Loading Dendritic Cells Research Articles

P03.08 * IMPROVED YIELD OF STEM-LIKE CELLS USING GLIOBLASTOMA SUSPENSIONS FROM CAVITRON ULTRASONIC SURGERY ASPIRATOR (CUSA) BAGS AS STARTING MATERIAL

Absence of serum and presence of growth factors (mostly EGF and bFGF) has been used to culture cancer stem cells (CSC). In case of GB the term neurospheres (NS) has been considered as synonymous of glioma stem-like cells (GSCs). In our laboratories we isolate and culture GSC from fresh GB specimens growing in the absence of serum and in the presence of EGF and bFGF as neurospheres after mechanical and enzymatic dissociation. Previous data and our experience suggest that NS may mirror more closely than previous, serum-based glioma cell lines, the actual biology of GB. Tumor fragments were used to start the culture. The Cavitation Ultrasonics Surgical Aspirator (CUSA) is increasingly used for GB surgery, decreasing the availability of tumor fragments. CUSA delivers an irrigating solution that converts the fragmented tissue into an emulsion and then aspirates the particles directly into a sterile bag. The bag contains some larger pieces of tissue, debris and high amounts of erythrocytes, and occasionally necrotic or reactive tissue. Here we describe our experience with CUSA bags as the staring material for GSC preparation. Tissue fragments in CUSA bags after several rounds of spinning ad washing are dissociated using the GentleMacs Dissociator (Miltenyi Biotec) that provides a closed system and reproducible results. We have optimized a gentle and effective protocol starting from appropriate gentleMACS programs, allowing to obtain a high yield of viable tumor cells. After processing, cell suspensions are cultured as neurospheres in DMEM/F12, B27 supplement, human recombinant b-FGF and EGF. During the last year we obtained from the Department of Neurosurgery of Istituto Besta a total of 54 CUSA. Primary GB were the most represented brain tumors (87%). Using tumor fragments obtained from surgery and combining mechanical dissociation with enzymatic disaggregation on a series of primary GB we previously obtained GSC in 52% of the cases. This percentage is now increased to 71% with the use of surgical material from CUSA and the optimized protocol. Proliferation kinetics was studied by plating three primary cell lines obtained in parallel from GB specimen and from CUSA material at density of 15,000-30,000 cells/cm2. Cultures were collected every 5 days and the total number of viable cells assessed at each passage by Trypan Blue exclusion. A long term proliferation of cell lines at low sub-culturing stages (3 to 10 passages), showed that proliferation increased exponentially, but the proliferation index of NS from CUSA materials was higher that cells from specimens (1.32 vs 1.18 respectively, p = 0.03). These improvements and the use of a closed system providing a wider margin of safety support the incorporation of this process in a clinical trial protocol which plans to use GSC as a source of antigens for dendritic cell loading, replacing the whole lysate from GB specimens used in current immunotherapy protocols.

Fc receptor-targeting of immunogen as a strategy for enhanced antigen loading, vaccination, and protection using intranasally administered antigen-pulsed dendritic cells

Dendritic cells (DCs) play a critical role in the generation of adaptive immunity via the efficient capture, processing, and presentation of antigen (Ag) to naïve T cells. Administration of Ag-pulsed DCs is also an effective strategy for enhancing immunity to tumors and infectious disease organisms. Studies have also demonstrated that targeting Ags to Fcγ receptors (FcγR) on Ag presenting cells can enhance humoral and cellular immunity in vitro and in vivo. Specifically, our studies using a Francisella tularensis (Ft) infectious disease vaccine model have demonstrated that targeting immunogens to FcγR via intranasal (i.n.) administration of monoclonal antibody (mAb)-inactivated Ft (iFt) immune complexes (ICs) enhances protection against Ft challenge. Ft is the causative agent of tularemia, a debilitating disease of humans and other mammals and a category A biothreat agent for which there is no approved vaccine. Therefore, using iFt Ag as a model immunogen, we sought to determine if ex vivo targeting of iFt to FcγR on DCs would enhance the potency of i.n. administered iFt-pulsed DCs. In this study, bone marrow-derived DCs (BMDCs) were pulsed ex vivo with iFt or mAb-iFt ICs. Intranasal administration of mAb-iFt-pulsed BMDCs enhanced humoral and cellular immune responses, as well as protection against Ft live vaccine strain (LVS) challenge. Increased protection correlated with increased iFt loading on the BMDC surface as a consequence of FcγR-targeting. However, the inhibitory FcγRIIB had no impact on this enhancement. In conclusion, targeting Ag ex vivo to FcγR on DCs provides a method for enhanced Ag loading of DCs ex vivo, thereby reducing the amount of Ag required, while also avoiding the inhibitory impact of FcγRIIB. Thus, this represents a simple and less invasive strategy for increasing the potency of ex vivo-pulsed DC vaccines against chronic infectious diseases and cancer.

