http://informahealthcare.com/imt ISSN: 1547-691X (print), 1547-6901 (electronic) J Immunotoxicol, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/1547691X.2013.865094

A REVIEW ARTICLE BASED UPON A PRESENTATION AT THE 3RD INTERNATIONAL CONFERENCE ON CANCER IMMUNOTHERAPY AND IMMUNOMONITORING (CITIM), KRAKOW, POLAND, APRIL 2013

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Breaking immunotolerance of tumors: A new perspective for dendritic cell therapy Jacek Rolinski and Iwona Hus 1

Chair and Department of Clinical Immunology and 2Department of Clinical Transplantology, Medical University of Lublin, Lublin, Poland

Abstract

Keywords

The use of dendritic cells (DC) in cancer immunotherapy is based on their potent abilities to present antigens, so they can act as ‘natural adjuvants’ to enhance immunogenicity of tumor antigens and stimulate specific cytotoxic T-cells. Large amounts of DC can be generated from bone marrow, neonatal cord blood, and peripheral blood CD34þ hematopoietic stem cells, or from peripheral blood monocytes. The DC can then be pulsed with tumor antigens and reinfused. In vitro, antigen-pulsed DC can stimulate allogeneic T-cell proliferation and induction of autologous specific cytotoxic T-cells; in vivo, the cells inhibit the growth of tumors or protect hosts (i.e. mice) from development of inoculated tumors. The results of preliminary clinical trials have shown that DC vaccines are safe and elicit immune responses; however, the rates of clinical responses are low. It has become quite clear that one key reason for unsatisfactory clinical results is tumor-induced immunosuppression. Among the factors contributing to this type of immunosuppression are populations of regulatory cells including: T-regulatory (Treg) cells, myeloid-derived suppressor cells (MDSC), tumor-associated macrophages (TAM), and DC expressing 2,3-dioxygenase indoleamine (IDO-DC). This review presents an overview of the current understanding about populations of regulatory cells and the most current research efforts directed to overcome immunosuppressive activity due to the tumor microenvironment.

Cancer, dendritic cells, immunotherapy, tumor-induced immunosuppression

Introduction Dendritic cells (DC) were first described in 1973 by Ralph Steinmann and colleagues as a distinct sub-population of murine spleen cells and named after their characteristic tree-like morphology (Greek dendreon ¼ tree) (Steinmann & Cohn, 1973). Over time, many studies have reported on the origin, phenotype, and function of different sub-populations of-as well as use of-DC in immunotherapy. The great importance of all those research efforts was confirmed in that the Nobel Prize in Medicine and Physiology was awarded posthumously to Dr Steinmann who died on 30 September 2011, 3 days before announcement of the winners’ names. Much of the interest associated with DC ever since their discovery is connected with their specific abilities to present antigens, including tumor-associated antigens. The concept of a potential efficacy for using DC in fighting cancers arose from two main facts. First, a significant cause of ‘escape’ of cancer cells from immune system control is a lack of effective presentation of tumor antigens and, second, that DC have the most potent antigen presentation properties among all antigenpresenting cells, including unique capacities to activate naive T-lymphocytes and overcome T-lymphocyte non-responsiveness

Address for correspondence: Professor Jacek Rolinski, Chair, Department of Clinical Immunology, Medical University of Lublin, Chodzki 4a Street, 20-093 Lublin, Poland. Tel: 48817564840. Fax: 48817564840. E-mail: [email protected]

History Received 1 October 2013 Revised 4 November 2013 Accepted 8 November 2013 Published online 4 February 2014

