Cellular immunotherapy for ovarian cancer

doi:10.1517/14712590902932897

Expert Opin Biol Ther. Author manuscript; available in PMC 2010 June 1.

Published in final edited form as:

Expert Opin Biol Ther. 2009 June; 9(6): 677–688.

doi: 10.1517/14712590902932897

PMCID: PMC2718679

NIHMSID: NIHMS124041

Cellular immunotherapy for ovarian cancer

Martin J Cannon and Timothy J O'Brien

Obstetrics and Gynecology, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205, USA

The publisher's final edited version of this article is available at Expert Opin Biol Ther

See other articles in PMC that cite the published article.

Abstract

Background

Ovarian cancer is frequently diagnosed at an advanced stage, and although initially responsive to surgery and chemotherapy, has a high rate of recurrence and mortality. Cellular immunotherapy may offer the prospect of treatment to prevent or delay recurrent metastatic disease.

Objective

To provide an overview of current innovations in cellular immunotherapy for ovarian cancer, with an emphasis on dendritic cell vaccination and adoptive T cell immunotherapy.

Methods

Three key areas are explored in this review. First, an appraisal of the current state of the art of cellular immunotherapy for treatment of ovarian cancer. Second, a discussion of the immunological defenses erected by ovarian cancer to prevent immunological attack, with an emphasis on the role of tumor-associated regulatory T cells. Third, an exploration of innovative techniques that may enhance the ability of cellular immunotherapy to overcome ovarian tumor-associated immune suppression.

Results/conclusion

Ovarian cancer is recognized as a paradigm for tumor-associated immune suppression. Innovative approaches for antagonism of tumor-associated regulatory T cell infiltration and redirection of self antigen-driven regulatory T cell activation may provide the key to development of future strategies for cellular immunotherapy against ovarian cancer.

Keywords: Ovarian cancer, Regulatory T cells, CD4⁺ T cells, Th17 T cells, Dendritic cells, Interleukin-2, Interleukin 1β, Interleukin-15

1. Introduction

The search for effective treatments for ovarian cancer has been a source of frustration for decades, and ovarian cancer remains the disease with the highest mortality rate among gynecological tumors. Patients with advanced disease have a response rate of over 80% following surgery and adjuvant chemotherapy with platinum-taxane, with a median progression-free interval of 18 months [1]. Unfortunately, the majority of these patients will recur, and the overall 5-year survival rate for patients with advanced stage disease is only 23-30% [2]. One of the major reasons why the survival rates are so poor is that as many as three-quarters of patients present with advanced disease at the time of diagnosis [1], indicating that there is a clear need for techniques that would facilitate earlier diagnosis of ovarian cancer. The need for development of novel treatments that prevent disease recurrence or progression following first-line treatment is also a matter of some urgency. Immunotherapy based on the induction of tumor-specific helper T cell and cytotoxic T lymphocyte (CTL) responses may have potential efficacy, but ovarian cancer presents many barriers to the development of successful cellular immunotherapies. However, improved understanding of the mechanisms of tumor-associated immune regulation has raised the prospects for immunological treatment of ovarian cancer, based in part on manipulation of dendritic cells (DC) to facilitate programming of antitumor effector T cell responses. This review will discuss the barriers to cellular immunotherapy that are imposed by ovarian tumor-associated immunosuppression, and will consider potential strategies by which these barriers may be overcome, with an emphasis on the central role of DC in immune regulation.

2. Dendritic cell immunotherapy

Although DC-based immunotherapy or vaccination has been widely explored for the treatment of many other malignancies, investigation of the potential for DC immunotherapy of ovarian cancer has been relatively limited. Schlienger and colleagues have shown that DC pulsed with killed autologous primary ovarian tumor cells induced antigen-specific T cells that secreted IFNγ upon stimulation with autologous tumor cells [3], suggesting that antigen-pulsed DC may be a viable option for therapeutic vaccination against ovarian cancer. Various reports showing that DC loaded with tumor lysates, DC pulsed with acid-eluted peptides from ovarian cancer cells, DC fused with ovarian tumor cells, or DC loaded with ovarian tumor cells killed by oxidation could induce HLA class I-restricted CTL responses against autologous ovarian tumor cells [4-8] support this position. A limitation of these approaches is that the identity of the tumor antigens recognized by DC-stimulated CTL is not well defined, and it is not clear that T cell responses retain specificity for the tumor. Therapeutic vaccination with DC loaded with defined ovarian tumor antigens of known tissue distribution would be preferable, both in terms of clinical benefit and limitation of autoimmune toxicity, particularly for long-term control of disease progression.

In the clinical setting, a phase I trial of autologous tumor antigen-loaded DC vaccination in 6 patients with ovarian cancer revealed no significant toxicity and 3 of 6 patients showed stable disease lasting 25 to 45 weeks [9]. Lymphoproliferative responses to tumor antigen were detected in 2 patients. The most durable clinical response to DC vaccination was described in a case report of a patient with recurrent metastatic ovarian cancer, who received 10 vaccinations of autologous DC loaded with mRNA encoding folate receptor-α [10]. Follow-up CT at 5 months after the last vaccination showed a partial response, and CT at 16 months showed greater than 50% remission of lymph node metastases. CA-125 levels were greatly reduced after the 1^st vaccination (from 640 U/mL to 60 U/mL) and remained at baseline 11 months after completion of vaccination. A clinical trial of MUC1 and HER2/neu peptide-pulsed DC vaccination in patients with advanced ovarian or breast cancer reported peptide-specific CTL responses in 5 of 10 patients [11], and also showed evidence of epitope spreading. In one patient vaccinated with MUC1 peptides, carcinoembryonic antigen and MAGE3 peptide-specific T-cell responses were detected, and in a second patient, MUC1-specific T-cell responses were detected after seven vaccinations with HER2/neu peptide-pulsed DC [11]. These results are interesting and encouraging, notwithstanding the limitations of HER2/neu as a target antigen for ovarian tumor immunotherapy, since this antigen is expressed in only about 10% of ovarian cancers.

Probably the best known ovarian tumor antigen is CA125, but it has received surprisingly little attention as a target for cellular immunotherapy. Possible reasons may have included a lack of information on the primary sequence, and the consequent difficulty of producing CA125 in recombinant form. However, the primary sequence and structure of CA125 has now been elucidated, revealing a core protein with a mass exceeding 2 million Da [12]. Knowledge of the sequence has facilitated identification of HLA class I-restricted epitopes, resulting in DC-driven generation of CA-125-specific CD8⁺ CTL responses capable of killing ovarian tumors that express CA125 [13].

Serine proteases represent a group of antigens with potential as targets for cellular immunotherapy of ovarian cancer [14]. Of these antigens, stratum corneum chymotryptic enzyme (SCCE) in particular holds promise, based on a high frequency of expression by ovarian tumors and limited distribution in normal tissues. Quantitative RT-PCR analysis indicated that SCCE was overexpressed in >88% of serous ovarian tumors, and 100% of endometrioid and clear cell tumors, respectively [15]. Using computer analysis of algorithms derived for estimation of epitope binding affinities to HLA DR molecules [16], peptide sequences with multiple DR-binding motifs have been identified within SCCE. DC loaded with one such peptide, SCCE 110-139, were capable of stimulating CD4⁺ Th1 T cell responses from individuals of diverse HLA class II backgrounds, and were also capable of cross-priming CD8⁺ CTL responses restricted by HLA A2.1 and other class I molecules [17]. This approach thus has the virtue of stimulating both CD4⁺ helper T cell responses and CD8⁺ CTL responses in a broad patient population, without suffering from the limitations of HLA type that are usually associated with peptide-based strategies.