In situ loading of skin dendritic cells with apoptotic bleb-derived antigens for the induction of tumor-directed immunity

The generation and loading of dendritic cells (DC) ex-vivo for tumor vaccination purposes is laborious and costly. Direct intradermal (i.d.) administration of tumor-associated antigens could be an attractive alternative approach, provided that efficient uptake and cross-presentation by appropriately activated skin DCs can be achieved. Here, we compare the efficiency of i.d. delivery of relatively small apoptotic blebs (diameter ∼0.1–1 μm) derived from MART-1 transduced acute myeloid leukemia (AML) HL60 cells, to that of larger apoptotic cell remnants (ACR; 2–10 μm) in a physiologically highly relevant human skin explant model. Injection of either fluorescently-labelled ACRs or blebs alone did not affect the number or distribution of migrated DC subsets from skin biopsies after 48 hours, but resulted in a general up-regulation of the co-stimulatory molecules CD83 and CD86 on skin DCs that had ingested apoptotic material. We have previously shown that i.d. administration of GM-CSF and IL-4 resulted in preferential migration of a mature and highly T cell-stimulatory CD11hiCD1a+CD14− dermal DC subset. Here, we found that co-injection of GM-CSF and IL-4 together with either ACRs or blebs resulted in uptake efficiencies within this dermal DC subset of 7.6% (±6.1%) and 19.1% (±15.9%), respectively, thus revealing a significantly higher uptake frequency of blebs (P < 0.02). Intradermal delivery of tumor-derived blebs did not affect the T-cell priming and TH-skewing abilities of migratory skin DC. Nevertheless, in contrast to i.d. administration of ACR, the injection of blebs lead to effective cross-presentation of MART-1 to specific CD8+ effector T cells. We conclude that apoptotic bleb-based vaccines delivered through the skin may offer an attractive, and broadly applicable, cancer immunotherapy.

The research of antitumor activities in vitro of DCs loading antigen prouced by radiofrequency ablation of tumor combined with CIK cells

Objective To study the in vitro anti-tumor activity of dendritic cells (DCs) loading with antigen produced by radiofrequency ablation of tumor lysate in situ combined with cytokine-induced killer cells (CIK).Methods CIK ceils derived from BALB/C mouse spleen and DCs derived from bone marrow were prepared,and experimental model of murine colon carcinoma were established for radiofrequency ablation.The supernatant of tumor tissue in situ lysis after repeated freezing and thawing were tested by lowry protein quantitative statutory,amounting to a final concentration of 5 μg/ml,then load to the first 5 days of culture DCs (Ag-DC),2 days later,co-cultured with CIK cells after the first seven days of culture 48 h (Ag-DC-CIK).Flow cytometry was used to analyze costimulatory molecules on the surface of the cells,and CCK-8 assay to detect in vitro cytotoxic activity.Results The DCs loading with antigen resulted in an increase in the proportion of CD86 + CD11 c +,MHC Ⅱ + CD11 c + and MHC Ⅱ + CD80 + cells.The main effector cells of CIK cells were CD3 + NK1.1 + cells.The percentage of CD3 + NK1.1 + cells was 1.45％ on the first day of the culture ; while when they had been cultured for 7 days,the percentage CD3 + NK1.1 + significantly increased to 36.9％.The cytotoxicity of Ag-DC-CIK cells toward C26 cells was much more efficient than that of DC-CIK,CIK cells.The cytotoxic activity of the former was significantly lower than the latter and the same target ratio.When the ratios of effector cells to target cells were 5 ∶ 1,the cytotoxic activity of Ag-DC-CIK cells against C26 cells was (74.9 ± 3.5) ％,; while the DC-CIK was (71.2 ± 2.1) ％ and the CIK cells was (68.7 ± 2.9) ％.The difference was statistically significant(F =7.007,P =0.007).When the ratios of effector cells to target cells were 10 ∶ 1,the cytotoxic activity of Ag-DC-CIK cells against C26 cells was (82.3 ± 4.5) ％,while the DC-CIK cells was (77.1 ± 5.1) ％,and the CIK cells was (72.7 ± 2.8) ％.The difference was statistically significant (F =7.727,P =0.005).When the ratios of effector cells to target cells were 20 ∶ 1,the cytotoxic activity of Ag-DC-CIK cells against C26 cells was (83.2 ± 1.9) ％,while the DC-CIK cells was (77.2 ± 4.2) ％,and the CIK cells was (73.0 ± 2.6) ％.The difference was statistically significant (F =16.594,P =0.000).Conclusion DCs loading with antigen produced by radiofrequency ablation of tumor in situ pyrolysis products can improve in vitro cytotoxic activity combined with CIK cells,which can provide a new comprehensive cancer treatment strategy. Key words: Dendritic cells; Colonic neoplasms; Radiofrequency ablation