in vivo (Santambrogio et al., 1999). It has been shown in vitro that DC stimulated by antigens have an ability to induce cytotoxic CD8þ T-lymphocytes directed against tumor cells (Albert et al., 1998; Hoffmann et al., 2000) and that one ‘educated’ DC might activate hundreds of tumor-specific T-lymphocytes. Studies in animals, both in the case of inoculated solid tumors (e.g. melanoma, fibrosarcoma, adenocarcinoma) (Fields et al., 1998; Lambert et al., 2001; Wang et al., 1998) as well as leukemias (He et al., 2001) confirmed the beneficial effects of DC vaccines in inhibiting the development of, and enhancing the regression of, implanted tumors. The idea of implementation of immunotherapy to the armatorium of anti-cancer treatments is based on evidence from murine models showing that immunocompromised mice have increased incidence of cancers (Dighe et al., 1994; Schwartz, 2001) and, on the other side, that immunization could induce tumor-specific immunity and reduce tumor mass and growth (Kugler et al., 2000; Lin et al., 2002; Saleh et al., 2001; Shimizu et al., 1989; Vonderheide et al., 2004). The hypothesis for a key role of the immune system in combating cancer is further supported by clinical observations on spontaneous regressions of tumor in humans (Bodey, 2002; del Giudice et al., 2009; Oquin˜ena et al., 2009) and an increased risk of some cancer development in immunodeficient patients (Jagadeesh et al., 2012; Kubica & Brewer, 2012). It has also been shown that a presence of immune cell infiltrates in cancer tissues correlate with a better prognosis (Movassagh et al., 2004; Page`s et al., 2005; Wansom et al., 2012). Further, tumor-specific T-lymphocytes could not

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only be isolated from cancer patients, but adoptive transfer of autologous anti-tumor lymphocytes could induce rapid regression of nodes, as well as liver and adrenal metastases, in patients with melanoma (Rosenberg et al., 2008). Apart from the adoptive transfer of T-lymphocytes, the other intensively explored method of immunotherapy is in the form of cancer vaccines. Since vaccination is the greatest success in the field of clinical immunology, current immunotherapeutic approaches seek to continue this success to treat cancer. Anticancer vaccines might be used both in primary prophylaxis (i.e. vaccines against human papilloma virus and hepatitis B virus) as well as in anti-cancer therapy. Among therapeutic vaccines, one of the most important forms is the DC vaccine. In contrast to the other treatments (e.g. chemotherapy, radiation therapy, adoptive immunotherapy), immunization with DC, if effective, might induce a durable-even lifelong-specific antitumor response in the form of memory T-lymphocytes. Since 1996, when Hsu et al. reported the results of the first clinical trial with the use of DC vaccines in patients with B-cell non-Hodgkin lymphoma (Hsu et al., 1996), attempts to use DC immunotherapy have been undertaken in patients with many types of cancers, including both solid tumors and hematological malignancies (Galluzzi et al., 2012). Nevertheless, currently, no DC vaccination regimen is accepted as a standard anti-cancer therapy and no anti-cancer vaccines have been effective enough to be implemented in the routine clinical setting. Conclusions driven from early clinical trials were that DC vaccines were safe and elicited immune responses, but rates of clinical responses were low. Results of select (since 2004) early-phase therapeutic vaccine trials in patients with metastatic solid cancers show there were 13 (3.7%) responses in 347 patients receiving peptide vaccines, 10 (4.2%) in 240 patients receiving DC vaccines, one (0.9%) response in 108 patients receiving recombinant viral vaccines, two (2%) responses in 98 patients receiving tumor cell vaccines, and four (6.7%) responses in 60 patients receiving DNA plasmid vaccines (Klebanoff et al., 2011). It is difficult to compare results from DC immunotherapy due to the various approaches used in clinical studies, namely: different number of cells/ vaccine, number of vaccines, length of vaccine program, site of vaccination, frozen preservation of vaccine, methods of DC maturation, etc. (Ridgway, 2003). A trial watch summarizing the results of recently-completed clinical trials, and discussing the progress of ongoing studies that have evaluated DC-based cancer immunotherapy, revealed that only two studies had even entered Phase III (Galluzzi et al., 2012). This led to the questions: why do DC-based vaccines-being immunogenic-have limited clinical responsiveness and whether anti-cancer active specific DC immunotherapies have reached a plateau (of results)? Still, in the literature, one could find data on successful DC therapies too. Retrospective analyses of overall survival in a cohort of 66 patients treated during 1999–2003, reported by the Banchereau group, showed a 20% long-term survival (Ueno et al., 2010). In one of the last clinical trials, Oshita et al. (2012) vaccinated 24 metastatic melanoma patients with DC pulsed with a cocktail of five melanoma-specific synthetic peptides, and subsequently observed significant prolongation in overall survival as compared to that among non-vaccinated patients (i.e. 13.6 vs 7.3 months). Moreover, specific immune response measured by ELISPOT significantly correlated with overall survival time. In the patients with high immune response, overall survival time was 21.9 vs 8.1 months in patients with low response, suggesting that monitoring of immune response might be important for predicting a clinical response. The authors of that study did not evaluate regulatory

