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Nov 24, 2013 - Cellular and molecular pathways in the tumor immunoenvironment: 3rd Cancer Immunotherapy and Immunomonitoring (CITIM) meeting, ...

Cancer Immunol Immunother (2014) 63:73–80 DOI 10.1007/s00262-013-1501-z

Meeting Report

Cellular and molecular pathways in the tumor immunoenvironment: 3rd Cancer Immunotherapy and Immunomonitoring (CITIM) meeting, 22–25 April 2013, Krakow, Poland Michael R. Shurin · Viktor Umansky · Anatoli Malyguine · Arthur A. Hurwitz · Ron N. Apte · Theresa Whiteside · Anahid Jewett · Yasmin Thanavala · William J. Murphy  Received: 1 October 2013 / Accepted: 10 November 2013 / Published online: 24 November 2013 © Springer-Verlag Berlin Heidelberg 2013

Keywords  Cancer · Immunotherapy · Immunomonitoring · Tumor microenvironment · CITIM

Introduction The treatment of cancer using the body’s own immune system has long been a goal of both tumor immunologists and oncologists. Given the complex interactions between immune effector and regulatory cells in the tumor microenvironment, it is reasonable that understanding the pathways responsible for both induction and suppression of antitumor immunity would be a crucial step in designing efficacious and feasible approaches to cancer immunotherapy. This meeting report is a summary of presentations from the Third International Conference on Cancer Immunotherapy and Immunomonitoring, CITIM-2013, published together with a series of Focussed Research Reviews based on lectures given at the conference.

Revealing and harnessing the bidirectorial interactions between multiple immune cell subsets at the tumor site was the primary focus of 3rd International Conference “Cancer Immunotherapy and Immunomonitoring (CITIM)” which was held in Krakow, Poland, in April 2013. CITIM-2013 Conference attracted about 200 participants from the USA, Europe, South America and Asia and provided a platform for discussions of more than 40 full and short presentations by experts and young investigators from the USA, Germany, Italy, France, the Netherlands, Belgium, Spain, Poland, Czech Republic, Hungary, Denmark, Sweden, Norway, Israel, Russia, the UK and China. The numerous discussions following each presentation and during session breaks as well as many new collaborations that were formed confirmed the great success of CITIM series of conferences.

M. R. Shurin · T. Whiteside  University of Pittsburgh Medical Center, Pittsburgh, PA, USA

A. A. Hurwitz  NIH, Frederick, MD, USA

M. R. Shurin · T. Whiteside  University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

R. N. Apte  Ben Gurion University of Negev, Beersheba, Israel

V. Umansky (*)  Skin Cancer Unit, German Cancer Research Center, INF 280, 69120 Heidelberg, Germany e-mail: [email protected]

A. Jewett  Jonsson Comprehensive Cancer Center, UCLA School of Dentistry and Medicine, Los Angeles, CA, USA

V. Umansky  University Medical Center Mannheim, Ruprecht-Karl University of Heidelberg, Heidelberg, Germany A. Malyguine  Frederick National Laboratory for Cancer Research, SAICFrederick Inc., Frederick, MD, USA

Y. Thanavala  Roswell Park Cancer Institute, Buffalo, NY, USA W. J. Murphy  School of Medicine, University of California Davis, Davis, CA, USA