Dendritic Cell-Based Approaches in the Fight Against Diseases

DENDRITIC CELL ROLE IN THE IMMUNE SYSTEM AND ITS MANIPULATION The immune system works to contain infections through activation of different molecules and cell types. Correct presentation of antigens by antigen-presenting cells (APCs) is a critical step necessary to initiate an immune response. APCs have the ability to take-up and process antigens, and express high levels of co-stimulatory and major histocompatibility complex (MHC) molecules bound to antigens (1). Dendritic cells (DCs) are innate immune cells first characterized and reported by Ralph Steinman in 1973 (2). For their unique properties and features, DCs are the most important APCs acting at the interface of innate and adaptive immunity, which results in the activation of immune responses in the body. Distinct subsets of DCs are associated with lineage and receptor expression patterns (3). Dendritic cells have different roles in the immune system, such as activation and regulation of adaptive immune responses, and other opposing functions in the induction of tolerance and anergy (4). During immune responses, DCs are crucial decision makers toward the development of naive T cells to T helper type 1 (Th1) or type (Th2) profile (5). Among the different families of molecules expressed by DC to aid in their function, one of them is the family of toll-like receptors (TLR). TLR which are expressed by different types of DCs, and bind to common molecules associated with pathogens. Once bound, molecules such as bacterial lipopolysaccharide and hypomethylated CpG DNA, can induce activation of biochemical cell pathways, resulting in overexpression of MHC, co-stimulatory molecules (CD80, CD86), and cytokines (6). In this context, a number of methods have been available to manipulate DCs from diverse sites in the body resulting in activated cells for therapy. These methods include reinfusion of unloaded DCs; reinfusion of DCs co-cultured with peptides or proteins of interest; in vivo DC loading; DC transfection with antigen-encoding viruses or nucleic acids; and DC-derived exosomes (7, 8). After this, DCs might be ready to promote protection or treat specific diseases. In this context, the availability of methods to manipulate DCs in laboratory, arise as an important tool for immunointerventions in different diseases. In this opinion article, we focused on the basis of DC approaches already available in the field of cancer currently in test for infectious diseases, and future interventions that are needed.

Abstract P4-13-04: Autologous dendritic cells vaccines combined with neoadjuvant chemotherapy increase total pCR in stages II-III non-overexpressing HER2 breast cancer patients and induce phenotypic changes in peripheral blood

