different experimental systems or different CD4+ Treg cell populations in these studies. Nonetheless, more than one mechanism of CD4+ Treg cell-mediated suppression appears to operate in vitro and in vivo. Most tumor-specific CD4+ Treg cells suppress immune responses (proliferation and IL-2 secretion of na¨ıve or effector T cells) through a cell-contact mechanism [17,18]. Although membrane-bound TGF-␤ expressed on the surface of Treg cells has been implicated as a molecule required for immune suppression, the precise mechanism and molecules responsible for Treg cell-mediated suppression remain to be identified. In addition, tumor-specific CD4+ Treg cells secrete an unidentified soluble factor(s) rather than IL-10 to mediate the suppression of proliferation of both na¨ıve CD4+ T cells and CD8+ T cells (R. Wang, unpublished data).

110

R.-F. Wang / Seminars in Cancer Biology 16 (2006) 106–114

7. Linking of TLR signaling to the functional control of Treg cells Toll-like receptors (TLRs) have recently emerged as a critical component of the innate immune system for detecting microbial infection and activation of DC maturation programs to induce adaptive immune responses [107,108]. At least 11 TLRs have been identified in humans and mice. They recognize a limited but highly conserved set of molecular structures, so called pathogenassociated molecular patterns (PAMPs). For example, TLR4 recognizes LPS, which is unique to gram-negative bacteria, while TLR2 recognizes peptidoglycan found in gram-positive bacteria. TLR3 recognizes double-stranded RNA produced during viral infection, while TLR9 recognizes an unmethylated CpG DNA motif of prokaryotic genomes and DNA viruses [108]. TLR7 and 8 recognize single-stranded viral RNAs or guanosinerelated analogs (loxoribine and imidazoquinoline) [109–111], however, murine TLR8 is not functional [112]. TLR expression has been detected in many types of cells, including different subsets of DCs, T cells, neutrophils, eosinophils, mast cell, monocytes and epithelial cells [107]. Some TLRs have been reported to be expressed in mouse CD4+ CD25+ Treg cells [113]. TLR2, 4, 5 and 6 are expressed on the cell surface, while TLR3, 7, 8 and 9 reside in endosomal compartments [112,114,115]. In particular, TLR7, 8 and 9 form an evolutionary cluster [108,116]. The current model of TLR signaling predicts that TLR2, 5, 7, 8, and 9 use MyD88 as their sole receptor-proximal adaptor to transduce signals, while TLR3 relies on a TRIF and interferonregulated factor 3 (IRF3) mediated pathway for the production of IFN-␤ in response to pathogen recognition [108,115]. TLR4 is linked to both MyD88-dependent and -independent pathways [108,115]. Thus, MyD88 is essential for the signaling activities of most TLRs to MyD88-IRKA4 and other downstream molecules. Recent studies suggest that MyD88 may also interact with IRF-5 and -7 for gene induction of proinflammatory cytokines or the type-I interferon (IFN-alpha/beta) response [117–119]. 7.1. Triggering of TLRs on DCs shifts the balance between Treg and effector T cells Despite the important role of TLRs in sensing microbial infection and initiating both innate and adaptive immunity through defined signaling pathways, it is not clear whether TLR signaling is directly or indirectly involved in the regulation of Treg cell function. CD4+ Treg cells not only suppress the proliferative response of na¨ıve or effector T cells and the IL-2 secretion of antigen-specific effector T cells, but also inhibit the maturation of DCs and induce IDO expression [120–122]. Both CD40 ligation and TLR triggering on DCs have been demonstrated to release immature DCs from the suppression of CD4+ Treg cells [120]. Similarly, the reversal of DC function was observed after in vitro or in vivo stimulation of DCs with CpG and/or anti-IL10R antibody [123]. Although it was reported that TLR signaling on DCs by CpG or lipopolysaccharide (LPS) renders effector cells refractory to Treg cell-mediated suppression [86], recent studies demonstrated that stimulation