Immunity and the malignant process While considerable attention has been paid to the microenvironment of developing primary tumors over the past several years, considerably less is known about the metastatic microenvironment. In the Keynote Presentation, I. Witz (Tel Aviv University, Tel Aviv, Israel) presented his laboratory’s studies on the interactions between tumor cells and distant metastatic sites. In a model of neuroblastoma, tumors form metastases in the lung with varying degrees of seeding efficiency, and the gene expression patterns differed based on the success of metastasis. In tumors which form dormant micro-metastases that develop slowly, upregulation of genes associated with cell death and growth arrest predominated. In contrast, larger, macro-metastatic tumors expressed genes associated with invasion (MMPs) and survival (hexokinase, MAPK). In a melanoma brain metastasis model, diminished expression of Claudin I (a structural protein involved in tight junction formation) was associated with increased metastatic potential, and over-expression of Claudin I reduced brain metastasis and improved survival in mouse models. Taken together, these studies demonstrated the intricate balance between tumor cells looking for residence (or “niche”) in distant tissues and the role of parenchymal cells, which can regulate tumor survival and invasion. In his Keynote Presentation, R. Wiltrout (NCI, Bethesda, MD, USA) described novel models in which hepatocellular carcinoma (HCC) was induced by hydrodynamic delivery of oncogenic transgenes. Delivery of c-met and activated β-catenin resulted in an aggressive phenotype, whereas AKT and catenin initially presented with a steatotic adenoma phenotype, which progresses following serial passage. Elegant studies with mice deficient in various immune and inflammatory parameters demonstrated a role for B cell-derived cytokines and cytokine responsiveness in tumor formation. These models will undoubtedly be useful for future studies of immune-based therapies and the role of inflammatory mediators in tumor formation. New data from the laboratory of W. H. Fridman (Cordeliers Research Centre, Paris, France) reported in his Keynote Presentation have also revealed the importance of immune responses to cancer. This group reported that the reactivity of T cells infiltrating colorectal tumors was correlative of a favorable prognosis, findings which led to the “Immunoscore” for grading tumors. More recent results extended this finding to the lung metastases from colorectal tumors, showing that tumor infiltration with CD8+ and DCLamp+ cells can be predictive of longer survival. In renal cell carcinoma, a different profile may exist, where CD8+ and DC-Lamp+ cells were predictive of poor outcome and NK cells numbers correlate with more favorable outcomes. Further studies in lung cancers suggested that following


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chemotherapy, patient prognosis was most favorable when the tumor viability was low and when tumors contained infiltrating DC-Lamp+ cells. Studies from the laboratory of Y. Thanavala (Roswell Park Cancer Institute, Buffalo, NY, USA) also demonstrated the presence of FOXP3+GARP+CTLA-4+ regulatory T cells (Tregs) as well as myeloid-derived suppressor cells (MDSCs) in hepatocellular carcinoma patients and that targeting these cells may improve the clinical outcome. Studies reported by C. Sautès-Fridman (Cordeliers Research Centre, Paris, France) indicated the importance of the presence of tertiary lymphoid structures (TLS) containing DC-Lamp+ in lung tumors for the development of a coordinated local Th1CD8+ T cell responses. These findings may be critically important as prognostic indicators, and they may provide a basis for determining the patients with the greatest potential for successful immune-based therapies. In his talk, W. Zou (University of Michigan, Ann Arbor, MI, USA) shared novel results on “stemness” characteristics in both T cells and tumor cell subpopulations. Th17 cells promoted potent antitumor immunity when transferred with CD8+ T cells and also possessed stem cell-like properties, expressing Nanog/Sox3/Oct3, being long-lived and apoptosis-resistant. It was also demonstrated that MDSCs could promote stem cell-like properties in tumor cells (i.e., STAT3 and Notch activation) and induce the expression of MiR101, which can regulate “stemness.” These studies lay the groundwork for understanding how MDSCs, which regulate tumor immunity, can also control tumor self-renewal. Studies from the laboratory of E. Yefenof (Hebrew University, Jerusalem, Israel) demonstrated an intriguing response to glucocorticoids. Hematopoietic tumors, which respond to glucocorticoids, up-regulated a pro-apoptotic profile dominated by the expression of miR-103, which promoted apoptosis, and led to elevated expression of Bim and reduced levels of Myc. A link between glucocorticoidinduced regulation of GSK3 activity and Myc down-regulation may serve as a novel target for promoting apoptosis in hematopoietic malignancies. Tumors can evade the recognition by T cells through limiting expression of antigen/MHC molecules on their surface. That was the theme of the presentation by the laboratory of F. Garrido (University Hospital Virgen de las Nieves, Granada, Spain) who previously described two types of MHC lesions affecting expression: “soft,” where MHC expression can be recovered and “hard,” where expression is permanently extinguished. They further described a unique connection between the MHC expression and oncogenicity in the fibrosarcoma model where there was an inverse correlation between MHC levels and primary tumor formation. Studies from the laboratory of B. Seliger (University Halle-Wittenberg, Halle, Germany) also examined MHC Class I “abnormalities.” Genes

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associated with antigen presentation were dysregulated in tumors, and this may be controlled at the level of oncogenes and tumor suppressor genes. Expression of MHC by tumors also may be under the control of distinct signaling pathways. The link between T cell responsiveness and metastasis is an important area and may be relevant for the ongoing clinical trials that target metastatic lesions with ex vivo activated T cells.

