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The aim of the present study was to enhance the efficiency of leukemia immunotherapy by increasing the antigen-specific cytotoxic T lymphocyte-inducing ability of leukemia cells. The leukemic plasmacytoid dendritic cell line PMDC05 containing the HLA-A02/24 antigen, which was previously established in our laboratory (Laboratory of Hematology and Oncology, Graduate School of Health Sciences, Niigata University, Niigata, Japan), was used in the present study. It exhibited higher expression levels of CD80 following transduction with lentiviruses encoding the CD80 gene. This CD80-expressing PMDC05 was named PMDC11. In order to establish a more potent antigen-presenting cell for cellular immunotherapy of tumors or severe infections, PMDC11 cells were transduced with a constitutively active (ca) toll-like receptor 4 (TLR4) gene using the Tet-On system (caTLR4-PMDC11). CD8(+) T cells from healthy donors with HLA-A02 were co-cultured with mutant WT1 peptide-pulsed PMDC11, lipopolysaccharide (LPS)-stimulated PMDC11 or caTLR4-PMDC11 cells. Interleukin (IL)-2 (50 IU/ml) and IL-7 (10 ng/ml) were added on day three of culture. Priming with mutant WT1 peptide-pulsed PMDC11, LPS-stimulated PMDC11 or caTLR4-PMDC11 cells was conducted once per week and two thirds of the IL-2/IL-7 containing medium was replenished every 3-4 days. Immediately prior to the priming with these various PMDC11 cells, the cultured cells were analyzed for the secretion of interferon (IFN)-γ in addition to the percentage and number of CD8(+)/WT1 tetramer(+) T cells using flow cytometry. caTLR4-PMDC11 cells were observed to possess greater antigen-presenting abilities compared with those of PMDC11 or LPS-stimulated PMDC11 cells in a mixed leukocyte culture. CD8 T cells positive for the WT1 tetramer were generated following 3-4 weeks of culture and CD8(+)/WT1 tetramer+ T cells were markedly increased in caTLR4-PMDC11-primed CD8(+) T cell culture compared with PMDC11 or LPS-stimulated PMDC11-primed CD8(+) T cell culture. These CD8(+) T cells co-cultured with caTLR4-PMDC11 cells were demonstrated to secrete IFN-γ and to be cytotoxic to WT1-expressing target cells. These data suggested that the antigen-specific cytotoxic T lymphocyte (CTL)-inducing ability of PMDC11 was potentiated via transduction of the caTLR4 gene. The present study also suggested that caTLR4-PMDC11 cells may be applied as potent antigen-presenting cells for generating antigen-specific CTLs in adoptive cellular immunotherapy against tumors and severe viral infections.

PURPOSE: Melanoma patients with a high risk of recurrence may benefit from immunotherapy with mRNA-electroporated autologous monocyte-derived dendritic cells (DCs). Further benefit may be found in combining DC-therapy with interferon alfa-2b. PATIENTS AND METHODS: The long-term clinical outcome of AJCC stage III/IV melanoma patients who had no evidence of disease at the time of treatment with autologous mRNA-electroporated DCs in a single-center pilot clinical trial was analyzed. Antigen loading was accomplished by co-electroporation of mRNA encoding a fusion protein between MAGE-A1, -A3, -C2, Tyrosinase, MelanA/MART-1, or gp100, and an HLA class II-targeting sequence. DCs were administered by 4-6 bi-weekly intradermal injections. IFN-α-2b (5 MIU TIW) was initiated either at recurrence (cohort 1), concomitant with DCs (cohorts 2 and 3), or following the fourth DC administration (cohort 4). RESULTS: Thirty melanoma patients were recruited between April 2006 and June 2009. DC-related adverse events included grade 2 local injection site reactions in all patients, grade 2 fever and flu-like symptoms in one patient, and skin depigmentation in seven patients. After a median follow-up of over 6 years, the median relapse-free survival is 22 months (95% CI 12-32 months). Twelve patients have died. The median overall survival has not been reached; the 2-year and 4-year survival rates are 93 and 70%, respectively. CONCLUSIONS: Adjuvant therapy following the resection of melanoma metastases with autologous mRNA-electroporated DCs, combined with interferon alfa-2b, is tolerable and results in encouraging long-term overall survival rates justifying further evaluation in a randomized clinical trial.

Since decades, the main goal of tumor immunologists has been to increase the capacity of the immune system to mediate tumor regression. In this regard, one of the major focuses of cancer immunotherapy has been the design of vaccines promoting strong tumor-specific cytotoxic T lymphocyte responses in cancer patients. Here, dendritic cells (DCs) play a pivotal role as they are regarded as nature's adjuvant and as such have become the natural agents for antigen delivery in order to finally elicit strong T cell responses (Villadangos and Schnorrer in Nat Rev Immunol 7:543-555, 2007; Melief in Immunity 29:372-383, 2008; Palucka and Banchereau in Nat Rev Cancer 12:265-277, 2012; Vacchelli et al. in Oncoimmunology 2:e25771, 2013; Galluzzi et al. in Oncoimmunology 1:1111-1134, 2012). Therefore, many investigators are actively pursuing the use of DCs as an efficient way of inducing anticancer immune responses. Nowadays, DCs can be generated at a large scale in closed systems, yielding sufficient numbers of cells for clinical application. In addition, with the identification of tumor-associated antigens, which are either selectively or preferentially expressed by tumors, a whole range of strategies using DCs for immunotherapy have been designed and tested in clinical studies. Despite the evidence that DCs loaded with tumor-associated antigens can elicit immune responses in vivo, clinical responses remained disappointingly low. Therefore, optimization of the cellular product and route of administration was urgently needed. Here, we review the path we have followed in the development of TriMixDC-MEL, a potent DC-based cellular therapy, discussing its development as well as further modifications and applications.

Dendritic cell therapy has been optimized a lot aiming to induce a strong and broad immune response in terms of the recognized epitopes by both CD8+ and CD4+ T cells and the use of the patients' complete unique set of HLA molecules. We here give an overview of our approach for antigen loading and maturation of dendritic cells and describe the consequences to evaluate the immune response after treatment as well as the Brussels experience in clinical trials.

Dendritic cells (DCs) are the orchestrators of the immune system and are frequently used in clinical trials in order to boost the immune system in cancer patients. Among several available techniques for DC modification, mRNA electroporation is an interesting technique due to the favorable characteristics of mRNA. Antigen expression level and duration can be increased by multiple optimizations of an antigen-encoding mRNA template. Here, we describe different molecular modifications to a WT1-encoding mRNA construct in order to increase antigen expression and the subsequent introduction of mRNA into DCs.

