Generation Of Dendritic Cells Research Articles

Minocycline promotes the generation of dendritic cells with regulatory properties

Abstract Minocycline, which has long been used as a broad-spectrum antibiotic, also exhibits non-antibiotic properties such as inhibition of inflammation and angiogenesis. In this study, we show that minocycline significantly enhances the generation of dendritic cells (DCs) from mouse (BM) cells when used together with GM-CSF and IL-4. DCs generated from BM cells in the presence of minocycline (Mino-DCs) demonstrate the characteristics of regulatory DCs. Compared with control DCs, Mino-DCs are resistant to subsequent maturation stimuli, impaired in MHC class II-restricted exogenous Ag presentation, and show decreased cytokine secretion and allogeneic T cell stimulation in vitro. Mino-DCs also have a decreased ability to prime alloantigen-specific T cells when transferred to allogeneic mice. Splenic Mino-DCs generated in mice by injecting minocycline together with GM-CSF are also severely impaired in priming alloantigen-specific T cells in allogeneic mice. In allogeneic mice, Mino-DCs induce expansion of CD4+CD25+Foxp3+ T regulatory cells. Our study identifies minocycline as a new pharmacological agent that could be potentially used to increase the production of regulatory DCs for cell therapy to treat autoimmune disorders, allergy, and transplant rejection.

An animal component-free, serum-free culture method for generation of human dendritic cells

Abstract Dendritic cells (DCs) are central regulators of adaptive immune responses that potentially have major applications in the immunotherapy of cancer and autoimmune disorders. DCs are rare in peripheral blood (PB) and are therefore often generated in vitro from PB monocytes. We developed an animal component-free (ACF) and serum-free medium, ImmunoCult-ACF DC, that supports the generation of DCs from monocytes in culture. CD14+ monocytes were isolated using EasySep immunomagnetic separation and cultured for 5 days in ImmunoCult-ACF DC medium and cytokines (GM-CSF and IL-4) to promote their differentiation into CD14−CD83− immature DCs. The immature DCs were then stimulated for 2 days with the ImmunoCult-DC Maturation Supplement, a combination of cytokines and pro-inflammatory mediators, to promote their maturation to CD14− CD83+ DCs (93 ± 5% CD83+, n=23). The yield of mature DCs was 42 ± 23% (n=23), similar to the yield in a control serum-free medium (49 ± 24%, n=16). Mature DCs produced high levels of IL-12, on average 371 pg/mL (range: 27–1756, n=9 in cultures initiated with 106 monocytes). Mature DCs loaded with CMV, EBV and Flu virus peptides efficiently stimulated the proliferation of autologous CD8+ T cells in 7 day co-cultures. An 18-fold increase in T cell numbers (range: 5–31, n=4) was observed when compared to control culture conditions containing only T cells without mature DCs. In conclusion, functional DCs can be efficiently generated by differentiation of monocytes in a completely animal (including human) component-free and serum-free medium. This medium and culture method will enable further research into the development and application of DCs for cellular therapy.

Differential Expression of the Transcription Factor SPI1 (PU.1) in Dendritic Cell Subsets

Abstract SPI1 belongs to the E26-transformation-specific (Ets) family of transcription factors and is exclusively expressed in hematopoietic cells. Originally identified as an oncogene that blocks erythroblast differentiation, SPI1 plays a critical role in the development and lineage commitment of both myeloid and lymphoid cells. Gene knockout studies show that spi1−/− mice exhibit a multi-lineage defect in the generation of monocytes, granulocytes, B, T, and dendritic cells. While SPI1 expression is required for normal hematopoietic cell development, the level of SPI1 expression influences specific lineage commitment. Using in-house generated species-specific clones, human peripheral blood mononuclear cells (PMBCs) and mouse splenocytes were evaluated for SPI1 expression by flow cytometric analysis. In human PMBCs, Lin− HLA-DR+ CD11c+ CD303− monocyte-derived dendritic cells (mDCs) exhibit high levels of expression while Lin− HLA-DR+ CD11cint CD303+ plasmacytoid dendritic cells (pDCs) do not express SPI1 protein. These data suggest SPI1 expression is required for the development and function of human mDCs, while SPI1 expression is not necessary for terminally differentiated human pDCs. Interestingly, SPI1 expression is detected in both murine DC subsets: Lin− I-A/I-E+ CD11c+ CD317− mDCs and Lin− I-A/I-E+ CD11cint CD317+ pDCs, although the expression level in pDCs is significantly lower than in mDCs. These observations indicate expression patterns differ between mouse and human cells, and demonstrate that DC subsets exhibit differential expression of SPI1 protein.