of DCs with TLR ligands significantly enhances the proliferation of na¨ıve and effector T cells, making it harder for Treg cells to inhibit them [124,125]. TLR-matured DCs are also capable of stimulating the proliferation of Treg cells [95,97,124–127]. Thus, it appears that the manipulation of TLR signaling pathways in murine DCs can stimulate both na¨ıve CD4+ CD25− T cells and CD4+ CD25+ Treg cells to proliferate; however, stimulation of human DCs with LPS is not sufficient to activate na¨ıve CD4+ T cells to proliferate in the presence of Treg cells. 7.2. Human TLR 8 directly reverses the suppressive function of Treg cells We recently demonstrated that CpG-A and Poly-G10 oligonucleotides can directly reverse the suppressive function of Treg cells in the absence of DCs [128]. Further experiments indicated that short Poly-G oligonucleotides, but not the CpG motif in CpG-A, were responsible for the observed reversal effect. Indeed, shorter oligonucleotides (Poly-G2, Poly-G3 and Poly-G4 with phosphorothioate linkages) proved more potent than longer oligonucleotides (Poly-G5, PolyG7 and Poly-G10) reversing Treg cell function. A stretch of guanosines (G5) with a regular phosphodiester backbone failed to reverse the suppressive activity of Treg cells, most likely because of its rapid degradation by nucleases [128]. To demonstrate the ability of effective Poly-G oligonucleotides to reverse the suppressive function of naturally occurring CD4+ CD25+ Treg cells, we showed that the suppressive activity of the purified naturally occurring CD4+ CD25+ Treg cells could be reversed by Poly-G5 oligonucleotides, but not by Poly-T10. Using na¨ıve CD4+ T cells or Treg cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), we found that the CFSE-labeled na¨ıve CD4+ T cells could proliferate in response to OKT3 antibody stimulation, whereas the CFSE-labeled Treg cells could not, regardless of the presence or absence of Poly-G2 oligonucleotides [128]. The proliferation of CFSE-labeled na¨ıve CD4+ T cells was completely suppressed by naturally occurring CD4+ CD25+ Treg cells as well as by Treg 102 cells, but this effect could be reversed by treatment with Poly-G2 oligonucleotides [128]. To identify the receptor for Poly-G2 oligonucleotides, we used RNA inteference to knock down TLR8, MyD88 and IRAK4 in Treg cells, which abolished the ability of Treg cells to respond to Poly-G oligonucleotides [128]. Consistent with this result, reverse-transcription (RT)-PCR revealed that TLR8 was the only receptor that was consistently expressed by naturally occurring as well as antigen-specific Treg cells. We further demonstrated that natural ligands for human TLR8, ssRNA40 and ssRNA33, in a complex with cationic lipid [109], completely reversed the suppressive function of Treg cells, while ligands for other TLRs failed to do so. These results indicate that the TLR8-MyD88 signaling pathway controls the suppressive function of both human antigen-specific and naturally occurring CD4+ CD25+ Treg cells without the involvement of DCs.