Antitumor cytolytic T lymphocytes (CTLs) recognize peptides derived from cellular proteins and presented on MHC class I. One category of peptides recognized by these CTLs is derived from proteins encoded by "cancer-germline" genes, which are specifically expressed in tumors, and therefore represent optimal targets for cancer immunotherapy. Here, we identify an antigenic peptide, which is derived from the MAGE-A1-encoded protein (160-169) and presented to CTLs by HLA-B*44:02. Although this peptide is encoded by MAGE-A1, processed endogenously and presented by tumor cells, the corresponding synthetic peptide is hardly able to sensitize target cells to CTL recognition when pulsed exogenously. Endogenous processing and presentation of this peptide is strictly dependent on the presence of tapasin, which is believed to help peptide loading by stabilizing a peptide-receptive form of HLA-B*44:02. Exogenous loading of the peptide can be dramatically improved by paraformaldehyde fixation of surface molecules or by peptide loading at acidic pH. Either strategy allows efficient exogenous loading of the peptide, presumably by generating or stabilizing a peptide-receptive, empty conformation of the HLA. Altogether, our results indicate a potential drawback of short peptide-based vaccination strategies and offer possible solutions regarding the use of problematic epitopes such as the one described here.

Cancer immunotherapy aims to establish immune-mediated control of tumor growth by priming T-cell responses to target tumor-associated antigens. Three signals are required for T-cell activation: (i) presentation of cognate antigen in self MHC molecules; (ii) costimulation by membrane-bound receptor-ligand pairs; and (iii) soluble factors to direct polarization of the ensuing immune response. The ability of dendritic cells (DCs) to provide all three signals required for T-cell activation makes them an ideal cancer vaccine platform. Several strategies have been developed to enhance and control antigen presentation, costimulation, and cytokine production. In this review, we discuss progress toward developing DC-based cancer vaccines by genetic modification using RNA, DNA, and recombinant viruses. Furthermore, the ability of DC-based vaccines to activate natural killer (NK) and B-cells, and the impact of gene modification strategies on these populations is described. Clinical trials using gene-modified DCs have shown modest results, therefore, further considerations for DC manipulation to enhance their clinical efficacy are also discussed.

Melanoma metastases are characterized by pronounced neo-angiogenesis and spontaneous bleeding frequently occurring within central nervous system metastases. Clinically apparent spontaneous hemorrhage within subcutaneous melanoma metastases, however, is a rare event that coincides with progression of such metastases. We report, to our knowledge the first observation, on regression of subcutaneous metastases with hemorrhage of the overlying skin in three patients with stage IV melanoma who participated in clinical trials on therapeutic vaccination. In two patients, loss of arterial flow on Doppler ultrasound imaging was documented in the metastasis at the time of hematoma formation. One patient suffered from an intracranial hemorrhage in a subcentimetric brain metastasis coincident with the hemorrhagic regression of some of his skin metastases.

Dendritic cells (DCs) are the orchestrators of the immune system and are frequently used in clinical trials in order to boost the immune system in cancer patients. Among several available techniques for DC modification, mRNA electroporation is an interesting technique due to the favorable characteristics of mRNA. Antigen expression level and duration can be increased by multiple optimizations of an antigen-encoding mRNA template. Here, we describe different molecular modifications to a WT1-encoding mRNA construct in order to increase antigen expression and the subsequent introduction of mRNA into DCs.

Cancer immunotherapy has been proposed as a powerful treatment modality. Active immunotherapy aspires to stimulate the patient's immune system, particularly T cells. These cells can recognize and kill cancer cells and can form an immunological memory. Dendritic cells (DCs) are the professional antigen-presenting cells of our immune system. They take up and process antigens to present them to T cells. Consequently, DCs have been investigated as a means to stimulate cancer-specific T-cell responses. An efficient strategy to program DCs is the use of mRNA, a well-defined and safe molecule that can be easily generated at high purity. Importantly, vaccines consisting of mRNA-modified DCs showed promising results in clinical trials. Therefore, we will introduce cancer immunotherapy and DCs and give a detailed overview on the application of mRNA to generate cancer-fighting DC vaccines.

Dendritic cells (DC) are professional antigen-presenting cells that are used in vaccine approaches to cancer. Classically, mature monocyte-derived DC are generated in vitro in the presence of interleukin (IL)-4, granulocyte macrophage-colony stimulating factor, and inflammatory cytokines (G4-DC). Recently, it has been described that DC can also be generated in the presence of IL-3 and interferon (IFN)-β and that these DC are efficiently matured using polyriboinosinic polyribocytidylic acid (I3-DC). In this study, a series of in vitro experiments was performed to compare side-by-side I3-DC and G4-DC as vaccine adjuvants. Phenotypic characterization of the DC revealed differences in the expression of the monocyte marker CD14 and the maturation marker CD83. Low expression of CD14 and high expression of CD83 characterized G4-DC, whereas I3-DC displayed intermediate expression of CD14 and CD83. Both types of DC were as potent in the induction of allogeneic T cell proliferation. Upon CD40 ligation, G4-DC produced lower amounts of IFN-α and pulmonary and activation-regulated chemokine, similar amounts of IL-6, macrophage-inflammatory protein (MIP)-1α, and MIP-1β, and higher amounts of IL-12 p70, tumor necrosis factor α, and MIP-3β than I3-DC. We further evaluated whether the DC could be frozen/thawed without loss of cell number, viability, phenotype, and function. After freezing/thawing, 56.0% ± 9.0% of I3-DC and 77.0% ± 3.0% of G4-DC (n=9) were recovered as viable cells, displaying the same phenotype as their fresh counterparts. Finally, in vitro stimulations showed that fresh and frozen peptide-loaded I3-DC are more potent inducers of Melan-A-specific CD8+ T cell responses than G4-DC. The antigen-specific T cells were functional as shown in cytotoxicity and IFN-γ secretion assay.

An optimal anticancer vaccine probably requires the cooperation of both CD4+ Th cells and CD8+ CTLs. A promising tool in cancer immunotherapy is, therefore, the genetic modification of dendritic cells (DCs) by introducing the coding region of a tumor Ag, of which the antigenic peptides will be presented in both HLA class I and class II molecules. This can be achieved by linking the tumor Ag to the HLA class II-targeting sequence of an endosomal or lysosomal protein. In this study we compared the efficiency of the targeting signals of invariant chain, lysosome-associated membrane protein-1 (LAMP1) and DC-LAMP. Human DCs were electroporated before or after maturation with mRNA encoding unmodified enhanced green fluorescent protein (eGFP) or eGFP linked to various targeting signals. The lysosomal degradation inhibitor chloroquine was added, and eGFP expression was evaluated at different time points after electroporation. DCs were also electroporated with unmodified MAGE-A3 or MAGE-A3 linked to the targeting signals, and the presentation of MAGE-A3-derived epitopes in the context of HLA class I and class II molecules was investigated. Our data suggest that proteins linked to the different targeting signals are targeted to the lysosomes and are indeed presented in the context of HLA class I and class II molecules, but with different efficiencies. Proteins linked to the LAMP1 or DC-LAMP signal are more efficiently presented than proteins linked to the invariant chain-targeting signal. Furthermore, DCs electroporated after maturation are more efficient in Ag presentation than DCs electroporated before maturation.