Abstract Background Based on the synergistic effect between immuno- and chemotherapy (CT), we have elaborated an autologous vaccine with dendritic cells loaded with patients´ own tumor antigens (lysate), and we have already demonstrated that the addition of the vaccines to a standard neoadjuvant CT schedule has increased total pCR (breast+ axilla) in stages II-III non-overexpressing HER2 breast cancer patients (Santisteban M, SABCS 2012). Both cohorts, the control (C) and the vaccinated (V) were well balanced related to demographic characteristics. Toxicity has been similar in both the C and the V cohorts. Moreover, we have analyzed the phenotypic changes in peripheral blood induced by the vaccine and its correlation with pathologic responses. Indeed, we have studied if the amount of lysate used to load the dendritic cells or the total dendritic cell numbers received by the patients in the first five doses (before surgery) is correlated with pCR Methods Twenty-eight patients with stage II-III HER2 negative breast cancer have started on sequential neoadjuvant CT based on dose dense antracyclines (E 100mg/m2 and C 600 mgr/m2) x4 cycles plus GM-CSF followed by taxanes (DOC 75-100 mgr/m2) x4 cycles plus vaccination. The C historic cohort was composed of thirty patients who received the same treatment except for the absence of the vaccines. Vaccine calendar was started after the 4th EC and alternated with DOC and as maintenance up to a maximum of a 2 year-period. The first 5 vaccines were administered before breast surgery. Changes in different lymphocytes populations were measured in peripheral blood of patients at different points by flow cytometry (absolute cell counts). To date, twenty-one patients have both determinations of lymphocyte subpopulations before the 1st and the 6th vaccine. Paired samples t-tests and Fisher exact were used Results pCR was superior in the V cohort (24% versus 3.3%, p = 0.04). Lymphocyte subpopulations were measured in peripheral blood (cells/uL) and a stimulation of the immune system was found after the 5 vaccines schedule at the time of surgery as follows: NK (p&amp;lt;0.001), T cytotoxic CD8 (p = 0.018), T helper CD4 (p = 0.04), CD19 (p = 0.001), HLADRCD8 (p = 0.007), CD16CD8 (p = 0.003), HLADRCD4 (p&amp;lt;0.001), CD16CD4 (p = 0.008) and T regulators lymphocytes (p = 0.004). We did not find any differences among CD57CD8 (p = 0.17), CD56CD8 (p = 0.11), CD57CD4 (p = 0.45) and CD56CD4 (p = 0.65). We neither see correlation among the amount of lysate to load dendritic cells and the tpCR (p = 0.09) nor the amount of dendritic cells (summatory of 5 vaccines) administered intradermally and the pCR (p = 0.59) Conclusions Immunotherapy added to standard neoadjuvant CT could improve total pCR in stage II-III non-overexpressing HER2 breast cancer patients. After 5 doses of vaccination plus chemotherapy, we can observe phenotypic changes in peripheral blood: some immune system subpopulations increased statistically after the treatment in vaccinated patients. Neither the amount of lysate nor the number of dendritic cells used in the five first vaccines significantly correlated with the pRC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-13-04.

CD8(+) T-cell priming and boosting: more antigen-presenting DC, or more antigen per DC?

RNA transfection is a standard method to load dendritic cells (DC) with antigen for therapeutic cancer vaccination. While electroporation yields high transfection efficiency and satisfying expression levels, lipofection results in only few cells expressing high amounts of antigen. We compared antigen loading of human monocyte-derived DC by MelanA RNA electroporation and lipofection. No differences in phenotype or migrational capacity were detected, but lipofected DC induced stronger cytokine secretion by antigen-specific T cells and were superior in priming and boosting of MelanA-specific CD8(+) T cells. Interestingly, T cells stimulated with the differently transfected DC did not differ in their functional avidity. To determine whether the amount of antigen per cell is indeed responsible for the superiority of the lipofected DC, we increased the amount of MelanA RNA fivefold and mixed those DC with mock-electroporated ones to mimic the antigen distribution of lipofected cells. This significantly improved the stimulatory capacity, indicating that indeed the amount of antigen per cell seems to be the responsible feature for the observed superiority of lipofected DCs. These data suggest that a few DC that express high amounts of antigen are more immunogenic than many DC expressing lower amounts, although this needs to be tested in a two-armed immunogenicity trial.

Loading of dendritic cells for immunotherapy

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.

Immunosurgical resection of host tissues mediated by a novel dendritic cell-based vaccine proficient in the promulgation of TH1 immunity by exploitation of the p43/sCTLA-4 axis (P4248)

Abstract The current understanding of TH1 polarization and the central role dendritic cells (DC) play in skewing such a response engender hope that DC-based vaccines may ultimately be capable of promoting therapeutic efficacy against recalcitrant pathogens and neoplastic self. However, to date the development of such a vaccine has been unsuccessful presumably because the full panoply of signals required for promulgation of bona fide TH1 immunity remains uncharacterized. Recently, our group demonstrated that synergistic loading of DC with homologous MHC class I and II antigens generates a powerful TH1 response significantly stronger and more specific than that generated by alternatively-loaded DC. Using this approach, we developed ‘TH1 DC’ vaccines against both autologous prostate and seminal vesicle (SV) in the wild type mouse. The data indicate that vaccines generated by homologous antigenic matching can overcome peripheral tolerance, producing a clinical result reminiscent of surgical resection. Translation of this platform to cancer immunotherapy has shown preliminary success against established tumors in inbred mouse models as well as spontaneous canine oligodendrocytomas. We demonstrate that these results are in part dependent upon the newly-characterized AIMp1(p43)/sCTLA-4 axis and comment upon the important role of this axis in the development of TH1 immunity as well as its applicability to cancer immunotherapy.

