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Invasion, immune modulation, and angiogenesis are crucial in melanoma progression. Studies based on animals or two-dimensional cultures poorly recapitulate the tumor-microenvironmental cross-talk found in humans. This highlights a need for more physiological human models to better study melanoma features. Here, six melanoma cell lines (A375, COLO829, G361, MeWo, RPMI-7951, and SK-MEL-28) were used to generate an in vitro three-dimensional human melanoma-in-skin (Mel-RhS) model and were compared in terms of dermal invasion and immune modulatory and pro-angiogenic capabilities. A375 displayed the most invasive phenotype by clearly expanding into the dermal compartment, whereas COLO829, G361, MeWo, and SK-MEL-28 recapitulated to different extent the initial stages of melanoma invasion. No nest formation was observed for RPMI-7951. Notably, the integration of A375 and SK-MEL-28 cells into the model resulted in an increased secretion of immune modulatory factors (e.g., M-CSF, IL-10, and TGFβ) and pro-angiogenic factors (e.g., Flt-1 and VEGF). Mel-RhS-derived supernatants induced endothelial cell sprouting in vitro. In addition, observed A375-RhS tissue contraction was correlated to increased TGFβ release and α-SMA expression, all indicative of differentiation of fibroblasts into cancer-associated fibroblast-like cells and reminiscent of epithelial-to-mesenchymal transition, consistent with A375′s most prominent invasive behavior. In conclusion, we successfully generated several Mel-RhS models mimicking different stages of melanoma progression, which can be further tailored for future studies to investigate individual aspects of the disease and serve as three-dimensional models to assess efficacy of therapeutic strategies.

Preclinical studies show that locoregional CTLA-4 blockade is equally effective in inducing tumor eradication as systemic delivery, without the added risk of immune-related side effects. This efficacy is related to access of the CTLA-4 blocking antibodies to tumor-draining lymph nodes (TDLNs). Local delivery of anti-CTLA-4 after surgical removal of primary melanoma, before sentinel lymph node biopsy (SLNB), provides a unique setting to clinically assess the role of TDLN in the biological efficacy of locoregional CTLA-4 blockade. Here, we have evaluated the safety, tolerability, and immunomodulatory effects in the SLN and peripheral blood of a single dose of tremelimumab [a fully human immunoglobulin gamma-2 (IgG2) mAb directed against CTLA-4] in a dose range of 2 to 20 mg, injected intradermally at the tumor excision site 1 week before SLNB in 13 patients with early-stage melanoma (phase 1 trial; NCT04274816). Intradermal delivery was safe and well tolerated and induced activation of migratory dendritic cell (DC) subsets in the SLN. It also induced profound and durable decreases in regulatory T cell (T

Purpose:Adenocarcinoma of the uterine cervix is the second most common type of cervical cancer after squamous cell carcinoma (SCC). Although both subtypes are treated similarly, patients with adenocarcinoma have a worse prognosis. In this study, immunologic features of the tumor microenvironment in these two subsets were pursued with potential therapeutic implications.Experimental Design:The immune microenvironment of primary tumors and nonmetastatic tumor-draining lymph nodes (TDLN) was compared between patients with cervical adenocarcinoma (n = 16) and SCC (n = 20) by polychromatic flow cytometry and by transcriptional profiling of the primary tumors (n = 299) using publicly available data from The Cancer Genome Atlas (TCGA).Results:Flow cytometric analyses revealed intact T-cell differentiation in TDLNs, but hampered effector T-cell trafficking to the primary tumors in adenocarcinoma, as compared with SCC. TCGA analysis demonstrated higher expression of chemokines involved in effector T-cell homing (CXCL9/10/11) in SCC primary tumors as compared with adenocarcinoma primary tumors, which was highly correlated to a transcriptional signature for type I conventional dendritic cells (cDC1). This was consistent with elevated frequencies of CD141/BDCA3+cDC1 in primary tumor SCC samples relative to adenocarcinoma and correspondingly elevated levels of CXCL9 and CXCL10 in 24-hour ex vivo cultures. Hampered cDC1 recruitment in adenocarcinoma was in turn related to lower transcript levels of cDC1-recruiting chemokines and an elevated β-catenin activation score and was associated with poor overall survival.Conclusions:Our data have identified an opportunity for the investigation of potentially novel therapeutic interventions in adenocarcinoma of the cervix, that is, β-catenin inhibition and cDC1 mobilization.

For the treatment of metastatic renal cell cancer several strategies are used among which the mTOR inhibitor everolimus. As mTOR plays an important role in the immune system, e.g., by controlling the expression of the transcription factor FoxP3 thereby regulating regulatory T cells (Tregs), it plays a key role in the balance between tolerance and inflammation. Previous reports showed stimulatory effects of mTOR inhibition on the expansion of Tregs, an effect that can be considered detrimental in terms of cancer control. Since metronomic cyclophosphamide (CTX) was shown to selectively deplete Tregs, a phase 1 clinical trial was conducted to comprehensively investigate the immune-modulating effects of several dosages and schedules of CTX in combination with the standard dose of everolimus, with the explicit aim to achieve selective Treg depletion. Our data show that 50 mg of CTX once daily and continuously administered, in combination with the standard dose of 10 mg everolimus once daily, not only results in depletion of Tregs, but also leads to a reduction in MDSC, a sustained level of the CD8+ T-cell population accompanied by an increased effector to suppressor ratio, and reversal of negative effects on three peripheral blood DC subsets. These positive effects on the immune response may contribute to improved survival, and therefore this combination therapy is further evaluated in a phase II clinical trial. Electronic supplementary material The online version of this article (10.1007/s00262-018-2288-8) contains supplementary material, which is available to authorized users.

Germline mutations in the Folliculin (FLCN) tumor suppressor gene cause Birt–Hogg–Dubé (BHD) syndrome, a rare autosomal dominant disorder predisposing carriers to kidney tumors.FLCNis a conserved, essential gene linked to diverse cellular processes but the mechanism by whichFLCNprevents kidney cancer remains unknown. Here, we show that disruptingFLCNin human renal tubular epithelial cells (RPTEC/TERT1) activates TFE3, upregulating expression of its E-box targets, including RRAGD and GPNMB, without modifying mTORC1 activity. Surprisingly, the absence of FLCN or its binding partners FNIP1/FNIP2 induces interferon response genes independently of interferon. Mechanistically, FLCN loss promotes STAT2 recruitment to chromatin and slows cellular proliferation. Our integrated analysis identifies STAT1/2 signaling as a novel target of FLCN in renal cells and BHD tumors. STAT1/2 activation appears to counterbalance TFE3-directed hyper-proliferation and may influence immune responses. These findings shed light on unique roles of FLCN in human renal tumorigenesis and pinpoint candidate prognostic biomarkers.