Cancer, inflammation and immunosuppression The issue of contribution of inflammation to carcinogenesis and tumor invasiveness was discussed at length during the meeting. Excessive inflammation appears primarily mediated by different myeloid cell subsets and promotes tumor invasiveness by enhancing the proliferation of malignant cells, promoting angiogenesis, invasiveness, tumor cell migration and dissemination to remote organs. In the target organs, inflammatory molecules produced by the primary tumor “prepare” the pre-metastatic niche, which serves as the “soil” for the “seeded” malignant cells that facilitates metastasis formation. Inflammation also contributes to the malignant phenotype by inducing tumor-mediated immune suppression, primarily mediated by MDSCs and Tregs. S. Ostrand-Rosenberg (University of Maryland Baltimore County, Baltimore, MD, USA) in her Keynote Lecture summarized the current knowledge of MDSC biology. She emphasized that MDSCs are immunosuppressive also by the induction of other regulatory cells such as Tregs, pro-tumorigenic M2 macrophages and possibly regulatory dendritic cells (DCs). She suggested that HMBG1 is a common inducer of inflammation at the tumor site. When released from necrotic cells, HMBG1 acts as an alarmin and initiates inflammation. HMBG1 signaling also leads to the secretion of a cascade of pro-inflammatory molecules, such as IL-1β, TNF-α, IL-6 and angiogenic factors. R. Apte (Ben Gurion University, Beer-Sheva, Israel) described a novel cross talk between IL-1β and VEGF that is active in initiating tumor-mediated angiogenesis. IL-1β is secreted by MDSCs in the tumor microenvironment, where endothelial cells secrete VEGF after being activated by IL-1β. Inhibition of either IL-1β or VEGF completely abrogated the angiogenic response at the tumor site. Furthermore, E. Voronov (Ben Gurion University, Beer-Sheva, Israel) has demonstrated the distinct role of the IL-1 agonistic pathways in colon inflammation using a mouse model of colitis. In IL-1Ra-deficient mice, which lack the inhibitor of the IL-1R signaling receptor (IL-1R1), tumors were induced only after chronic DSS application, indicating that excessive IL-1 can induce colon chronic inflammation, in which MDSCs were dominant and display the potential to propagate colon carcinogenesis.


M. Baniyash (Hebrew University, Jerusalem, Israel) emphasized the role of another “alarm” cytokine—TNF-α in the MDSC biology. She reported that TNF-α in the tumor microenvironment induced MDSC-mediated inflammatory responses, which could be abrogated by Etanercept, a TNF-α antagonist. Etanercept administration reduced the immunosuppressive function of MDSCs and induced their maturation to functional macrophages and DCs that were devoid of suppressive activity. Interestingly, MDSC infiltration in the inflamed colon predisposed mice to tumor development, whereas the depletion of MDSCs reduced the frequency of colon tumor development and induced shrinkage and even a disappearance of tumors. To delineate the mechanisms of MDSC generation in humans, R. Kiessling (Karolinska Institute, Stockholm, Sweden) has devised a system of incubation of CD14+ monocytes from healthy donors with freshly isolated melanoma cell lines, which resulted in the appearance of myeloid cells resembling MDSCs. COX-2 and STAT3 signaling were essential for the suppressive activity of the in vitro generated MDSCs, suggesting that antagonizing COX-2 and STAT3 signaling may serve as a novel mean for inhibiting tumor-mediated suppression. A. Ben-Baruch (Tel Aviv University, Tel Aviv, Israel) discussed the role of inflammatory cytokines IL-1β and TNF-α in the invasiveness of breast cancer. Around 90 % of tumors obtained from patients with relapsed disease coexpressed both cytokines. Tumor-derived IL-1β and TNF-α promoted the release of pro-tumoral chemokines by cancer cells and stroma cells, acting in concert with oncogenic signals such as hormones and growth factors to activate cell remodeling in the malignant cells that amplify tumor progression. V. Umansky (German Cancer Research Center, Heidelberg, Germany) reported a potentially new pathway of “educating” CD11b+Gr1+-immature myeloid cells from normal mice and converting them into MDSCs by the treatment with exosomes derived from melanoma cells. This resulted in prolonging the MDSC lifespan, higher expression of arginase (ARG)-1 and production of nitric oxide (NO) as well as in acquiring immunosuppressive activity both in vitro and in vivo. Similarly, M. Zembala (Jagiellonian University Medical College, Krakow, Poland) used microvesicles derived from tumor cells and recovered from the plasma of patients with solid tumors. He presented data that they could activate tumor cells and cells from the microenvironment, resulting in broad effects on tumor progression. The impact of immunotherapy in aged versus adult tumor-bearing mice was studied by W. Murphy (UC Davis Health System, Sacramento, CA, USA). In aged mice, immunotherapy with IL-2 combined with antiCD40 monoclonal antibodies (mAbs) induced a systemic cytokine storm followed by multiple organ failure