Dendritic cells (DC) are key players in several types of cancer vaccines. Large numbers of DC can easily be generated in closed systems from the monocyte fraction of the peripheral blood. They are the professional antigen-presenting cells, and electroporation of mRNA-encoding tumor antigens is a very efficient and a relatively simple way to load the DC with antigen. The co-electroporation of a tumor antigen of choice and the combination of 3 costimulatory molecules, including CD70, caTLR4, and CD40L (TriMix-DC), leads to fully potent antigen-presenting DC able to generate a broad immune response.Here we describe the in vitro transcription of the mRNA and the subsequent generation and electroporation of autologous DC used for the treatment of melanoma patients.

Dendritic cells (DC) are key players in several types of cancer vaccines. Large numbers of DC can easily be generated in closed systems from the monocyte fraction of the peripheral blood. They are the professional antigen-presenting cells, and electroporation of mRNA-encoding tumor antigens is a very efficient and a relatively simple way to load the DC with antigen. The co-electroporation of a tumor antigen of choice and the combination of 3 costimulatory molecules, including CD70, caTLR4, and CD40L (TriMix-DC), leads to fully potent antigen-presenting DC able to generate a broad immune response.Here we describe the in vitro transcription of the mRNA and the subsequent generation and electroporation of autologous DC used for the treatment of melanoma patients.

Regulatory T cells (Tregs) counteract anticancer immune responses through a number of mechanisms, limiting dendritic cell (DC)–based anticancer immunotherapy. In this study, we investigated the influence of various DC activation stimuli on the Treg functionality. We compared DCs activated by electroporation with mRNA encoding constitutively active TLR4 (caTLR4) and CD40 ligand (DiMix-DCs), or these factors together with mRNA encoding the costimulatory molecule CD70 (TriMix-DCs) with DCs maturated in the presence of a mixture of inflammatory cytokines (DCs maturated with a combination of the cytokines IL-1β, IL-6, TNF-α, and PGE2) for their ability to counteract Tregs on different levels. We first demonstrated that there was no difference in the extent of Treg induction starting from CD4+CD25− T cells under the influence of the different DC maturation stimuli. Second, we showed that both DiMix- and TriMix-DCs could partly alleviate Treg inhibition of CD8+ T cells. Third, we observed that CD8+ T cells that had been precultured with DiMix-DCs or TriMix-DCs were partially protected against subsequent Treg suppression. Finally, we showed that Tregs cocultured in the presence of TriMix-DCs, but not DiMix-DCs, partially lost their suppressive capacity. This was accompanied by a decrease in CD27 and CD25 expression on Tregs, as well as an increase in the expression of T-bet and secretion of IFN-γ, TNF-α, and IL-10, suggesting a shift of the Treg phenotype toward a Th1 phenotype. In conclusion, these data suggest that TriMix-DCs are not only able to suppress Treg functions, but moreover could be able to reprogram Tregs to Th1 cells under certain circumstances.

Background Autologous monocyte-derived dendritic cells (DCs) electroporated with synthetic messenger RNA (mRNA) encoding a CD40 ligand, a constitutively active Toll-like receptor 4 and CD70, together with mRNA encoding fusion proteins of a human leukocyte antigen (HLA)-class II targeting signal (DC-LAMP) and a melanoma-associated antigen (MAA); either MAGE-A3, MAGE-C2, tyrosinase or gp100) (TriMixDC-MEL) are superiorly immunogenic. Patients and methods In this phase IB clinical trial, 24 million viable DCs were administered by four biweekly combined intradermal (id) and intravenous (iv) administrations, and a fifth administration on week 16. The number of iv-administered DCs was escalated in four sequentially treated cohorts. Immune responses were assessed by analysis of antigen specificity of blood-derived T-cells and skin infiltrating lymphocytes (SKILs). Results Fifteen patients with pretreated advanced melanoma tolerated administration of TriMixDC-MEL well. Two patients achieved a complete response and two patients a partial response. All objective responders are progression-free after a follow-up of, respectively, 24+, 28+, 33+, and 34+ months. Post-therapy antigen-specific SKILs were documented in 6 of 12 patients, and antigen-specific CD8+ T-cells were detected in the blood of 4 of 5 patients. Conclusions Cellular immunotherapy with TriMixDC-MEL is safe and immunogenic. Antitumor activity with durable disease control is observed across the investigated iv-dose levels. ClinicalTrials.gov Identifier NCT01066390.

Treatment of melanoma patients with mRNA electroporated dendritic cells (TriMixDC-MEL) stimulates T-cell responses against the presented tumor-associated antigens (TAAs). In the current clinical trials, melanoma patients with systemic metastases are treated, requiring priming and/or expansion of preexisting TAA-specific T cells that are able to migrate to both the skin and internal organs. We monitored the presence of TAA-specific CD8+T cells infiltrating the skin at sites of intradermal TriMixDC-MEL injection (SKILs) and within the circulation of melanoma patients treated in two clinical trials. In 10 out of fourteen (71%) patients screened, CD8+T cells recognizing any of the four TAA presented by TriMixDC-MEL cellular vaccine were found in both compartments. In total, 30 TAA-specific T-cell responses were detected among the SKILs and 29 among peripheral blood T cells, of which 24 in common. A detailed characterization of the antigen specificity of CD8+T-cell populations in four patients indicates that the majority of the epitopes detected were only recognized by CD8+T cells derived from either skin biopsies or peripheral blood, indicating that some compartmentalization occurs after TriMix-DC therapy. To conclude, functional TAA-specific CD8+T cells distribute both to the skin and peripheral blood of patients after TriMixDC-MEL therapy.

Tumor antigen-encoding mRNA for dendritic cell (DC)-based vaccination has gained increasing popularity in recent years. Within this context, two main strategies have entered the clinical trial stage: the use of mRNA for ex vivo antigen loading of DCs and the direct application of mRNA as a source of antigen for DCs in vivo. DCs transfected with mRNA-encoding Wilms' tumor 1 (WT1) protein have shown promising clinical results. Using a stepwise approach, we re-engineered a WT1 cDNA-carrying transcription vector to improve the translational characteristics and immunogenicity of the transcribed mRNA. Different modifications were performed: (i) the WT1 sequence was flanked by the lysosomal targeting sequence of dendritic cell lysosomal-associated membrane protein to enhance cytoplasmic expression; (ii) the nuclear localization sequence (NLS) of WT1 was deleted to promote shuttling from the nucleus to the cytoplasm; (iii) the WT1 DNA sequence was optimized in silico to improve translational efficiency; and (iv) this WT1 sequence was cloned into an optimized RNA transcription vector. DCs electroporated with this optimized mRNA showed an improved ability to stimulate WT1-specific T-cell immunity. Furthermore, in a murine model, we were able to show the safety, immunogenicity, and therapeutic activity of this optimized mRNA. This work is relevant for the future development of improved mRNA-based vaccine strategies K.Molecular Therapy-Nucleic Acids (2013) 2, e134; doi:10.1038/mtna.2013.54; published online 19 November 2013.