Background The sentinel lymph node (SLN) is the first node to receive lymphatic drainage from the primary tumor and the site where naive T cells are first primed. As such it is of great importance in initiating an effective anti-tumor immune response and an attractive target for immunomodulatory agents. Pre-clinical studies have reported that i.t. administration of anti-CTLA-4 is as effective in inducing tumor eradication as systemic delivery, without the risk of treatment related side effects. However, it remains unclear whether this is due primarily to modulation of the tumor microenvironment or of tumor-draining lymph nodes (TDLN). Here, we have evaluated the safety, tolerability and immunomodulatory effects in the SLN and peripheral blood mononuclear cells (PBMC) of anti-CTLA-4/tremelimumab, delivered locally at the tumor excision site in patients with early-stage melanoma. This unique setting (post tumor excision but prior to SLN biopsy) allowed us to clinically assess the role of TDLN in the biological efficacy of CTLA-4 blockade. Methods In this phase I dose-escalation trial, patients with clinical stage I-II melanoma received one intradermal injection of tremelimumab at four dose levels (2, 5, 10 [n=3 each] or 20 mg [n=4]) around the primary excision site of the tumor, seven days prior to re-excision and SLN biopsy. Flow cytometry was performed to study viable cells from melanoma SLN and PBMC (prior to tremelimumab administration [day 0], and at 7 days, 3 weeks and 3 months after tremelimumab injection). Systemic melanoma antigen (MART-1/NY-ESO-1)-specific T cells responses were assessed by IFN-γ ELISPOT assay. Results Intradermal delivery of tremelimumab was safe and well tolerated. In terms of biological efficacy it selectively induced profound and durable decreases in Treg frequencies in both SLN and PBMC, decreased systemic MDSC rates, activated migratory dendritic cell subsets in the SLN, and induced T cell activation (by HLA-DR and ICOS up-regulation), both in SLN and PBMC. Moreover, systemic anti-melanoma T cell responses were induced (n=5) or boosted (n=2), in association with T cell activation and central-memory T cell differentiation. Of note, tumor recurrences so far were only observed in two patients who did not develop a systemic anti-tumor T cell response. Conclusions These findings indicate that i.d. administration of anti-CTLA-4 may offer a safe and promising adjuvant treatment strategy for patients with early-stage melanoma. Moreover, they demonstrate a central role for TDLN in the biological efficacy of CTLA-4 blockade and warrant the development of TDLN-targeted delivery methods for anti-CTLA-4. Acknowledgements This study received funding from the Harry J. Lloyd Charitable Trust; tremelimumab was provided by Pfizer Inc. Trial Registration NCT04274816 Ethics Approval The study was approved by the Medical Ethics Committee of the VU University Medical Center and Spaarne Gasthuis.

Background: For patients with metastatic renal cell cancer (mRCC) who progressed on vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor therapy, the orally administered mammalian target of rapamycin (mTOR) inhibitor everolimus has been shown to prolong progression free survival. Intriguingly, inhibition of mTOR also promotes expansion of immunosuppressive regulatory T cells (Tregs) that can inhibit anti-tumor immune responses in a clinically relevant way in various tumor types including RCC. This study intends to investigate whether the antitumor efficacy of everolimus can be increased by preventing the detrimental everolimus induced expansion of Tregs using a metronomic schedule of cyclophosphamide. Methods/design: This phase I-II trial is a national multi-center study of different doses and schedules of low-dose oral cyclophosphamide in combination with a fixed dose of everolimus in patients with mRCC not amenable to or progressive after a VEGF-receptor tyrosine kinase inhibitor containing treatment regimen. In the phase I part of the study the optimal Treg-depleting dose and schedule of metronomic oral cyclophosphamide when given in combination with everolimus will be determined. In the phase II part of the study we will evaluate whether the percentage of patients progression free at 4 months of everolimus treatment can be increased from 50% to 70% by adding metronomic cyclophosphamide (in the dose and schedule determined in the phase I part). In addition to efficacy, we will perform extensive immune monitoring with a focus on the number, phenotype and function of Tregs, evaluate the safety and feasibility of the combination of everolimus and cyclophosphamide, perform monitoring of selected angiogenesis parameters and analyze everolimus and cyclophosphamide drug levels. Discussion: This phase I-II study is designed to determine whether metronomic cyclophosphamide can be used to counter the mTOR inhibitor everolimus induced Treg expansion in patients with metastatic renal cell carcinoma and increase the antitumor efficacy of everolimus. Trial Registration: ClinicalTrials.gov Identifier NCT01462214, EudraCT number 2010-024515-13, Netherlands Trial Register number NTR3085.

Contains fulltext : 215594.pdf (Publisher’s version ) (Open Access) In patients with cancer, the functionality of Dendritic Cells (DC) is hampered by high levels of tumor-derived suppressive cytokines, which interfere with DC development and maturation. Poor DC development can limit the efficacy of immune checkpoint blockade and in vivo vaccination approaches. Interference in intracellular signaling cascades downstream from the receptors of major tumor-associated suppressive cytokines like IL-10 and IL-6, might improve DC development and activation, and thus enhance immunotherapy efficacy. We performed exploratory functional screens on arrays consisting of >1000 human kinase peptide substrates to identify pathways involved in DC development and its inhibition by IL-10 or IL-6. The resulting alterations in phosphorylation of the kinome substrate profile pointed to glycogen-synthase kinase-3beta (GSK3beta) as a pivotal kinase in both DC development and suppression. GSK3beta inhibition blocked human DC differentiation in vitro, which was accompanied by decreased levels of IL-12p70 secretion, and a reduced capacity for T cell priming. More importantly, adenoviral transduction of monocytes with a constitutively active form of GSK3beta induced resistance to the suppressive effects of IL-10 and melanoma-derived supernatants alike, resulting in improved DC development, accompanied by up-regulation of co-stimulatory markers, an increase in CD83 expression levels in mature DC, and diminished release of IL-10. Moreover, adenovirus-mediated intratumoral manipulation of this pathway in an in vivo melanoma model resulted in DC activation and recruitment, and in improved immune surveillance and tumor control. We propose the induction of constitutive GSK3beta activity as a novel therapeutic means to bolster DC functionality in the tumor microenvironment.

Contains fulltext : 207393.pdf (Publisher’s version ) (Open Access) INTRODUCTION: Metastatic renal cell cancer (mRCC) patients have a median overall survival (mOS) of approximately 28 months. Until recently, mammalian target of rapamycin (mTOR) inhibition with everolimus was the standard second-line treatment regimen for mRCC patients, improving median progression-free survival (mPFS). Treatment with everolimus supports the expansion of immunosuppressive regulatory T cells (Tregs), which exert a negative effect on antitumor immune responses. In a phase 1 dose-escalation study, we have recently demonstrated that a low dose of 50 mg oral cyclophosphamide once daily can be safely combined with everolimus in mRCC patients and prevents the everolimus-induced increase in Tregs. MATERIALS AND METHODS: In a multicenter phase 2 study, performed in patients with mRCC not amenable to or progressive on a vascular endothelial growth factor (VEGF)-receptor tyrosine kinase inhibitor (TKI) containing treatment regimen, we assessed whether the addition of this metronomic dosing schedule of cyclophosphamide to therapy with everolimus could result in an improvement of progression-free survival (PFS) after 4 months of treatment. RESULTS: Though results from this study confirmed that combination treatment effectively lowered circulating levels of Tregs, addition of cyclophosphamide did not improve the PFS rate at 4 months. For this reason, the study was abrogated at the predefined interim analysis. CONCLUSION: Although the comprehensive immunomonitoring analysis performed in this study provides relevant information for the design of future immunotherapeutic approaches, the addition of metronomic cyclophosphamide to mRCC patients receiving everolimus cannot be recommended.

Though Vγ9Vδ2-T cells constitute only a small fraction of the total T cell population in human peripheral blood, they play a vital role in tumor defense and are therefore of major interest to explore for cancer immunotherapy. Vγ9Vδ2-T cell-based cancer immunotherapeutic approaches developed so far have been generally well tolerated and were able to induce significant clinical responses. However, overall results were inconsistent, possibly due to the fact that these strategies induced systemic activation of Vγ9Vδ2-T cells without preferential accumulation and targeted activation in the tumor. Here we show that a novel bispecific nanobody-based construct targeting both Vγ9Vδ2-T cells and EGFR induced potent Vγ9Vδ2-T cell activation and subsequent tumor cell lysis both in vitro and in an in vivo mouse xenograft model. Tumor cell lysis was independent of KRAS and BRAF tumor mutation status and common Vγ9Vδ2-T cell receptor sequence variations. In combination with the conserved monomorphic nature of the Vγ9Vδ2-TCR and the facile replacement of the tumor-specific nanobody, this immunotherapeutic approach can be applied to a large group of cancer patients.

Purpose: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets. Experimental Design: By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)–educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography. Results: We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFβ, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFβ-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFβ are increased in osteosarcoma patients. Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFβ-blocking agents as new therapeutic options for osteosarcoma patients. Clin Cancer Res; 23(14); 3721–33. ©2017 AACR.