R.-F. Wang / Seminars in Cancer Biology 16 (2006) 106–114

8. Summary Despite the important roles of CD4+ Treg cells in controlling immune responses to self- and non-self-antigens, their natural ligands remain largely unknown. Identification of tumorspecific ligands for antigen-specific CD4+ Treg cells provides compelling evidence that tumor-specific CD4+ Treg cells may suppress immune responses elicited by peptide- or DC-based vaccines. Similar strategies may be applied to the identification of autoantigens or pathogenic antigens that are capable of stimulating antigen-specific CD4+ Treg cells. New strategies that simultaneously stimulate CD4+ effector T cells while inhibiting or depleting CD4+ Treg cells are needed to improve the outcome of cancer immunotherapy. The opposite strategy, inducing expansion of the CD4+ Treg population at the expense of the effector cell population, might enhance the treatment of autoimmune diseases. Many factors – including antigen dose, mode of antigen presentation, DCs, cytokines, and TLR signalling – contribute to the generation and function of CD4+ Treg cells. Our recent studies demonstrate that the suppressive function of Treg cells can be reversed through manipulation of TLR signaling, thus linking this pathway to the control of Treg cell function. Further studies are needed to evaluate the clinical potential of shifting the balance between CD4+ T-helper and Treg cells via TLR stimulation. Acknowledgements The author apologizes to those researchers whose work has not been cited due to space limitations. This work is in part supported by grants from NIH, American Cancer Society and Cancer Research Institute. References [1] Dunn GP, Old LJ, Schreiber RD. The three Es of cancer immunoediting. Annu Rev Immunol 2004;22:329–60. [2] Old LJ, Chen YT. New paths in human cancer serology. J Exp Med 1998;187:1163–7. [3] Wang RF, Rosenberg SA. Human tumor antigens for cancer vaccine development. Immunol Rev 1999;170:85–100. [4] Houghton AN, Gold JS, Blachere NE. Immunity against cancer: lessons learned from melanoma. Curr Opin Immunol 2001;13:134–40. [5] Rosenberg SA. Progress in human tumour immunology and immunotherapy. Nature 2001;411:380–4. [6] Wang RF. Enhancing antitumor immune responses: intracellular peptide delivery and identification of MHC class II-restricted tumor antigens. Immunol Rev 2002;188:65–80. [7] Berzofsky JA, Ahlers JD, Janik J, Morris J, Oh S, Terabe M, et al. Progress on new vaccine strategies against chronic viral infections. J Clin Invest 2004;114:450–62. [8] Sakaguchi S, Sakaguchi N, Shimizu J, Yamazaki S, Sakihama T, Itoh M, et al. Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol Rev 2001;182:18–32. [9] Wood KJ, Sakaguchi S. Regulatory T cells in transplantation tolerance. Nat Rev Immunol 2003;3:199–210. [10] Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor foxp3. Science 2003;299:1057–61.

111