It is generally thought that dendritic cells (DCs) loaded with full-length tumor antigen could improve immunotherapy by stimulating broad T-cell responses and by allowing treatment irrespective of the patient's human leukocyte antigen (HLA) type. To investigate this, we determined the specificity of T cells from melanoma patients treated with DCs loaded with mRNA encoding a full-length tumor antigen fused to a signal peptide and an HLA class II sorting signal, allowing presentation in HLA class I and II. In delayed-type hypersensitive (DTH)-biopsies and blood, we found functional CD8(+) and CD4(+) T cells recognizing novel treatment-antigen-derived epitopes, presented by several HLA types. Additionally, we identified a CD8(+) response specific for the signal peptide incorporated to elicit presentation by HLA class II and a CD4(+) response specific for the fusion region of the signal peptide and one of the antigens. This demonstrates that the fusion proteins contain newly created immunogenic sequences and provides evidence that ex vivo-generated mRNA-modified DCs can induce effector CD8(+) and CD4(+) T cells from the naive T-cell repertoire of melanoma patients. Thus, this work provides definitive proof that DCs presenting the full antigenic spectrum of tumor antigens can induce T cells specific for novel epitopes and can be administered to patients irrespective of their HLA type.

The use of tumor-associated antigen (TAA) mRNA for therapeutic purposes is under active investigation. To be effective, mRNA vaccines need to deliver activation stimuli in addition to TAAs to dendritic cells (DC). In this study, we evaluated whether intranodal delivery of TAA mRNA together with TriMix, a mix of mRNA encoding CD40 ligand, constitutive active Toll-like receptor 4 and CD70, results in the in situ modification and maturation of DCs, hence, priming of TAA-specific T cells. We showed selective uptake and translation of mRNA in vivo by lymph node resident CD11c+ cells. This process was hampered by codelivery of classical maturation stimuli but not by TriMix mRNA. Importantly, TriMix mRNA induced a T-cell–attracting and stimulatory environment, including recruitment of antigen-specific CD4+ and CD8+ T cells and CTLs against various TAAs. In several mouse tumor models, mRNA vaccination was as efficient in CTL induction and therapy response as vaccination with mRNA-electroporated DCs. Together, our findings suggest that intranodal administration of TAA mRNA together with mRNA encoding immunomodulating molecules is a promising vaccination strategy. Cancer Res; 72(7); 1661–71. ©2012 AACR.

Dendritic cells (DCs) electroporated with mRNA encoding CD70, CD40L and a constitutively active toll-like receptor 4 (TriMix-DC) have an increased T-cell stimulatory capacity. In a prospective phase IB clinical trial, we treated melanoma patients with intradermal and intravenous injections of autologous TriMix-DC co-electroporated with mRNA encoding full-length MAGE-A3, MAGE-C2, tyrosinase and gp100. We report here the immunological and clinical results obtained in one patient with a particularly favorable outcome. This patient had stage IV-M1c melanoma with documented progression during dacarbazine chemotherapy and received 5 TriMix-DC injections. Following DC therapy, a broad CD8(+) T-cell response against multiple epitopes derived from all four treatment antigens was found in the blood and among T cells derived from DTH biopsy. In addition, CD4(+) T cells recognizing different MAGE-A3-derived epitopes were detected in DTH-derived cells. A spontaneous anti-MAGE-C2 CD8(+) T-cell response was present prior to TriMix-DC therapy and increased during treatment. The tumor response was assessed with 18-fluorodeoxyglucose-positron emission/computed tomography. We documented a partial tumor response according to RECIST criteria with a marked reduction in (18)F-FDG-uptake by lung, lymph node and bone metastases. The patient remains free from progression after 12 months of follow-up. This case report indicates that administration of autologous TriMix-DC by the combined intradermal and intravenous route can mediate a durable objective tumor response accompanied by a broad T-cell response in a chemorefractory stage IV-M1c melanoma patient.

Background: Autologous monocyte derived dendritic cells (DC) electroporated with synthetic messenger RNA (smRNA) encoding CD40 ligand, a constitutively active TLR4, and CD70 (TriMixDC), together with smRNA encoding fusion-proteins of a HLA-class II targeting signal and a melanoma associated antigen are immunogenic when administered ID (Wilgenhof et al. ASCO AM 2009). The TriMixIDIV clinical trial investigates the safety, immunogenicity, and activity of combined ID and IV administration in patients with pretreated melanoma. Methods: Following leukapheresis, immature DC (derived from adherent PBMC cultured for 6 days in IL-4/GM-CSF supplemented medium) are electroporated with smRNA encoding MAGE-A3, MAGE-C2, Tyrosinase and gp100 linked to DC-LAMP, and TriMix smRNA. TriMix-DC are cryopreserved and 24.106 viable DC are administered by 4 ID/IV-injections q2w, and a 5th-injection on w16. The number of IV administered DC was escalated from 4.106, over 12.106, to 20.106 in cohort 1, 2 and 3 respectively. Tumor response assessments are performed by 18F-PET/CT at baseline and in w8, 16 and 24. Immunomonitoring is performed by analysis of DTH infiltrating lymphocytes (DIL) at an ID injection site. Results: 11 pts (9M/2F; med age 54, range 40-77) with stage IV pretreated melanoma, have initiated study treatment. TriMix-DC related AE's: gr2 local skin injection site reactions (all pts); fever & lethargy (gr1, 1 pt in cohort 1); chills (gr2, 2 pts in cohort 3). Vaccine-specific DIL were documented in 6/7 pts (5/7 pts had a CD137+CD8+ and 4/7 pts a CD4+ T-cell response). As of Jan 2011, 8 pts were evaluated for response (RECISTv1.1): 1 CR, 1 PR, 3 SD, and 3 PD; regression of metastases occurred in lung- (2 pts) and lymph node metastases (3 pts); 4/8 pts remain progression-free after respectively 3.1, 3.6, 7.7 and 8.2 mths of follow-up. Conclusions: TriMix-DC therapy by combined IV/ID administration is feasible, safe, and immunogenic. Durable anti-melanoma activity is observed across the investigated IV-dose levels and compares favorably with our prior observations with TriMix-DC administered ID-only.