concomitantly with an increase in TNF-α level in serum and tissue. Macrophages from aged mice were also more potent than cells from young mice in secretion of proinflammatory cytokines such as TNF-α and IL-6, and depletion of macrophages could result in protection from toxicity. In addition, aged mice contained large amounts of visceral fat tissue compared to younger counterparts, which was responsible for the production of pro-inflammatory cytokines contributing to the cytokine storm following immunotherapy. In young obese mice (Ob/Ob), an increase in pro-inflammatory cytokines was observed after immunotherapy but with lesser pathology, indicating that fat and age act in synergy to induce multiple organ failure following therapy. Taken together, the reported data showed that the regulation of the tumor microenvironment by different cellular pathways (both normal and neoplastic) plays a key role in tumor progression and immune evasion. M. Shurin (University of Pittsburgh Medical Center, Pittsburgh, PA, USA) analyzed how acute bacterial infection that occurs during tumor development could affect the tumor immunoenvironment and how it altered myeloid and lymphoid regulatory cells. In tumor-bearing mice infected with Ehrlichia muris, a significant reduction in tumor burden in the lungs with a concomitant increase in the mouse survival was observed. This was associated with the down-regulation of both MDSC and Treg numbers and an increase in activated CD8+ T cells co-expressing granzyme B and IFN-γ. Thus, pathogens that infect tumor-bearing hosts might dramatically affect the course of tumor progression in part due to immune modulation.

Regulatory cells and tumor‑induced immunosuppression Tumor-induced immunosuppression remains a significant problem in cancer, which confounds clinicians and interferes with the efficacy of immune therapies. Among the broad variety of mechanisms that tumors utilize to disarm the host immune system, Tregs and MDSCs have been most extensively investigated recently. T. Whiteside (University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA) has described inducible (i) Tregs expressing ectonucleotidases, CD39 and CD73, which hydrolyze ATP to 5′-AMP and adenosine (ADO) and thus suppress functions of leukocytes expressing ADO receptors. Other subsets of iTregs may preferentially produce PGE2, TGF-β or IL-10. While iTregs accumulating in solid tumors are thought to be responsible for contributing to tumor escape, in cancers characterized by an intense inflammatory reaction, they may protect the host from tumor progression. In this context, P. Kraj (Georgia Health Sciences University, Augusta,


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GA, USA) found that Bone Morphogenic Protein Receptor 1α (BMPR1α, Alk-3, a member of the TGF-β family) modulated functions of effector T cells as well as Tregs and regulated thymic development of T lymphocytes. Mice in which BMPR1α was deleted had fewer Tregs, more active effector T cells, and tumors grew slowly. BMPR1α might control the migration of Tregs to tumors, and its inhibitors might be useful in immunotherapy. MDSCs play a detrimental role in cancer due to immunosuppression, and as reported by I. Kryczek (University of Michigan School of Medicine, Ann Arbor, MI, USA), they also activate cancer stem cells (CSCs) in breast carcinoma. MDSCs can initiate STAT3 phosphorylation via IL-6, sustain its prolonged activation and also cross talk with the Notch pathway. The MDSC frequency and activity correlate with CSC numbers and could predict survival of breast cancer patients. G. Shurin (University of Pittsburgh Medical Center, Pittsburgh, PA, USA) reported that CD11b+Gr1+ MDSCs in the tumor environment can differentiate into regulatory DCs (regDCs) in mice. These regDCs, when co-cultured with activated T cells, became strongly immunosuppressive. Their polarization was mediated by the small Rho GTPase signaling and could be interrupted by ultralow doses of paclitaxel. HspB5 or αβcrystallin was found by A. Dimberg (Rudbeck laboratory, Uppsala, Sweden) to be expressed on CD11b+Gr1+ immature myeloid cells. This may serve as a marker for these cells, regulating their systemic expansion. Beyond cytotoxicity, NK cells can also regulate immune responses, and the concept that CD16+CD56dimCD69neg NK cells mediate antitumor and anti-CSC cytotoxicity, while CD16dim/negCDCD56dim/+CD69+ NK cells serve as regNK was introduced by A. Jewett (UCLA School of Dentistry and Medicine, Los Angeles, CA, USA). These regNK cells modulate inflammatory responses and tissue regeneration by the promotion of cellular differentiation and subsequent resistance of differentiated cells to NK and T cell-mediated cytotoxicity. One of the important questions about the accumulation of regulatory immune cells within the tumor concerns the mechanisms responsible for their homing and functions in situ. Using murine lung cancer model, D. Gutkin (University of Pittsburgh School of Medicine, Pittsburgh, PA, USA) demonstrated that the inhalation of nonmaterials accelerated immune cell infiltration and growth of Lewis lung carcinoma cells in vivo. Exposure to carbon nanotubes, which accelerated tumor growth, was mediated by MDSCs and TGF-β, emphasizing the critical role of these cells and their products in promoting tumor progression. It is now apparent that the tumor microenvironment, including its metabolic phenotype such as hypoxia or acidity, can affect the extent of immune suppression. C. Castelli (Istituto Nazionale Tumori, Milan, Italy) reported