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cells; however, in our opinion, measures of their numbers might have provided additional predictive information. In one of our heavily pre-treated (i.e. ipilimumab, IFN, chemotherapy) melanoma patients with progressive disease who also presented with multiple metastases to liver, lung, and skinand who was currently being treated (since July 2013) with DC pulsed with metastatic tumor lysates (i.e. two melanoma-specific peptides [gp100, tyrosinase]) and KLH combined with systemic IFN [6  106 U/3 times a week), we observed stable disease and concurrent increases in the percentages of T-regulatory (Treg) cells with each consecutive vaccination. After six treatments (one given/2 week), it was decided to 3-fold increase the dose of DC in one vaccine (up to 70  106 DC/vaccine) and to use lysates from new metastases for DC stimulation; this resulted in partial response with regression of most pulmonary metastases (evaluated by CT), normalization of liver enzymes, and decreases in Treg cell levels (unpublished data). To the best of our knowledge, this was the first clinical trial where melanoma synthetic peptides and lysate-pulsed DC were combined with an IFN treatment. We believe improved results with DC vaccinations can and will be achieved by combining various methods of immunotherapy, including, for example, the use of DC in combination with PD-1 blocking agents. In another study (Palucka et al., 2006), significant clinical responses were observed in two of 20 patients with Stage IV melanoma (with resistance to current treatment) treated with DC generated from peripheral blood monocytes stimulated with allogeneic melanoma cells. The first patient achieved complete remission (CR) lasting 18 months; a second one had a near CR lasting &55 months. van Tendeloo et al. (2010) reported results of an immunotherapy with DC transfected with mRNA tumor antigen WT1 (Wilms’ Tumor 1) in 10 patients with acute myeloid leukemia (AML) that had been treated initially with chemotherapy. Two patients with partial responses achieved complete responses; in three patients with complete response, conversion to molecular response (defined as normalization of WT1 mRNA levels in blood) was achieved. One of the most important studies in the field of DC immunotherapy was a randomized Phase III clinical trial performed in 127 patients with metastatic hormone-refractory prostate cancer (Kantoff et al., 2010). This study reported a significant effect on overall survival of patients that received the vaccine sipuleucel-T (ProvengeÕ , Dendreon), a DC-stimulated autologous fusion protein consisting of a specific antigen of prostate cancer cells, i.e. prostatic acid phosphatase (prostate phospatase acid, PAP) coupled with GM-CSF (granulocytemacrophage colony stimulating factor). Median overall survival in the treated group was significantly longer (25.8 vs 21.7 months) and estimated probability of survival 36 months after randomization was 31.7% in the sipuleucel-T group (23.0% in placebo). Based on the study results, Sipuleucel-T acquired (in April 2010) a registration by the US Food and Drug Administration (FDA) as the first therapeutic cancer vaccine for use in humans. These data suggested that DC anti-cancer immunotherapy had the potential to be effective. However, after more than 15 years since the first reported clinical studies, it became quite clear that one key reason for unsatisfactory clinical results was a tumor-induced immunosuppression. Therefore, current research efforts were increasingly directed at not only being able to mount the most effective specific immune response, but to also overcome immunosuppressive activity attributable to the tumor and its microenvironment. Among the various factors contributing to this immunosuppression, there are several populations of regulatory cells including: T-regulatory (Treg) cells, myeloid derived suppressor cells (MDSC), tumor

New perspectives for dendritic cell anti-cancer therapy

DOI: 10.3109/1547691X.2013.865094

associated macrophages (TAM), and DC expressing 2,3dioxygenase indoleamine (IDO-DC).