Previous studies showed that stimulation of T cells derived from HIV-1-infected patients with autologous dendritic cells electroporated with mRNA encoding HIV antigens can induce antigen-specific T cell responses in vitro. Linking the antigen to an MHC class II-targeting sequence, such as dendritic cell lysosome-associated membrane protein (DC-LAMP), in the mRNA construct results in presentation of antigenic peptides in both MHC class I and class II molecules and therefore enhances the induced T cell responses. To analyze whether the lumenal domain of DC-LAMP is required for optimal induction of cellular immunity against HIV antigens, we compared fusion constructs with or without the lumenal domain of the DC-LAMP protein. A human codon-optimized consensus Gag sequence and a chimeric cDNA sequence encompassing Tat, Rev, and Nef codons (TaReNef ) were cloned into a vector containing the DC-LAMP sequence with or without its lumenal domain. The Gag protein lacking the DC-LAMP-derived sequence altogether elicited only weak T cell responses. DCs electroporated with Gag or TaReNef linked to DC-LAMP were able to elicit similar levels of antigen-specific CD4(+) and CD8(+) T cell responses for both Gag and TaReNef, irrespective of the addition of the DC-LAMP lumenal domain. These data show that DC-LAMP-mediated antigen targeting is absolutely required for optimal T cell stimulation, but that in our experimental setup, the lumenal part of DC-LAMP does not improve the overall induction of antigen-specific T cell responses.

Since decades, the main goal of tumor immunologists has been to increase the capacity of the immune system to mediate tumor regression. Considerable progress has been made in enhancing the efficacy of therapeutic anticancer vaccines. First, dendritic cells (DCs) have been identified as the key players in orchestrating primary immune responses. A better understanding of their biology and the development of procedures to generate vast amounts of DCs in vitro have accelerated the development of potent immunotherapeutic strategies for cancer. Second, tumor-associated antigens have been identified which are either selectively or preferentially expressed by tumor cells and can be recognized by the immune system. Finally, several studies have been performed on the genetic modification of DCs with tumor antigens. In this regard, loading the DCs with mRNA, which enables them to produce/process and present the tumor antigens themselves, has emerged as a promising strategy. Here, we will first overview the different aspects that must be taken into account when generating an mRNA-based DC vaccine and the published clinical studies exploiting mRNA-loaded DCs. Second, we will give a detailed description of a novel procedure to generate a vaccine consisting of tumor antigen-expressing dendritic cells with an in vitro superior capacity to induce anti-tumor immune responses. Here, immature DCs are electroporated with mRNAs encoding a tumor antigen, CD40 ligand (CD40L), CD70, and constitutively active (caTLR4) to generate mature antigen-presenting DCs.

9024 Background: Electroporation of dendritic cells (DC) with mRNA encoding fusion-proteins of a HLA-class II targeting signal and a melanoma associated antigen (MAA) together with mRNA encoding CD40 ligand, a constitutively active TLR4 and CD70 (TriMix) improves the immunostimulatory capacity of autologous DC. Methods: Following leukapheresis, immature DCs (derived from adherent PBMC cultured for 6 days in IL-4 / GM-CSF supplemented medium) are electroporated with mRNA encoding MAGE-A3, MAGE-C2, Tyrosinase and gp100 linked to DC-LAMP, and TriMix mRNA. TriMix-DC (12.5 106/antigen) are cryopreserved and administered by 4 ID-injections q2w, and q8w thereafter. After the 4th vaccination, interferon alfa-2b (IFN- a2b, 5 MIU TIW) is initiated. Immune monitoring is performed by skin biopsy of a vaccine injection site. Biopsies are investigated by IHC and by analyzing the activation (CD137+), cytolytic capacity (CD107a+), and cytokine release (IFN-γ and TNF-α) of DTH infiltrating T-cells in response to autologous EBV-B cells expressing MAA. Results: 29 pts (17M/12F; med age 49, range 28–75) with stage III/IV melanoma, nl LDH, and no CNS metastases were recruited. Vaccine related AE's (first 24 pts): gr2 local injection site reactions (all pts); fever & lethargy (gr2, 1 pt). Pts (20) who initiated IFN-a2b experienced constitutional side effects (gr3, 1 pt). Vaccine-specific DTH infiltrating T cells were documented post-vaccination in 13/17 pts (10/13 pts had a CD137+CD8+ and 2/13 pts a CD4+ response). Out of the 11 pts without evaluable disease, 2 had a local recurrence (salvaged by surgery). After a mFU of 7.8 mths (range 4.3–13.7) all pts remain disease-free. Out of the 13 pts with measurable disease, BOR (RECIST) was 8 SD and 5 PD; 1 pt with initial PD subsequently obtained a PR. Regression of metastases occurred in lung- (2 pts), orbita- (1 pt) and lymph node metastases (3 pts). After a mFU of 7 mths (range 1–14), the mPFS is 3,1 mths (95% CI 2,29–4,08); 4 pts remain progression-free after respectively 5, 8, 10 and 11 mths of follow-up. Conclusions: Therapeutic vaccination with TriMix-DC combined with sequential IFN-a2b is feasible, safe, immunogenic and associated with anti-tumor activity in patients with advanced melanoma. [Table: see text]

The effectiveness of the dendritic cell (DC) vaccination protocols that are currently in use could be improved by providing the DCs with a more potent maturation signal. We therefore investigated whether the T-cell stimulatory capacity of human monocyte-derived DCs could be increased by co-electroporation with different combinations of CD40L, CD70, and constitutively active toll-like receptor 4 (caTLR4) encoding mRNA. We show that immature DCs electroporated with CD40L and/or caTLR4 mRNA, but not those electroporated with CD70 mRNA, acquire a mature phenotype along with an enhanced secretion of several cytokines/chemokines. Moreover, these DCs are very potent in inducing naive CD4(+) T cells to differentiate into interferon-gamma (IFN-gamma)-secreting type 1 T helper (Th1) cells. Further, we assessed the capacity of the electroporated DCs to activate naive HLA-A2-restricted MelanA-specific CD8(+) T cells without the addition of any exogenous cytokines. When all three molecules were combined, a >500-fold increase in MelanA-specific CD8(+) T cells was observed when compared with immature DCs, and a >200-fold increase when compared with cytokine cocktail-matured DCs. In correlation, we found a marked increase in cytolytic and IFN-gamma/tumor necrosis factor-alpha (TNF-alpha) secreting CD8(+) T cells. Our data indicate that immature DCs genetically modified to express stimulating molecules can induce tumor antigen-specific T cells in vitro and could prove to be a significant improvement over DCs matured with the methods currently in use.