In vitro impact of bisphenols BPA, BPF, BPAF and 17β-estradiol (E2) on human monocyte-derived dendritic cell generation, maturation and function

Bisphenols (BPs) are widely spread pollutants that act as estrogen-like endocrine disruptors and are potentially affecting human health on a long run. We explored the effects of BPA, BPF and BPAF, on in vitro differentiation and maturation of MDDCs. Monocytes were treated with 17β-estradiol (E2) and each BP at the beginning of their differentiation into iMDDCs. We found that 10 and 50μM of BPA and BPF, 10 and 30μM of BPAF and 10 and 50nM of E2 did not affect cell viability. However, 50μM of BPA and BPF, as well as 10 and 30μM of BPAF, significantly decreased the endocytotic capacity of iMDDCs. Both, BPA (50μM) and BPAF (30μM) decreased the expression of CD1a and increased the amount of DC-SIGN molecules on iMDDCs. The E2 pre-treatment moderately decreased expression of CD80, CD86 and CD83 co-stimulatory molecules while increasing the numbers of HLA-DR on mMDDCs. Only BPAF significantly influenced the expression of CD80 and CD86 (both decreased), as well as CD83 and HLA-DR molecules (both increased) on mMDDCs. In addition, BPAF modulated DC maturation signaling pathways by lowering the phosphorylation of p65 NF-κB (nuclear factor-kappaB) and ERK (extracellular signal regulated kinase) 1/2 proteins. Consequently, the in vitro proliferation of allogeneic T cells, stimulated with differently pre-treated iMDDCs and mMDDCs, was significantly reduced only in case of BPAF.

Generation of mouse and human dendritic cells in vitro

Dendritic cells (DC) that can orchestrate immune responses and maintain host homeostasis, are indispensable components of the immune system. Although distributed widely in many lymphoid and non-lymphoid tissues, their rarity in number has become a limiting factor for DC related research and therapies. Therefore, methods for efficiently generating large numbers of DC resembling their in vivo counterparts are urgently needed for DC related research and therapies. Herein we summarize the current methods for generating mouse and human DC in vitro and hope that these will facilitate both studies of DC biology and their clinical applications.

Hepatic carcinoma-associated fibroblasts induce IDO-producing regulatory dendritic cells through IL-6-mediated STAT3 activation.

Although carcinoma-associated fibroblasts (CAFs) in tumor microenvironments have a critical role in immune cell modulation, their effects on the generation of regulatory dendritic cells (DCs) are still unclear. In this study, we initially show that CAFs derived from hepatocellular carcinoma (HCC) tumors facilitate the generation of regulatory DCs, which are characterized by low expression of costimulatory molecules, high suppressive cytokines production and enhanced regulation of immune responses, including T-cell proliferation impairment and promotion of regulatory T-cell (Treg) expansion via indoleamine 2,3-dioxygenase (IDO) upregulation. Our findings also indicate that STAT3 activation in DCs, as mediated by CAF-derived interleukin (IL)-6, is essential to IDO production. Moreover, IDO inhibitor, STAT3 and IL-6 blocking antibodies can reverse this hepatic CAF-DC regulatory function. Therefore, our results provide new insights into the mechanisms by which CAFs induce tumor immune escape as well as a novel cancer immunotherapeutic approach (for example, targeting CAFs, IDO or IL-6).

Subgroup J avian leukosis virus infection of chicken dendritic cells induces apoptosis via the aberrant expression of microRNAs.