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that tumor acidosis accompanied by the accumulation of carbonic anhydrase IX provided a selective growth advantage to tumors at the expense of infiltrating T cells, which cannot survive in this hostile environment. Several presentations dealt with the potential strategies for altering tumor microenvironment to make it more permissive to immune cell activity. A. Hurwitz (NCI, NIH, Frederick, MD, USA) considered the generation and adoptive transfer of high-avidity CD8+ T cell receptor (TCR)hi T cells that could resist detrimental factors in the tumor environment. However, his data clearly showed in mice that such highavidity T cells were easily tolerized and were not effective in eliminating tumor cells. Adoptive transfer of genetically engineered T cells was described by M. Essand (Uppsala University, Uppsala, Sweden), who has cloned a TCR recognizing the TARP4–13 epitope expressed in normal prostate epithelium, prostate and breast adenocarcinomas. He also produced T cells with a 3rd generation CAR recognizing PSCA and successfully used these cells for therapy of PSCA-expressing tumors in nude mice. Numerous immunotherapy approaches aimed at altering tumor microenvironment were discussed. L. Vannucci (Institute of Microbiology, Prague, Czech Republic) focused on cytokine-induced changes in the collagen architecture and immunological responses in the microenvironment of colorectal carcinoma and melanoma. The collagen- and tumor-targeting agent that was used consisted of human ferritin nanoparticles decorated with melanocytestimulating hormone. In mice with B16F10 melanoma, this type of targeting resulted in high uptake of nanoparticles by the tumor cells, suppressing tumor growth. Other immunotherapeutic strategies included the use of Vγ9 Vδ2 T cells for recognition and elimination of malignant B cells in multiple myeloma or CLL. M. Massaia (San Giovanni Battista Hospital, Torino, Italy) demonstrated these cells can kill tumor cells via TCR-dependent and TCRindependent mechanisms. However, these cells represent a small subset of lymphocytes (1–5 %) and are especially sensitive to tumor-induced suppression in the bone marrow microenvironment which may limit clinical utility.

Novel strategies for tumor immunotherapy—preclinical studies Using an orthotopic mouse tumor models, J. Weiss (NCI, NIH, Frederick, MD, USA) reported that the treatment with IL-2 in combination with anti-CD40 mAbs, but not IL-2 or anti-CD40 mAbs alone, resulted in significant antitumor activity and induced NO synthase (NOS2) expression within the tumor microenvironment through an IFNγ-dependent pathway. The NOS2 inhibition in the treated mice completely abrogated the ability of immunotherapy