Antigen-loaded dendritic cells (DCs) have been intensively investigated as potential cellular antitumor vaccines. Several recent reports have indicated that loading DCs with whole tumor derived mRNA or defined tumor-antigen-encoding mRNA represents an effective nonviral strategy to stimulate T cell responses both for in vitro and in vivo models. Here, we describe the electroporation method as a tool for introducing in vitro transcribed capped mRNA into human DCs for tumor vaccination. We use MART-1/Melan-A as a model tumor-associated antigen for the generation of a DC-based vaccine against melanoma cancer. In addition to efficient antigen loading, it is important to obtain a maximal number of potent antigen-presenting cells. Another prerequisite for the development of a DC-based cancer vaccine is to obtain mature DCs. In this chapter, we describe the basic techniques required for the successful genetic modification of DCs by using the mRNA electroporation method.

Antigen-loaded dendritic cells (DCs) have been intensively investigated as potential cellular antitumor vaccines. Several recent reports have indicated that loading DCs with whole tumor derived mRNA or defined tumor-antigen-encoding mRNA represents an effective nonviral strategy to stimulate T cell responses both for in vitro and in vivo models. Here, we describe the electroporation method as a tool for introducing in vitro transcribed capped mRNA into human DCs for tumor vaccination. We use MART-1/Melan-A as a model tumor-associated antigen for the generation of a DC-based vaccine against melanoma cancer. In addition to efficient antigen loading, it is important to obtain a maximal number of potent antigen-presenting cells. Another prerequisite for the development of a DC-based cancer vaccine is to obtain mature DCs. In this chapter, we describe the basic techniques required for the successful genetic modification of DCs by using the mRNA electroporation method.

The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutic strategies. One of the approaches that has gained prominence in recent years is therapeutic vaccination. We decided to assess the capacity of mature dendritic cells, derived from blood monocytes of HIV-1 infected patients, to generate functional T-cell responses. For this purpose, we constructed a chimeric mRNA encoding the proteins Tat, Rev and Nef. The TaReNef encoding information was linked to the HLA class II-targeting sequence of DC-LAMP. Broadly directed HIV-specific CD4(+) and CD8(+) cytotoxic T cells exhibiting a poly-functional cytokine secretion pattern were generated by co-culturing with autologous chimeric mRNA electroporated dendritic cells. Thus, administration of ex vivo generated dendritic cells expressing the early proteins Tat, Rev and Nef might offer a promising approach for therapeutic vaccination in HIV-1 infection. (C) 2008 Elsevier Ltd. All rights reserved.

CD4+CD25+ regulatory T cells (Treg) have been described as an important hurdle for immunotherapy. Engagement of glucocorticoid-induced TNF receptor-related protein (GITR) has emerged recently as an important mechanism to control the suppression of CD4+CD25+ Treg. Furthermore, it has been documented extensively that GITR ligation is costimulatory for naive and activated T cells in the murine setting. However, little is known about the role of the human GITR ligand (huGITRL). We wanted to explore whether huGITRL could enhance antigen-specific T cell priming by dendritic cells (DC). First, we confirmed the endogenous expression of GITRL on HUVEC. We also detected GITRL expression on EBV-B cell lines, whereas no GITRL expression was observed on human monocyte-derived DC. Electroporation of GITRL mRNA in monocyte-derived DC resulted in a strong and long-lasting surface expression of GITRL. In contrast to data obtained in mice, no significant abrogation of Treg suppression by GITRL-expressing human DC was observed. Consistent with our mouse data, we showed that huGITRL is costimulatory for responder T cells. Furthermore, we found that GITRL-expressing DC primed increased numbers of Melan-A-specific CD8+ T cells. We conclude that although huGITRL is not capable of alleviating Treg suppression of responder T cells, huGITRL overexpression on monocyte-derived DC enhances their capacity to induce antigen-specific T cell responses. Thus, GITRL incorporation in DC might improve the antitumor immune response after vaccination.

Ex vivo lentivirally transduced dendritic cells (DC) have been described to induce CD8+ and CD4+ T-cell responses against various tumor-associated antigens (TAAs) in vitro and in vivo. We report here that direct administration of ovalbumin (OVA) encoding lentiviral vectors caused in vivo transduction of cells that were found in draining lymph nodes (LNs) and induced potent anti-OVA cytotoxic T cells similar to those elicited by ex vivo transduced DC. The cytotoxic T-lymphocyte (CTL) response following direct injection of lentiviral vectors was highly effective in eliminating target cells in vivo up to 30 days after immunization and was efficiently recalled after a boost immunization. Injection of lentiviral vectors furthermore activated OVA-specific CD4+ T cells and this CD4 help was shown to be necessary for an adequate primary and memory CTL response. When tested in therapeutic tumor experiments with OVA+ melanoma cells, direct administration of lentiviral vectors slowed down tumor growth to a comparable extent with the highest dose of ex vivo transduced DC. Taken together, these data indicate that direct in vivo administration of lentiviral vectors encoding TAAs has strong potential for anticancer vaccination.

Until now, studies utilizing mRNA electroporation as a tool for the delivery of tumor antigens to human monocyte-derived dendritic cells (DC) have focused on DC electroporated in an immature state. Immature DC are considered to be specialized in antigen capture and processing, whereas mature DC present antigen and have an increased T-cell stimulatory capacity. Therefore, the consensus has been to electroporate DC before maturation. We show that the transfection efficiency of DC electroporated either before or after maturation was similarly high. Both immature and mature electroporated DC, matured in the presence of an inflammatory cytokine cocktail, expressed mature DC surface markers and preserved their capacity to secrete cytokines and chemokines upon CD40 ligation. In addition, both immature and mature DC can be efficiently cryopreserved before or after electroporation without deleterious effects on viability, phenotype or T-cell stimulatory capacity including in vitro antigen-specific T-cell activation. However, DC electroporated after maturation are more efficient in in vitro migration assays and at least as effective in antigen presentation as DC electroporated before maturation. These results are important for vaccination strategies where an optimal antigen presentation by DC after migration to the lymphoid organs is crucial.

Recently, it has become more and more obvious that not only CD8+ cytotoxic T lymphocytes, but also CD4+ T helper cells are required for the induction of an optimal, long-lasting anti-tumor immune response. CD4+ T helper cells, and in particular IFN-gamma-secreting type 1 T helper cells, have been shown to fulfill a critical function in the mounting of a cancer-specific response. Consequently, targeting antigens into MHC class II molecules would greatly enhance the efficacy of an anti-cancer vaccine. The dissection of the MHC class II presentation pathway has paved the way for rational approaches to achieve this goal: novel systems have been developed to genetically manipulate the MHC class II presentation pathway. First, different genetic approaches have been used for the delivery of known epitopes into the MHC class II processing pathway or directly onto the peptide-binding groove of the MHC molecules. Second, several strategies exist for the targeting of whole tumor antigens, containing both MHC class I andclass II restricted epitopes, to the MHC class II processing pathway. We review these data and describe how this knowledge is currently applied in vaccine development. Copyright (c) 2005 John Wiley & Sons, Ltd.