3. Adoptive T cell immunotherapy

The presence of tumor-infiltrating T cells in ovarian cancer has been associated with improved survival [18], suggesting that T cell immunotherapy may afford clinical benefit. This idea is reinforced by studies demonstrating expansion of tumor-infiltrating lymphocytes (TIL) for treatment of ovarian cancer patients [19, 20] and in vitro activation of tumor-specific T cells derived from peripheral blood or TIL [4, 5, 21]. More recent studies have explored the potential of adoptive transfer with T cells engineered to express receptors that endow antitumor activity. Drawing on the observation that over 80% of ovarian tumors express receptors for NKG2D (an agonist NK receptor), Barber and colleagues transduced T cells with a chimeric receptor linking NKG2D with the CD3ζ chain [22], and showed that NKG2D/CD3ζ T cells expressed inflammatory cytokines when cultured with autologous tumor cells from ovarian cancer patients. They further showed that chimeric receptor-expressing T cells could be used therapeutically in a mouse model of ovarian cancer [22]. Another promising strategy embodies the use of T cells engineered to express chimeric receptors composed of tumor antigen-specific single chain variable antibody fragments linked to T cell signaling domains. A very recent report has described genetically redirected T cells expressing a single chain antibody specific for mesothelin (an important ovarian tumor antigen) coupled to signaling domains from the T cell receptor (CD3) ζ chain, CD28 and CD137. These mesothelin-specific T cells could lyse mesothelin-expressing ovarian and mesothelioma tumor cells in vitro, and could control established mesothelioma tumor xenografts in NOD/SCID mice [23]. T cells engineered to express a single chain antibody specific for folate receptor have been tested in a phase I clinical trial for treatment of metastatic ovarian cancer [24]. Some patients experienced grade 3 to grade 4 toxicity, probably attributable to co-administration of IL-2, but otherwise treatment was well tolerated. The transferred T cells did not persist in the circulation, being barely detectable one month after transfer, and tracking studies with In¹¹¹-labeled T cells showed a failure of T cells to home to the tumor [24]. From this experience, one may conclude that improved T cell persistence and homing ability are needed for adoptive T cell transfer to offer the prospect of clinical benefit. However, it is possible that adoptively transferred T cells may still afford some benefit through recruitment of more durable endogenous anti-tumor immune responses, as suggested by the studies of Sentman and colleagues [22].

3.1 The increasing significance of CD4⁺ T cells in cellular immunotherapy

Although the conventional wisdom of cellular immunotherapy dictates that an emphasis should be placed on stimulation of tumor-specific CD8⁺ CTL responses, accumulating evidence points to a critical, and possibly dominant, role for tumor antigen-specific CD4⁺ T cell responses in effective anti-tumor immunity [25-28]. A notable recent study showed that CD4⁺ T cells could eliminate tumors that are resistant to CD8⁺ T cell-mediated rejection, even when the tumors expressed MHC class I, but not MHC class II, suggesting that CD4⁺ T cell responses could outperform CD8+ CTL in mediating antitumor effector function [29]. The significance of these findings is underlined by a novel report of successful treatment of metastatic melanoma by tumor antigen-specific CD4⁺ T cell immunotherapy [30]. From these observations, we propose that, in contrast with most prior studies, cellular immunotherapy or antitumor vaccination should place an emphasis on activation of ovarian tumor antigen-specific CD4⁺ T cell responses. In addition to the above noted work on HLA class I and class II-binding epitopes within an extended peptide from SCCE [17], this principle has been recognized by two recent studies, the first of which describes activation of both CD4⁺ and CD8⁺ T cell responses following vaccination of ovarian cancer patients with a peptide containing both HLA class I and class II-binding epitopes from NY-ESO [31]. Extending this work, these investigators have identified two novel HLA class I-restricted NY-ESO epitopes embedded within a region containing promiscuous HLA DR-binding epitopes, thus broadening the potential number of ovarian cancer patients that may benefit from vaccination [32].

4. Barriers to cellular immunotherapy for ovarian cancer

4.1 Tumor-infiltrating regulatory T cells

The first evidence that ovarian tissues may be immunogenic was provided by a study published in 1969, revealing that neonatal day 3 thymectomy of female mice resulted in loss of the ovaries [33]. This curious result was first interpreted as evidence for an endocrine link between the thymus and ovarian development, but subsequent studies revealed heavy lymphocytic infiltration and autoimmune destruction of the ovaries, and further showed that if the mice were reconstituted with normal T cells, the autoimmune process was inhibited [34]. The most probable mechanism is that neonatal thymectomy resulted in loss of production of innate, thymus-derived regulatory T cells (Treg) that would normally block autoimmune T cell responses to ovarian antigens. However, more recent studies have shown that day 3 thymectomized mice in fact possess functional Treg in ovarian draining lymph nodes, but they are not sufficient to control fully the development of autoimmune ovarian disease [35]. A second, and non-exclusive, mechanism is that day 3 thymectomy results in enrichment of autoreactive, pathogenic CD4⁺ T cells that escape deletion [36].

An important conclusion is that ovarian tissues, and by inference ovarian tumors, are intrinsically immunogenic, but that homeostatic mechanisms effectively block immune reactivity. The clinical significance of tumor-associated CD4⁺ Treg in ovarian cancer is highlighted by the work of Curiel and colleagues, who showed that Treg are recruited to ovarian tumors by the chemokine CCL22 (predominantly expressed by ovarian tumors), and that the presence of Treg confers immune privilege and is associated with a poor prognosis and increased mortality [37]. These observations are underlined by further studies showing that high expression of the forkhead box transcription factor foxp3, which is preferentially expressed by CD4⁺ Treg, is an independent prognostic factor for reduced overall survival in ovarian cancer [38]. Such findings lend credence to the notion that strategies for depletion of tumor-associated Treg, or inhibition of Treg function, may be of therapeutic benefit, particularly in conjunction with active, tumor-specific immunotherapy.

4.2 Inhibitory B7 family receptors

The role of the classical B7.1 and B7.2 receptors in regulation of T cell activation, either delivering a positive costimulatory signal through CD28 on T cells, or delivering an anergic signal through engagement of CTLA-4, has been established for over 20 years. More recently, new members of the B7 family have been described, at least two of which, B7-H1 and B7-H4, are likely to play important roles in regulation of antitumor immunity in ovarian cancer.

B7-H1 (also known as PD-L1) is expressed on ovarian cancer cells and infiltrating myeloid DC in the tumor microenvironment, and can promote T cell anergy or apoptosis through engagement of PD-1 (or possibly CD80) expressed by T cells [39-41]. B7-H1 expression by DC has also been implicated in DC-driven conversion of naïve CD4⁺ T cells to adaptive foxp3⁺ Treg, a process that can be blocked by DC maturation with anti-CD40 and a TLR4 ligand (see section 6.4). Blockade of B7-H1 enhances DC-mediated T cell activation and boosts the efficacy of adoptive T cell immunotherapy against autologous ovarian tumors engrafted in xenogeneic mice [40]. Of particular clinical interest, a retrospective analysis of human ovarian cancers revealed that patients with higher B7-H1 expression had a significantly poorer prognosis than those for whom the tumors had lower B7-H1 expression [42]. From these observations, blockade of B7-H1/PD-1 interaction may augment the efficacy of cellular immunotherapy for ovarian cancer. Alternatively, generation of antitumor effector T cells with diminished expression of PD-1 may allow the circumvention of B-H1-induced anergy or apoptosis upon tumor T cell infiltration.

Several studies have described high expression of B7-H4 in ovarian tumors and tumor-associated macrophages [43-45]. Kryczek and colleagues further showed that blockade of B7-H4 restored the T cell-stimulatory capacity of ovarian tumor-associated macrophages and contributed to tumor regression in vivo [43]. Related studies by these authors showed that Treg could stimulate antigen-presenting cell expression of B7-H4 [46], and further demonstrated that the intensity of B7-H4 expression by ovarian tumor-associated macrophages correlated with Treg numbers in the tumor [47]. Of particular clinical significance, B7-H4 expression by macrophages (but not by tumor cells) correlated inversely with patient outcome [47]. The mechanism of action of B7-H4 in inhibition of antitumor immunity is obscure, largely because a ligand for B7-H4 has not yet been identified. Nevertheless, the weight of experimental and clinical evidence indicates that blockade of B7-H4 may be a valuable adjunct to immunotherapy of ovarian cancer.