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T-regulatory (Treg) cells Treg cells, as a lymphocyte population with tolerogenic properties, were first identified from among the family of T-lymphocytes by Gershon et al. (1974). Treg cells include both CD4þ, as well as CD8þ T-cells; however the most studied population are those CD4þ T-lymphocytes that co-express CD25 (IL-2R)þ, Foxp3þ CTLA (cytotoxic T-lymphocyte-associated antigen)-4þ, and GITR (glucocorticoid-induced TNFR family-related) geneþ molecules (Read et al., 2000; McHugh et al. 2002). Treg cells were subsequently found to be responsible for immune tolerance to tumor development and to contribute to tumor growth in most cancer types (Dietl et al., 2007; Linehan & Goedegebuure, 2005; Oleinika et al., 2013; Watanabe et al., 2010). Along these lines, increased numbers of CD4þ/CD25þ Treg cells have been reported in patients with advanced cancers (Ichihara et al., 2003; Javia and Rosenberg, 2003; Ormandy et al., 2005; Schaefer et al., 2005; Woo et al., 2001) and there was a correlation seen between high percentages of Treg cells and shorter survival times of patients (Curiel et al., 2004). In patients with chronic lymphocytic leukemia (CLL) or multiple myeloma (MM) resistant to anticancer vaccines, there was an increase in circulating Treg cells levels during immunotherapy (Abdalla et al., 2008; Biagi et al., 2005). In two of our own studies, we used vaccines with allogeneic or autologous DC pulsed ex vivo with tumor cell lysates or apoptotic bodies to stimulate anti-tumor immunity in patients with earlystage CLL (Hus et al., 2005, 2008). Vaccination was feasible and safe, and a significant increase in levels of specific cytotoxic Tlymphocytes against both RHAMM/CD168 and fibro-modulin (a recently-characterized leukemia-associated antigen) led to some notable hematologic improvements. However, no objective response was noted and higher levels of Treg cells were noted in the patients not responding to the therapy as compared to the patients with hematologic improvements (Hus et al., 2008). In the context of the immunosuppressive properties of Treg cells, it is important to choose the method of DC generation and stimulation with tumor antigens that not only achieves an immune response, but allows for avoiding activation of Treg cells. Muthuswamy et al. (2008) showed that using DC1-polarizing cocktail (containing tumor necrosis factor [TNF]-, interleukin [IL]-1b, interferon [IFN]-, IFNg, and Poly-I:C) allowed one to obtain monocyte-derived DC (MoDC) with a lower expression of

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Treg cell-attracting chemokine CCL22 in comparison to what is obtained with a standard cocktail (containing TNF-, prostaglandin [PGE]-2, IL-1b, and IL-6). Many different methods of Treg cell depletion have also been described, including: low-dose cyclophosphamide (CYP; Liu et al., 2007), low-dose fludarabine (Hegde et al., 2008), as well as use of danileukin diftitox (ONTAK)-a fusion protein of IL-2 and diphtheria toxin (Dannull et al., 2005), anti-CTLA-4 antibody (Phan et al., 2003), or a specific antibody directed against IL-2R on Treg cells (PC61) (Matsushita et al., 2008). Some of these have already been implemented in clinical studies. The alkylating agent CYP, when given in low doses, selectively induces Treg cell depletion (Berd & Mastrangelo, 1987; Greten et al., 2010). Since the immunomodulatory properties of CYP include also DC activation and polarization of immune response to T-helper (TH)-1 cytokine secretion, the drug has been used in combination with both tumorcell vaccines and, more recently, with DC vaccines (Radojcic et al., 2010; Tzai et al., 1996; Wada et al. 2009; Wersa¨ll & Mellstedt, 1995). However, the efficacy of CYP in improving the effects of DC vaccines is still an open issue. For example, Alfaro et al. (2011) vaccinated 24 metastatic cancer patients using DC vaccines in combination with CYP, GM-CSF, and pegylated IFN. CYP was administered 7 days before each vaccination, causing a decrease of elevation of Treg cell levels; however, no objective responses were noted. In studies by Chu et al. (2012), patients with advanced ovarian cancer in remission were given peptideloaded DC with or without low-dose CYP; no changes in total Tlymphocyte or Treg cell numbers were seen. Similarly, Ellebaek et al. (2012) did not note Treg cell number decreases during immunotherapy with DC combined with IL-2, celecoxib, and/or metronomic doses of CYP. However, in that study, the number of patients achieving stable disease (SD) was higher and overall survival was longer compared to the patients treated previously only with DC. Addition of CYP to cellular immunotherapy became even more controversial in light of recent data, showing it could stimulate MDSC (Mikysˇkova´ et al., 2012; Sevko et al., 2013). To better evaluate the role of CYP in DC immunotherapy, we are currently conducting a clinical trial that uses CYP and celecoxib in patients with early-stage CLL. The immunotherapy scheme is presented in Figure 1. In comparison to our previous studies wherein DC monotherapy was applied in 10 patients with CLL in its early clinical stages (Hus et al., 2008), here we did not note any significant differences in Treg cell numbers before and after vaccination with DC combined with CYP and celecoxib (unpublished data). These results indicated to us that addition of CYP