The use of tumor antigen-loaded dendritic cells (DC) is one of the most promising approaches to inducing a tumor-specific immune response. We compared electroporation of mRNA to lentiviral transduction for the delivery of tumor antigens to human monocyte-derived and murine bone marrow-derived DC. Both lentiviral transduction and mRNA electroporation induced eGFP expression in on average 81% of human DC. For murine DC, eGFP mRNA electroporation (62%) proved to be more efficient than lentiviral transduction (47%). When we used tNGFR as a transgene we observed lentiviral pseudotransduction that overestimated lentiviral efficiency. Neither gene transfer method had an adverse effect on viability, phenotype, or allostimulatory capacity of either human or murine DC. Yet, the mRNA-electroporated DC showed a reduced production of IL-12p70 compared to their lentivirally transduced and unmodified counterparts. Human Ii80MAGE-A3-modified DC and murine Ii80tOVA-modified DC were able to present antigenic epitopes in the context of MHC class I and class II. Both types of modified murine DC were able to induce OVA-specific cytotoxic T cells in vivo; however, the mRNA-electroporated DC were less potent. Our data indicate that this may be related to their impaired IL-12 production.

Genetically modified dendritic cells (DC) constitute a promising approach in cancer immunotherapy. Viral gene delivery systems have been shown to be very efficient strategies, but safety concerns for their clinical use in immunotherapy remain an important issue. Recently, the technique of mRNA electroporation was described as a very efficient tool for the genetic modification of human monocyte-derived DC. Here, we show that transgene expression can be modulated by varying the amount of mRNA used for electroporation. We document that CD40 ligation leads to a significant production of IL-12 by the electroporated DC, although the level of IL-12 production is somewhat lower than for non- or mock-electroporated DC. Furthermore, we show that the electroporated DC can be frozen and thawed without loss of viability or function and that Influenza virus Matrix Protein 1 mRNA electroporated DC are capable of inducing a memory cytotoxic T lymphocyte response and are more potent in doing so than mRNA-pulsed DC. Similar results were obtained with MelanA/MART-1 mRNA electroporated DC. These results clearly indicate that mRNA-electroporated DC represent powerful candidates for use as tumor vaccines and could constitute an improvement compared with vaccines using peptide-pulsed DC.

Leukemic plasmacytoid dendritic cell (pDC) line with HLA-A02/24, PMDC05, which was established in our laboratory, acquired a higher antigen presenting ability by being transduced with lentiviruses encoding the CD80 gene. CD80 gene-transduced PMDC05 was named PMDC11. In order to establish a more potent antigen presenting cells for cellular immunotherapy in tumors, PMDC11 cells were transduced with a constitutively active (ca) TLR4 gene by using the Tet-On system (caTLR4-PMDC11). CD8+ T cells from healthy donors with HLA-A24 were co-cultured with WT1 peptide-pulsed caTLR4-PMDC11 cells. Priming with WT1 peptide-pulsed caTLR4-PMDC11 cells was performed every week. Immediately before the priming with caTLR4-PMDC11 cells, cultured cells were analyzed for the percentage and number of CD8+/WT1 tetramer+ T cells. CD8+/WT1 tetramer+ T cells were generated after 2-3 weeks of culture and much increased in caTLR4-PMDC11-primed CD8+ T cell culture compared with parental PMDC11-primed CD8+ T cell culture. These CD8+ T cells co-cultured with caTLR4-PMDC11 cells were demonstrated to be cytotoxic to WT1-expressing HLA-A24+ target cells. These data revealed that the antigen-specific cytotoxic T lymphocyte (CTL) inducing ability of PMDC11 was enhanced by being transduced with caTLR4 gene, and that caTLR4-PMDC11 cells could be applied as potent antigen presenting cells for generating antigen-specific CTLs in adoptive cellular immunotherapy against tumors and severe viral infection.

2507 Background: Autologous monocyte-derived DC electroporated with synthetic mRNA encoding CD40 ligand, a constitutively active TLR4, and CD70 (TriMix-DC) have superior T-cell stimulatory capacity. In a pilot clinical trial, ID administration of TriMixDC-MEL (a mixture of TriMix-DC co-electroporated with mRNA encoding a fusion of DC.LAMP and 1 of 4 melanoma associated antigens) was immunogenic but resulted in limited anti-tumor activity in patients (pts) with advanced melanoma. Ipilimumab (IPI) is a CTLA-4 blocking mAb with established activity in pts with advanced melanoma. Methods: TriMixDC-MEL by the IV and ID-route was investigated as a single-agent or in combination with IPI (10 mg/kg q3wks x4, allowing for maintenance with IPI-alone q12wks in pts PFS at >24 wks). Ratio of ID/IV administered DC: Cohort-1: 20.106/4.106 DC [2pts], -2: 12.106/12.106 DC [3pts], -3: 4.106/20.106 [6pts], and -4: 0/24.106 DC [4pts]; DC were administered 4x q2w, and a 5th administration on w16. in cohort-5, DC (4.106-ID/20.106-IV) were first administered alone and 2w thereafter in combination with IPI for a total of 4 administrations [6pts]. Results: Local skin reactions (gr1-2) were observed in all pts receiving DC ID, flu-like symptoms (< gr2) were observed in 12/21 pts, post IV-infusion chills (gr2) in 7/21 pts. Cytokines release (including a >2-fold rise in the serum levels of IL-1RA, IL-6, IL-8, IL-17, G-CSF, IFN-g, MIP-1a, MIP-1b, TNF-a) was documented during chills. Best objective tumor response: 2x PR + 2x CR (by RECIST) out of 15 pts (27%) treated with DC-only (ongoing after 10+, 14+, 19+, and 20+ mths) and 3 PR out of 6 pts (all stage IV-M1c) treated with DC+IPI (ongoing after 5+, 5+, 6+ mths). Conclusions: ID/IV-administration of TriMixDC-MEL as a single-agent cellular immunotherapy or combined with IPI is associated with distinct but manageable side-effects and has clinical activity against pretreated advanced melanoma. Activity compares favorably with TriMixDC-MEL by ID-administration-only. Combined ID/IV administration of TriMixDC-MEL with IPI is currently under further evaluation in a phase II trial.

8547 Background: mRNA electroporated autologous DC are being developed as therapeutic cancer vaccines. Potential advantages include the presentation of all potential tumor antigen confined epitopes...

