Mycobacterium abscessus MAB2560 induces maturation of dendritic cells via Toll-like receptor 4 and drives Th1 immune response

In this study, we showed that Mycobacterium abscessus MAB2560 induces the maturation of dendritic cells (DCs), which are representative antigen-presenting cells (APCs). M. abscessus MAB2560 stimulate the production of pro-inflammatory cytokines [interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β, and IL-12p70] and reduce the endocytic capacity and maturation of DCs. Using TLR4^-/- DCs, we found that MAB2560 mediated DC maturation via Toll-like receptor 4 (TLR4). MAB2560 also activated the MAPK signaling pathway, which was essential for DC maturation. Furthermore, MAB2560-treated DCs induced the transformation of naïve T cells to polarized CD4⁺ and CD8⁺ T cells, which would be crucial for Th1 polarization of the immune response. Taken together, our results indicate that MAB2560 could potentially regulate the host immune response to M. abscessus and may have critical implications for the manipulation of DC functions for developing DC-based immunotherapy. [BMB Reports 2014;47(9): 512-517]     

Induction of cellular immune responses against carcinoembryonic antigen in patients with metastatic tumors after vaccination with altered peptide ligand-loaded dendritic cells

Purpose: Dendritic cells (DCs) are characterized by their extraordinary capacity to induce T-cell responses, providing the opportunity of DC-based cancer vaccination protocols. In the present study, we conducted a phase I/II clinical trial to determine the capability of DCs differentiated from immunomagnetically isolated CD14+ monocytes and pulsed with a carcinoembyonic antigen-derived altered peptide (CEAalt) to induce specific CD8+ T cells in cancer patients. Experimental design: Nine patients with CEA-positive colorectal cancer (n=7) or lung cancer (n=2) were enrolled in this study. Autologous CD14+ monocytes were isolated by large-scale immunomagnetic separation and differentiated to mature DCs in sufficient numbers and at high purity. After incubation with the CEAalt peptide and keyhole limpet hemocyanin, DCs were administered to patients intravenously at dose levels of 1×10⁷ and 5×10⁷ cells. Patients received four immunizations every second week. Results: ELISPOT analysis revealed a vaccine-induced increase in the number of CEAalt peptide-specific Interferon (IFN)-gamma producing CD8+ T cells in five of nine patients and of CD8+ T lymphocytes recognizing the native CEA peptide in three of nine patients. In addition, CD8+ T lymphocytes derived from one patient exhibiting an immunological response after vaccination efficiently lysed peptide-loaded T2 cells and tumor cells. Immunization was well tolerated by all patients without severe signs of toxicity. Conclusion: Vaccination with CEAalt-pulsed DCs derived from immunomagnetically isolated CD14+ monocytes efficiently expand peptide-specific CD8+ T lymphocytes in vivo and may be a promising alternative for cancer immunotherapy.     

Large-scale production and directed induction of functional dendritic cells ex vivo from serum-free expanded human hematopoietic stem cells

BackgroundDendritic cells (DCs) that are derived from hematopoietic stem cells (HSCs) are the most potent antigen-presenting cells and play a pivotal role in initiating the immune response. Hence, large-scale production and direct induction of functional DCs ex vivo from HSCs are crucial to HSC research and clinical potential, such as vaccines for cancer and immune therapy.MethodsIn a previous study, we developed a serum-free HSC expansion system (SF-HSC medium) to expand large numbers of primitive HSCs ex vivo. Herein, a DC induction and expansion medium (DC medium) was proposed to further generate large numbers of functional DCs from serum-free expanded HSCs, which were developed and optimized by factorial design and the steepest ascent method.ResultsThe DC medium is composed of effective basal medium (Iscove's modified Dulbecco's medium [IMDM]) and cytokines (2.9 ng/mL stem cell factor [SCF], 2.1 ng/mL Flt-3 ligand, 3.6 ng/mL interleukin [IL]-1β, 19.3 ng/mL granulocyte-macrophage colony-stimulating factor [GM-CSF] and 20.0 ng/mL tumor necrosis factor-α [TNF-α]). After 10-day culture in DC medium, the maximum fold expansion for accumulated CD1a⁺CD11c⁺ DCs was more than 4000-fold, and the induced DCs were characterized and confirmed by analysis of growth kinetics, surface antigen expression, endocytosis ability, mixed lymphocyte reaction, specific cytokine secretion and lipopolysaccharide stimulation.DiscussionIn conclusion, the combination of DC medium and SF-HSC medium can efficiently induce and expand a large amount of functional DCs from a small scale of HSCs and might be a promising source of DCs for vaccine and immune therapy in the near future.     