Figure 1. A scheme for DC-based immunotherapy.

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and celecoxib, at least in the presented schedule, did not influence Treg cell number in CLL patients. Treg cell depletion was also achieved through the use of denileukin or diftitox, but no correlation between depletion and immune responses was noted (Dannull et al., 2005). Jacobs et al. (2010) presented similar observations using daclizumab (a humanized antibody against IL-2R -chain [CD25]) in combination with DC vaccines. However, despite effective Treg cell depletion, there was no enhancement of DC vaccine efficacy. A quite different concept was to re-program FoxP3þ regulatory cells into competent effector TH17 cells using DC activated by a novel immunomodulant, i.e. human B7-DC crosslinking antibody (B7- DC XAb). In mice, administration of B7DC XAb prevented tumor growth (Radhakrishnan et al., 2008); a Phase I trial is in progress to evaluate the potential for use of B7-DC XAb as a therapeutic immune-modulator in advanced melanoma. The results of so-far reported clinical studies showing no substantial improvement from DC immunotherapy after Treg cell depletion might suggest a more substantial role for other populations of regulatory cells (except Treg cells) during tumorinduced immunosuppression.

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4 6 months) in another two patients. Other agents found to reduce MDSC numbers in murine models include: 5-fluorouracil (Vincent et al., 2010), docetaxel (Kodumudi et al., 2010, 2012), and tyrosine kinase inhibitors like sunitinib or axitinib (Bose et al., 2010; Ko et al., 2009; Ozao-Choy et al., 2009). The third strategy, i.e. to inhibit MDSC function, has been attempted using phosphodiesterase type 5 (PDE-5) (Serafini et al., 2006) or anti-inflammatory drugs like cyclooxygenase-2 (COX-2) inhibitors (Veltman et al., 2010) or synthetic triterpenoid (CDDO-Me; bardoxolone methyl) (Nagaraj et al., 2010). CDDO-Me inhibits immunosuppressive effects of MDSC and improves immune responses in tumor-bearing mice and cancer patients (Nagaraj et al., 2010). Inhibition of MDSC immunosuppression could also be achieved by use of a nanoparticulated vaccine adjuvant, i.e. very small size proteo-liposomes (VSSP), a combination of outer membrane vesicles (OMP) from Neisseria meningitidis with GM3 ganglioside (Ferna´ndez et al., 2011). VSSP seem to be especially attractive candidates for use as a DC vaccine adjuvant since at the same time as enhancing T-lymphocyte immunity they down-regulate MDSC (Ferna´ndez et al., 2011).