4.3 Tumor-associated DC

Ovarian tumor cells express significant levels of CXCL12 (stromal cell-derived factor 1), which drives infiltration of plasmacytoid precursor DC with immunosuppressive function [48]. Further investigation revealed that plasmacytoid DC recovered from ovarian tumor ascites induced tumor antigen-specific regulatory CD8⁺ T cells that were capable of inhibiting myeloid DC-driven effector T cell responses by an IL-10-dependent mechanism [49]. Plasmacytoid DC are thus likely to make a significant contribution to the immunosuppressive infrastructure of ovarian tumors. Other recent studies in a mouse model of ovarian cancer have shown that DC expressing alpha-smooth muscle actin and VE-cadherin home to perivascular areas in ovarian tumors, and are important for maintenance of tumor vasculature. Selective depletion of DC bearing these markers led to vascular apoptosis and delayed growth of established tumors [50]. From these observations, selective depletion of tumor-associated DC may hold promise as an adjunct to adoptive T cell immunotherapy or tumor vaccination strategies.

4.4 Dendritic cell induction of regulatory T cells

While there is an increasing consensus that active immunotherapy or antitumor vaccination should be supported by selective and efficient depletion of Treg, there is also a new appreciation that vaccination or cellular immunotherapies may themselves induce or expand Treg, thus promoting tumor-specific tolerance. For example, vaccination with recombinant vaccinia virus in a mouse tumor model system resulted in expansion of both effector and regulatory T cells, with Treg function being dominant, blocking effector function in vitro and in vivo [51]. With respect to cellular immunotherapy, a notable clinical study showed that injection of DC matured with inflammatory cytokines (TNFα, IL-1, IL-6 and PGE₂) expanded foxp3⁺CD4⁺ Treg in 3 of 3 myeloma patients tested [52]. While it is well known that immature DC induce Treg and peripheral tolerance [53, 54], the finding that mature DC can also expand Treg clearly raises a barrier for current approaches to DC vaccination. From these observations, it is apparent that although depletion of Treg prior to vaccination may be necessary, Treg depletion alone will not be sufficient to lead to optimal effector function and antitumor immunity. Redirection of DC-driven maturation and function will also be required to prevent de novo induction of vaccine antigen-specific Treg.

5. Strategies to overcome tumor-associated immune suppression

Cellular suppression of immunosurveillance and tumor immunotherapy was clearly demonstrated several decades ago by Berendt & North [55]. Importantly, the North laboratory also played a pioneering role in demonstrating the value of combining cyclophosphamide with cellular tumor immunotherapy, showing that cyclophosphamide eliminated tumor-induced suppressor L3T4⁺ (CD4⁺) T cells [56, 57]. Many other studies have since confirmed the value of low dose cyclophosphamide as an adjuvant for tumor vaccines and adoptive immunotherapy [58-61]. Although low dose cyclophosphamide has long been known for its ability to boost cellular immunity, its mechanism of action in alleviating T cell-mediated suppression (Treg function) has been obscure until recent studies revealed that inhibition of inducible nitric oxide synthase (iNOS) was directly linked to its immunostimulatory effects [59]. Cyclophosphamide also possesses anti-angiogenic properties, mediated by upregulation of thrombospondin-1 (Tsp-1), which induces apoptosis of endothelial cells [62, 63] and sequesters vascular endothelial growth factor [64]. It is tempting to view the anti-angiogenic activity of cyclophosphamide as an added bonus for cancer treatment, but this may be offset by the recent finding that Tsp-1 promotes the generation of Treg from naïve or memory CD4⁺CD25^- T cells, through interaction with CD47 [65].

In vivo depletion of Treg can also be achieved by targeting CD25, which is preferentially expressed by CD4⁺ Treg. Antibodies against CD25 can deplete Treg and enhance antitumor immunity in animal models [66-68], and an IL-2 immunotoxin fusion protein (denileukin diftitox, also known as ONTAK) depletes Treg and contributes to durable immune control of breast tumors in neu-transgenic mice [69]. ONTAK is an FDA-approved drug (for treatment of cutaneous T cell lymphoma), and thus has broad appeal for clinical applications. A clinical trial of ONTAK treatment in combination with tumor RNA-transfected DC vaccination for patients with renal cell carcinoma resulted in reduced numbers of Treg in the peripheral blood and enhanced the generation of tumor-specific T cell responses [70]. However, these encouraging results were counterbalanced by studies in melanoma patients, in which ONTAK failed to deplete Treg numbers, as measured by foxp3 expression, and did not diminish Treg activity of CD4⁺CD25⁺ T cells in co-culture suppression assays [71]. In addition, a practical limitation is that although CD25 may be a marker for Treg among resting T cell populations, it is also broadly expressed by activated T cells, and thus treatments that target CD25 run the risk of depleting activated, tumor-reactive effector T cells in addition to removal of Treg. Notwithstanding these concerns, ONTAK is currently being tested in a clinical trial enrolling patients with epithelial carcinoma, including ovarian cancer [72].

Other drug treatments may also have clinical utility for depletion of Treg, or inhibition of Treg function. The application of fludarabine combined with cyclophosphamide as a conditioning regimen for Treg depletion prior to adoptive immunotherapy is well established [73, 74], and other studies have indicated that fludarabine may have clinical value as a stand-alone agent for in vivo inhibition of Treg [75], or as an adjuvant for DC-driven generation of tumor-specific T cells for adoptive transfer [76]. A notable recent study showed that imatinib mesylate (Gleevec) administered at clinical concentrations in mice inhibited foxp3 expression and Treg suppression, without altering expression of TGFβ and IL-10 [77]. Of particular interest, Gleevec enhanced antitumor responses following DC vaccination against a Gleevec-resistant lymphoma, suggesting that Gleevec may have direct applicability as an adjuvant for cellular immunotherapy.

6. Prevention of Treg recruitment by redirection of DC maturation

Mature DC activate and expand CD4⁺foxp3⁺ Treg in vitro [78], supporting the clinical observation that vaccination with cytokine-matured DC expands CD4⁺foxp3⁺ Treg in myeloma patients [52]. It is thus probable that DC activation of anti-tumor effector T cell responses would be seriously compromised by concomitant activation of Treg. For DC vaccination to be clinically effective, the new challenge is to identify alternative pathways of DC differentiation that bias T cell responses away from Treg homeostatis and in favor of active anti-tumor immunity.

6.1 Inhibition of p38 mitogen-activated protein kinase (MAPK) signaling as a mechanism for attenuation of Treg activation

Recent studies have pointed to a role for p38 MAPK signaling in DC-driven induction of Treg responses. Jarnicki and colleagues have shown that TLR agonists promote the induction of Treg through DC production of IL-10 consequent upon p38 MAPK signaling [79]. These investigators further demonstrated that inhibition of p38 MAPK signaling attenuated Treg induction and enhanced the efficacy of DC pulsed with antigen and CpG (a TLR9 agonist) as an antitumor cellular therapy, demonstrating an increased frequency of IFNγ-expressing T cells and diminished Foxp3⁺ Treg infiltration of tumors [79]. In addition, clinical studies in myeloma patients have shown that inhibition of p38 MAPK signaling could correct defects in DC function, restoring their ability to activate alloreactive and tumor antigen-specific T cells [80]. Activation of p38 MAPK may also play a direct role in Treg function. Adler and colleagues have shown that p38 activation in inducible Treg is critical for maintenance of anergy dependent on elevated expression of the cell cycle inhibitor p27^Kip1, and have further shown that p38 inhibition results in complete loss of regulatory function [81]. These findings suggest that the use of p38 MAPK inhibitors may be a valuable addition to our armory in overcoming Treg suppression of cellular immunotherapy and other antitumor vaccination strategies.