[11] Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD4(+)CD25(+) T regulatory cells. Nat Immunol 2003;4:337–42. [12] Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4(+)CD25(+) regulatory T cells. Nat Immunol 2003;4:330–6. [13] Sakaguchi S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 2004;22:531–62. [14] Roncarolo MG, Levings MK. The role of different subsets of T regulatory cells in controlling autoimmunity. Curr Opin Immunol 2000;12:676–83. [15] Francois Bach J. Regulatory T cells under scrutiny. Nat Rev Immunol 2003;3:189–98. [16] Weiner HL. Induction and mechanism of action of transforming growth factor-beta-secreting Th3 regulatory cells. Immunol Rev 2001;182:207–14. [17] Wang HY, Lee Deen A, Guangyong Peng, Zhong Guo, Yanchun Li, Yukiko Kiniwa, et al. Tumor-specific human CD4+ regulatory T cells and their ligands: implication for immunotherapy. Immunity 2004;20:107–18. [18] Wang HY, Peng G, Guo Z, Shevach EM, Wang R-F. Recognition of a new ARTC1 peptide ligand uniquely expressed in tumor cells by antigen-specific CD4+ gegulatory T cells. J Immunol 2005;174:2661–70. [19] Shevach EM. CD4+ CD25+ suppressor T cells: more questions than answers. Nat Rev Immunol 2002;2:389–400. [20] Levings MK, Sangregorio R, Sartirana C, Moschin AL, Battaglia M, Orban PC, et al. Human CD25+CD4+ T suppressor cell clones produce transforming growth factor beta, but not interleukin 10, and are distinct from type 1 T regulatory cells. J Exp Med 2002;196:1335–46. [21] Dieckmann D, Bruett CH, Ploettner H, Lutz MB, Schuler G. Human CD4(+)CD25(+) regulatory, contact-dependent T cells induce interleukin 10-producing, contact-independent type 1-like regulatory T cells [corrected]. J Exp Med 2002;196:247–53. [22] Jonuleit H, Schmitt E, Kakirman H, Stassen M, Knop J, Enk AH. Infectious tolerance: human CD25(+) regulatory T cells convey suppressor activity to conventional CD4(+) T helper cells. J Exp Med 2002;196:255–60. [23] Walker MR, Kasprowicz DJ, Gersuk VH, Benard A, Van Landeghen M, Buckner JH, et al. Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+CD25- T cells. J Clin Invest 2003;112:1437–43. [24] Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, et al. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med 2003;198:1875–86. [25] Jordan MS, Boesteanu A, Reed AJ, Petrone AL, Holenbeck AE, Lerman MA, et al. Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat Immunol 2001;2:301–6. [26] Cozzo C, Larkin IIIrd J, Caton AJ. Cutting edge: self-peptides drive the peripheral expansion of CD4(+)CD25(+) regulatory T cells. J Immunol 2003;171:5678–82. [27] Apostolou I, Sarukhan A, Klein L, von Boehmer H. Origin of regulatory T cells with known specificity for antigen. Nat Immunol 2002;3:756–63. [28] Boonstra A, Asselin-Paturel C, Gilliet M, Crain C, Trinchieri G, Liu YJ, et al. Flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing T helper type 1 and 2 cell development: dependency on antigen dose and differential toll-like receptor ligation. J Exp Med 2003;197:101–9. [29] Apostolou I, Von Boehmer H. In vivo instruction of suppressor commitment in naive T cells. J Exp Med 2004;199:1401–8. [30] Chen Y, Kuchroo VK, Inobe J, Hafler DA, Weiner HL. Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 1994;265:1237–40. [31] Jonuleit H, Schmitt E, Schuler G, Knop J, Enk AH. Induction of interleukin 10-producing, nonproliferating CD4(+) T cells with regulatory