The immunostimulatory capacity of dendritic cells is improved by co-electroporation with mRNA encoding CD40 ligand, constitutively active toll-like receptor 4, and CD70 (TriMix-DC). This pilot clinical trial evaluated the feasibility, safety, and immunogenicity of a therapeutic vaccination containing autologous TriMix-DC co-electroporated with mRNA encoding a human leukocyte antigen class II-targeting signal linked to 1 of 4 melanoma-associated antigens (MAGE-A3, MAGE-C2, tyrosinase, and gp100) in patients with advanced melanoma. Thirty-five American Joint Committee on Cancer stage III/IV melanoma patients received autologous TriMix-DC (4 administrations 2 weeks apart). Immune monitoring was performed by evaluating skin biopsies of delayed type IV hypersensitivity (DTH) reactions for presence of vaccinal antigen-specific DTH-infiltrating lymphocytes (DIL). Thereafter, patients could receive interferon-alpha-2b (IFN-alpha-2b) 5 MU subcutaneously 3 times weekly and additional TriMix-DC every 8 weeks. TriMix-DC-related adverse events comprised grade 2 local injection site reactions (all patients), and grade 2 fever and lethargy (2 patients). Vaccinal antigen-specific DIL were found in 0/6 patients tested at vaccine initiation and in 12/21 (57.1%) assessed after the fourth vaccine. A positive postvaccination DTH test correlated with IL-12p70 secretion capacity of TriMix-DC. No objective responses to TriMix-DC alone were seen according to RECIST. Twenty-nine patients received IFN-?-2b after the fourth vaccine without unexpected adverse events. During TriMix-DC/IFN-alpha-2b combination therapy, 1 partial response and 5 stable disease (disease control of >6 months with regression of metastases) were observed in 17 patients with evaluable disease at baseline. In conclusion, this study demonstrated that therapeutic vaccination with autologous TriMix-DC is feasible, safe, and immunogenic and can be combined with sequential IFN-alpha-2b.

Purpose: A critical factor determining the effectiveness of currently used dendritic cell (DC)–based vaccines is the DC activation or maturation status. We have recently shown that the T-cell stimulatory capacity of DCs pulsed with tumor-antigen–derived peptides can be considerably increased by activating the DCs through electroporation with mRNA encoding CD40 ligand, CD70, and a constitutively active Toll-like receptor 4 (TriMix DCs). Here, we investigate whether TriMix DCs can be coelectroporated with whole tumor-antigen–encoding mRNA. Experimental Design: The T-cell stimulatory capacity of TriMix DCs pulsed with the immunodominant MelanA-A2 peptide and that of TriMix DCs coelectroporated with MelanA mRNA were compared in vitro. TriMix DCs were also coelectroporated with mRNA encoding Mage-A3, Mage-C2, tyrosinase, or gp100. The capacity of these DCs to stimulate tumor-antigen–specific T cells in melanoma patients was investigated both in vitro before vaccination and after DC vaccination. Results: Like peptide-pulsed TriMix DCs, TriMix DCs coelectroporated with MelanA mRNA are very potent in inducing MelanA-specific CD8+ T cells in vitro. These T cells have an activated phenotype, show cytolytic capacity, and produce inflammatory cytokines in response to specific stimulation. TriMix DCs coelectroporated with tyrosinase are able to stimulate tyrosinase-specific CD8+ T cells in vitro from the blood of nonvaccinated melanoma patients. Furthermore, TriMix DCs coelectroporated with Mage-A3, Mage-C2, or tyrosinase are able to induce antigen-specific CD8+ T cells through therapeutic DC vaccination. Conclusions: TriMix DCs coelectroporated with whole tumor-antigen mRNA stimulate antigen-specific T cells in vitro and induce antigen-specific T-cell responses in melanoma patients through vaccination. Therefore, they represent a promising new approach for antitumor immunotherapy.

Human CD83 is a marker molecule for mature dendritic cells (DC) and is also expressed on activated B and T cells. Although CD83 has been implicated in immune responses, its function on DC and T cells remains unclear. In this study, we wanted to assess the role of CD83 expressed on DC and T cells in the immune response. Down-regulation of CD83 expression on human DC through RNA interference (RNAi) results in a less potent induction of allogeneic T cell proliferation, reduced IFN-gamma secretion by established T cells and decreased capacity in the priming of functional tumor antigen-specific CD8(+) T lymphocytes. In addition, CD83 mRNA-electroporated DC are stronger T cell stimulators. However, CD83 overexpression on Melan-A/MART-1-specific tumor-infiltrating lymphocytes (TIL) circumvents the need for CD83 expression on DC. Co-culture of immature DC with TIL or K562 cells overexpressing CD83 results in the production of enhanced levels of pro-inflammatory cytokines, whereas this production is less pronounced oreven absent in co-cultures with non-modified TIL or K562 cells. In conclusion, we demonstrate that CD83 expression on T cells and DC modulates the immune response by activating DC and by delivering costimulatory signals for the stimulation of naive and memory T cells, respectively.

The discovery of tumor-associated antigens, which are either selectively or preferentially expressed by tumors, together with an improved insight in dendritic cell biology illustrating their key function in the immune system, have provided a rationale to initiate dendritic cell-based cancer immunotherapy trials. Nevertheless, dendritic cell vaccination is in an early stage, as methods for preparing tumor antigen presenting dendritic cells and improving their immunostimulatory function are continuously being optimized. In addition, recent improvements in immunomonitoring have emphasized the need for careful design of this part of the trials. Still, valuable proofs-of-principle have been obtained, which favor the use of dendritic cells in subsequent, more standardized clinical trials. Here, we review the recent developments in clinical DC generation, antigen loading methods and immunomonitoring approaches for DC-based trials.

In this study, we compared dendritic cells (DCs) differentiated from positively selected monocytes (CD14-DCs) to DCs differentiated from adherence-selected monocytes (adh-DCs) with emphasis on lentiviral transduction. Using a second-generation, triple-helix containing, self-inactivating lentiviral vector at a multiplicity of infection (MOI) of 15, we observed enhanced transduction of CD14-DCs (72.8 +/- 5.3%, mean fluorescence intensity [MFI] = 166 +/- 76) compared to adh-DCs (32.3 +/- 13.1%, MFI = 119 +/- 76, n = 5). More importantly, the efficiency to transduce adh-DCs was significantly increased when monocytes were incubated with antiCD14 antibody coupled beads, anti-CD14 antibodies, or lipopolysaccharide (LPS), reaching transduction efficiencies up to 86.6%, 53.3%, and 80.9%, respectively. We showed that this enhanced transduction was correlated to an activation of the monocytes, characterized by the up regulation of the cytokines interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha and the de novo synthesis of IL-6 and IL-10. However, the enhanced transduction of immature CD14-DCs was not correlated with a progression in the cell cycle from G(0) to G(1). We further showed that CD14-DCs were phenotypically comparable to adh-DCs. Functional analysis revealed that there were no differences in allostimulatory capacity, production of IL-12 p70 on CD40 ligation or expression of IL-1beta, IL-6, IL-8, IL-10, IL-12, and TNF-alpha as evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Finally, we showed that lentivirally transduced CD14-DCs were equally capable as adh-DCs in stimulating MAGE-A3 antigen-specific CD4(+) and CD8(+) T cells in vitro.