Tumor-associated macrophages (TAM) Myeloid-derived suppressive cells (MDSC) MDSC are a subject of intensive studies in the context of their immunosuppressive role in cancer. MDSC are a heterogenous population of immature myeloid mononuclear cells, including precursors of granulocytes, macrophages, and DC. In mice, they are characterized by an expression of CD11bþ i Gr-1þ molecules and in humans by expression of myeloid markers CD33 and CD11b and a lack of markers of lymphocytes, NK cells, monocytes, and DC (Waldron et al., 2013; Youn & Gabrilovich, 2010). MDSC are activated by T-lymphocytes, then inhibit the function of both CD4þ and CD8þ T-lymphocytes, NK cells (through production of arginase-1 and inducible NO synthase [iNOS] in a manner independent of MHC) and induce Treg cell development (Ostrand-Rosenberg & Sinha, 2009). MDSC numbers are increased in patients with different cancers, and these increases correlate with a poor prognosis (Diaz-Montero et al., 2009; Gabitass et al., 2011; Huang et al., 2013; Ochoa et al. 2007; Ohki et al., 2012; Zhang et al., 2013). For these reasons, this is why MDSC have become an important new target in strategies aimed at improving the efficacy of DC immunotherapies. There are three main methods to inhibit MDSC: induction of differentiation, depletion, and inhibition of function. While some methods are still mostly under pre-clinical evaluation, there are also some being evaluated in early clinical studies. For example, both in vitro and in animal models, all trans-retinoid acid (ATRA) has been used to induce MDSC differentiation into mature myeloid cells (Kusmartsev et al. 2008; Mirza et al., 2006). More recently, paclitaxel (at ultra-low non-cytotoxic doses) was shown to stimulate differentiation of MDSC into DC (Michels et al., 2012). Another method to reduce MDSC counts was the use of cytostatic agents like gemcitabine; in a mouse model, this drug was shown to reduce GR-1þ/CD11bþ MDSC levels (Le et al., 2009; Suzuki et al., 2005). Hirooka et al. (2009) reported the first clinical trial involving a combination therapy of gemcitabine during DC immunotherapy. Here, five patients with inoperable locally-advanced pancreatic cancer received a treatment combining gemcitabine, OK432-pulsed DC injected into the tumor, and lymphokine-activated killer (LAK) cells that had been stimulated with anti-CD3 monoclonal antibody (CD3-LAK). The therapy resulted in partial remission with a concurrent increase in levels of tumor antigen-specific CTL in one patient and a stabilizing of the disease (lasting for

TAM, derived from monocytoid precursors recruited to tumor sites (Allavena and Mantovani, 2012; Sica et al. 2012), dampen anti-tumor immunity and promote cancer growth and metastases by inhibiting cancer cell apoptosis and promoting angiogenesis. TAM also release chemokines that attract Treg cells (Liu et al., 2011). As such, TAM are currently considered an important component of a immunosuppressive microenvironment favoring tumor growth. In many cancer types, a correlation has been found between high TAM numbers and poor prognosis (Chen et al., 2011; Kennedy et al., 2013; Medrek et al., 2012; Pin˜a et al., 2010). TAM resemble the so-called M2 population of macrophages stimulated by TH2 cytokines in that they have scavenging activity, stimulate wound healing, and suppress TH1 immune responses; this is in contrast to M1 macrophages, that are stimulated by bacterial products and TH1 cytokines (like IFN ) to enhance the host immune response (Allavena & Mantovani, 2012; Sica et al., 2012). Both oppositely polarized populations are able to be reprogrammed into each other (Allavena & Mantovani, 2012; Sica et al., 2012). Like other regulatory cells, TAM also seems to be a novel potential important target in strategies aimed at improving the efficacy of DC therapies. Preliminary in vitro studies showed that selective depletion of macrophages and TAM could be achieved by use of the cytostatic agent trabectidin (Allavena et al., 2005). Interestingly, bisphosphonates (like zoledronic acid) also induced apoptosis and inhibited proliferation of macrophages, probably because they belong to the same cell lineage as osteoclascts, the main therapeutic target of bisphosphonates (Rogers & Holen, 2011). An alternative approach to depletion could be to re-program TAM into M1-polarized macrophages. Peng et al. (2013) showed that selective TLR7 stimulation with transforming growth factor (TGF)-b inhibition could re-direct TAM towards an M1-like phenotype. Similarly, COX-2 inhibition with celecoxib changed a TAM phenotype from M2 to M1, and concomitantly up-regulated the expression of M1-related IFN (Nakanishi et al., 2011). According to Duluc et al. (2009), IFN could not only elicit a switch among immunosuppressive TAM isolated from ovarian cancer to M1-polarized macrophages, but also directed the differentiation of peripheral blood monocytes attracted to the tumor into M1 macrophages. This suggested a potential use for IFN to augment DC immunotherapies.