112

[32]

[33] [34] [35]

[36]

[37]

[38] [39]

[40]

[41] [42]

[43] [44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

R.-F. Wang / Seminars in Cancer Biology 16 (2006) 106–114 properties by repetitive stimulation with allogeneic immature human dendritic cells. J Exp Med 2000;192:1213–22. Sundstedt A, O’Neill EJ, Nicolson KS, Wraith DC. Role for IL-10 in suppression mediated by Peptide-induced regulatory T cells in vivo. J Immunol 2003;170:1240–8. Sarantopoulos S, Lu L, Cantor H. Qa-1 restriction of CD8+ suppressor T cells. J Clin Invest 2004;114:1218–21. Jiang H, Chess L. An integrated view of suppressor T cell subsets in immunoregulation. J Clin Invest 2004;114:1198–208. Vlad G, Cortesini R, Suciu-Foca N. License to heal: bidirectional interaction of antigen-specific regulatory T cells and tolerogenic APC. J Immunol 2005;174:5907–14. Jiang H, Chess L. The specific regulation of immune responses by CD8+ T cells restricted by the MHC class Ib molecule, Qa-1. Annu Rev Immunol 2000;18:185–216. Hu D, Ikizawa K, Lu L, Sanchirico ME, Shinohara ML, Cantor H. Analysis of regulatory CD8 T cells in Qa-1-deficient mice. Nat Immunol 2004;5:516–23. Cantor H. Reviving suppression? Nat Immunol 2004;5:347–9. Brenner MB, McLean J, Dialynas DP, Strominger JL, Smith JA, Owen FL, et al. Identification of a putative second T-cell receptor. Nature 1986;322:145–9. Shin S, El-Diwany R, Schaffert S, Adams EJ, Garcia KC, Pereira P, et al. Antigen recognition determinants of gammadelta T cell receptors. Science 2005;308:252–5. Brandes M, Willimann K, Moser B. Professional antigen-presentation function by human gammadelta T Cells. Science 2005;309:264–8. Seo N, Tokura Y, Takigawa M, Egawa K. Depletion of IL-10- and TGF-beta-producing regulatory gamma delta T cells by administering a daunomycin-conjugated specific monoclonal antibody in early tumor lesions augments the activity of CTLs and NK cells. J Immunol 1999;163:242–9. Lamb Jr LS, Lopez RD. gammadelta T cells: a new frontier for immunotherapy? Biol Blood Marrow Transplant 2005;11:161–8. Berendt MJ, North RJ. T-cell-mediated suppression of anti-tumor immunity. An explanation for progressive growth of an immunogenic tumor. J Exp Med 1980;151:69–80. North RJ. Cyclophosphamide-facilitated adoptive immunotherapy of an established tumor depends on elimination of tumor-induced suppressor T cells. J Exp Med 1982;155:1063–74. North RJ. Gamma-irradiation facilitates the expression of adoptive immunity against established tumors by eliminating suppressor T cells. Cancer Immunol Immunother 1984;16:175–81. North RJ, Awwad M. T cell suppression as an obstacle to immunologically-mediated tumor regression: elimination of suppression results in regression. Prog Clin Biol Res 1987;244:345–58. Awwad M, North RJ. Immunologically mediated regression of a murine lymphoma after treatment with anti-L3T4 antibody. A consequence of removing L3T4+ suppressor T cells from a host generating predominantly Lyt-2+ T cell-mediated immunity. J Exp Med 1988;168:2193–206. North RJ, Awwad M. Elimination of cycling CD4+ suppressor T cells with an anti-mitotic drug releases non-cycling CD8+ T cells to cause regression of an advanced lymphoma. Immunology 1990;71:90–5. Turk MJ, Guevara-Patino JA, Rizzuto GA, Engelhorn ME, Houghton AN. Concomitant tumor immunity to a poorly immunogenic melanoma is prevented by regulatory T cells. J Exp Med 2004;200:771–82. Mukherji B, Guha A, Chakraborty NG, Sivanandham M, Nashed AL, Sporn JR, et al. Clonal analysis of cytotoxic and regulatory T cell responses against human melanoma. J Exp Med 1989;169:1961– 76. Chakraborty NG, Twardzik DR, Sivanandham M, Ergin MT, Hellstrom KE, Mukherji B. Autologous melanoma-induced activation of regulatory T cells that suppress cytotoxic response. J Immunol 1990;145:2359–64. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL2 receptor alpha-chains (CD25). Breakdown of a single mechanism