Different types of cells infected with Epstein-Barr virus (EBV) can release exosomes containing viral components that functionally affect neighboring cells. Previously, we found that EBV was localized mostly in infiltrating lymphocytes within the stromal layer of cervical lesions. In this study, we aimed to determine effects of exosome-transferred EBV-encoded RNAs (EBERs) on keratinocytes expressing human papillomavirus (HPV) 16 E6/E7 (DonorI-HPV16 HFKs). Lipid transfection of in vitro-transcribed EBER1 molecules (ivt EBER1) into DonorI-HPV16 HFKs caused strong induction of interferon (IFN)-related genes and interleukin 6 (IL-6). To gain insights into the physiological situation, monocyte-derived dendritic cells (moDCs), low passage DonorI-HPV16 HFKs and primary keratinocytes were used as recipient cells for internalization of exosomes from wild-type EBV (wt EBV) or B95-8 EBV-infected lymphoblastoid cell lines (LCLs). qRT-PCR was used to determine the expression of EBER1, HPV16 E6/E7, IFN-related genes and IL-6 in recipient cells. The secretion of inflammatory cytokines was investigated using cytometric bead array. Wt EBV-modified exosomes induced both IFN-related genes and IL-6 upon uptake into moDCs, while exosomes from B95-8 EBV LCLs induced only IL-6 in moDCs. Internalization of EBV-modified exosomes was demonstrated in DonorI-HPV16 HFKs, yielding only EBER1 but not EBER2. However, EBER1 transferred by exosomes did not induce IFN-related genes or IL-6 expression and inflammatory cytokine secretion in DonorI-HPV16 HFKs and primary keratinocytes. EBER1 copy numbers in exosomes from wt EBV-infected LCLs were 10-fold higher than in exosomes from B95-8 LCLs (equal cell equivalent), whereas ivt EBER1 was used at approximately 100-fold higher concentration than in exosomes. These results demonstrated that the induction of IFN-related genes and IL-6 by EBER1 depends on quantity of EBER1 and type of recipient cells. High levels of EBER1 in cervical cells or infiltrating dendritic cells may play a role in the inflammation-to-oncogenesis transition of HPV-associated cervical cancer through modulation of innate immune signals.

Autologous tumor cell-based vaccines provide a wide range of tumor antigens and personalized neo-epitopes based on individual tumors’ unique antigenic mutanome signatures. However, tumor-derived factors may hamper in situ maturation of dendritic cells (DC) and thus interfere with the generation of effective anti-tumor immunity. As the skin is a preferred site for tumor vaccine delivery, we investigated the influence of primary colon carcinoma-derived soluble factors on the maturation state of migrating DC in a human skin explant model. Primary tumor-derived supernatants (TDSN) enhanced the phenotypic maturation state of skin-emigrated DC, resulting in an increased T-cell stimulatory ability in an allogeneic mixed leukocyte response. In case of monocyte-derived DC a similar TDSN-induced maturation induction was found to entirely depend on cyclooxygenase (COX)-regulated prostaglandins. In contrast, the increase in skin-emigrated DC maturation was completely prostaglandin-independent, as evidenced by the inability of the COX inhibitor indomethacin to abrogate this TDSN-induced effect. Although TDSN conditioning affected a drop in IL-12p70 release by the skin-emigrated DC and induced a predominant Th17/Th22 transcriptional profile in subsequently stimulated T-cells, Th cell subset differentiation, as assessed by intracellular cytokine expression upon polyclonal priming and re-stimulation, was not affected. Comparative analysis of phenotypic and transcriptional profiles suggests that the observed maturational effects in skin-derived DC may have been induced by tumor-derived GM-CSF. In conclusion, soluble factors derived from whole-cell colon tumor vaccines will not negatively impact DC migration and maturation in human skin, but rather induce DC maturation that will facilitate the priming of a poly-functional Th cell response.

// Gijs G. Zom 1 , Marij J.P. Welters 2 , Nikki M. Loof 2 , Renske Goedemans 2 , Sinead Lougheed 3 , Rob R.P.M. Valentijn 4 , Maarten L. Zandvliet 4 , Nico J. Meeuwenoord 5 , Cornelis J.M. Melief 1, 6 , Tanja D. de Gruijl 3 , Gijsbert A. Van der Marel 5 , Dmitri V. Filippov 5 , Ferry Ossendorp 1, * , Sjoerd H. Van der Burg 2, * 1 Department of Immunohematology and Blood Transfusion, Section Tumorimmunology, Leiden University Medical Center, Leiden, The Netherlands 2 Department of Clinical Oncology, Leiden University Medical Center, Leiden, The Netherlands 3 Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands 4 Department of Clinical Pharmacy and Toxicology, Leiden University Medical Center, Leiden, The Netherlands 5 Leiden Institute of Chemistry, University of Leiden, Leiden, The Netherlands 6 ISA Pharmaceuticals BV, Leiden, The Netherlands * These authors have contributed equally to this work Correspondence to: Ferry Ossendorp, email: f.a.ossendorp@lumc.nl Sjoerd H. Van der Burg, email: shvdburg@lumc.nl Keywords: Toll-like receptor ligand, conjugate, synthetic long peptide, cancer vaccine, cervical cancer Received: April 19, 2016 Accepted: July 10, 2016 Published: August 23, 2016 ABSTRACT The potency of human papillomavirus type 16 (HPV16)-encoded synthetic long peptides (SLP), conjugated to an optimized Toll-like receptor 2 ligand (TLR2-L), was assessed in ex vivo activation of HPV16 + cancer patient-derived T cells. Two highly immunogenic SLP sequences derived from the oncogenic E6 protein of HPV16 were selected and conjugated to a Pam3CSK4-based TLR2-L under GMP conditions. Both conjugates were able to mature human DCs in vitro and to activate human skin-derived antigen-presenting cells (APCs) upon intradermal injection in an ex vivo skin model, associated with induction of a favorable chemokine profile to attract and activate T cells. The conjugated SLPs were efficiently processed by APCs, since HPV16-specific CD4 + and CD8 + T-cell clones isolated from HPV16+ cervical tumors proliferated in response to both conjugates. The TLR2-L SLP conjugates significantly enhanced ex vivo T helper type 1 T-cell activation in cell suspensions obtained from tumor-draining lymph nodes (LN) resected during hysterectomy of HPV16 + cervical cancer patients. These results show that TLR2-L SLP conjugates can activate circulating or LN-derived tumor-specific T cells, a promising outcome for studying these two conjugates in a phase I/II clinical safety and immunogenicity trial.

Item does not contain fulltext Immune checkpoint blockade enhances antitumor responses, but can also lead to severe immune-related adverse events (IRAE). To avoid unnecessary exposure to these potentially hazardous agents, it is important to identify biomarkers that correlate with clinical activity and can be used to select patients that will benefit from immune checkpoint blockade. To understand the consequences of CTLA-4 blockade and identify biomarkers for clinical efficacy and/or survival, an exploratory T cell monitoring study was performed in a phase I/II dose escalation/expansion trial (n = 28) of combined Prostate GVAX/ipilimumab immunotherapy. Phenotypic T cell monitoring in peripheral blood before and after Prostate GVAX/ipilimumab treatment revealed striking differences between patients who benefited from therapy and patients that did not. Treatment-induced rises in absolute lymphocyte counts, CD4(+) T cell differentiation, and CD4(+) and CD8(+) T cell activation were all associated with clinical benefit. Moreover, significantly prolonged overall survival (OS) was observed for patients with high pre-treatment frequencies of CD4(+)CTLA-4(+), CD4(+)PD-1(+), or differentiated (i.e., non-naive) CD8(+) T cells or low pre-treatment frequencies of differentiated CD4(+) or regulatory T cells. Unsupervised clustering of these immune biomarkers revealed cancer-related expression of CTLA-4(+) in CD4(+) T cells to be a dominant predictor for survival after Prostate GVAX/ipilimumab therapy and to thus provide a putative and much-needed biomarker for patient selection prior to therapeutic CTLA4 blockade.