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IDO-DC

References

Another population of immunosuppressive cells being targeted in new immunotherapy strategies are so called IDO-DC, DC characterized by an expression of tryptophan catalyzing enzyme 2,3-indoleamine dioxygenase (IDO) (Harden & Egilmez, 2012). A tolerogenic activity of IDO, resulting from tryptophan degradation, is to inhibit proliferation and induce apoptosis of effector T-lymphocytes, while simultaneously stimulating FoxP3þ Treg cell generation (Godin-Ethier et al., 2011). High IDO expression in cancer tissues is associated with lower numbers and impaired function of CD8þ T-lymphocytes and NK cells (Ino et al., 2008; Liu et al. 2009; Wang et al., 2012; Zhang et al., 2011) and, in turn, correlated with a poorer patient prognosis (Inaba et al., 2009; Smith et al., 2012; Yu et al., 2011). A presence of IDO-DC in tumor-draining lymph nodes might contribute to the immunologic unresponsiveness in cancer patients (Godin-Ethier et al., 2011). Therefore, special attention should be paid to observations that expression of IDO might also be induced by agents used with the aim of increasing antitumor response, i.e. adjuvants like TLR9 agonist CpG (Mellor et al., 2005; Wingender et al. 2006). Moreover, it was reported by Wobser et al. (2007) that the use of IL-1b, TNF, IL-6, and PGE2 to stimulate in vitro DC generation resulted in upregulation of IDO expression in DC. On the other hand, inhibition of IDO activity by small molecules analogous to tryptophan, like 1-MT, elicited anti-tumor responses in experimental animal models and enhanced the effects of chemotherapy. By combining 1-MT with DC/tumor cell fusion, this led to an enhanced immune response in lung as well as in breast cancer murine models (Ou et al., 2008). As IDO expression could also be blocked by COX-2 inhibitors like celecoxib (Basu et al., 2006; Lee et al., 2009), this could be an especially attractive approach to use in anti-cancer immunotherapy, as this would lead to a simultaneous inhibition of (except for IDO-DC) T-regulatory cells, TAM, and MDSC.