Subgroup J avian leukosis virus (ALV-J) is an oncogenic retrovirus that causes immunosuppression and enhances susceptibility to secondary infection. The innate immune system is the first line of defense in preventing bacterial and viral infections, and dendritic cells (DCs) play important roles in innate immunity. Because bone marrow is an organ that is susceptible to ALV-J, the virus may influence the generation of bone marrow-derived DCs. In this study, DCs cultured in vitro were used to investigate the effects of ALV infection. The results revealed that ALV-J could infect these cells during the early stages of differentiation, and infection of DCs with ALV-J resulted in apoptosis. miRNA sequencing data of uninfected and infected DCs revealed 122 differentially expressed miRNAs, with 115 demonstrating upregulation after ALV-J infection and the other 7 showing significant downregulation. The miRNAs that exhibited the highest levels of upregulation may suppress nutrient processing and metabolic function. These results indicated that ALV-J infection of chicken DCs could induce apoptosis via aberrant microRNA expression. These results provide a solid foundation for the further study of epigenetic influences on ALV-J-induced immunosuppression.

Satb1 Overexpression Drives Tumor-Promoting Activities in Cancer-Associated Dendritic Cells.

Special AT-rich sequence-binding protein 1 (Satb1) governs genome-wide transcriptional programs. Using a conditional knockout mouse, we find that Satb1 is required for normal differentiation of conventional dendritic cells (DCs). Furthermore, Satb1 governs the differentiation of inflammatory DCs by regulating major histocompatibility complex class II (MHC II) expression through Notch1 signaling. Mechanistically, Satb1 binds to the Notch1 promoter, activating Notch expression and driving RBPJ occupancy ofthe H2-Ab1 promoter, which activates MHC II transcription. However, tumor-driven, unremitting expression of Satb1 in activated Zbtb46(+) inflammatory DCs that infiltrate ovarian tumors results in an immunosuppressive phenotype characterized by increased secretion of tumor-promoting Galectin-1 and IL-6. Invivo silencing of Satb1 in tumor-associated DCs reverses their tumorigenic activity and boosts protective immunity. Therefore, dynamic fluctuations in Satb1 expression govern the generation and immunostimulatory activity of steady-state and inflammatory DCs, but continuous Satb1 overexpression in differentiated DCs converts them into tolerogenic/pro-inflammatory cells that contribute to malignant progression.

Sirt6 regulates dendritic cell differentiation, maturation, and function.

Dendritic cells (DCs) are antigen-presenting cells that critically influence decisions about immune activation or tolerance. Impaired DC function is at the core of common chronic disorders and contributes to reduce immunocompetence during aging. Knowledge on the mechanisms regulating DC generation and function is necessary to understand the immune system and to prevent disease and immunosenescence. Here we show that the sirtuin Sirt6, which was previously linked to healthspan promotion, stimulates the development of myeloid, conventional DCs (cDCs). Sirt6-knockout (Sirt6KO) mice exhibit low frequencies of bone marrow cDC precursors and low yields of bone marrow-derived cDCs compared to wild-type (WT) animals. Sirt6KO cDCs express lower levels of class II MHC, of costimulatory molecules, and of the chemokine receptor CCR7, and are less immunostimulatory compared to WT cDCs. Similar effects in terms of differentiation and immunostimulatory capacity were observed in human monocyte-derived DCs in response to SIRT6 inhibition. Finally, while Sirt6KO cDCs show an overall reduction in their ability to produce IL-12, TNF-α and IL-6 secretion varies dependent on the stimulus, being reduced in response to CpG, but increased in response to other Toll-like receptor ligands. In conclusion, Sirt6 plays a crucial role in cDC differentiation and function and reduced Sirt6 activity may contribute to immunosenescence.