Abstract 1253: Improvement of the potency of the dendritic cell-based vaccine immunotherapy through local inhibition of TGF-β in tumor-draining lymph nodes.

Abstract The dendritic cell (DC) -based vaccine immunotherapy has been a promising anti-cancer immunotherapy, but has been unable to elicit anti-tumor immune responses enough to eradicate the tumor in patients with advanced cancer. In order to develop the potent DC-based vaccine immunotherapy, it should be required to focus on the unique environment implicated in the induction of anti-tumor immune responses. Transforming growth factor-β (TGF-β), an immunosuppressive cytokine, is produced in the tumor microenvironment, and flows into tumor-draining lymph nodes (TDLs), primary priming sites for generation of anti-tumor immune responses. In TDLs, TGF-β plays a critical role in suppressing the induction of anti-tumor immune responses. In this study, we focused on TGF-β-mediated immunosuppression in TDLs, and examined whether local inhibition of TGF-β in TDLs could improve the potency of the DC-based vaccine immunotherapy in tumor-bearing mouse model. C57BL/6 mice bearing Lewis lung carcinoma (LLC1) subcutaneously were vaccinated with DCs loading LLC1-derived antigen. In order to inhibit TGF-β in TDLs, those mice were administered the plasmid DNA encoding the extracellular domain of TGF-β type II receptor fused to the human IgG heavy chain(TGFR DNA) near the established tumor intramuscularly. Soluble TGF-β type II receptor secreted from TGFR DNA-transfected cells is expected to function to attenuate TGF-β level in TDLs. After this combination therapy, anti-tumor immune responses elicited in those mice were evaluated. As results, the level of TGF-β in TDLs was decreased by administrations of TGFR DNA near the established tumor. The attenuation of TGF-β level in TDLs by TGFR DNA was able to suppress the proliferative activity of Foxp3+ regulatory T cells in TDLs. Regarding the induction of effector cells in TDLs, tumor antigen-specific CD8+ cells producing interferon-gamma were significantly increased by this combination therapy. Next, we evaluated the association between the local inhibition of TGF-β in TDLs and systemic anti-tumor immune responses. Tumor antigen-specific cytotoxic responses as well as natural killer activities were significantly enhanced through the local inhibition of TGF-β in TDLs, leading to effective suppression of tumor growth. Such potent anti-tumor immune responses were not achieved in mice that had been administered DC-based vaccine alone. Improvement of the induction of anti-tumor immune responses in TDLs by the local inhibition of TGF-β contributed to enhancement of systemic anti-tumor immune responses. In conclusion, inhibition of TGF-β-mediated immunosuppression in TDLs can enhance the potency of DC-based vaccine immunotherapy. This animal model provides for a novel rationale with DC-based vaccine immunotherapy. Citation Format: Koji Teramoto, Yasuhiko Ohshio, Takuya Fujita, Jun Hanaoka, Yataro Daigo. Improvement of the potency of the dendritic cell-based vaccine immunotherapy through local inhibition of TGF-β in tumor-draining lymph nodes. [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 1253. doi:10.1158/1538-7445.AM2013-1253

Design and evaluation of theranostic perfluorocarbon particles for simultaneous antigen-loading and 19F-MRI tracking of dendritic cells

Perfluorocarbon (PFC) particles are currently on the rise as cell labeling agents for 19F-MRI tracking of dendritic cell (DC)-based vaccines. In this work, we design theranostic PFC particles for single-step loading of DCs with both antigenic protein and with a liquid PFC for 19F-MRI detection of the antigen-loaded cells. Upon addition to DCs in vitro, the antigen-loaded PFC particles are efficiently internalized, resulting in intracellular presence of up to 40pmol 19F atoms per cell. At the same time, the DCs become loaded with antigenic proteins, that can be efficiently processed, without important effects on cell viability or altering the DC's phenotype and the cell's capacity to respond to danger signals. In addition, antigen-loaded PFC particle containing DCs are capable of inducing extensive proliferation of antigen-specific CD8+ T cells in vitro. Importantly, the antigen-coated PFC particles allow in vitro19F-MRI-based detection of the antigen-containing DCs with detection limits as low as 103cellsμl−1. The dual-modality characteristics of the designed particles could assure that only those DCs that have taken up the antigen, and hence are responsible for an immune response, are traceable via 19F-MRI. Taken together, these novel dual-modality particles represent an interesting strategy in the development of a traceable DC vaccine.