Dendritic cell-based vaccines suppress metastatic liver tumor via activation of local innate and acquired immunity

Background  Dendritic cell (DC)-based vaccines have been applied clinically in the setting of cancer, but tumor-associated antigens (TAAs) have not yet been enough identified in various cancers. In this study, we investigated whether preventive vaccination with unpulsed DCs or peptide-pulsed DCs could offer anti-tumor effects against MC38 or BL6 liver tumors. Methods  Mice were subcutaneously (s.c.) immunized with unpulsed DCs or the recently defined TAA EphA2 derived peptide-pulsed dendritic cells (Eph-DCs) to treat EphA2-positive MC38 and EphA2-negative BL6 liver tumors. Liver mononuclear cells (LMNCs) from treated mice were subjected to ⁵¹Cr release assays against YAC-1 target cells. In some experiments, mice were injected with anti-CD8, anti-CD4 or anti-asialo GM1 antibody to deplete each lymphocyte subsets. Results  Immunization with unpulsed DCs displayed comparable efficacy against both MC38 and BL6 liver tumors when compared with Eph-DCs. Both DC-based vaccines significantly augmented the cytotoxicity of LMNCs against YAC-1 cells. In vivo antibody depletion studies revealed that NK cells, as well as, CD4+ and CD8+ T cells play critical roles in the anti-tumor efficacy associated with either DC-based modality. Tumor-specific cytotoxic T lymphocyte (CTL) activity was generally higher if mice had received Eph-DCs versus unpulsed DCs. Importantly, the mice that had been protected from MC38 liver tumor by either unpulsed DCs or Eph-DCs became resistant to s.c. MC38 rechallenge, but not to BL6 rechallenge. Conclusions  These results demonstrate that unpulsed DC vaccines might serve as an effective therapy for treating metastatic liver tumor, for which TAA has not yet been identified. Shinjiro Yamaguchi and Tomohide Tatsumi contributed equally to this work.     

Canine Recombinant Adenovirus Vector Induces an Immunogenicity-Related Gene Expression Profile in Skin-Migrated CD11b+ -Type DCs

Gene expression profiling of the blood cell response induced early after vaccination has previously been demonstrated to predict the immunogenicity of vaccines. In this study, we evaluated whether the analysis of the gene expression profile of skin-migrated dendritic cells (DCs) could be informative for the in vitro prediction of immunogenicity of vaccine, using canine adenovirus serotype 2 (CAV2) as vaccine vector. CAV2 has been shown to induce immunity to transgenes in several species including sheep and is an interesting alternative to human adenovirus-based vectors, based on the safety records of the parental strain in dogs and the lack of pre-existing immunity in non-host species. Skin-migrated DCs were collected from pseudo-afferent lymph in sheep. Both the CD11b⁺ -type and CD103⁺ -type skin-migrated DCs were transduced by CAV2. An analysis of the global gene response to CAV2 in the two skin DC subsets showed that the gene response in CD11b⁺ -type DCs was far higher and broader than in the CD103⁺ -type DCs. A newly released integrative analytic tool from Ingenuity systems revealed that the CAV2-modulated genes in the CD11b⁺ -type DCs clustered in several activated immunogenicity-related functions, such as immune response, immune cell trafficking and inflammation. Thus gene profiling in skin-migrated DC in vitro indicates that the CD11b⁺ DC type is more responsive to CAV2 than the CD103⁺ DC type, and provides valuable information to help in evaluating and possibly improving viral vector vaccine effectiveness.     

Autologous dendritic cell vaccine for estrogen receptor (ER)/progestin receptor (PR) double-negative breast cancer

Purpose

A wealth of preclinical information, as well as a modest amount of clinical information, indicates that dendritic cell vaccines have therapeutic potential. The aim of this work was to assess the immune response, disease progression, and post-treatment survival of ER/PR double-negative stage II/IIIA breast cancer patients vaccinated with autologous dendritic cells pulsed with autologous tumor lysates.

Methods

Dendritic cell (DC) vaccines were generated from CD14+ precursors pulsed with autologous tumor lysates. DCs were matured with defined factors that induced surface marker and cytokine production. Individuals were immunized intradermally four times. Specific delayed type IV hypersensitivity (DTH) reaction, ex vivo cytokine production, and lymphocyte subsets were determined for the evaluation of the therapeutic efficiency. Overall survival and disease progression rates were analyzed using Kaplan–Meier curves and compared with those of contemporaneous patients who were not administered DC vaccines.

Results

There were no unanticipated or serious adverse effects. DC vaccines elicited Th1 cytokine secretion and increased NK cells, CD8+ IFN-γ+ cells but decreased the percentage of CD3+ T cells and CD3+ HLA-DR+ T cells in the peripheral blood. Approximately 58% (18/31) of patients had a DTH-positive reaction. There was no difference in overall survival between the patients with and without DC vaccine. The 3-year progression-free survival was significantly prolonged: 76.9% versus 31.0% (with vs. without DC vaccine, p?Conclusion Our findings strongly suggest that tumor lysate-pulsed DCs provide a standardized and widely applicable source of breast cancer antigens that are very effective in evoking anti-breast cancer immune responses.     