We have been studying the re-activation of tumor-associated antigen (TAA)-specific CD8 + T cells in sentinel lymph nodes (SLN) of melanoma patients upon intradermal administration of the CpG-B oligodeoxynucleotide PF-3512676. To facilitate functional testing of T cells from small SLN samples, high-efficiency polyclonal T cell expansion is required. In this study, SLN cells were expanded via classic methodologies with plate- or bead-bound anti-CD3/CD28 antibodies and with the K562/CD32/4-1BBL artificial APC system (K32/4-1BBL aAPC) and analyzed for responsiveness to common recall or TAA-derived peptides. K32/4-1BBL-expanded T cell populations contained significantly more effector/memory CD8 + T cells. Moreover, recall and melanoma antigen-specific CD8 + T cells were more frequently detected in K32/4-1BBL-expanded samples as compared with anti-CD3/CD28-expanded samples. We conclude that K32/4-1BBL aAPC are superior to anti-CD3/CD28 antibodies for the expansion of in vivo -primed specific CD8 + T cells and that their use facilitates the sensitive monitoring of functional anti-tumor T cell immunity in SLN.

The adoptive transfer of in vitro-induced and expanded tumor-specific cytotoxic T lymphocytes (CTL) presents a promising immunotherapeutic approach for the treatment of cancer. The in vitro induction of tumor-reactive CTL requires repeated stimulation of CTL precursors with dendritic cells (DC). To circumvent problems like scarcity of blood DC precursors and donor variability, it would be attractive to use DC from a non-autologous, unlimited source. DCs derived from the human acute myeloid leukemia (AML) cell line MUTZ-3 are attractive candidates since these DCs closely resemble monocyte-derived DC (MoDC) in terms of phenotype and T cell stimulatory capacity. Here we demonstrate that functional CTL clones could be generated against multiple tumor-associated antigens, i.e., human telomerase reverse transcriptase (hTERT), ErbB3-binding protein-1 (Ebp1), carcinoembryonic antigen (CEA) and Her-2/neu, by stimulating CD8β+ CTL precursors with peptide-loaded allogeneic, HLA-A2-matched MUTZ-3-derived DC. A consistent induction capacity, as determined by MHC tetramer-binding, was found in multiple donors and comparable to autologous peptide-loaded MoDC. Functional characterization at the clonal level revealed the priming of CTL that recognized endogenously processed epitopes on tumor cell lines in an HLA-A2-restricted fashion. Our data indicate that MUTZ-3-derived DC can be used as stimulator cells for in vitro priming and expansion of functional TAA-specific effector CTL. MUTZ-3-derived DCs thus represent a ready and standardized source of allogeneic DC to generate CTL for therapeutic adoptive transfer strategies.

TLR agonists are attractive candidate adjuvants for therapeutic cancer vaccines as they can induce a balanced humoral and T cell–mediated immune response. With a dense network of dendritic cells (DCs) and draining lymphatics, the skin provides an ideal portal for vaccine delivery. Beside direct DC activation, TLR agonists may also induce DC activation through triggering the release of inflammatory mediators by accessory cells in the skin microenvironment. Therefore, a human skin explant model was used to explore the in vivo potential of intradermally delivered TLR agonists to stimulate Langerhans cells and dermal DCs in their natural complex tissue environment. The skin-emigrated DCs were phenotyped and analyzed for T cell stimulatory capacity. We report that, of six tested TLR-agonists, the TLR2 and -3 agonists peptidoglycan (PGN) and polyribosinic-polyribocytidylic acid (Poly I:C) were uniquely able to enhance the T cell–priming ability of skin-emigrated DCs, which, in the case of PGN, was accompanied by Th1 polarization. The enhanced priming capacity of Poly I:C–stimulated DCs was associated with a strong upregulation of appropriate costimulatory molecules, including CD70, whereas that of PGN-stimulated DCs was associated with the release of a broad array of proinflammatory cytokines. Transcriptional profiling further supported the notion that the PGN- and Poly I:C–induced effects were mediated through binding to TLR2/nucleotide-binding oligomerization domain 2 and TLR3/MDA5, respectively. These data warrant further exploration of PGN and Poly I:C, alone or in combination, as DC-targeted adjuvants for intradermal cancer vaccines.

While systemic immune checkpoint blockade is unsurpassed in the induction of clinically effective T cell immunity in advanced-stage melanoma patients, the risk of autoimmune-related side effects precludes its application in earlier stages. In a Phase-1 dose escalation study we have studied the safety, feasibility and immunological effects of intradermal delivery of a single low dose of anti-CTLA-4/tremelimumab at the primary tumor excision site of patients with clinically Stage I-II melanoma. Dose escalation (4x3 cohorts of 2, 5, 10 and 20 mg) has been concluded with one additional patient enrolled in a 20 mg expansion cohort. Breslow thickness ranged from 0.9 to 6 mm (mean 1.98 mm). Examination of the sentinel lymph node (SLN) revealed (micro-)metastases in five of the patients. The tremelimub administration was found to be safe without apparent side effects except for one case of localized vitiligo at the highest (20 mg) dose level. Of note, potent immunological effects were observed in equal measure at all dose levels. In all patients a decrease in systemic rates of activated Tregs (aTregs, defined as CD4+CD25hiFoxP3hi T cells) was observed after 7 days of treatment (p Specific T cell reactivity against long peptides derived from the melanoma antigens NY-ESO-1 and MART-1 was tested after in vitro re-stimulation in an IFNγ Elispot assay. In 7/13 patients evidence was found of tremelimumab-induced increases in systemic T cell rates, which remained elevated up to 12 weeks post-treatment. Remarkably, 5/13 patients showed pre-existent NY-ESO-1 reactivity, which in all cases had decreased by day 7, and in 3/5 patients had increased over baseline levels by day 21. T cell reactivity to either NY-ESO-1 or MART-1 was observed in 4/4 tested SLN. Of note, both NY-ESO-1 and MART-1 reactive T cells were detected in the SLN of the patient experiencing an isolated incidence of vitiligo after receiving 20 mg tremelimumab. In conclusion, local administration of a single low dose of anti-CTLA-4/tremelimumab has proven safe and effective in terms of attenuating aTreg levels in peripheral blood and boosting systemic antitumor immunity. As such it may offer an attractive, and up to now sorely lacking, adjuvant treatment option for early-stage melanoma patients at risk for tumor recurrence. Citation Format: Anita G.M. Stam, Kim M. van Pul, Bas D. Koster, Dafni Chondronasiou, Sinéad M. Lougheed, M. Petrousjka van den Tol, Karin Jooss, Ronald J.C.L.M. Vuylsteke, Alfons J.M. van den Eertwegh, Tanja D. de Gruijl. Single low-dose administration of anti-CTLA-4/tremelimumab at the tumor excision site boosts systemic antitumor immunity in early-stage melanoma patients: Results from a Phase-1 study. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B138.

Objective We have previously shown that homocysteine (Hcy) induces phosphatidylserine (PS) exposure, apoptosis and necrosis in human endothelial cells. Since it has been suggested that S -Adenosylhomocysteine (SAH) is the main causative factor in Hcy-induced pathogenesis of cardiovascular disease, we evaluate here whether the cytotoxic Hcy effect in endothelial cells is also SAH dependent. Methods and results Human umbilical vein endothelial cells (HUVECs) were exposed to the following conditions: (1) non-treated control (resulting in 2.8 nM intracellular SAH and 3.1 μM extracellular l -Hcy); and incubation with (2) 50 μM adenosine-2,3-dialdehyde (ADA; resulting in 17.7 nM intracellular SAH and 3.1 μM extracellular l -Hcy), (3) 2.5 mM Hcy (resulting in 20.9 nM intracellular SAH and 1.8 mM extracellular l -Hcy), and (4) 1, 10 and 100 μM SAH. We then determined the effect of treatment on annexin V-positivity, caspase-3 activity, cytochrome c release (sub)cellular expression of NOX2, NOX4, p47 phox and nitrotyrosine, and H 2 O 2 . Both Hcy and ADA significantly increased PS exposure ( n = 5), caspase-3 activity ( n = 6) and cytochrome c release ( n = 3). Incubation with extracellular SAH alone did not affect cell viability. Both Hcy and ADA also induced similar increases in nuclear NOX2 and (peri)nuclear NOX4, coinciding with (peri)nuclear p47 phox expression and local reactive oxygen species (ROS) ( n = 3). Inhibition of NOX-mediated ROS by the flavoenzyme inhibitor diphenylene iodonium (DPI) significantly decreased apoptosis induction ( n = 3) and ROS production ( n = 3). Conclusion SAH induces PS exposure and apoptosis in endothelial cells independently of Hcy. Our study therefore shows that Hcy-mediated endothelial dysfunction, as determined in the cell model used, is mainly due to SAH accumulation.