[54]

[55]

[56]

[57]

[58]

[59]

[60] [61]

[62] [63] [64] [65]

[66] [67] [68]

[69]

[70]

[71] [72]

[73]

[74]

of self-tolerance causes various autoimmune diseases. J Immunol 1995;155:1151–64. Sutmuller RP, van Duivenvoorde LM, van Elsas A, Schumacher TN, Wildenberg ME, Allison JP, et al. Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25(+) regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses. J Exp Med 2001;194:823–32. Tanaka H, Tanaka J, Kjaergaard J, Shu S. Depletion of CD4+ CD25+ regulatory cells augments the generation of specific immune T cells in tumor-draining lymph nodes. J Immunother 2002;25:207–17. Woo EY, Chu CS, Goletz TJ, Schlienger K, Yeh H, Coukos G, et al. Regulatory CD4(+)CD25(+) T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer. Cancer Res 2001;61:4766–72. Liyanage UK, Moore TT, Joo HG, Tanaka Y, Herrmann V, Doherty G, et al. Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma. J Immunol 2002;169:2756–61. Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 2004;10:942–9. Viguier M, Lemaitre F, Verola O, Cho MS, Gorochov G, Dubertret L, et al. Foxp3 expressing CD4(+)CD25(high) regulatory T cells are overrepresented in human metastatic melanoma lymph nodes and inhibit the function of infiltrating T cells. J Immunol 2004;173:1444–53. Shevach EM. Regulatory T cells in autoimmmunity. Annu Rev Immunol 2000;18:423–49. Taguchi O, Kojima A, Nishizuka Y. Experimental autoimmune prostatitis after neonatal thymectomy in the mouse. Clin Exp Immunol 1985;60:123–9. McCullagh P. Interception of the development of self tolerance in fetal lambs. Eur J Immunol 1989;19:1387–92. McCullagh P. The significance of immune suppression in normal self tolerance. Immunol Rev 1996;149:127–53. Seddon B, Mason D. Peripheral autoantigen induces regulatory T cells that prevent autoimmunity. J Exp Med 1999;189:877–82. Chatenoud L, Salomon B, Bluestone JA. Suppressor T cells—they’re back and critical for regulation of autoimmunity! Immunol Rev 2001;182:149–63. Curotto de Lafaille MA, Lafaille JJ. CD4(+) regulatory T cells in autoimmunity and allergy. Curr Opin Immunol 2002;14:771–8. Maloy KJ, Powrie F. Regulatory T cells in the control of immune pathology. Nat Immunol 2001;2:816–22. Hori S, Haury M, Coutinho A, Demengeot J. Specificity requirements for selection and effector functions of CD25+4+ regulatory T cells in anti-myelin basic protein T cell receptor transgenic mice. Proc Natl Acad Sci USA 2002;99:8213–8. Takahashi T, Kuniyasu Y, Toda M, Sakaguchi N, Itoh M, Iwata M, et al. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int Immunol 1998;10:1969–80. Hsieh CS, Liang Y, Tyznik AJ, Self SG, Liggitt D, Rudensky AY. Recognition of the peripheral self by naturally arising CD25+ CD4+ T cell receptors. Immunity 2004;21:267–77. Baecher-Allan C, Hafler DA. Suppressor T cells in human diseases. J Exp Med 2004;200:273–6. Nishikawa H, Kato T, Tanida K, Hiasa A, Tawara I, Ikeda H, et al. CD4+ CD25+ T cells responding to serologically defined autoantigens suppress antitumor immune responses. Proc Natl Acad Sci USA 2003;100:10902–6. Nishikawa H, Kato T, Tawara I, Saito K, Ikeda H, Kuribayashi K, et al. Definition of target antigens for naturally occurring CD4(+) CD25(+) regulatory T cells. J Exp Med 2005;201:681–6. Fontenot JD, Rasmussen JP, Williams LM, Dooley JL, Farr AG, Rudensky AY. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 2005;22:329–41.