to reduce lung metastases but had no effect on its capacity to reduce primary kidney tumors. He further demonstrated that IL-2/anti-CD40 mAbs induced an NO-dependent decrease in the matrix metalloproteinase (MMP) expression and activity, concomitant with an increase in the expression of tissue inhibitor of metalloproteinase (TIMP)-1 within the tumor microenvironment. P. Beckhove (German Cancer Research Center, Heidelberg, Germany) reported that appropriately activated tumor-associated macrophages (TAMs) represent an important step in the effective recruitment and in situ activity of tumor-specific T cells. The use of spontaneous tumor mouse models indicated that the activation of TAMs in situ through local irradiation resulted in profound changes of the tumor vasculature, leading to an efficient effector T cell recruitment and tumor rejection. He further demonstrated that these events were mediated by inducible NO synthase. To improve a genetically attenuated Salmonella Typhimurium strain VNP20009 for potential tumor targeting, J. Bereta and colleagues (Jagiellonian University, Krakow, Poland) expressed the carcinoembryonic antigen (CEA)-specific single chain antibody fragment (scFv) on the bacteria surface together with an additional copy of the gene coding for SipB, a pro-apoptotic protein. They observed that the presence of anti-CEA scFv on the surface of Salmonella improved bacterial targeting of CEA-positive tumor cells. In addition, an increased SipB expression led to the enhanced apoptosis of infected tumor cells and macrophages. L. Eisenbach (Weizmann Institute of Science, Rehovot, Israel) demonstrated an interesting method, which allowed for an increase in the affinity of the TCR to its ligand by the induction of somatic hypermutation directed by the mutator enzyme activationinduced cytidine deaminase (AID). Affinity maturation reactions were performed ex vivo on transfectable cells, and affinity-maturated TCRs were selected by the tetramer staining followed by FACS sorting. The affinity-maturated TCRs were used to create antitumor reactive T cells, which were functionally tested in vitro and in vivo using murine melanoma models and demonstrated greater activity. Y. Keisari (Tel Aviv University, Tel Aviv, Israel) presented two in situ tumor ablation treatments for solid tumors. The first treatment, pulsed electric current tumor ablation (PECTA), was applied through electrodes and cured 100 % of tumor-bearing mice. The second treatment, diffusing alpha-emitters radiation therapy (DaRT), was based on insertion of 224Ra-loaded wires into the tumor. He concluded that the ablation of primary antigenic tumors by either PECTA or DaRT released tumor-derived antigenic material that induced T cell-dependent antitumor immunological responses. U. Gaipl (University Hospital Erlangen-Nürnberg, Erlangen, Germany) presented the use of the combination of radiotherapy and chemotherapy (CRT) to induce



immune activation. Supernatants from melanoma cells, which were treated with CRT plus hyperthermia as immune stimulator and the pan-caspase inhibitor zVAD-fmk as cell death modulator, could influence the activation of antigenpresenting cells. Furthermore, the experiments where local radiotherapy was combined with autologous tumor vaccines generated by high hydrostatic pressure technology were presented. He concluded that the induction of antitumor immune responses by standard tumor therapies often required additional immune stimulation and/or tumor cell death induction. W. Lasek (Medical University of Warsaw, Warsaw, Poland) presented a promising IL-12-based immunotherapy, in which immortalized C57BL/6 murine bone marrow-derived DCs and IL-12 were combined in a mouse melanoma model. The vaccination with these DCs pulsed with tumor-cell lysates in combination with IL-12 was superior to the treatment with either agent alone and led to the tumor eradication in more than 50 % of mice.

New approaches and targets for immunotherapy of cancer patients With regard to adoptive immunotherapy strategies involving the transfer of effector cells, researchers reported new approaches and indicated improved results. P. thor Straten (University Hospital Herlev, Herlev, Denmark) presented the transfer of tumor-infiltrating lymphocytes (TIL) in melanoma patients, who were injected with lower doses of IL-2, resulting in high response rates with lower side effects. Similarly, a presentation by B. Chmielowski (UCLA, Los Angeles, CA, USA) used MART-1 TCR transgenic lymphocyte transfer in patients with melanoma to improve the targeting and efficacy. While responses were observed, significant toxicities were also observed. The feasibility of the ex vivo transduction of the T cells for therapy was also demonstrated. A. Lange (Institute of Immunology and Experimental Therapy, Wroclaw, Poland) presented data, in which the donor lymphocytes following an allogeneic bone marrow transplant were given via intrabone injections in a patient with acute lymphoblastic leukemia (ALL), and a significant bone lesion regression was observed. W. Wei (Sun Yat-sen University Cancer Center, Guangzhou, China) presented results of a clinical trial, in which cytokine-induced killer cells (CIK) were given to patients with hepatocellular carcinoma following resection. Adverse events were minimal; however, no differences in disease-free survival were observed. G. Anderson (University Hospital Herlev, Herlev, Denmark) presented intriguing preclinical data on the use of a γδT cell population that can act as both antigen-presenting cells and killer cells. Thus, while adoptive cellular therapy has a promise, better targeting and the reduction in side effects are still needed to be optimized.