6.2 Polarization of DC with type I and type 2 interferons

Several studies have shown that maturation of DC in the presence of IFNγ induces high expression of IL-12p70 and favors activation of strong CD4⁺ Th1 responses and CD8⁺ CTL responses [82-84]. More recent observations from Kalinski and colleagues have also pointed to a valuable role for IFNα in modulating DC interactions with Treg. These authors found that DC matured in the presence of PGE₂ attract Treg through secretion of CCL22, and further showed that DC maturation in the presence of IFNα inhibited CCL22 production and Treg recruitment, while promoting expression of Th1-attracting chemokines [85].

6.3 Inhibition of A20 expression in DC

A20 is a zinc-finger protein that negatively regulates the Toll-like receptor and TNF receptor signaling pathways through dual ubiquitinase and de-ubiquitinase activity. A20-silenced mouse DC inhibit Treg activation and strongly enhance T helper cell and CTL responses that are refractory to Treg suppression [86]. The proposal that A20 silencing in DC could be a powerful approach to boosting antitumor immunity is supported by studies with human DC, showing that A20 is a negative regulator of NF-κB and AP-1 activation, and that its downregulation enhances the ability of DC to stimulate tumor antigen-specific CTL responses [87].

6.4 Maturation of DC with anti-CD40 and TLR4 agonists

Recent studies in mice have shown that CD8α⁺ DC induce conversion of naïve CD4⁺ T cells into adaptive foxp3⁺ Treg in the presence of TGFβ, and have further shown that multiple coinhibitory pathways regulate foxp3 induction [88]. Blockade of PD-L1 (B7-H1), CTLA4 or GITR were all capable of inhibiting DC-driven conversion of naïve CD4⁺ T cells to Foxp3⁺ Treg. Notably, PD-L1^-/- DC were severely impaired in their ability to induce foxp3 expression in the presence of TGFβ, suggesting that PD-L1 signaling is critical for the induction of adaptive foxp3⁺ Treg. Maturation of DC with agonist anti-CD40 antibody or the TLR4 ligand LPS, and particularly a combination of both markedly diminished DC-driven foxp3 expression, suggesting that adaptive Treg induction could be regulated by manipulation of DC maturation [88]. Further studies will be needed to determine whether similar mechanisms are operative with human DC activation and expansion of foxp3⁺ Treg.

6.5 Opposing roles for IL-2 and IL-15 in regulation of tumor immunity

Under normal conditions, innate thymus-derived CD4⁺ Treg play an important role in controlling autoimmune pathology, chiefly through maintenance of peripheral tolerance to self antigens. Conversely, the pathology of cellular autoimmune diseases involves loss of tolerance and abrogation of Treg suppression of autoreactive T cells. Dysregulation of cytokine expression plays a major role in autoimmune pathogenesis, and increasing evidence points to overexpression of IL-15 as a key factor in a variety of autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, ulcerative colitis, celiac syndrome, psoriasis and sarcoidosis [89]. It is thus possible that IL-15 contributes to reduced Treg activity.

IL-15 shares some functions with IL-2, but also has some important opposing actions. Whereas IL-2 is critical for maintenance of CD4⁺ Treg and is involved in activation-induced cell death (AICD) of autoreactive T cells, IL-15 inhibits AICD and promotes survival of memory CD8⁺ T cells [89]. Recent studies have shown that treatment with IL-2 results in expansion of Treg in patients with melanoma [90] and ovarian cancer [91]. IL-2 also promotes Treg trafficking to tumors by inducing expression of CCR4 and CXCR4, which bind CCL22 and CXCL12 (stromal cell-derived factor-1), respectively [37, 91], both of which are abundantly produced in ovarian tumors [37, 48]. In contrast with the Treg-promoting properties of IL-2, IL-15 enhances T cell adoptive immunotherapy of cancer [92], in vivo reactivity of antitumor CD8⁺ T cells [93] and boosts antigen-specific primary CD8⁺ T cell responses to DC vaccination [94]. An important recent study has shown that IL-15 can abrogate tolerance in tumor-antigen-specific CD8⁺ T cells, rendering them effective for immunotherapy of established tumors [95]. These latter observations provide a direct link between the known activity of IL-15 in cellular autoimmune disease and the potential for IL-15 to boost anti-tumor immunity through breakdown of peripheral tolerance to self tumor antigens. IL-15 can also act directly on DC, conferring a survival benefit [96] and skewing differentiation towards a Langerhans cell phenotype [97]. DC generated in the presence of IL-15 induce more potent CD8⁺ T cell responses, characterized by higher expression of Th1 cytokines, than DC generated with GM-CSF alone or GM-CSF plus IL-4 [98], suggesting that IL-15-treated DC may be more effective than standard DC for tumor immunotherapy. Collectively, these observations discourage the use of IL-2 as an adjuvant for cellular immunotherapy, and suggest that IL-15 treated DC would diminish activation and expansion of Treg and favor stimulation of effector T cell responses.

7. Subversion of regulatory T cell responses to self antigens as a novel stratagem for cellular immunotherapy

Accumulating evidence has clearly shown that the distribution of self-reactive T cells with high affinity is heavily skewed in favor of the CD4⁺ Treg compartment, as a result of thymic selection that is biased toward direction of autoreactive T cells into that compartment [99-102]. In addition, self-antigens drive peripheral expansion of Treg [103], and direct their accumulation in sites where the antigen is processed and presented [104, 105]. Furthermore, remarkably few Treg appear to be required to suppress effector T cell responses in vivo [106]. In the face of this reality, it is probable that most tumor vaccination strategies will face defeat, because the frequency of self-reactive effector T cells will be markedly lower than the frequency of antigen-specific Treg, and because the affinity of self-reactive effector T cells will also likely be lower than the affinity of their Treg counterparts. How can this seemingly intractable problem be resolved? Novel approaches to Treg depletion have gained some currency, but these strategies do nothing to improve the repertoire or affinity of self-reactive effector T cells. Perhaps a better option is not to deplete Treg, but to tap into the Treg compartment as a source of high affinity self-reactive (and tumor-reactive) T cells, and to reprogram their function such that they act as antitumor effector T cells. The developing consensus that Treg and Th17 differentiation and expansion are reciprocally regulated [107, 108] affords such an opportunity. The idea that subversion of Treg responses in favor of Th17 responses may have therapeutic benefit for cellular immunotherapy of ovarian cancer is strongly supported by clinical evidence that the presence of infiltrating Th17 cells in ovarian tumors has a pronounced positive correlation with prolonged overall survival (Kryczek et al, Th17 cells in ovarian cancer patients, 23^rd Annual meeting of the International Society for Biological Therapy of Cancer, San Diego, October 31-November 2, 2008).

Redirection of Treg responses to self antigen-reactive Th17 responses may be achieved by activation of T cells in the presence of cytokines that drive Th17 differentiation. The exact nature of the requirements for Th17 differentiation has been blurred by some apparently conflicting reports [109], but recent work has pointed to a central role for IL-1β [110-112]. In contrast, IL-2 signaling inhibits Th17 T cell responses [113], and important studies from Kryczek and colleagues have shown that IL-2 and IL-1 play opposing roles in regulating Th17 differentiation, with IL-2 blocking Th17 differentiation and IL-1 playing a dominant role in Th17 induction [114]. These authors have also shown that IL-2 reduces Th17 differentiation in the tumor microenvironment and promotes Treg expansion [115], pointing to dynamic regulation of Treg and Th17 responses, and lending further credence to the position that adjuvant IL-2 administration may be counterproductive for tumor vaccination or immunotherapy (see above).

The ability to manipulate Treg versus Th17 responses with cytokines may be advantageous for adoptive T cell immunotherapy, in which tumor-specific T cells are expanded in vitro, but is less helpful for DC vaccination, since the adjuvant use of inflammatory cytokines such as IL-1β or IL-15 (see above) is likely to incur unacceptable off-target toxicity. The challenge, then, is to drive Th17 differentiation at the expense of autoreactive Treg responses by manipulation of DC alone. Evidence that this may be achievable has been provided by the observation that DC treated with an antibody that cross-links the B7-DC receptor are capable of reprogramming Foxp3⁺ Treg to become Th17 autoimmune effectors [116].