Design of an Optimized Wilms’ Tumor 1 (WT1) mRNA Construct for Enhanced WT1 Expression and Improved Immunogenicity In Vitro and In Vivo

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.

Stimulation of dendritic cells with vaccine and vaccine–antibody complex and effect on immune response

Dendritic cell (DC) vaccines are a promising and potent therapeutic tool for chronic diseases, autoimmune diseases, and cancer because of the unique ability of DCs to stimulate T cells. The challenge of DC vaccines is to find an effective form for antigen presentation. Although pure antigens, antigen complexes, plasmids, and mRNA have been used in different studies, no proper application to overcome this problem has been found yet. In this study, we investigated the eligibility of a commercial hepatitis B virus (HBV) vaccine or a vaccine-monoclonal antibody complex for antigen loading of DCs for a therapeutic purpose. DCs were derived from the bone marrow of transgenic hepatitis B (HBV-tg) mice using a granulocyte macrophage-colony stimulating factor and interleukin-4, and then loaded with a commercial HBV vaccine (containing hepatitis B virus surface antigens and aluminum hydroxide adjuvant) or a vaccine-antibody complex. HBV-tg mice were immunized with the vaccine and vaccine-antibody loaded DCs. Optimum HBV vaccine concentration and loading time were determined by 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) methods. Therapeutic effects of vaccine-antibody loaded DCs were determined by the evaluation of antibody response and hepatitis B surface expression levels in HBV-tg mice. Our results showed that commercial HBV vaccine loaded DCs induced humoral response in HBV-tg mice but had no effect on cellular immunity.

Differential Glioma-Associated Tumor Antigen Expression Profiles of Human Glioma Cells Grown in Hypoxia

Human U251 and D54 glioma cells were tested for expression of 25 glioma-associated tumor antigen precursor proteins (TAPP) under hypoxic (1% O2) or normoxic (21% O2) conditions. Hypoxic glioma cell lines increased their mRNA expression for nine TAPP (Aim2, Art-4, EphA2, EZH2, Fosl1, PTH-rP, Sox 11, Whsc2 and YKL-40), as assessed by quantitative reverse transcriptase real-time/polymerase chain reaction (qRT-PCR). Increased differences with three hypoxic-induced TAPP: EZH2, Whsc2 and YKL-40 were shown at the protein levels by fluorescent antibody staining and quantitative electrophoretic analysis. Two TAPP (MRP3 and Trp1) were down-regulated by hypoxia in glioma cell lines. Growing the glioma cells under hypoxia for 13 days, followed by returning them back to normoxic conditions for 7 days, and restored the original normoxic TAPP profile. Thus, hypoxia was an environmental factor that stimulated the transient expression of these antigens. Intracranial xenografts grown in nude mice derived from U251 cells that had been cultured under neurosphere stem cell conditions showed increased expression of Whsc2 or YKL-40, demonstrating that these in vitro properties of glioma also occur in vivo. Whsc2-specific cytotoxic T lymphocytes killed the hypoxic U251 glioma cells better than normoxic glioma cells. The antigens expressed by hypoxic tumor cells may be a better source of starting tumor material for loading dendritic cells for novel immunotherapy of glioma using tumor-associated antigens.

Loading dendritic cells from HIV-1 infected patients with PLA-p24 nanoparticles or MVA expressing HIV genes induces HIV-1-specific T cell responses

Background The evaluation of the interaction of new immunogens with dendritic cells is important for new vaccine strategies. We used polylactic acid (PLA) colloidal biodegradable particles, coated with HIV Gag antigens (p24), and Modified Vaccinia Ankara (MVA) expressing Gag or Tat, Nef and Rev in an ex vivo model of human monocyte-derived dendritic cells (MDDC) to compare two different loading strategies,either viral or synthetic derived.