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Another approach that has been very promising in cancer immunotherapy involved the vaccination although the efficacy of such approaches needs to be improved. Various speakers discussed the latest results in improving efficacy in a number of cancers. G. Chatta (Virginia Mason Medical Center, Seattle, WA, USA) presented an update of several approaches being explored to assess the efficacy of vaccines targeting various tissue-specific proteins in prostate cancer and ways to overcome the significant barrier of immunosuppression. Similarly, J. Rolinski (Medical University of Lublin, Lublin, Poland) reported that immunosuppression represented a major barrier impeding vaccination efficacy and that inhibiting inflammatory processes and Treg effects could improve clinical results. B. Kotlan (National Institute of Oncology, Budapest, Hungary) discussed a new approach to identify antigen targets in melanoma by using punch biopsy specimens. This strategy made feasible the selection of cancer-initiating cells in metastatic melanomas by providing novel markers for this minor cell population and making possible their further characterization by gene profiling. A presentation by A. Mackiewicz (University of Medical Sciences, Poznan, Poland) dealt with a novel vaccine for melanoma using the over-expression of IL-6 as a tool to promote cancer stem cell targeting. Six clinical trials consisting of 492 patients were reported with a 57 % response rate. These results suggest that cancer stem cells targeting may provide new opportunities in cancer therapy. E. Tartour (Hôpital Européen Georges Pompidou, Paris, France) reported that the route of vaccine administration can impact the localization of immune responses. Using a vaccine administered intranasally, he demonstrated greater lymphocytic infiltration in mucosal tumors compared to an intramuscular administration due to differential expression of integrins on the CD8+ T cells. S. van der Burg (Leiden University Medical Centre, Leiden, The Netherlands) presented results of clinical trial using HPV vaccines in cancer. Upon treatment with the HPV16 E6/ E7 peptide vaccine, immune responses were observed but clinical regressions of the tumor were not, suggesting that immune suppression barriers need to be overcome. Overall, the results suggested that vaccine approaches need to take into consideration the presence of cancer stem cells, to increase immune responses toward the vaccine antigens, allowing sustained tumor infiltration with immune sites and to overcome the immunosuppression at the tumor site.

Novel techniques and immunomonitoring in clinical trials and preclinical studies The conference provided a great opportunity for attendees to learn about novel techniques and immunomonitoring strategies employed in clinical trials and preclinical studies.

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The presentation by A. Malyguine (NCI, Frederick, MD, USA) highlighted not only the importance of immunomonitoring during clinical trials of cancer vaccines but also that the measurement of any single immunological parameter was insufficient and could not represent the complex interactions between different tumor and immune cells which impacts efficacy. Additional efforts are also needed to better characterize the phenotypic, biochemical and genetic characteristics of tumor-infiltrating lymphocytes. Collectively such approach should provide a better correlation between clinical endpoint and immune responses. G. Pawelec (University of Tübingen Medical School, Tübingen, Germany) discussed their data on immunological correlates associated with long-term survival of late-stage melanoma patients regardless of the treatment. In peripheral blood mononuclear cells (PBMCs) from 84 patients with stage IV melanoma evaluated before the treatment, the presence of T cells responding to NY-ESO-1 and/or Melan-A correlated with superior survival at 5-year followup. However, this correlation did not exist for MAGE-3 or survivin-reactive T cells. The phenotype of T cells responding to NY-ESO-1 was mainly CD4+, and those responding to Melan-A CD8+. In a separate study, it was found that the presence of NY-ESO-1-reactive T cells together with low percentages of MDSCs was predictive of long-term survival, and this combination tended to be better than either biomarker alone. Preliminary evidence of successful treatment of patients with ipilimumab being associated with lower levels of MDSCs was also discussed. Methods that can predict the immunogenicity of cancer vaccine antigens would certainly be valuable, and this was the theme of the presentation by S. Walter (Immatics biotechnologies GmbH, Tübingen, Germany). He discussed the merits of a novel in vitro platform that they have developed. The technique uses artificial antigen-presenting cells to prime CD8+ T cells in vitro against tumor-associated peptides (TUMAPs) that are potential candidate cancer vaccine epitopes. The method was utilized to validate three multi-peptide vaccines for renal cell cancer, colorectal cancer and glioblastoma. Out of four Phase I-II studies in 211 patients, the in vivo responses represented by the percentage of patients with vaccine-induced responses correlated with the in vitro immunogenicity of the TUMAPs in three trials. A. Porgador (Ben Gurion University, Beer Sheva, Israel) discussed in his presentation the critical role of NK cells in the host defense against cancer. He presented that proliferating cell nuclear antigen (PCNA), which is highly expressed in proliferating cells including cancer cells, interacts with NKp44, and this result in the inhibition of NK cell function. This paradoxical reduction in NK cell function allowed an increased tumor survival and was achieved through the ITIM motif present in the cytoplasmic domain