Targeting dendritic cells (DC) through the release of suppressive factors is an effective means for tumors to escape immune control. We assessed the involvement of downstream signaling through the JAK2/STAT3 and p38 MAPK pathways in tumor-induced suppression of human DC development. Whereas the JAK2/STAT3 pathway has been pinpointed in mouse studies as a key regulator of myeloid suppression, in human DC this is less well established. We studied the effects of STAT3 inhibition on the suppression of monocyte-derived DC differentiation mediated by a short-list of four predominant suppressive factors and found that pharmacological STAT3 inhibition could only counteract the effects of IL-6. Accordingly, in testing a panel of supernatants derived from 11 cell lines representing various types of solid tumors, STAT3 inhibition only modestly affected the suppressive effects of a minority of supernatants. Importantly, combined interference in the STAT3 and p38 pathways completely prevented inhibition of DC differentiation by all tested supernatants and effected superior DC function, evidenced by increased allogeneic T cell reactivity with elevated IL-12p70/IL-10 ratios and Th1 skewing. Combined STAT3 and p38 inhibition also afforded superior protection against the suppressive effects of primary glioma and melanoma supernatants and induced a shift from CD14(+) cells to CD1a(+) cells in metastatic melanoma single-cell suspensions, indicating a potential for improved DC differentiation in the tumor microenvironment. We conclude that combined interference in the STAT3 and p38 MAPK signaling pathways is a promising approach to overcome tumor-induced inhibitory signaling in DC precursors and will likely support clinical immunotherapeutic strategies.

Aims: Langerhans cell (LC) infiltration has been observed in glioblastoma, but the glioblastoma microenvironment may be conditioned to resist antitumor immune responses. As little is known about how glioblastoma may affect dendritic cell differentiation, here we set out to delineate the effects of glioblastoma-derived soluble factors on LC differentiation. Methods: CD34+ precursor cells of the human myeloid cell line MUTZ-3 were differentiated into LC in the presence of conditioned media of the human glioblastoma cell lines U251 or U373 and phenotypically and functionally characterized. Results: Glioblastoma-conditioned media inhibited LC differentiation, resulting in functional impairment, as determined by allogeneic mixed leukocyte reactivity, and induction of STAT3 activation. IL-6 blockade completely abrogated these glioblastoma-induced immunosuppressive effects and reduced STAT3 phosphorylation. However, neither addition of JSI-124 (cucurbitacin-I; a JAK2/STAT3 inhibitor), nor of GW5074 (a Raf-1 inhibitor), both of which interfere with signaling pathways reported to act downstream of the IL-6 receptor, prevented the observed inhibitory effects on LC differentiation. Conclusion: Glioblastoma-derived IL-6 is responsible for the observed suppression of LC differentiation from CD34+ precursors but appears to exert this effect in a STAT3 and Raf-1 independent fashion.

Item does not contain fulltext CD1d restricted, alpha-galactosylceramide (alphaGC) responsive invariant (i)NKT cells positively regulate immune responses. Both intravenous and intradermal administered alphaGC are known to activate iNKT cells. iNKT cells become unresponsive to a second intravenous alphaGC injection, whereas no data are available regarding potential anergy upon intradermal administration. Here, comparative analysis of two intradermal versus two intravenous injections in mice demonstrated that iNKT cell anergy was prevented by intradermal injection and when combined with a vaccine, superior tumor protection afforded by intradermally administered alphaGC. Moreover, human skin dendritic cells (DC) took up intradermally injected alphaGC and activated iNKT cells upon migration, while iNKT cells in human skin-draining lymph nodes expanded in response to alphaGC presented either by exogenously added DC or by CD1d positive antigen presenting cells in the lymph nodes. In conclusion, glycolipids such as alphaGC may greatly improve the efficacy of skin immunization strategies, targeting cutaneous and lymph node DC. 01 september 2010

Background Increasing evidence suggests that immune regulated pathways influence both breast cancer (BrC) development and response to (neo) adjuvant chemotherapeutic treatment. The sentinel lymph node (SLN) is the first site of BrC induced suppression of immune effector subsets, rendering them unable to mount an effective anti-tumor response. Since detailed knowledge of the functional status of these immune effector subsets is lacking, we compared the immune status of BrC SLN with healthy lymph nodes (HLN) using viable cell samples and correlated our findings to several clinico-pathological characteristics. Additionally we investigated if ex vivo conditioning with the Toll-like receptor-9 ligand CpG-B with or without simultaneous inhibition of JAK2/STAT3 signaling (a downstream signaling pathway implicated as the ‘master switch’ in tumor induced immune suppression) could overcome the observed immune suppression in BrC SLN. Methods Viable SLN cells were obtained from 40 clinically node negative BrC patients. Axillary HLN were obtained from 17 BRCA-1 or -2 patients undergoing a prophylactic mastectomy. Frequencies and activation state of dendritic cell (DC) subsets and regulatory T cells (Treg) were determined by extensive multi-color flowcytometric analyses. Additionally, SLN cells from 12 BrC patients were cultured in absence and presence of CpG-B (PF-3512676) and the combination of CpG-B with a JAK2-STAT3 inhibitor (AG-490). Results Of the 40 BrC SLN 11 contained metastasis. We found clear evidence of BrC-related immune suppression, as significantly higher Treg frequencies were observed in metastasis negative BrC SLN as compared to HLN. These frequencies further increased in metastasis positive BrC SLN. Activation state (by expression of the co-stimulatory molecules CD40, CD83 and CD86) of lymph node resident (LNR)-DC subsets (both CD11c+ myeloid DC and CD123+BDCA2+ plasmacytoid DC), but not of CD1a+ migratory subsets, was significantly lower in BrC SLN as compared to HLN. Additionally, in BrC SLN, activation state of these LNR-DC subsets was further reduced upon metastatic involvement and in non-luminal BrC subtypes (triple negative and HER-2+) as compared to luminal BrC subtypes (ER and/or PR positive). We previously showed in early-stage melanoma patients that local treatment with CpG-B led to the preferential activation of LNR-DC subsets. Similarly, ex vivo targeting of BrC SLN with CpG and CpG + JAK2/STAT3 inhibition resulted primarily in significantly enhanced activation of LNR-DC subsets, with superior effects of combined CpG and JAK2/STAT3 targeting evidenced by significantly higher CD83 expression. Conclusion BrC induced immune suppression in SLN seems to be primarily mediated by hampered activation of LNR-DC subsets, which was most effectively overcome (ex-vivo) with the immune activating compound CpG-B in combination with a small-molecule inhibitor of the immune suppressive JAK2/STAT3 signaling pathway (AG490). This combined immune targeting strategy might be of clinical benefit in enhancing effectiveness of conventional (neo)adjuvant chemotherapeutic treatment, especially in -more immune suppressed- non-luminal BrC subtypes with known poor prognosis. Citation Format: Kim M van Pul, Ronald JCLM Vuylsteke, Sinéad M Lougheed, Lisette EA te Velde, Petrousjka van den Tol, Emiel J Th Rutgers, Hein BAC Stockmann, Tanja D de Gruijl. Breast cancer related immune suppression in the sentinel lymph node can be effectively countered by combined CpG-B administration and JAK2/STAT3 inhibition [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-04-01.