Apart from inhibition of Treg activation and induction of high affinity tumor antigen-specific effector T cell responses, other benefits may accrue from Th17 induction. Perhaps most important, a considerable weight of evidence suggests that Th17 responses are markedly less sensitive than Th1 responses to suppression by Treg [117-120], which may remain a barrier to cellular immunotherapy in ovarian cancer patients, even in those pre-treated with cyclophosphamide or ONTAK to deplete tumor-associated Treg.

8. Expert Opinion

Although various studies have suggested that ovarian tissues are intrinsically immunogenic, and that cellular immune responses can develop against ovarian tumors, it has become increasingly apparent that ovarian tumors have recruited diverse and powerful defensive strategies to protect against immunological attack. The critical role played by tumor-infiltrating Treg in protection against antitumor T cell immunity is now widely recognized for many types of cancer, and Treg represent a particularly important defense mechanism in ovarian cancer. The clinical significance of tumor-associated Treg has been highlighted by Curiel and colleagues, who showed that Treg are recruited to ovarian tumors by the chemokine CCL22, and that the presence of Treg confers immune privilege and is associated with a poor prognosis and increased mortality [37]. Diligent investigation has revealed that other defense mechanisms are also operative in the ovarian tumor microenvironment, notably the expression of chemokines that recruit suppressor cell populations, including Treg and plasmacytoid DC, and the expression of inhibitory members of the B7 family of receptors (B7-H1 and B7-H4). Collectively, these immunosuppressive mechanisms present a truly formidable barrier to cellular immunotherapy.

The identification of drugs that may selectively deplete tumor-associated Treg has raised the prospect of their use in cancer treatment, either as free-standing agents or as adjuvants in support of tumor vaccination or cellular immunotherapy. Cyclophosphamide has long been recognized for its immunopotentiating qualities, and more recently the use of denileukin diftitox, which binds CD25 on Treg, has also come into vogue. Both these agents, and other drugs discussed in this review, may help to break down a major barrier to successful immunotherapy. Blockade of tumor-associated chemokines that recruit suppressor cells, or blockade of inhibitory coreceptors may also facilitate a counterattack by vaccine-induced antitumor immunity or cellular immunotherapy.

From the above discussion, one can conclude that considerable progress has been made in identifying mechanisms of tumor-associated immunosuppression, and in developing strategies that may surmount these barriers. Unfortunately, the recent recognition that antitumor vaccination or immunotherapy itself may contribute to activation and expansion of tumor antigen-specific Treg responses poses a further challenge that demands a highly creative solution. The root of the problem lies with the mechanisms by which the thymus shapes the peripheral T cell repertoire. Self-reactive precursor T cells may suffer one of two fates, either negative selection and apoptosis, or differentiation into peripheral Treg with high affinity for self antigen. As a result, a majority of self-reactive T cells are suppressor cells, with the primary role of preventing autoimmune disease. Antitumor vaccination or cellular immunotherapy may be capable of activating a relatively low affinity effector T cell response, but is also likely to activate and expand high affinity self-reactive Treg that will clearly impose major constraints on the efficacy of the treatment. In the light of this reality, it is hardly surprising that most attempts at tumor vaccination or immunotherapy have failed to live up to expectations in clinical trials.

A possible solution to the challenge posed by thymic selection of self-reactive Treg lies in the increasing body of evidence showing that Treg and Th17 T cell responses are reciprocally regulated, and that it is possible to reprogram Treg and generate effector Th17 responses that may support strong antitumor immunity. Important recent studies have shown that IL-2 and IL-1β may play key roles in direction of Treg and Th17 responses, with IL-2 favoring Treg activation and expansion and IL-1β playing a dominant role in Th17 differentiation. Apart from suggesting that the longstanding clinical use of IL-2 as a freestanding or adjuvant antitumor agent may in fact be counterproductive, these observations point to the use of IL-1β for generation of antitumor T cells from a high affinity pool of self–reactive T cells for adoptive immunotherapy. The use of IL-1β (or other proinflammatory cytokines such as IL-15) may have utility for ex vivo applications, but would likely incur unacceptable toxicities in the adjuvant setting. An alternative and more attractive approach may be to endow DC with the capacity to reprogram Treg and induce Th-17 driven antitumor immunity, as described recently by Radhakrishnan and colleagues [116].

In closing, there are two further points in favor of the generation of antitumor Th17 responses from the high affinity self-reactive Treg repertoire. First, Th17 responses are broadly refractile to Treg suppression, suggesting that antitumor effector T cells would be less prone to inhibition in the tumor microenvironment. This is a quality that is conspicuously not shared by CD4⁺ Th1 responses, which for many years have been regarded as desirable for antitumor immunity. Second, the recent clinical evidence that IL-17 expression in ovarian tumors has a strong positive correlation with improved overall survival provides a convincing rationale for Th17-driven cellular immunotherapy for ovarian cancer.

Acknowledgments

This work was supported by NIH grants CA 108257 and CA 098927.

References

1. Yap TA, Carden CP, Kaye SB. Beyond chemotherapy: targeted therapies in ovarian cancer. Nature Rev Cancer. 2009;9:167–81. [PubMed]

2. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2008. CA Cancer J Clin. 2008;58:71–96. [PubMed]

3. Schlienger K, Chu CS, Woo EY, et al. TRANCE- and CD40 ligand-matured dendritic cells reveal MHC class I-restricted T cells specific for autologous tumor in late-stage ovarian cancer patients. Clin Cancer Res. 2003;9:1517–1527. [PubMed]

4. Santin AD, Hermonat PL, Ravaggi A, et al. Induction of tumor-specific HLA class I-restricted CD8⁺ cytotoxic T lymphocytes by ovarian tumor antigen-pulsed autologous dendritic cells in patients with advanced ovarian cancer. Am J Obstet Gynecol. 2000;183:601–9. [PubMed]

5. Santin AD, Bellone S, Ravaggi A, et al. Induction of ovarian tumor-specific CD8⁺ cytotoxic T lymphocytes by acid-eluted peptide-pulsed autologous dendritic cells. Obstet Gynecol. 2000;96:422–30. [PubMed]

6. Zhao X, Wei YQ, Peng ZL. Induction of T cell responses against autologous ovarian tumors with whole tumor cell lysate-pulsed dendritic cells. Immunol Invest. 2001;30:33–45. [PubMed]

7. Gong J, Nikrui N, Chen D, et al. Fusions of human ovarian carcinoma cells with autologous or allogeneic dendritic cells induce antitumor immunity. J Immunol. 2000;165:1705–11. [PubMed]

8. Chiang CLL, Ledermann JA, Rad AN, et al. Hypochlorous acid enhances immunogenicity and uptake of allogeneic ovarian tumor cells by dendritic cells to cross-prime tumor-specific T cells. Cancer Immunol Immunother. 2006;55:1384–95. [PubMed]

9. Hernando JJ, Park TW, Kubler K, et al. Vaccination with autologous tumour antigen-pulsed dendritic cells in advanced gynaecological malignancies: clinical and immunological evaluation of a phase I trial. Cancer Immunol Immunother. 2002;51:45–52. [PubMed]

10. Hernando JJ, Park TW, Fischer HP, et al. Vaccination with dendritic cells transfected with mRNA-encoded folate-receptor-α for relapsed metastatic ovarian cancer. Lancet Oncol. 2007;8:451–4. [PubMed]

11. Disis ML, Gooley TA, Rinn K, et al. Generation of T-cell immunity to the HER-2/neu protein after active immunization with HER-2/neu peptide-based vaccines. J Clin Oncol. 2002;20:2624–32. [PubMed]

12. O'Brien TJ, Beard JB, Underwood LJ, Shigemasa K. The CA125 gene: a newly discovered extension of the glycosylated N-terminal domain doubles the size of this extracellular superstructure. Tumour Biol. 2002;23:154–69. [PubMed]

13. Bellone S, Anfossi S, O'Brien TJ, et al. Generation of CA125-specific cytotoxic T lymphocytes in human leukocyte antigen A2-positive healthy donors and patients with advanced ovarian cancer. Am J Obstet Gynecol. 2009;200:75.e1–10. [PubMed]