of NKp44. Since PCNA contributes to several survival processes, the over-expression of PCNA by tumor cells correlates with increased malignancy. The interaction of PCNA with NKp44 and the resulting inhibition of NK cell activity may be another mechanism by which PCNA facilitates immune evasion and tumor survival. The presentation by P. Lehmann (Cellular Technology Limited, Shaker Heights, OH, USA) was focused on the analysis of the discrepant results that were obtained when trying to equate T cell-monitoring data and clinical outcome. Parameters that help define the ability of T cells to efficiently kill tumors include effector cell lineage, the avidity of T cells for the tumor antigen and the nature and magnitude of the cytokine response. Additionally, since determinant or epitope spreading occurs as the response progresses, the responses to vaccine-unrelated tumor antigens also need to be evaluated. The cross talk that occurs between both immune and cancer cells was discussed in a presentation by F. Mattei (Istituto Superiore di Sanità Viale, Rome, Italy). In order to visualize these interactions in real time, they employed a cell-on-chip system that is comprised of three wide, parallel fluidic channels connected by short capillary microchannels. The microfluidic devices were loaded with spleen cell suspensions from wild-type or IRF-8 knockout mice and from B16 melanoma cells The tumor cells migrated in the presence of spleen cells from IRF 8 KO mice, suggesting a more invasive phenotype. The presentation by D. Klysz (IGMM-UMR 5535-CNRS, Montpellier, France) stressed that all lymphopenia-inducing regimens are not equal in their ability to support T cell expansion and effector T cell differentiation. They found that rendering mice lymphopenic by sublethal irradiation resulted in a skewed population of donor CD8+ T cells, whereas the use of a chemotherapy regimen involving bisulphan/cyclophosphamide induced IL-7-independent proliferation of donor CD4+ T cells, which was also associated with expansion of transferred Foxp3+ Treg cells. The presentation by V. Baldan (University of Manchester, Manchester, United Kingdom) dealt with issues pertaining to the successful expansion of TILs for adoptive immunotherapy in cancer patients. The protocol involved the enzymatic digestion of tumor samples using the gentle MACS dissociator. Activating beads coated with anti-CD3 and anti-CD28 antibodies were used to expand the TILs followed by the expansion with IL-2 treatment.

Concluding remarks The primary goals of CITIM-2013 Conference were the presentation of new insights into the mechanisms of neoplastic, stromal and immune cell interactions in the tumor



microenvironment and extensive discussions of recent results of preclinical and clinical testing of innovative immunotherapeutic approaches. An additional emphasis was made on evaluating the role of immunomonitoring of patients with cancer receiving different therapeutic modalities in predicting the efficacy of immunotherapies and patient selection for specific therapeutic interventions. Wide-ranging discussions of new data and ideas, as well as intensive communications between researchers and clinicians, should support and hasten further development and evaluation of efficient therapeutic strategies for cancer treatment. For instance, the feasibility of targeting immune regulatory cell activity by the neutralization of immunosuppressive pathways within the tumor milieu should be more extensively evaluated in the preclinical and clinical settings. Potential clinical significance of this and other


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directions will be discussed at CITIM-2015 Conference (www.CancerITIM.org). Acknowledgments  We thank CTL (Cellular Technology Ltd), Europe GmbH, Bonn, Germany; GTCbio (Global Technology Community), Monrovia, CA, USA; Immatics Biotechnologies GmbH, Tübingen, Germany; PIVAC; Springer Science + Business Media B. V.; Cancer Immunology and Immunotherapy; The Journal of Immunotoxicology; Targi w Krakowie, Krakow, Poland; Lab-JOT Ltd, Poland; and Jagiellonian Centre of Innovation, Poland, for their generous support of the CITIM meeting. Anatoli Malyguine has been funded by the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Conflict of interest The authors state no conflict of interests.

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