The study of early events in dendritic cell (DC) differentiation is hampered by the lack of homogeneous primary cell systems that allow the study of cytokine-driven, transitional DC differentiation steps. The CD34+ acute myeloid leukemia cell line MUTZ-3 displays a unique ability to differentiate into interstitial DC (IDC) and Langerhans cells (LC) in a cytokine-dependent manner. Phenotypic characterization revealed MUTZ-3 to consist of three distinct subpopulations. Small CD34+CD14 -CD11b- progenitors constitute the proliferative compartment of the cell line with the ability to differentiate through a CD34-CD14- CD11b+ stage to ultimately give rise to a morphologically large, nonproliferating CD14+CD11bhi progeny. These CD14+CD11bhi cells were identified as common, immediate myeloid DC precursors with the ability to differentiate into LC and IDC, exhibiting characteristic and mutually exclusive expression of Langerin and DC-specific ICAM-grabbing nonintegrin, respectively. The identity of the MUTZ-3-derived LC subset was confirmed further by the presence of Birbeck granules. We conclude that the MUTZ-3 cell line provides a ready and continuous supply of common myeloid precursors, which should facilitate further study of the ontogeny of myeloid DC lineages.

Migration of dendritic cells (DC) to secondary lymphoid organs under proinflammatory conditions coincides with their maturation and acquisition of T cell stimulatory abilities. In contrast, impaired activation of DC, e.g., in tumor-conditioned environments, may hamper their activation and possibly their subsequent migration to lymph nodes, leading to either immunological tolerance or ignorance, respectively. In this study, the influence of cytokines in the peripheral skin microenvironment on the activation state of migrating cutaneous DC was assessed using an ex vivo human skin explant model. We observed a phenotypic shift from mature CD83+ DC to immature CD14+ macrophage-like cells within 7 days subsequent to migration from unconditioned skin. These macrophage-like cells displayed a poor T cell stimulatory ability and lacked expression of CCR7, thus precluding their migration to paracortical T cell areas in the lymph nodes. The balance of suppressive and stimulatory cytokines during the initiation of migration decided the postmigrational fate of DC with IL-10 accelerating and GM-CSF and IL-4 preventing the phenotypic switch, which proved irreversible once established. These observations indicate that, in immunosuppressed environments, a postmigrational DC-to-macrophage shift may hinder T cell activation, but also that it may be prevented by prior conditioning of the tissue microenvironment by GM-CSF and/or IL-4.

Adenoviral (Ad) transduction of dendritic cells (DC) is a promising vaccination strategy. However, clinical applicability of Ad vectors is hampered by the necessity to use high titers of infectious Ad particles for efficient DC transduction. Here, we report on the production of a bacterially expressed bispecific conjugate, consisting of a fusion of recombinant single-chain (sc) mAb Fv fragments, which bind and neutralize the Ad fiber knob (through the S11 mAb scFv) and retarget Ad to CD40 on the DC surface (through the G28-5 mAb scFv). We show that this bispecific scFv fusion protein significantly enhances transduction efficiency of monocyte-derived DC (MoDC), reduces the amount of virus needed for a given level of transduction, and increases the ability of MoDC to activate CTL in an antigen specific manner. This single-component conjugate may prove to be a valuable immunotherapeutic too] for the targeting of Ad to DC in vivo. (C) 2003 Elsevier Science Ltd. All rights reserved.

Therapeutic tumor vaccination with viral vectors or naked DNA, carrying the genetic code for tumor-associated Ags, critically depends on the in vivo transduction of dendritic cells (DC). Transfection of predominantly nonprofessional APC and only small numbers of DC may hamper proper T cell activation. Aim of this study was, therefore, the targeted, selective, and enhanced in situ transduction of DC. A human skin explant model was used to explore targeted transduction of cutaneous DC after intradermal injection of a bispecific Ab conjugate to link adenoviral (Ad) vectors directly to CD40 on the DC surface. A significantly enhanced transduction efficiency and selectivity, and an increased activation state of migrating DC were thus achieved. Moreover, DC transduced by CD40-targeted Ad maintained their Ag-specific CTL-stimulatory ability for up to 1 wk after the start of migration, in contrast to DC transduced by untargeted Ad, which had lost this capacity by that time. Because DC targeting in vivo might obviate the need for the in vitro culture of autologous DC for adoptive transfer, CD40-targeted Ad vectors constitute a promising new vaccine modality for tumor immunotherapy.

Many human myeloid leukemia–derived cell lines possess the ability to acquire a dendritic cell (DC) phenotype. However, cytokine responsiveness is generally poor, requiring direct manipulation of intracellular signaling mechanisms for differentiation. In contrast, the CD34+ human acute myeloid leukemia cell line MUTZ-3 responds to granulocyte macrophage– colony-stimulating factor (GM-CSF), interleukin 4 (IL-4), and tumor necrosis factor alpha (TNFα), cytokines known to be pivotal both in vivo and in vitro for DC generation from monocytes and CD34+ stem cells. In all respects, MUTZ-3 cells behave as the immortalized equivalent of CD34+ DC precursors. Upon stimulation with specific cytokine cocktails, they acquire a phenotype consistent with either interstitial- or Langerhans-like DCs and upon maturation (mDC), express CD83. MUTZ-3 DC display the full range of functional antigen processing and presentation pathways. These findings demonstrate the unique suitability of MUTZ-3 cells as an unlimited source of CD34+DC progenitors for the study of cytokine-induced DC differentiation.

We have been studying the safety and clinical efficacy of the Prostate GVAX vaccine (consisting of two allogeneic GM-CSF secreting tumor cell lines: LNCaP and PC3) and the CTLA-4 blocking antibody ipilimumab in prostate cancer patients in a phase I/II dose escalation/expansion trial. To assess immune reactivity against the GVAX vaccine, immune infiltrate analysis of the vaccination sites was performed. Punch biopsies of the vaccination site were taken 48 hours after the 1st, 5th, and 11th injections and analyzed by immunohistochemical staining. Injection of the GVAX vaccine evoked striking local reactions that were characterized by local erythema, induration and swelling in all patients. Analysis of the vaccination site biopsies revealed that the GVAX/ipilimumab combination resulted in an inflammatory response in the skin with intradermal infiltrates of CD1a+ dendritic cells (DC), DC-SIGN+/CD68+ immature DC/macrophages, CD14+ monocyte-like cells, eosinophils, and of CD4+, CD8+, and Granzyme-B+ T lymphocytes. Strikingly, CD56+ NK cells were completely absent. The inflammatory response significantly increased over the course of treatment and resulted in accelerated clearance of the vaccine in vivo. Moreover, analysis revealed that the PC3 vaccine was more immunogenic, and induced a more potent anti-vaccine T cell response. The latter was confirmed in an in vitro cytotoxicity assay. Upon multiple vaccinations T cell reactivity to PC3 was significantly enhanced in the PC3 but not in the LNCaP vaccination sites, whereas reactivity to LNCaP was not enhanced in either vaccination site. We conclude that the GVAX/ipilimumab combination elicits a local immune infiltrate that increases over time, consistent with an induced CD8+ effector T cell mediated cytolytic response, only in vaccination sites of the PC3 component of the vaccine. Citation Format: Saskia J. Santegoets, Leonieke JJ van Mens, Jeroen AM Beliën, Anita GM Stam, Sinead M. Lougheed, Rik J. Scheper, Shabnam Samiei, Helen Gall, Karin Jooss, Jean-Marie Cuillerot, Natalie Sacks, Kirsten Hege, Winald R. Gerritsen, Alfons JM van den Eertwegh, Tanja D. de Gruijl. Inflammatory responses with infiltrating DC and functional cytotoxic T lymphocytes in the dermal vaccination sites of GVAX/ipilimumab treated prostate cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 496. doi:10.1158/1538-7445.AM2013-496