14. Cannon MJ, Santin AD, O'Brien TJ. Immunological treatment of ovarian cancer. Curr Opin Obstet Gynecol. 2004;16:87–92. [PubMed]

15. Tanimoto H, Underwood LJ, Shigemasa K, et al. The stratum corneum chymotryptic enzyme which mediates shedding and desquamation of skin cells is highly overexpressed in ovarian tumor cells. Cancer. 1999;86:2074–82. [PubMed]

16. Southwood S, Sidney J, Kondo A, et al. Several common HLA-DR types share largely overlapping peptide binding repertoires. J Immunol. 1998;160:3363–73. [PubMed]

17. Bondurant KL, Crew MD, Santin AD, et al. Definition of an immunodominant region within the ovarian tumor antigen stratum corneum chymotryptic enzyme. Clin Cancer Res. 2005;11:3446–54. [PubMed]

18. Zhang L, Conejo-Garcia JR, Katsaros D, et al. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med. 2003;343:203–13. [PubMed]

19. Fujita K, Ikarishi H, Takakuwa K, et al. Prolonged disease-free period in patients with advanced epithelial ovarian cancer after adoptive transfer of tumor-infiltrating lymphocytes. Clin Cancer Res. 1995;1:501–7. [PubMed]

20. Freedman RS, Platsoucas CD. Immunotherapy for peritoneal ovarian carcinoma metastasis using ex vivo expanded tumor infiltrating lymphocytes. Cancer Treat Res. 1996;82:115–46. [PubMed]

21. Santin AD, Bellone S, Palmieri M, et al. Restoration of tumor specific human leukocyte antigens class I-restricted cytotoxicity by dendritic cell stimulation of tumor infiltrating lymphocytes in patients with advanced ovarian cancer. Int J Gynecol Cancer. 2004;14:64–75. [PubMed]

22. Barber A, Zhang T, DeMars LR, et al. Chimeric NKG2D receptor-bearing T cells as immunotherapy for ovarian cancer. Cancer Res. 2007;67:5003–8. [PubMed]

23. Carpenito C, Milone M, Hassan R, et al. Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. Proc Natl Acad Sci USA. 2009;106:3360–3365. [PubMed]

24. Kershaw MH, Westwood JA, Parker LL, et al. A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer. Clin Cancer Res. 2006;12:6106–15. [PMC free article] [PubMed]

25. Baxevanis CN, Voutsas IF, Tsitsilonis OE, et al. Tumor-specific CD4⁺ T lymphocytes from cancer patients are required for optimal induction of cytotoxic T cells against the autologous tumor. J Immunol. 2000;164:3902–12. [PubMed]

26. Knutson KL, Disis ML. Tumor antigen-specific T helper cells in cancer immunity. Cancer Immunol Immunother. 2005;54:721–8. [PubMed]

27. Hwang M, Lukens JR, Bullock TNJ. Cognate memory CD4⁺ T cells generated with dendritic cell priming influnce the expansion, trafficking, and differentiation of secondary CD8⁺ T cells and enhance tumor control. J Immunol. 2007;179:5829–38. [PubMed]

28. Kennedy R, Celis E. Multiple roles for CD4⁺ T cells in anti-tumor immune responses. Immunol Rev. 2008;222:129–44. [PubMed]

29. Perez-Diez A, Joncker NT, Choi K, et al. CD4 T cells can be more efficient at tumor rejection than CD8 T cells. Blood. 2007;109:5346–54. [PubMed]

30. Hunder NN, Wallen H, Cao J, et al. Treatment of metastatic melanoma with autologous CD4⁺ T cells against NY-ESO-1. N Engl J Med. 2008;358:2698–703. [PMC free article] [PubMed]

31. Odunsi K, Qian F, Matsuzaki J, et al. Vaccination with an NY-ESO-1 peptide of HLA class I/II specificities induces integrated humoral and T cell responses in ovarian cancer. Proc Natl Acad Sci USA. 2007;104:12837–42. [PubMed]

32. Matsuzaki J, Qian F, Luescher I, et al. Recognition of naturally processed and ovarian cancer reactive CD8⁺ T cell epitopes within a promiscuous HLA class II T-helper region of NY-ESO-1. Cancer Immunol Immunother. 2008;57:1185–95. [PubMed]

33. Nishizuka Y, Sakakura T. Thymus and reproduction: sex-linked dysgenesis of the gonad after neonatal thymectomy in mice. Science. 1969;166:753–755. [PubMed]

34. Nishizuka Y, Sakakura T, Taguchi O. Mechanism of ovarian tumorigenesis in mice after neonatal thymectomy. Natl Cancer Inst Monogr. 1979;51:89–96. [PubMed]

35. Samy ET, Wheeler KM, Roper RJ, et al. Autoimmune disease in day 3 thymectomized mice is actively controlled by endogenous disease-specific regulatory T cells. J Immunol. 2008;180:4366–70. [PubMed]

36. Smith H, Chen IM, Kubo R, Tung KS. Neonatal thymectomy results in a repertoire enriched in T cells deleted in adult thymus. Science. 1989;245:749–52. [PubMed]

37. Curiel TJ, Coukos G, Zou L, et al. Specific recruitment of regulatory T cells in ovarian cancer fosters immune privilege and predicts reduced survival. Nature Med. 2004;10:942–49. [PubMed]

38. Wolf D, Wolf AM, Rumpold H, et al. The expression of the regulatory T cell-specific forkhead box transcription factor FoxP3 is associated with poor prognosis in ovarian cancer. Clin Cancer Res. 2005;11:8326–8331. [PubMed]

39. Dong H, Strome SE, Salomao DR, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: A potential mechanism of immune evasion. Nature Med. 2002;8:793–800. [PubMed]

40. Curiel TJ, Wei S, Dong H, et al. Blockade of B7-H1 improves myeloid dendritic cell-mediated antitumor immunity. Nature Med. 2003;9:562–7. [PubMed]

41. Zou W, Chen L. Inhibitory B7-family molecules in the tumour microenvironment. Nature Rev Immunol. 2008;8:467–77. [PubMed]

42. Hamanishi J, Mandai M, Iwasaki M, et al. Programmed cell death 1 ligand 1 and tumor-infiltrating CD8⁺ T lymphocytes are prognostic factors of human ovarian cancer. Proc Natl Acad Sci USA. 2007;104:3360–5. [PubMed]

43. Kryczek I, Zou L, Rodriguez P, et al. B7-H4 expression identifies a novel suppressive macrophage population in human ovarian cancer. J Exp Med. 2006;203:871–81. [PMC free article] [PubMed]

44. Simon I, Zhuo S, Corrai L, et al. B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer. Cancer Res. 2006;66:1570–5. [PubMed]

45. Tringler B, Liu W, Corrai L, et al. B7-H4 overexpression in ovarian tumors. Gynecol Oncol. 2006;100:44–52. [PubMed]

46. Kryczek I, Wei S, Zou L, et al. Induction of B7-H4 on APCs through Il-10: Novel suppressive mode for regulatory T cells. J Immunol. 2006;177:40–4. [PubMed]

47. Kryczek I, Wei S, Zhu G, et al. Relationship between B7-H4, regulatory T cells, and patient outcome in human ovarian carcinoma. Cancer Res. 2007;67:8900–5. [PubMed]

48. Zou W, Machelon V, Coulomb-L'Hermin A, et al. Stromal-derived factor-1 in human tumors recruits and alters the function of plasmacytoid precursor dendritic cells. Nature Med. 2001;7:1339–46. [PubMed]

49. Wei S, Kryczek I, Zou L, et al. Plasmacytoid dendritic cells induce CD8⁺ regulatory T cells in human ovarian carcinoma. Cancer Res. 2005;65:5020–6. [PubMed]

50. Huarte E, Cubillos-Ruiz JR, Nesbeth YC, et al. Depletion of dendritic cells delays ovarian cancer progression by boosting antitumor immunity. Cancer Res. 2008;68:7684–7691. [PMC free article] [PubMed]