R.-F. Wang / Seminars in Cancer Biology 16 (2006) 106–114 [75] Wan YY, Flavell RA. Identifying Foxp3-expressing suppressor T cells with a bicistronic reporter. Proc Natl Acad Sci USA 2005;102: 5126–31. [76] Sakaguchi S. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 2005;6:345–52. [77] Schwartz RH. Natural regulatory T cells and self-tolerance. Nat Immunol 2005;6:327–30. [78] Voo KS, Peng G, Guo Z, Fu T, Li Y, Frappier L, et al. Functional characterization of EBV-encoded nuclear antigen 1-specific CD4+ helper and regulatory T cells elicited by in vitro peptide stimulation. Cancer Res 2005;65:1577–86. [79] Lerman MA, Larkin IIIrd J, Cozzo C, Jordan MS, Caton AJ. CD4+ CD25+ regulatory T cell repertoire formation in response to varying expression of a neo-self-antigen. J Immunol 2004;173:236–44. [80] Kohyama M, Sugahara D, Sugiyama S, Yagita H, Okumura K, Hozumi N. Inducible costimulator-dependent IL-10 production by regulatory T cells specific for self-antigen. Proc Natl Acad Sci USA 2004;101:4192–7. [81] Papiernik M, de Moraes ML, Pontoux C, Vasseur F, Penit C. Regulatory CD4 T cells: expression of IL-2R alpha chain, resistance to clonal deletion and IL-2 dependency. Int Immunol 1998;10:371–8. [82] Setoguchi R, Hori S, Takahashi T, Sakaguchi S. Homeostatic maintenance of natural Foxp3(+) CD25(+) CD4(+) regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J Exp Med 2005;201:723–35. [83] Groux H, O’Garra A, Bigler M, Rouleau M, Antonenko S, de Vries JE, et al. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 1997;389:737–42. [84] Levings MK, Bacchetta R, Schulz U, Roncarolo MG. The role of IL-10 and TGF-beta in the differentiation and effector function of T regulatory cells. Int Arch Allergy Immunol 2002;129:263–76. [85] O’Garra A, Vieira P. Regulatory T cells and mechanisms of immune system control. Nat Med 2004;10:801–5. [86] Pasare C, Medzhitov R. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science 2003;299:1033–6. [87] Zheng SG, Wang JH, Gray JD, Soucier H, Horwitz DA. Natural and induced CD4+CD25+ cells educate CD4+CD25- cells to develop suppressive activity: the role of IL-2, TGF-beta, and IL-10. J Immunol 2004;172:5213–21. [88] Peng Y, Laouar Y, Li MO, Green EA, Flavell RA. TGF-beta regulates in vivo expansion of Foxp3-expressing CD4+CD25+ regulatory T cells responsible for protection against diabetes. Proc Natl Acad Sci USA 2004;101:4572–7. [89] Marie JC, Letterio JJ, Gavin M, Rudensky AY. TGF-beta1 maintains suppressor function and Foxp3 expression in CD4+CD25+ regulatory T cells. J Exp Med 2005;201:1061–7. [90] Moser M. Dendritic cells in immunity and tolerance-do they display opposite functions? Immunity 2003;19:5–8. [91] Steinman RM, Hawiger D, Nussenzweig MC. Tolerogenic dendritic cells. Annu Rev Immunol 2003;21:685–711. [92] Kurts C, Kosaka H, Carbone FR, Miller JF, Heath WR. Class Irestricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8(+) T cells. J Exp Med 1997;186:239–45. [93] Probst HC, Lagnel J, Kollias G, van den Broek M. Inducible transgenic mice reveal resting dendritic cells as potent inducers of CD8+ T cell tolerance. Immunity 2003;18:713–20. [94] Mahnke K, Qian Y, Knop J, Enk AH. Induction of CD4+/CD25+ regulatory T cells by targeting of antigens to immature dendritic cells. Blood 2003;101:4862–9. [95] Yamazaki S, Iyoda T, Tarbell K, Olson K, Velinzon K, Inaba K, et al. Direct expansion of functional CD25+ CD4+ regulatory T cells by antigen-processing dendritic cells. J Exp Med 2003;198:235–47. [96] Oldenhove G, de Heusch M, Urbain-Vansanten G, Urbain J, Maliszewski C, Leo O, et al. CD4+ CD25+ regulatory T cells control T helper cell type 1 responses to foreign antigens induced by mature dendritic cells in vivo. J Exp Med 2003;198:259–66.