The effects of a GM-CSF-secreting allogeneic prostate cancer vaccine (Prostate GVAX) and the anti-CTLA4 antibody ipilimumab were investigated in a Phase I dose escalation/expansion trial of patients with metastatic castration resistant prostate cancer (van den Eertwegh et al, Lancet Oncology, 13: 509-17 2012). The GVAX and ipilimumab combination treatment led to PSA declines of more than 50% (Partial Response, PR) in 5 of 28 patients, PSA stabilizations (Stable Disease, SD) in 12 of 28 patients and disease progression (PD) in 11 of 28 patients. Regressing bone and lymph node metastases were observed in 2/5 PR patients. As an indication of tumor antigen-specific responsiveness we analyzed serologic and T cell responses against NY-ESO-1 before, during and after treatment. NY-ESO-1 seroreactivity (determined by ELISA) was observed in seven of 28 patients (25%). Five patients were seropositive at baseline and 2 became seropositive following treatment; no significant correlations were found with clinical benefit or survival. NY-ESO-1 T cell reactivity was determined in 19 patients by IFNγ ELISPOT after stimulation with a peptide pool of overlapping 20-mer peptides covering the immunodominant region aa121-180. NY-ESO-1-specific T cell responses were detected in 13 of 19 patients (68%) at any time before or during treatment. Increases in the frequency of NY-ESO-1 specific T cells following treatment were only observed in five of these (26%). A remarkably high number of nine out of 19 patients (47%) already exhibited NY-ESO-1-specific T cell reactivity before treatment. Interestingly, three of four (75%) PR patients displayed pre-existing frequencies of NY-ESO-1 T cells, whereas these cells were only detected in six of 15 SD/PD (40%) patients. Moreover, patients with pre-existing NY-ESO-1 T cell reactivity demonstrated a significantly prolonged overall survival (p=0.044, log-rank test). These data suggest that baseline NY-ESO-1 T cell reactivity may have predictive value for the clinical outcome of prostate GVAX and/or ipilimumab treatment. Citation Format: Anita GM Stam, Saskia JAM Santegoets, Alfons JM van den Eertwegh, Rik J. Scheper, Sinéad M. Lougheed, Helen Gall, Karin Jooss, Natalie Sacks, Thomas Harding, Kristen Hege, Israel Lowy, Sacha Gnjatic, Jedd D. Wolchok, Winald R. Gerritsen, Tanja D. de Gruijl. Pre-existing NY-ESO-1-specific T cell reactivity is associated with improved clinical outcome in castration resistant prostate cancer patients treated with Prostate GVAX and ipilimumab. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1229. doi:10.1158/1538-7445.AM2013-1229

Interleukin (IL)-10 is a major cancer-related immunosuppressive factor, exhibiting a unique ability to hamper the maturation of dendritic cells (DCs). We have previously reported that IL-10 induces the conversion of activated, migratory CD1a+ DCs found in the human skin to CD14+CD141+ macrophage-like cells. Here, as a model of tumor-conditioned DC maturation, we functionally assessed CD14- and CD14+ DCs that matured in vitro upon exposure to IL-10. IL-10-induced CD14+ DCs were phenotypically characterized by a low maturation state as well as by high levels of BDCA3 and DC-SIGN, and as such they closely resembled CD14+ cells infiltrating melanoma metastases. Compared with DC matured under standard conditions, CD14+ DCs were found to express high levels of B7-H1 on the cell surface, to secrete low levels of IL-12p70, to preferentially induce TH2 cells, to have a lower allogeneic TH cell and tumor antigen-specific CD8+ T-cell priming capacity and to induce proliferative T-cell anergy. In contrast to their CD14+ counterparts, CD14- monocyte-derived DCs retained allogeneic TH priming capacity but induced a functionally anergic state as they completely abolished the release of effector cytokines. Transcriptional and cytokine release profiling studies indicated a more profound angiogenic and pro-invasive signature of CD14+ DCs as compared with DCs matured in standard conditions or CD14− DCs matured in the presence of IL-10. Importantly, signal transducer and activator of transcription 3 (STAT3) depletion by RNA interference prevented the development of the IL-10-associated CD14+ phenotype, allowing for normal DC maturation and providing a potential means of therapeutic intervention.

Blockade of the CTLA-4 immune checkpoint can enhance anti-tumor responses and prolong survival, but it can also lead to the development of severe and potentially life-threatening immune-related adverse events (IRAE). To avoid unnecessary exposure to this risk, it is essential to identify biomarkers that correlate with clinical activity and can be used to recognize and select patients that will benefit from immune checkpoint blockade. We therefore performed extensive immune monitoring in a phase I/II dose escalation/expansion trial of combined Prostate GVAX (a GM-CSF-secreting allogeneic prostate cancer vaccine) and antiCTLA-4/ipilimumab immunotherapy in patients with Castration Resistant Prostate Cancer (CRPC). Here we report on the effects of the treatment on circulating myeloid cells and the identification of potential myeloid-related biomarkers. The GVAX/ipilimumab combination was clinically active with PSA declines of more than 50% in 5, and PSA stabilizations in 12 of 28 patients. Regressing bone and lymph node metastasis were observed in 2/5 PR patients. Flowcytometric monitoring of myeloid subsets in peripheral blood before and after Prostate GVAX/ipilimumab treatment revealed some striking differences between patients who benefited from therapy and patients who did not. Significant treatment-induced decreases of conventional and plasmacytoid Dendritic Cell subsets (cDC and pDC, respectively) were observed, which were paralleled by increased DC activation and recruitment to the vaccination sites. Treatment-induced activation of BDCA1/CD1c+ cDC and 6-sulfo LacNAc+ inflammatory DC was associated with significantly prolonged over-all survival (OS). In contrast, increased frequencies of CD11b+CD14−CD15+ granulocytic myeloid-derived suppressor cells (MDSC) and high pre-treatment levels of CD14+HLA-DRlo/− monocytic MDSC were associated with reduced OS. Together with similar analyses of T cell subsets, these studies have yielded an immune profile with predictive value for clinical outcome. The profile is characterized by pre- or on-treatment activation of immune effector subsets and low frequencies of regulatory/suppressive subsets. It may thus provide a potentially useful tool for patient selection and should be validated as such in other patient groups treated by antiCTLA-4 blockade. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5381. doi:1538-7445.AM2012-5381

The effects of Prostate GVAX and the anti-CTLA4 antibody Ipilimumab were investigated in a Phase I dose escalation/expansion trial of patients with castration resistant prostate cancer. Results showed that the GVAX/Ipilimumab combination was clinically active with PSA declines of more than 50% (Partial Response, PR) in 6 and PSA stabilizations (Stable Disease, SD) in 11 of 28 patients. Immune response monitoring was performed to identify changes that might predict or correlate with clinical efficacy. Upon administration of high Ipilimumab dosages (3 or 5 mg/kg), significant increases in frequencies of activated CD4+ and/or CD8+ T cells were observed by HLA-DR, PD-1, FoxP3 and ICOS expression. Monitoring over the course of treatment, revealed these markers to be differentially associatied with survival, as were levels of effector/memory T cells or Tregs. Early HLA-DR upregulation appeared useful as a marker for response prediction, since it was observed to significant levels in PR or SD, but not in PD patients. In addition, GVAX/Ipilimumab administration was found to induce Th2/Th17 cytokine profiles, as determined ex vivo by intracellular staining of peripheral T cells. Significantly increased levels of IL-4 in both CD4+ and CD8+ T cells were observed in patients with PR or SD, but not in patients with PD and, importantly, profound up-regulation of CD4+IL-5+ T cell frequencies was associated with improved overall survival (p=0.03). Similarly, significantly increased Th17 rates were only observed in patients with PR and SD (p As an indication of tumor-specific responsiveness, IgG antibody responses against 11 (prostate) tumor-associated antigens were determined by western blot and ELISA. Increased seroreactivity to prostate-specific membrane antigen (PSMA), pyridoxamine 5′-phosphate oxidase (PNPO) and/or Neuropilin-2 (NRP2) was significantly correlated with improved overall survival (p Our data indicate that changes in frequencies of activated memory/effector T cells and Tregs, Th2 and Th17 rates and seroconversion to multiple tumor antigens may define a predictive immune profile for patients with potential benefit from Prostate GVAX and/or anti-CTLA4 immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5512. doi:10.1158/1538-7445.AM2011-5512