51. Zhou G, Drake CG, Levitsky HI. Amplification of tumor-specific regulatory T cells following therapeutic cancer vaccines. Blood. 2006;107:628–36. [PubMed]

52. Banerjee DK, Dhodapkar MV, Matayeva E, et al. Expansion of FOXP3high regulatory T cells by human dendritic cells (DCs) in vitro and after injection of cytokine-matured DCs in myeloma patients. Blood. 2006;108:2655–61. [PubMed]

53. Jonuleit H, Schmitt E, Schuler G, et al. Induction of interleukin 10-producing, nonproliferating CD4⁺ T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J Exp Med. 2000;192:1213–22. [PMC free article] [PubMed]

54. Dhodapkar MV, Steinman RM, Krasovsky J, et al. Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J Exp Med. 2001;193:233–8. [PMC free article] [PubMed]

55. 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. [PMC free article] [PubMed]

56. 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. [PMC free article] [PubMed]

57. Awwad M, North RJ. Cyclophosphamide-induced immunologically mediated regression of a cyclophosphamide-resistant murine tumor: a consequence of eliminating precursor L3T4+ suppressor T-cells. Cancer Res. 1989;49:1649–54. [PubMed]

58. Ghiringhelli F, Larmonier N, Schmitt E, et al. CD4⁺CD25⁺ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative. Eur J Immunol. 2004;34:336–344. [PubMed]

59. Loeffler M, Kruger JA, Reisfeld RA. Immunostimulatory effects of low-dose cyclophosphamide are controlled by inducible nitric oxide synthase. Cancer Res. 2005;65:5027–30. [PubMed]

60. Ikezawa Y, Nakazawa M, Tamura C, et al. Cyclophosphamide decreases the number, percentage and the function of CD25⁺ CD4⁺ regulatory T cells, which suppress induction of contact hypersensitivity. Dermatol Sci. 2005;39:105–12. [PubMed]

61. Lutsiak ME, Semnani RT, De Pascalis R, et al. Inhibition of CD4⁺CD25⁺ T regulatory cell function implicated in enhanced immune response by low-dose cyclophosphamide. Blood. 2005;105:2862–68. [PubMed]

62. Nor JE, Mitra RS, Sutorik MM, et al. Thrombospondin-1 induces endothelial cells apoptosis and inhibits angiogenesis by activating the caspase death pathway. J Vasc Res. 2000;37:209–18. [PubMed]

63. Bocci G, Francia G, Man S, et al. Thrombospondin 1, a mediator of the antiangiogenic effects of low-dose metronomic chemotherapy. Proc Natl Acad Sci USA. 2003;100:12917–22. [PubMed]

64. Lawler J, Detmar M. Tumor progression: the effects of thrombospondin-1 and -2. Int J Biochem Cell Biol. 2004;36:1038–45. [PubMed]

65. Grimbert P, Bouguermouh S, Baba N, et al. Thrombospondin/CD47 interaction: a pathway to generate regulatory T cells from human CD4+ CD25- T cells in response to inflammation. J Immunol. 2006;177:3534–41. [PubMed]

66. Shimizu J, Yamazuki S, Sakaguchi S. Induction of tumor immunity by removing CD25⁺CD4⁺ T cells: a common basis between tumor immunity and autoimmunity. J Immunol. 1999;163:5211–8. [PubMed]

67. Antony PA, Restifo NP. CD4⁺CD25⁺ T regulatory cells, immunotherapy of cancer, and Interleukin-2. J Immunother. 2005;28:120–8. [PMC free article] [PubMed]

68. Imai H, Saio M, Nonaka K, et al. Depletion of CD4⁺CD25⁺ regulatory T cells enhances interelukin-2-induced antitumor immunity in a mouse model of colon adenocarcinoma. Cancer Sci. 2007;98:416–23. [PubMed]

69. Knutson KL, Dang Y, Lu H, et al. IL-2 immunotoxin therapy modulates tumor-associated regulatory T cells and leads to lasting immune-mediated rejection of breast cancers in neu-transgenic mice. J Immunol. 2006;177:84–91. [PubMed]

70. Dannull J, Su Z, Rizzieri D, et al. Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletion of regulatory T cells. J Clin Invest. 2005;115:3623–33. [PMC free article] [PubMed]

71. Attia P, Maker AV, Haworth LR, et al. Inability of fusion protein of IL-2 and diphtheria toxin (Denileukin diftitix, DAB₃₈₉IL-2, ONTAK) to eliminate regulatory T lymphocytes in patients with melanoma. J Immunother. 2005;28:582–92. [PMC free article] [PubMed]

72. Curiel TJ. Tregs and rethinking cancer immunotherapy. J Clin Invest. 2007;117:1167–74. [PMC free article] [PubMed]

73. Muranski P, Boni A, Wrzesinski C, et al. Increased intensity lymphodepletion and adoptive immunotherapy – how far can we go? Nature Clin Pract Oncol. 2006;3:668–81. [PMC free article] [PubMed]

74. Thistlethwaite FC, Elkord E, Griffiths RW, et al. Adoptive transfer of T(reg) depleted autologous T cells in advanced renal cell carcinoma. Cancer Immunol Immunother. 2008;57:623–34. [PubMed]

75. Beyer M, Kochanek M, Darabi K, et al. Reduced frequencies and suppressive function of CD4⁺CD25^hi regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine. Blood. 2005;106:2018–25. [PubMed]

76. Hegde U, Chhabra A, Chattopadhyay S, et al. Presence of low dose of fludarabine in cultures blocks regulatory T cell expansion and maintains tumor-specific cytotoxic T lymphocyte activity generated with peripheral blood lymphocytes. Pathobiol. 2008;75:200–8. [PubMed]

77. Larmonier N, Janikashvili N, LaCasse CJ, et al. Imatinib mesylate inhibits CD4⁺CD25⁺ regulatory T cell activity and enhances active immunotherapy against BCR-ABL^- tumors. J Immunol. 2008;181:6955–63. [PMC free article] [PubMed]

78. Yamazaki S, Iyoda T, Tarbell K, et al. Direct expansion of functional CD25⁺CD4⁺ regulatory T cells by antigen-processing dendritic cells. J Exp Med. 2003;198:235–47. [PMC free article] [PubMed]

79. Jarnicki AG, Conroy H, Brereton C, et al. Attenuating regulatory T cell induction by TLR agonists through inhibition of p38 MAPK signaling in dendritic cells enhances their efficacy as vaccine adjuvants and cancer immunotherapeutics. J Immunol. 2008;180:3797–806. [PubMed]

80. Wang S, Hong S, Yang J, et al. Optimizing immunotherapy in multiple myeloma: restoring the function of patients' monocyte-derived dendritic cells by inhibiting p38 or activating MEK/ERK MAPK and neutralizing interleukin-6 in progenitor cells. Blood. 2006;108:4071–7. [PubMed]

81. Adler HS, Kubsch S, Graulich E, et al. Activation of MAP kinase p38 is critical for the cell-cycle-controlled suppressor function of regulatory T cells. Blood. 2007;109:4351–9. [PubMed]

82. Xu S, Koski GK, Faries M, et al. Rapid high efficiency sensitization of CD8⁺ T cells to tumor antigens by dendritic cells leads to enhanced functional avidity and direct tumor recognition through an IL-12-dependent mechanism. J Immunol. 2003;171:2251–61. [PubMed]

83. Maillard RB, Wankowicz-Kalinska A, Cai Q, et al. Alpha-type-1 polarized dendritic cells: A novel immunization tool with optimized CTL-inducing activity. Cancer Res. 2004;64:5934–7. [PubMed]

84. ten Brinke A, Karsten ML, Dieker MC, et al. The clinical grade maturation cocktail monophosphoryl lipid A plus IFNgamma generates monocyte-derived dendritic cells with the capacity to migrate and induce Th1 polarization. Vaccine. 2007;25:7145–52. [PubMed]

85. Muthuswamy R, Urban J, Lee JJ, et al. Ability of mature dendritic cells to interact with regulatory T cells is imprinted during maturation. Cancer Res. 2008;68:5972–8. [PMC free article] [PubMed]