RETRACTION of: Optimizing the exogenous antigen loading of monocyte-derived dendritic cells

Dendritic cell (DC) vaccination, i.e. the adoptive transfer of antigen-loaded DC, is still at an early stage and requires standardization. In this study, we investigated the exogenous loading of monocytederived DCs with HLA class I- and II-restricted peptides, as despite widespread use, little effort has been put into its pre-clinical validation. We found that only mature DCs (m-DC) but not immature DCs (im-DC) could be sufficiently loaded with exogenous class I-restricted peptides and were by far superior in expanding CD8 1 primary (Melan-A.A2 peptide-specific) and recall [Influenza matrix peptide (IMP) A2-specific] T cell responses. Primary stimulation with peptide-loaded im-DCs even down-regulated antigen-specific T cell responses. Our results indicate that stimulation with m-DCs is superior in terms of quantity and quality compared with im-DCs, supporting their preferred use in clinical DC trials. Loading of m-DCs with high (10 lM) concentrations generated clearly more Melan-A effectors than loading with 1 or 0.1 lM without any negative effect on the quality (affinity) of the resulting T cells. In contrast to the findings with the Melan-A peptide loading with 10 lM IMP was counter-productive, induced apoptosis and yielded fewer specific T cells of inferior affinity as compared with loading with 1 or 0.1 lM. In sharp contrast to the situation for HLA class I, much higher levels and longer half-lives of peptide‐HLA class II complexes were obtainable upon loading of im-DCs with exogenous peptide, but m-DCs were functionally preferable to induce Th1 responses in vitro. Another surprising finding was that, while presentation to T cells upon simultaneous loading of several peptides with highly varying affinities and competing for the same class I or II molecule was possible, in priming experiments peptide competition clearly inhibited T cell induction. Although peptides will obviously vary in their individual properties, our study clearly points to some important principles that should be taken into account.

Dendritic Cells Loaded With mRNA Encoding Full-length Tumor Antigens Prime CD4+ and CD8+ T Cells in Melanoma Patients

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.

Immunization with MOG fused to a novel single chain fragment variable (scFv) for murine DEC-205 suppresses allergic experimental encephalomyelitis (EAE) in mice. (106.12)

Abstract The antigenreceptor DEC-205 is expressed by Dendritic cells (DC) and greatly increases antigen presentation. We have shown that in vivo loading of DC with antigen via anti-DEC-205:antigen conjugates induces immunosuppressive regulatory T-cells (Treg). Therefore we set out to create single chain fragment variables (scFv) specific for DEC-205 fused with the EAE antigen MOG, to target non-activated DC for induction of tolerance to EAE in vivo. A non-binding anti-b-gal scFv:MOG was created as control. ScFv:MOG fusions proteins were expressed and purified, and immunohistochemical staining of cytospins from isolated CD11c+ cells displayed a positive staining for DEC-scFv, colocalizing with MHC class II. For functional testing DEC-scFv:MOG was injected into mice. Lymph node DC were isolated thereafter and cocultivated with MOG-specific (2D2) T-cells. Strong T-cell proliferation was induced by DC from mice injected with DEC-scFv:MOG in contrast to controls. When analyzing the T-cell populations in DEC-scFv:MOG injected mice, we found enhanced numbers of CD4+CD25+FoxP3+ Treg (16% of CD4) as compared to control animals (10% of CD4). Most importantly, when EAE was induced in either DEC-scFv-MOG-injected or in control mice, none of the DEC-scFv:MOG injected mice developed EAE symptoms. In contrast, all animals in the control group developed a severe EAE. Thus, these data indicate that targeting of MOG to steady state DC in vivo prevents EAE by a Treg-driven mechanism.

Abstract 1574: Quantitation of HLA-A2-MAGE3271-279 complexes on the DC surface improves anticancer vaccine designs