The effects of a GM-CSF-secreting allogeneic prostate cancer vaccine (Prostate GVAX) and the anti-CTLA4 antibody Ipilimumab were investigated in a Phase I dose escalation/expansion trial of patients with metastatic, hormone-refractory prostate cancer (mHRPC). Patients received a 500 million cell GVAX priming dose on day 1 followed by 12 bi-weekly intradermal administrations of 300 million cells, while Ipilimumab was administered every 4 wks from day 1 for a total of 6 times. Initially, 12 patients were enrolled in cohorts of 3 and each cohort was assigned an escalating dose of Ipilimumab at 0.3, 1, 3 or 5 mg/kg. 16 additional patients were enrolled in the expansion cohort of 3 mg/kg Ipilimumab. Results showed that the GVAX and Ipilimumab combination was clinically active in mHRPC patients. PSA declines of more than 50% (Partial Response, PR) were observed in 5 of 22 patients in the 3–5 mg/kg Ipilimumab dose cohorts, and were associated with Autoimmune Breakthrough Events (ABE), including Grade 2 or 3 hypophysitis and Grade 3 alveolitis. PSA stabilizations (Stable Disease, SD) were observed in 1/3 patients in lower (0.3 and 1 mg/kg), and in 7 of 22 in the higher (3–5 mg/kg) dose levels. Moreover, regressing bone and lymph node metastases were observed in 2/5 PR patients. Immune response monitoring was performed to identify changes that might predict or correlate with clinical efficacy. Most notably, pronounced and significant increases in frequencies of activated and effector CD4+ and CD8+ T cells were observed by HLA-DR and ICOS expression upon administration of high (3 and 5 mg/kg) Ipilimumab doses; a relation to clinical behaviour was only observed for HLA-DR with earlier and more pronounced increases in patients with PR or SD. As an indication of tumor-specific responsiveness HLA-Tetramer (Tm) and seroreactivity to NY-ESO and PSMA were tested. For NY-ESO, therapy-induced increased seroreactivity (by Western Blot or ELISA) was observed in 6/28 patients. Of these 6 patients, three could be tested for Tm reactivity and in two (1SD, 1 Progressive Disease, PD) increased NY-ESO157 rates were found concomitant with increasing serum Ab levels, whereas no Tm reactivity was detected in 8 patients without seroconversions. PSMA seroconversions were observed in a total of 12/28 patients (and, of note, in 4/5 PR), but no Tm positivity at any time was found in a total of 15 patients tested. In the 3–5 mg/kg dose levels (n=22), PSMA seroconversion was associated with increased overall survival (P=0.062). In addition, combined GVAX/Ipilimumab administration was found to induce Type-2/Th17 profiles, as determined ex vivo by intracellular staining of peripheral T cells. Significantly increased levels of IL-4 in both CD4+ and CD8+ T cells were observed in patients with PR or SD (P In summary, our analyses so far reveal a relationship between clinical efficacy, PSMA seroconversion, and generalized T cell activation accompanied by Th2 and Th17 skewing. Together these data point to a mechanism of action whereby combined Prostate GVAX and anti-CTLA4 immunotherapy can induce both Th2/humoral and Th17/cell-mediated immune responses, resulting in tumor destruction and collateral autoimmunity. Supported by the Prostate Cancer Foundation and the Dutch Cancer Society (KWF-VU 2006-3697) Citation Information: Clin Cancer Res 2010;16(7 Suppl):B20

The effects of a GM-CSF-secreting allogeneic prostate cancer vaccine (Prostate GVAX) and the anti-CTLA4 antibody Ipilimumab were investigated in a Phase I dose escalation/expansion trial of patients with metastatic, hormone-refractory prostate cancer (mHRPC). All patients received a 500 million cell priming dose of GVAX on day 1 followed by bi-weekly intradermal treatments of 300 million cells over a 24-week period. Ipilimumab was administered every 4 wks from day 1 during the same period. Initially, 12 patients were enrolled in cohorts of 3 and each cohort was assigned an escalating dose of Ipilimumab at 0.3, 1, 3 or 5 mg/kg. 16 additional patients were enrolled in the expansion cohort of 3 mg/kg Ipilimumab. Results showed that the GVAX and Ipilimumab combination was active in mHRPC patients. PSA declines of more than 50% (Partial Response, PR) were observed in 5 of 22 patients in the 3-5 mg/kg Ipilimumab doses, and were associated with Autoimmune Breakthrough Events (ABE), including Grade 2 or 3 hypophysitis and Grade 3 alveolitis. PSA stabilizations (Stable Disease, SD) were observed in 1/3 pts in lower (0.3 and 1 mg/kg), and in 7 of 22 in the higher (3-5 mg/kg) dose levels. In this study, cytokine profiles of peripheral blood-derived T cells were determined ex vivo and after in vitro stimulation with PMA and ionomycin by intracellular staining for IL-2, −4, −5, −10, TNF-α, IFN-γ and IL-17A. Of these cytokines, only IL-4 was detectable ex vivo in CD4+ and CD8+ T cells; post-treatment increases were detectable in the higher (3-5 mg/kg) but not in the lower (0.3-1 mg/kg) Ipilimumab dose levels. After PMA/ionomycin stimulation, no consistent changes were observed in the frequencies of IFN-γ, IL-2 and TNF-α-producing type-1 T cells compared to pre-treatment values. In contrast, significantly increased rates of IL-4 and IL-5 producing CD4+ and CD8+ T cells were found upon treatment (visit 1 versus visit 5) at higher (3-5 mg/kg) Ipilimumab dose levels. Overall these data clearly demonstrate a shift in favor of a Type-2 cytokine profile due to the GVAX/Ipilimumab administration. The frequencies of IL-17A producing CD4+ T (Th17) cells increased over the course of treatment in 5 of 18 patients. Of note, in 3 (PR) out of 5 of these patients this increase of the Th17 subset coincided with onset of hypophysitis and/or adrenal insufficiencies, as well as the onset of PSA decline. The two patients with increased Th17 rates without these pituitary or adrenal-related ABE showed SD. In summary, our results show that combined GVAX/Ipilimumab administration gives rise to Type-2/Th17 cytokine profiles in mHRPC patients, but that only increases in Th17 show a relation to ABE and PSA responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2934.

Background Cancer-related disturbances in myeloid lineage development, marked by high levels of myeloid-derived suppressor cells (MDSC) and impaired dendritic cell (DC) development, are associated with poor clinical outcome due to immune escape and therapy resistance. Redressing this balance may therefore be of clinical benefit. Here we investigated the effects of combined Prostate GVAX/ipilimumab immunotherapy on myeloid subsets in peripheral blood of castration-resistant prostate cancer (CRPC) patients as well as the putative predictive value of baseline and on-treatment myeloid parameters on clinical outcome. Methods Patients with CRPC (n = 28) received thirteen intradermal administrations of Prostate GVAX, consisting of two allogeneic GM-CSF-transduced and irradiated prostate cancer cell lines (LN-CaP and PC3) and six infusions of escalating doses of anti-CTLA4/ipilimumab. Frequencies and activation status of peripheral blood DC (PBDC) and MDSC were determined before, during and after treatment by flowcytometric analysis and related to clinical benefit. Results Significant treatment-induced activation of conventional and plasmacytoid DC subsets (cDC and pDC) was observed, which in the case of BDCA1/CD1c+ cDC1 and MDC8+/6-sulfoLacNAc+ inflammatory cDC3 was associated with significantly prolonged overall survival (OS), but also with the development of autoimmune-related adverse events. High pre-treatment levels of CD14+HLA-DR−monocytic MDSC (mMDSC) were associated with reduced OS. Unsupervised clustering of these myeloid biomarkers revealed particular survival advantage in a group of patients with high treatment-induced PBDC activation and low pretreatment frequencies of suppressive mMDSC in conjunction with our previously identified lymphoid biomarker of high pretreatment CD4+CTLA4+ T cell frequencies. Conclusions Our data demonstrate that DC and MDSC subsets are affected by prostate GVAX/ipilimumab therapy and that myeloid profiling may contribute to the identification of patients with possible clinical benefit of Prostate GVAX/ipilimumab treatment. Electronic supplementary material The online version of this article (doi:10.1186/s40425-014-0031-3) contains supplementary material, which is available to authorized users.

Meeting abstracts Immunotherapeutic approaches such as vaccination or immune checkpoint blockade have proven to be clinically active in prostate cancer, but only in fractions of treated patients; this calls for personalized application of these novel therapies based on predictive biomarkers. Our