Abstract B057: Optimizing the high throughput in vitro priming of tumor specific T cells

Abstract Peptide-based vaccines are a promising strategy for immunotherapy against cancer. Our group has characterized large numbers of HLA ligands from tumor cells; among them many that might be used for in vivo induction of tumor-specific immune responses. For the development of peptide-based immunotherapies such peptides should be able to induce in vivo tumor-directed immune responses, especially by CD8+ T cells that have a key role in inducing death of tumor cells. The aim of our work is to establish a fast and efficient workflow for immunogenicity testing of tumor-extracted HLA ligands. T cells usually depend on professional antigen presenting cells such as autologous dendritic cells (DCs) for specific induction and expansion. Since the generation of dendritic cells is expensive and time consuming with varying quality and amount of differentiated DCs, we established an artificial system to replace cell-based in vitro priming of T cells, streptavidin-coated artificial antigen presenting cells loaded with defined amounts of recombinant HLA molecules and costimulatory antibodies such as anti-CD28. Using the standard protocol, CD8+ T cells are stimulated three times with artificial antigen presenting cells in weekly intervals in the presence of IL-2 and IL-12. Peptide-specific CD8+ T cells are identified by tetramer staining. Our group started to vary different parameters of this protocol to increase the frequency and number of antigen specific T cells. Alternate costimulatory antibodies and cytokines proved successful to reach that goal. Employing artificial APCs for in vitro priming of tumor-reactive T cells will foster immunogenicity analysis of novel peptides for immunotherapy. Citation Format: Lea Prokop, Stefanie Souczek, Janet Peper, Hans-Georg Rammensee, Stefan Stevanović. Optimizing the high throughput in vitro priming of tumor specific T cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B057.

Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation.

Human liver stem cells (HLSCs) are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs), and dendritic cells (DCs) in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2) and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs), HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response.

Delivery of Synthetic mRNA Encoding FOXP3 Antigen into Dendritic Cells for Inflammatory Breast Cancer Immunotherapy.

Dendritic cell (DC)-based vaccines are commonly used for cancer immunotherapy. To prepare vaccines, DCs are pulsed or transfected with either: (a) defined peptides of tumor-associated antigens, (b) total protein isolated from the tumor cell, (c) autologous total RNA isolated from the tumor cell, (d) synthetic tumor-antigen-encoding mRNA, or (e) genes that encode for specific tumor-associated antigens. Introduction of tumor-associated antigen(s) and subsequent generation of mature DCs that can stimulate tumor-antigen-specific cytotoxic T lymphocytes comprise the critical steps of cancer vaccine preparation. Here, we described a method of: (a) preparing and delivering synthetic FOXP3 mRNA into human DCs, (b) generating mature DCs,

Generation of regulatory dendritic cells after treatment with paeoniflorin.

Regulatory dendritic cells are a potential therapeutic tool for assessing a variety of immune overreaction diseases. Paeoniflorin, a bioactive glucoside extracted from the Chinese herb white paeony root, has been shown to be effective at inhibiting the maturation and immunostimulatory function of murine bone marrow-derived dendritic cells. However, whether paeoniflorin can program conventional dendritic cells toward regulatory dendritic cells and the underlying mechanism remain unknown. Here, our study demonstrates that paeoniflorin can induce the production of regulatory dendritic cells from human peripheral blood monocyte-derived immature dendritic cells in the absence or presence of lipopolysaccharide (LPS) but not from mature dendritic cells, thereby demonstrating the potential of paeoniflorin as a specific immunosuppressive drug with fewer complications and side effects. These regulatory dendritic cells treated with paeoniflorin exhibited high CD11b/c and low CD80, CD86 and CD40 expression levels as well as enhanced abilities to capture antigen and promote the proliferation of CD4(+)CD25(+) T cells and reduced abilities to migrate and promote the proliferation of CD4(+) T cells, which is associated with the upregulation of endogenous transforming growth factor (TGF)-β-mediated indoleamine 2,3-dioxygenase (IDO) expression. Collectively, paeoniflorin could program immature dendritic cells (imDCs) and imDCs stimulated with LPS toward a regulatory DC fate by upregulating the endogenous TGF-β-mediated IDO expression level, thereby demonstrating its potential as a specific immunosuppressive drug.