Abdalla, A. O., Kokhaei, P., Hansson, L., et al. 2008. Idiotype vaccination in patients with myeloma reduced circulating myeloma cells (CMC). Ann. Oncol. 19:1172–1179. Albert, M. L., Sauter, B., and Bhardwaj, N. 1998. Dendritic cells acquire antigen from apoptotic cells and induce Class I-restricted CTLs. Nature 392:86–89. Alfaro, C., Perez-Gracia, J. L., Suarez, N., et al. 2011. Pilot clinical trial of Type 1 dendritic cells loaded with autologous tumor lysates combined with GM-CSF, pegylated IFN, and cyclophosphamide for metastatic cancer patients. J. Immunol. 187:6130–6142. Allavena, P., and Mantovani, A. 2012. Immunology in the Clinic Review Series. Focus on cancer: Tumor-associated macrophages - Undisputed stars of inflammatory tumor microenvironment. Clin. Exp. Immunol. 167:195–205. Allavena, P., Signorelli, M., Chieppa, M., et al. 2005. Anti-inflammatory properties of the novel anti-tumor agent yondelis (trabectedin): Inhibition of macrophage differentiation and cytokine production. Cancer Res. 65:2964–2971. Basu, G. D., Tinder, T. L., Bradley, J. M., et al. 2006. Cyclooxygenase-2 inhibitor enhances the efficacy of a breast cancer vaccine: Role of IDO. J. Immunol. 177:2391–2402. Berd, D., and Mastrangelo, M. J. 1987. Effect of low-dose cyclophosphamide on the immune system of cancer patients: Reduction of T-suppressor function without depletion of the CD8þ subset. Cancer Res. 47:3317–3321. Biagi, E., Rousseau, R., Yvon, E., et al. 2005. Responses to human CD40 ligand/human IL-2 autologous cell vaccine in patients with B-cell chronic lymphocytic leukemia. Clin. Cancer Res. 11: 6916–6923. Bodey, B. 2002. Spontaneous regression of neoplasms: New possibilities for immunotherapy. Expert Opin. Biol. Ther. 2:459–476. Bose, A., Taylor, J. L., Alber, S., et al. 2010. Sunitinib facilitates the activation and recruitment of therapeutic anti-tumor immunity in concert with specific vaccination. Int. J. Cancer. 129:2158–2170. Chen, J., Yao, Y., Gong, C., et al. 2011. CCL18 from tumor-associated macrophages promotes breast cancer metastasis via PITPNM3. Cancer Cell 19:541–555. Chu, C. S., Boyer, J., Schullery, D. S., et al. 2012. Phase I/II randomized trial of dendritic cell vaccination with or without cyclophosphamide for consolidation therapy of advanced ovarian cancer in first or second remission. Cancer Immunol. Immunother. 61:629–641. Curiel, T. J., Coukos, G., Zou, L., et al. 2004. Specific recruitment of regulatory T-cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat. Med. 10:942–949. Dannull, J., Su, Z., Rizzieri, D., et al. 2005. Enhancement of vaccinemediated anti-tumor immunity in cancer patients after depletion of regulatory T-cells. J. Clin. Invest. 115:3623–3633. del Giudice, I., Chiaretti, S., Tavolaro, S., et al. 2009. Spontaneous regression of chronic lymphocytic leukemia: Clinical and biologic features of 9 cases. Blood 114:638–646. Diaz-Montero, C. M., Salem, M. L., Nishimura, M. I., et al. 2009. Increased circulating myeloid-derived suppressor cells correlate with clinical cancer stage, metastatic tumor burden, and doxorubicincyclophosphamide chemotherapy. Cancer Immunol. Immunother. 58: 49–59. Dietl, J., Engel, J. B., and Wischhusen, J. 2007. The role of regulatory T-cells in ovarian cancer. Int. J. Gynecol. Cancer 17:764–770. Dighe, A. S., Richards, E., Old, L. J., and Schreiber, R. D. 1994. Enhanced in vivo growth and resistance to rejection of tumor cells expressing dominant negative IFNg receptors. Immunity 6:447–456. Duluc, D., Corvaisier, M., Blanchard, S., et al. 2009. IFNg reverses the immunosuppressive and pro-tumoral properties and prevents the generation of human tumor-associated macrophages. Int. J. Cancer 125:367–373. Ellebaek, E., Engell-Noerregaard, L., Iversen, T. Z., et al. 2012. Metastatic melanoma patients treated with dendritic cell vaccination, IL-2, and metronomic cyclophosphamide: Results from a Phase II trial. Cancer Immunol. Immunother. 61:1791–1804. Ferna´ndez, A., Mesa, C., Marigo, I., et al. 2011. Inhibition of tumorinduced myeloid-derived suppressor cell function by a nanoparticulated adjuvant. J. Immunol. 186:264–274. Fields, R. C., Shimizu, K., and Mule, J. J. 1998. Murine dendritic cells pulsed with whole-tumor lysates mediate potent anti-tumor immune

Conclusions Dendritic cell (DC) vaccines still seem a promising approach in anti-cancer immunotherapy, although results of most previous clinical studies have not met expectations, even despite induction of immune responses. Recent years have brought much interesting data about the regulatory cells responsible for induction of immunosuppression in/around a tumor microenvironment that, in turn, allows the transformed cells to escape immunosurveillance and so lead to cancer growth and expansion. Among the most studied cells are the T-regulatory cells, myeloid-derived suppressive cells, tumor-associated macrophages, and IDO-DC. Their individual biology and roles in cancer is currently a subject of intense study, and many novel strategies to reverse the immunosuppression via regulatory cell depletion or inhibition of their function are under development. Ultimately, approaches that temper the down-regulation of immune functions in cancer-bearing hosts would also likely significantly improve the efficacy of DC immunotherapies in the near-term. Such changes would then be clearly evident in clinical studies as well.

Declaration of interest The work was supported by research grant: Development Grant GR831 from State Funds for National Centre for Research and Development (NCBR). The funder had no role in study design, decision to publish, or preparation of the manuscript. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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