86. Song XT, Evel-Kabler K, Shen L, et al. A20 is an antigen presentation attenuator, and its inhibition overcomes regulatory T cell-mediated suppression. Nat Med. 2008;14:258–65. [PubMed]

87. Breckpot K, Aerts-Toegaert C, Heirman C, et al. Attenuated expression of A20 markedly increases the efficacy of double-stranded RNA-activated dendritic cells as an anti-cancer vaccine. J Immunol. 2009;182:860–870. [PubMed]

88. Wang L, Pino-Lagos K, de Vries VC, et al. Programmed death 1 ligand signaling regulates the generation of adaptive Foxp3⁺CD4⁺ regulatory T cells. Proc Natl Acad Sci USA. 2008;105:9331–36. [PubMed]

89. Waldmann TA. Targeting the interleukin-15/interleukin-15 receptor system in inflammatory autoimmune diseases. Arth Res Ther. 2004;6:174–7. [PMC free article] [PubMed]

90. Ahmadzadeh M, Rosenberg SA. IL-2 administration increases CD4⁺CD25^hiFoxp3⁺ regulatory T cells in cancer patients. Blood. 2006;107:2409–14. [PubMed]

91. Wei S, Kryczek I, Edwards RP, et al. Interleukin-2 administration alters the CD4⁺Foxp3⁺ T-cell pool and tumor trafficking in patients with ovarian carcinoma. Cancer Res. 2007;67:7487–94. [PubMed]

92. Antony PA, Paulos CM, Ahmadzadeh M, et al. Interleukin-2-dependent mechanisms of tolerance and immunity in vivo. J Immunol. 2006;176:5255–66. [PMC free article] [PubMed]

93. Klebanoff CA, Finkelstein SE, Surman DR, et al. IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8+ T cells. Proc Natl Acad Sci USA. 2004;101:1969–74. [PubMed]

94. Rubinstein MP, Kadima AN, Salem ML, et al. Systemic administration of IL-15 augments the antigen-specific primary CD8⁺ T cell response following vaccination with peptide-pulsed dendritic cells. J Immunol. 2002;169:4928–35. [PubMed]

95. Teague RM, Sather BD, Sacks JA, et al. Interleukin-15 rescues tolerant CD8⁺ T cells for use in adoptive immunotherapy of established tumors. Nature Med. 2006;12:335–41. [PubMed]

96. Dubois SP, Waldmann TA, Muller JR. Survival adjustment of mature dendritic cells by IL-15. Proc Natl Acad Sci USA. 2005;102:8662–7. [PubMed]

97. Mohamadzadeh M, Berard F, Essert G, et al. Interleukin 15 skews monocyte differentiation into dendritic cells with features of Langerhans cells. J Exp Med. 2001;194:1013–9. [PMC free article] [PubMed]

98. Pulendran B, Dillon S, Joseph C, et al. Dendritic cells generated in the presence of GM-CSF plus IL-15 prime potent CD8⁺ Tc1 responses in vivo. Eur J Immunol. 2004;34:66–73. [PubMed]

99. Jordan MS, Boesteanu A, Reed AJ, et al. Thymic selection of CD4⁺CD25⁺ regulatory T cells induced by an agonist self peptide. Nature Immunol. 2001;2:301–6. [PubMed]

100. Romagnoli P, Hudrisier D, van Meerwijk JPM. Preferential recognition of self antigens despite normal thymic deletion of CD4⁺CD25⁺ regulatory T cells. J Immunol. 2002;168:1644–8. [PubMed]

101. Romagnoli P, Hudrisier D, van Meerwijk JPM. Molecular signature of recent thymic selection events on effector and regulatory CD4⁺ T lymphocytes. J Immunol. 2005;175:5751–8. [PMC free article] [PubMed]

102. Hsieh CS, Liang Y, Tyznik AJ, et al. Recognition of the peripheral self by naturally arising CD25⁺CD4⁺ T cell receptors. Immunity. 2004;21:267–77. [PubMed]

103. Cozzo C, Larkin J, III, Caton AJ. Self-peptides drive the peripheral expansion of CD4⁺CD25⁺ regulatory T cells. J Immunol. 2003;171:5678–82. [PubMed]

104. Fisson S, Darasse-Jeze G, Litvinova E, et al. Continuous activation of autoreactive CD4⁺CD25⁺ regulatory T cells in the steady state. J Exp Med. 2003;198:737–46. [PMC free article] [PubMed]

105. Walker LS, Chodos A, Eggena M, et al. Antigen-dependent proliferation of CD4⁺CD25⁺ regulatory T cells in vivo. J Exp Med. 2003;198:249–58. [PMC free article] [PubMed]

106. Green EA, Choi Y, Flavell RA. Pancreatic lympho-node-derived CD4⁺CD25⁺ Treg cells:highly potent regulators of diabetes that require TRANCE-RANK signals. Immunity. 2002;16:183–91. [PubMed]

107. Kryczek I, Wei S, Zou L, et al. Th17 and regulatory T cell dynamics and the regulation by IL-2 in the tumor microenvironment. J Immunol. 2007;178:6730–6733. [PubMed]

108. Bettelli E, Oukka M, Kuchroo VK. T_H-17 cells in the circle of immunity and autoimmunity. Nature Immunol. 2007;8:345–350. [PubMed]

109. Laurence A, O'Shea JJ. T_H-17 differentiation: of mice and men. Nature Immunol. 2007;8:903–5. [PubMed]

110. Acosta-Rodriguez EV, Napolitani G, Lanzavecchia A, Sallusto F. Interleukins 1β and 6 but not transforming growth factor-β are essential for the differentiation of interleukin 17-producing human T helper cells. Nature Immunol. 2007;8:942–9. [PubMed]

111. Wilson NJ, Boniface K, Chan JR, et al. Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nature Immunol. 2007;8:950–7. [PubMed]

112. Liu H, Rohowsky-Kochan C. Regulation of IL-17 in human CCR6⁺ effector memory T cells. J Immunol. 2008;180:7948–57. [PubMed]

113. Laurence A, Tato CM, Davidson TS, et al. Interleukin-2 signaling via STAT5 constrains T helper 17 cell generation. Immunity. 2007;26:371–81. [PubMed]

114. Kryczek I, Wei S, Vatan L, et al. Opposite effects of IL-1 and IL-2 on the regulation of IL-17⁺ T cell pool: IL-1 subverts IL-2-mediated suppression. J Immunol. 2006;179:1423–6. [PubMed]

115. Kryczek I, Wei S, Zou L, et al. Th17 and regulatory T cell dynamics and the regulation by IL-2 in the tumor microenvironment. J Immunol. 2007;178:6730–3. [PubMed]

116. Radhakrishnan S, Cabrera R, Schenk EL, et al. Reprogrammed FoxP3⁺ T regulatory cells become IL-17⁺ antigen-specific autoimmune effectors in vitro and in vivo. J Immunol. 2008;181:3137–47. [PMC free article] [PubMed]

117. Annunziato F, Cosmi L, Santarlasci V, et al. Phenotypic and functional features of human Th17 cells. J Exp Med. 2007;204:1849–61. [PMC free article] [PubMed]

118. Evans HG, Suddason T, Jackson I, et al. Optimal induction of T helper 17 cells in humans requires T cell receptor ligation in the context of Toll-like receptor-activated monocytes. Proc Natl Acad Sci USA. 2007;104:17034–9. [PubMed]

119. Stummvoll GH, DiPaolo RJ, Huter EN, et al. Th1, Th2, and Th17 effector T cell-induced autoimmune gastritis differs in pathological pattern and in susceptibility to suppression by regulatory T cells. J Immunol. 2008;181:1908–16. [PMC free article] [PubMed]

120. Chauhan SK, El Anaan J, Ecoiffier T, et al. Autoimmunity in dry eye is due to resistance of Th17 to treg suppression. J Immunol. 2009;182:1247–52. [PMC free article] [PubMed]