A Common Progenitor for Macrophages, Osteoclasts and Dendritic Cells Newly Discovered in Human Bone Marrow and Cord Blood Yields Dendritic Cells with T-Cell Priming Ability

Dendritic cells (DC) have potent antigen-presentation and T-cell priming ability and therefore hold great promise in cancer immunotherapy. However, DC vaccination has not yet delivered a reliable clinical response rate, despite great efforts. Since primary DCs are rare (up 0.2-1.5% of circulating leukocytes), therapeutic DCs are generally derived from peripheral blood monocytes by culture with GM-CSF (moDC). Vaccines composed of moDC loaded with tumor antigens can induce potent and long-lasting tumour-specific immune responses in patients, but such positive results are infrequent and unpredictable. To improve success rate, research has focused on moDC culture regimens, antigen loading and activation strategies and methods of DC injection. Nevertheless, to date clinical trials using moDC have not yielded statistically significant treatment benefits over conventional strategies. Current attention has therefore shifted to the rare primary DCs that circulate in the blood under homeostatic conditions.Knowing the identity of the precursors of these DCs may facilitate the ex-vivo or in-vivo generation of DCs via the homeostatic pathway, potentially yielding DCs with optimal T cell priming ability. We (Xiao et al. Stem Cell Rep. 2015) and others (Lee et al. J. Exp. Med. 2015) have recently identified a population with DC progenitor potential in human bone marrow and cord blood, respectively. This population can be isolated on basis of a CD34+ c-KIT+ FLT3+ IL3Rαhigh phenotype and is furthermore Lin- CD10- CD11b- CD45RA+ CD38+. We have shown that this population is highly enriched for or identical to a common progenitor (P) of macrophages (M), osteoclasts (O) and DCs (D) and termed it MODP. We also identified the progenitor directly upstream from the MODP that still has granulocyte (G) differentiation potential and termed it GMODP.We hypothesized that DCs generated from GMODP or MODP under homeostatic conditions would have superb T-cell priming capacity. To examine this, the progenitors were sorted by flow cytometry from human bone marrow or cord blood and cultured with Flt3 ligand, M-CSF and IL-3 to generate DCs. We also tested the effect of a mensenchymal stem cell (MSC) feeder layer. Within 2-3 weeks of culture, 1000 DC progenitors generated approximately 150,000-250,000 DCs. Co-culture with MSC increased DC output significantly, at least 2 fold. The progenitor-derived DCs could be discerned into CD141+ conventional (c)DC, CD1c+ cDC and CD303+ plasmacytoid (p)DC. To study T-cell priming capacity of progenitor-derived DCs, we set up an in vitro DC-T co-culture assay. CD141+ cDC, CD1c+ cDC and CD303+ pDC were generated from GMODP or MODP of HLA-A2+ donors, flow cytometrically purified, activated with lipopolysaccharide and loaded with MART-126-35 peptide that represents a melanoma-derived tumor antigen. Primary T cells from peripheral blood of unrelated donors were retrovirally transduced to express a T cell antigen receptor (TCR) ab specific for the HLA-A2/MART-126-35 peptide complex. The ability of the DCs to prime a T-cell response was read out by antigen-specific CD8+ T cell proliferation. All DC subsets were able to induce MART-1 specific T cell proliferation, with the CD1c+ cDCs being most potent and the CD303+ pDC being least potent.In conclusion: We have established a culture method to derive DCs with T-cell priming ability from a newly identified DC progenitor. These results are of value for improvement of DC-based immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

AML Patients in Minimal Residual Disease Vaccinated with a Novel Generation of Fast Dendritic Cells Expressing WT-1 and PRAME Mount Specific Immune Responses That Relate to Clinical Outcome