113

[97] Walker LS, Chodos A, Eggena M, Dooms H, Abbas AK. Antigendependent proliferation of CD4+ CD25+ regulatory T cells in vivo. J Exp Med 2003;198:249–58. [98] Tang Q, Henriksen KJ, Bi M, Finger EB, Szot G, Ye J, et al. In vitro-expanded antigen-specific regulatory t cells suppress autoimmune diabetes. J Exp Med 2004;199:1455–65. [99] Tarbell KV, Yamazaki S, Olson K, Toy P, Steinman RM. CD25+ CD4+ T cells, expanded with dendritic cells presenting a single autoantigenic peptide, suppress autoimmune diabetes. J Exp Med 2004;199:1467–77. [100] Groux H, Fournier N, Cottrez F. Role of dendritic cells in the generation of regulatory T cells. Semin Immunol 2004;16:99–106. [101] Tang Q, Henriksen KJ, Boden EK, Tooley AJ, Ye J, Subudhi SK, et al. Cutting edge: CD28 controls peripheral homeostasis of CD4+CD25+ regulatory T cells. J Immunol 2003;171:3348–52. [102] Salomon B, Lenschow DJ, Rhee L, Ashourian N, Singh B, Sharpe A, et al. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 2000;12:431–40. [103] Bour-Jordan H, Salomon BL, Thompson HL, Szot GL, Bernhard MR, Bluestone JA. Costimulation controls diabetes by altering the balance of pathogenic and regulatory T cells. J Clin Invest 2004;114:979–87. [104] Liang S, Alard P, Zhao Y, Parnell S, Clark SL, Kosiewicz MM. Conversion of CD4+ CD25- cells into CD4+ CD25+ regulatory T cells in vivo requires B7 costimulation, but not the thymus. J Exp Med 2005;201:127–37. [105] Lohr J, Knoechel B, Jiang S, Sharpe AH, Abbas AK. The inhibitory function of B7 costimulators in T cell responses to foreign and selfantigens. Nat Immunol 2003;4:664–9. [106] von Boehmer H. Mechanisms of suppression by suppressor T cells. Nat Immunol 2005;6:338–44. [107] Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol 2004;5:987–95. [108] Takeda K, Akira S. Toll-like receptors in innate immunity. Int Immunol 2005;17:1–14. [109] Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 2004;303:1526–9. [110] Diebold SS, Kaisho T, Hemmi H, Akira S. Reis e Sousa C, Innate antiviral responses by means of TLR7-mediated recognition of singlestranded RNA. Science 2004;303:1529–31. [111] Lund JM, Alexopoulou L, Sato A, Karow M, Adams NC, Gale NW, et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc Natl Acad Sci USA 2004;101:5598–603. [112] Jurk M, Heil F, Vollmer J, Schetter C, Krieg AM, Wagner H, et al. Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat Immunol 2002;3:499. [113] Caramalho I, Lopes-Carvalho T, Ostler D, Zelenay S, Haury M, Demengeot J. Regulatory T cells selectively express toll-like receptors and are activated by lipopolysaccharide. J Exp Med 2003;197:403–11. [114] Hemmi H, Kaisho T, Takeuchi O, Sato S, Sanjo H, Hoshino K, et al. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol 2002;3:196–200. [115] Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004;4:499–511. [116] Crozat K, Beutler B. TLR7: a new sensor of viral infection. Proc Natl Acad Sci USA 2004;101:6835–6. [117] Takaoka A, Yanai H, Kondo S, Duncan G, Negishi H, Mizutani T, et al. Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors. Nature 2005;434:243–9. [118] Honda K, Yanai H, Negishi H, Asagiri M, Sato M, Mizutani T, et al. IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature 2005;434:772–7. [119] Honda K, Ohba Y, Yanai H, Negishi H, Mizutani T, Takaoka A, et al. Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction. Nature 2005;434:1035–40. [120] Serra P, Amrani A, Yamanouchi J, Han B, Thiessen S, Utsugi T, et al. CD40 ligation releases immature dendritic cells from the control of regulatory CD4+CD25+ T cells. Immunity 2003;19:877–89.

114

R.-F. Wang / Seminars in Cancer Biology 16 (2006) 106–114

[121] Munn DH, Sharma MD, Lee JR, Jhaver KG, Johnson TS, Keskin DB, et al. Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. Science 2002;297:1867–70. [122] Fallarino F, Grohmann U, Hwang KW, Orabona C, Vacca C, Bianchi R, et al. Modulation of tryptophan catabolism by regulatory T cells. Nat Immunol 2003;4:1206–12. [123] Vicari AP, Chiodoni C, Vaure C, Ait-Yahia S, Dercamp C, Matsos F, et al. Reversal of tumor-induced dendritic cell paralysis by CpG immunostimulatory oligonucleotide and anti-interleukin 10 receptor antibody. J Exp Med 2002;196:541–9. [124] Fehervari Z, Sakaguchi S. Control of Foxp3+ CD25+CD4+ regulatory cell activation and function by dendritic cells. Int Immunol 2004;16:1769–80.

[125] Kubo T, Hatton RD, Oliver J, Liu X, Elson CO, Weaver CT. Regulatory T cell suppression and anergy are differentially regulated by proinflammatory cytokines produced by TLR-activated dendritic cells. J Immunol 2004;173:7249–58. [126] Klein L, Khazaie K, von Boehmer H. In vivo dynamics of antigenspecific regulatory T cells not predicted from behavior in vitro. Proc Natl Acad Sci USA 2003;100:8886–91. [127] Fisson S, Darrasse-Jeze G, Litvinova E, Septier F, Klatzmann D, Liblau R, et al. Continuous activation of autoreactive CD4+ CD25+ regulatory T cells in the steady state. J Exp Med 2003;198:737–46. [128] Peng G, Guo Z, Kiniwa Y, Voo KS, Peng W, Fu T, et al. Toll-Like receptor 8 mediated reversal of CD4+ regulatory T cell function. Science 2005;309:1380–4.