Abstract Background: Dendritic cells (DC) loaded with tumor antigen (TA)-derived peptides are used as vehicles for anti-cancer vaccines. HLA class-I restricted short synthetic peptides are commonly used to load DC for therapy. However, randomized clinical trials have failed to confirm the efficacy of DC-based therapies. We hypothesized that new methods for the quantification of peptide loading of DC are needed to improve the overall response rate to anti-tumor vaccines. Methods: An unlabeled monoclonal antibody (Ab), 12B6 Ab, recognizing HLA-A2-MAGE3271-279 complexes was generated by S. Ferrone. Monocyte-derived DC were generated from HLA-A2+ buffy coats of healthy donors in media containing IL-4 and GM-CSF. DC harvested on day 5 of culture were matured by further 48-h exposure to LPS or Interferon-α (IFN-γ). The HLA-A2+ cell line (T2) deficient for LMP2, TAP1 and TAP2 was maintained in a complete RPMI medium. Peptides were synthesized at the University of Pittsburgh Peptide Synthesis facility. An HLA-A2+ CTL clone specific for MAGE3271-279 was used in ELISPOT assays. Cells were stained with 12B6 Ab followed by a PE-labeled secondary Ab. Flow cytometry was used to measure mean fluorescence intensity (MFI) of the HLA class-I-peptide complexes and the loading kinetics. HLA class-I-peptide complexes were dissociated by treatment of cells with citric acid, pH 3.0. Results: The 12B6 Ab reacted with HLA-A2+DC or T2 cells loaded with MAGE3271-279 but not with HLA-A2− DC loaded with MAGE3271-279 or cells loaded with irrelevant peptides. The MFI obtained for the complexes positively correlated with the concentration of MAGE3271-279 used for loading. Maximum levels of HLA-A2-MAGE3271-279 were detectable on DC after 30min loading, while optimal loading of T2 cells required 4h incubation. The levels of detectable complexes correlated with the total expression levels of HLA-A2 molecules on the DC surface. However, donors with comparable levels HLA-A2 expression showed different levels HLA-A2-MAGE3271-279 expression after loading with the same peptide concentration. The MFI for HLA-A2-MAGE3271-279 on DC, but not on T2 cells, was increased when peptide loading was performed after low pH treatment. This treatment did not affect expression of co-stimulatory molecules on DC. Importantly, the peptide-specific stimulation of CTL generated with mDC loaded after low pH treatment resulted in a significantly increased percentage of IFN-γ secreting T cells. Conclusion: The 12B6 Ab is specific for HLA-A2-MAGE3271-279 complexes and allows for their quantification on the surface of DC. Loading of DC was increased by the removal of HLA class-I-self antigen complexes prior to the addition of the TA-derived exogenous peptide. The treated DC showed a higher density of HLA-A2-MAGE3271-279 by flow cytometry and were more effective in priming MAGE3271-279 specific CTL. 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 1574. doi:1538-7445.AM2012-1574

Abstract LB-132: The potential role of the multienzyme aminoacyl-tRNA synthetase complex in Th-1 immunity: implications for cancer immunotherapy

Abstract Recent advances in dendritic cell (DC)-based cancer vaccines have highlighted the potential utility of arming the immune system to target neoplastic self. A strong, yet controlled, TH1 response has typically been the goal of DC-based protocols, as it is cell-based adaptive immunity that possesses the ability to most-efficiently destroy the nascent tumor or occult micrometastases. Nonetheless, many DC-intrinsic factors have impeded the development of a vaccine strong enough to impart meaningful clinical results, and there remains a need for further study of the parameters required for maximal, targeted DC activity. Previously, we have shown that contemporaneous loading of DC with overlapping MHC class I and MHC class II peptide antigens leads to an enhanced TH1 response when compared to canonical DC stimulation as assayed by release of IL-12, sCTLA-4, proliferation of CD8+ CD25+, INF-γ release, and specific killing of target both in vitro and in vivo. These and other data have lead to the hypothesis that DC possess a sensor mechanism capable of discerning amino acid sequence similarities between peptides bound by MHC class I and class II and that tRNA synthetases may serve as critical components of the putative recognition machinery due to their inherent ability to recognize and distinguish between individual amino acid side chains. Here we report that the aminoacyl-tRNA synthetase complex (MACS), a large molecular complex comprised of at least nine aminoacyl-tRNA synthetases, possess interesting characteristics that might link it to this phenomenon. Components of the MACS complex, including the p43 structural subunit, were found in DC exosomes, the contents of which mimic those of the late endosome, a known cross-presentation compartment. MARS (methionyl-tRNA synthetase), a component of the MACS, as well as non-MACS components VARS (valyl-tRNA synthetase) and GARS (glycyl-tRNA synthetase) were found within exosomes as well. UVC cross-linking of exosomal extracts resulted in the incorporation of individual tRNA-synthetase isoforms into a very large complex of indeterminate size. p43 was differentially released from the MACS in response to the loading of DC only with matched class I (GFP mRNA) and class II (recombinant GFP protein) determinants. siRNA knockdown of MACS structural component p38 was able to ablate the ability of doubly-loaded DC to enhance the generation of CD8+CD25+ cells as well as the ability of these cells to secrete IFN-γ. Further, siRNA knockdown of p38 abrogated the release of p43 from DC loaded with matched class I and II determinants. With an enhanced understanding of these regulatory phenomena, the development of small molecule TH1 agonists and/or immunotherapeutic protocols with enhanced clinical efficacy become realistic and achievable goals. 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 LB-132. doi:1538-7445.AM2012-LB-132