AML is frequently diagnosed in elderly patients, with a median age of 69. Many older patients cannot tolerate intensive chemotherapy and/or stem cell transplantation, making curative treatment difficult and rates of early relapse high. Immunotherapy with dendritic cell (DC) vaccines after chemotherapy was shown by others to provide clinical benefit to some AML patients (van Tendeloo et al. 2010).Here we report results in four AML patients receiving DC vaccines targeting the antigens Wilm's tumor-1 (WT-1) and preferentially expressed antigen in melanoma (PRAME), applied in compassionate use, employing new generation monocyte-derived fast DCs, matured with a cocktail containing the TLR7/8 ligand R848. The mature DCs show high expression of CD83, strong up-regulation of HLA-DR and co-stimulatory molecules, down-regulation of CD14 and polarized release of IL-12p70, with no or low IL-10 secretion, upon T cell encounter.After informed consent and hematopoietic recovery from chemotherapy, mononuclear cells were collected by apheresis and mature DC vaccines were prepared to separately express full length mRNA encoding the two target antigens (Subklewe et al. Cancer Immunol. Immunother. 2014). DCs were administered intradermally, once weekly for 4 wks, at wk6 and then on a monthly basis. Blood and bone marrow (BM) samples were collected throughout treatment. Minimal residual disease (MRD) was measured in BM and blood by quantitative PCR of WT-1 expression and BM was monitored by morphology.Table 1 summarizes the salient features of the patients, treatment parameters, MRD monitoring and initial immune response assessment. DTH reactions were detected in all patients challenged with DCs at wk6. Immune responses of CD4 and CD8 T cells demonstrating intracellular interferon gamma (IFNg) expression were assessed by flow cytometry of PBL stimulated overnight with peptides spanning WT-1, PRAME, and hTERT and survivin as vaccine-unrelated antigens. Responses were scored positive when two-fold or greater frequencies of IFNg-expressing T cells were found compared to unstimulated controls.Patient (Pt.)CU030 and Pt.CU031 showed CD4 and CD8 responses to different test antigens. Pt.CU030 displayed strong and persistent CD8 responses to PRAME and a surprising increase in hTERT reactivity, potentially representing epitope spreading. The pt. continues to receive monthly vaccination and displays a low fluctuating WT-1 PCR signal in BM but no signal is seen in blood at wk61 after start of vaccination. Pt.CU031 displayed WT-1-specific immune responses until wk37 when responses decreased and WT-1 PCR signals increased in BM. The pt. developed Bell's palsy and immune responses were no longer detected after cortisone therapy. WT-1 signals then increased strongly in BM, accompanied by an increase of blasts. Pt. CU033 had no significant T cell response during 9 months (m) of vaccination. WT-1 signals now increase slowly in BM but relapse cannot be confirmed by morphology and WT-1 PCR remains negative in blood. Pt.CU040 has only received DC vaccines for 5 m, remains in morphological remission and immune response and MRD monitoring are ongoing.These results show that fast, TLR-polarized DCs induce or enhance specific T cell responses in elderly and undertreated AML patients, with individual strengths and specificities. Preliminary assessments suggest that changes in MRD are related to increase or loss of vaccine-associated immune responses.Table 1Characteristics of AML patients receiving DC vaccinesPatientCU030CU031CU033CU040Age57506873SexfmffAML ClassificationM4M2M1M1Risk ClassificationintermedintermedintermedgoodChemotherapy cycles Induction/Consolidation2/02/42/02/0Time between chemo-therapy and vaccination5 m8 m3 m7 mMonths of vaccination as of (08/2015)16 m10 m9 m5 mDTH responses at w6 toWT-1/PRAME DC challengepos/pospos/pospos/pospos/posIFNg-positive T cell responses to overlapping peptides of WT-1, PRAME, hTERT, and SurvivinStrong and persistent CD8 responses to PRAME and hTERTEarly CD4 & CD8 responses to WT-1; decrease at wk37; full loss after cortisone therapyNo significant responses detected up to wk33To be done after acquisition of further samplesMRD (WT-1 PCR) in BM/bloodfluctuating low /negrapid increase after cortisone /posslow increase /negongoingBM morphology (most recent test)negposnegnegTime since completion of chemotherapy21 m18 m12 m12 m DisclosuresEckl:Medigene Immunotherapies GmbH: Employment. Schendel:Medigene Immunotherapies GmbH: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: for DC maturation cocktail. Kvalheim:Medigene Immunotherapies GmbH: Other: Scientific collaboration.

Tolerogenic Dendritic Cells for Regulatory T Cell Induction in Man

Dendritic cells (DCs) are highly specialized professional antigen-presenting cells that regulate immune responses, maintaining the balance between tolerance and immunity. Mechanisms via which they can promote central and peripheral tolerance include clonal deletion, the inhibition of memory T cell responses, T cell anergy, and induction of regulatory T cells (Tregs). These properties have led to the analysis of human tolerogenic DCs as a therapeutic strategy for the induction or re-establishment of tolerance. In recent years, numerous protocols for the generation of human tolerogenic DCs have been developed and their tolerogenic mechanisms, including induction of Tregs, are relatively well understood. Phase I trials have been conducted in autoimmune disease, with results that emphasize the feasibility and safety of treatments with tolerogenic DCs. Therefore, the scientific rationale for the use of tolerogenic DCs therapy in the fields of transplantation medicine and allergic and autoimmune diseases is strong. This review will give an overview on efforts and protocols to generate human tolerogenic DCs with focus on IL-10-modulated DCs as inducers of Tregs and discuss their clinical applications and challenges faced in further developing this form of immunotherapy.

Effect of different culture media on isolation and differentiation of dendritic cells / Farklı besiyerlerinin dendritik hücre izolasyon ve farklılaşmasına etkisi

Abstract Objective: Dendritic cells (DCs) are members of the mammalian immune system and are considered to be the most powerful antigen presenting cells. They are responsible for the induction of T-cells or T-cell dependent immunity and tolerance. In this study we have investigated the effect of different serum supplements on generation and yield of mature dendritic cells isolated from peripheral blood mononuclear cells. Methods: Three different serum supplements (10% Fetal Bovine Serum, 1% Human Serum Albumin and 1% autologous serum) were compared with serum-free media to identify the role and importance of serum supplements on DC cultivation. Effect of different media on maturation signs (CD40, CD80, CD86, CD209a) and cytokine release (TNF-α, IL-10, IL-12, IL-6) was examined. Results: DCs generated in serum-free media was similar to those of cells in medium with autologous serum. Few dendritic-like cells were observed in fetal bovine serum and human serum albumin. The effect of different media on maturation of DCs was compared phenotypically and increased expression of CD80, CD86 and CD209a identified maturation and yield of DCs. Conclusion: Our results suggest that serum free media can be used to overcome potential drawbacks associated with different serum containing supplements.

IMPS-17BRANCHED-MULTIPEPTIDE IMMUNOTHERAPY FOR GLIOBLASTOMA USING HLA-A*0201-RESTRICTED CYTOTOXIC T-LYMPHOCYTE EPITOPE FROM BIRC5, CD99 AND ERBB2

BACKGROUND: Branched-multipeptide has the increased molecular weight of the peptide which can elevate stable immunogenicity in the body. We identified and synthesized BIRC5, CD99 and ERBB2 peptides and their corresponding branched-multipeptide for HLA-A*0201 molecules. We investigated the possibility of immunotherapy for glioblastoma using cytotoxic T lymphocytes (CTLs) generated by branched-multipeptides pulsed dendritic cells (DCs). MATERIALS AND METHODS: We selected three peptides (BIRC5, CD99 and ERBB2), on the basis of their binding affinity, as determined by a peptide-T2 binding assay and their corresponding branched-multipeptide was synthesized using mini-polyethylene glycol (mini-PEGs) spacer. The function of DCs pulsed with multipeptide- cocktail was compared with DCs pulsed with branched-multipeptide by immunephenotype and cytokine secretion. We measured the functional activity for CTLs by using IFN-γ-enzyme linked immune absorbent spot (ELISPOT) assay. RESULTS: The stability of branched-multipeptide-T2 cells binding was relatively well maintained up to 24 hours compared to multipeptide cocktail. Based on FACS analysis which compared the phenotypes of multipeptide-cocktail and branched-multipeptide pulsed DCs, both DCs exhibited high expression of CD80, CD83 and CD86, which meant that both DCs resulted in efficient generation of DCs without altering the phenotype of the mature DCs. The both multipeptides pulsed DCs showed similarly increased IL-12p70, IL-12p40and IL-10 production. The CTLs that were stimulated by both multipeptides pulsed DCs displayed a greater number of IFN-γ-secreting cells against T2 cells loaded with both multipeptides and glioblastoma cell lines compared to those stimulated by un-pulsed DCs. CONCLUSION: Branched-multipeptide of BIRC5, CD99 and ERBB2 showed the stable binding with T cells and similar cytotoxic activity to target cells compared with multipeptide-cocktail. Branched-multipeptide can be the candidate of useful immunotherapeutic modalities for glioblastoma. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2014R1A1A1004469).

Fast dendritic cells matured with Poly (I:C) may acquire tolerogenic properties

Background aimsBecause of the labor-intensive and time-consuming conventional protocols for the generation of dendritic cells (DCs) as the most promising tools for anti-cancer therapy that enable the induction of a T-helper (Th)1-mediated anti-tumor immune response, the use of short-term protocols has been proposed. However, data on the applicability of such protocols in cancer immunotherapy are quite limited. MethodsWe compared the phenotypic and functional capability of fast DCs (fDCs) differentiated for 24 h and then matured for 48 h with Poly (I:C), a strong Th1-promoting agent, with donor-matched conventional DCs (cDCs) differentiated for 5 days and matured likewise. ResultsOf 12 donors tested, we identified seven whose monocytes failed to develop into immunogenic DCs through the use of fDC protocol, on the basis of incomplete downregulation of CD14, low expression of CD1a and macrophage-like morphology. Such fDCs have significantly lower expression of CD83, CD86, CCR7 and CD40, weaker allo-stimulatory Th1- and Th17-polarizing capacity caused by poor production of interleukin (IL)-12p70 and IL-23 and high production of IL-10, and prominent Th2-polarizing capacity, compared with donor-matched cDCs. Furthermore, such fDCs had tolerogenic properties as judged by higher expression of indolamine dioxigenase-3, IDO-1 and IL-1β and induction of a higher percentage of CD4+CD25+FoxP3+ T cells. These findings correlated with increased transforming growth factor (TGF)-β production by fDC-primed CD3+T cells and their stronger anti-proliferative capacity. ConclusionsWe emphasize that although fDCs could probably be applied as an alternative to cDCs for cancer therapy, the fDC protocol should not be applied to donors whose DCs acquire tolerogenic capabilities.

Fms-like tyrosine kinase 3 ligand is required for thymic dendritic cell generation from bone marrow-derived CD117⁺ hematopoietic progenitor cells.

Thymic dendritic cells (TDCs) are a type of dendritic cell (DC) in the thymus, which can enhance the proliferation of thymic T lymphocytes, regulate negative selection and induce central tolerance through autoantigen presentation. However, further investigations using TDCs has been restricted due to insufficient numbers. Therefore, an effective expansion method for TDCs in vitro is urgently required to further examine their biological characteristics. In the present study, a novel system was established using fetal thymus organ culture (FTOC) and a hanging drop culture system in the presence of fms‑like tyrosine kinase 3 ligand (Flt3L), termed the Flt3L/FTOC system. TDCs were successfully generated and expanded from CD117+ bone marrow hematopoietic progenitor cells. Conventional DCs (cDCs; CD11c+B220‑ DCs) and plasmacytoid DCs (pDCs; CD11c+B220+ DCs) were found in the TDCs generated using the Flt3L/FTOC system. These cells exhibited the specific morphological features of DCs, which were confirmed using Giemsa staining. Furthermore, the cytokine and surface marker profiles were also analyzed. Higher expression levels of interferon‑α and interleukin‑12 were observed in the pDCs, compared with the cDCs, and higher expression levels of toll‑like receptor (TLR)7 and TLR9 were found in the pDCs than in the cDCs. In addition, the Flt3L/FTOC‑derived TDCs also exhibited the ability to stimulate the allogenic T cell response. In conclusion, a novel in vitro culture system of thymic cDCs and pDCs using Flt3L was established, and this may provide a methodological basis for understanding the properties of TDCs.