CD8+ T Cell Priming by Dendritic Cell Vaccines Requires Antigen Transfer to Endogenous Antigen Presenting Cells

Background

Immunotherapeutic strategies to stimulate anti-tumor immunity are promising approaches for cancer treatment. A major barrier to their success is the immunosuppressive microenvironment of tumors, which inhibits the functions of endogenous dendritic cells (DCs) that are necessary for the generation of anti-tumor CD8+ T cells. To overcome this problem, autologous DCs are generated ex vivo, loaded with tumor antigens, and activated in this non-suppressive environment before administration to patients. However, DC-based vaccines rarely induce tumor regression.

Methodology/Principal Findings

We examined the fate and function of these DCs following their injection using murine models, in order to better understand their interaction with the host immune system. Contrary to previous assumptions, we show that DC vaccines have an insignificant role in directly priming CD8+ T cells, but instead function primarily as vehicles for transferring antigens to endogenous antigen presenting cells, which are responsible for the subsequent activation of T cells.

Conclusions/Significance

This reliance on endogenous immune cells may explain the limited success of current DC vaccines to treat cancer and offers new insight into how these therapies can be improved. Future approaches should focus on creating DC vaccines that are more effective at directly priming T cells, or abrogating the tumor induced suppression of endogenous DCs.     

Interleukin-15 Dendritic Cells Harness NK Cell Cytotoxic Effector Function in a Contact- and IL-15-Dependent Manner

The contribution of natural killer (NK) cells to the treatment efficacy of dendritic cell (DC)-based cancer vaccines is being increasingly recognized. Much current efforts to optimize this form of immunotherapy are therefore geared towards harnessing the NK cell-stimulatory ability of DCs. In this study, we investigated whether generation of human monocyte-derived DCs with interleukin (IL)-15 followed by activation with a Toll-like receptor stimulus endows these DCs, commonly referred to as “IL-15 DCs”, with the capacity to stimulate NK cells. In a head-to-head comparison with “IL-4 DCs” used routinely for clinical studies, IL-15 DCs were found to induce a more activated, cytotoxic effector phenotype in NK cells, in particular in the CD56^bright NK cell subset. With the exception of GM-CSF, no significant enhancement of cytokine/chemokine secretion was observed following co-culture of NK cells with IL-15 DCs. IL-15 DCs, but not IL-4 DCs, promoted NK cell tumoricidal activity towards both NK-sensitive and NK-resistant targets. This effect was found to require cell-to-cell contact and to be mediated by DC surface-bound IL-15. This study shows that DCs can express a membrane-bound form of IL-15 through which they enhance NK cell cytotoxic function. The observed lack of membrane-bound IL-15 on “gold-standard” IL-4 DCs and their consequent inability to effectively promote NK cell cytotoxicity may have important implications for the future design of DC-based cancer vaccine studies.     

Enhancement of Th1 immune response by CD8α dendritic cells loaded with heat shock proteins enriched tumor extract in tumor-bearing mice

The discovery of dendritic cells (DCs) as professional antigen presenting cells has opened up new possibilities for their use in the development of tumor vaccines. We investigated the effect of the CD8α⁺ DCs loaded with heat-treated tumor lysate (HTL) as a vaccine in tumor immunotherapy. The HTL loaded CD8α⁺ DCs, TL loaded CD8α⁺ DCs and unloaded CD8α⁺ DCs were subcutaneously injected in the fibrosarcoma-bearing mice. The splenocyte proliferation and the shifting of Th1/Th2 response were measured. The results indicated a significant increase in the lymphocytes proliferation and the IFN-γ production in the test group of mouse splenocytes. According to the results, HTL loaded CD8α⁺ DCs vaccine significantly decreased tumor growth and longer survival than the other immunized animals. These findings show that anti-tumor immune response against the fibrosarcoma can be induced by HTL loaded CD8α⁺ DCs and may provide a useful therapeutic model for development of approaches to tumor treatments.     

Immunization with a GM3 ganglioside nanoparticulated vaccine confers an effector CD8<Superscript>+</Superscript> T cells-mediated protection against melanoma B16 challenge

Preventive immunotherapy is an attractive strategy for patients at a high risk of having cancer. The success of prophylactic cancer vaccines would depend on the selection of target antigens that are essential for tumour growth and progression. The overexpression of GM3 ganglioside in murine and human melanomas and its important role in tumour progression makes this self antigen a potential target for preventive immunotherapy of this neoplasm. We have previously shown that preventive administration of a GM3-based vaccine to C57BL/6 mice elicited the rejection of the GM3 positive-B16 melanoma cells in most of the animals. Despite the crucial role of cellular immune response in tumour protection, the involvement of T cells in anti-tumour immunity of ganglioside vaccines is not described. Here, we examined the mechanisms by which this immunogen confers tumour protection. We have found that induction of anti-GM3 IgG antibodies correlated with tumour protection. Surprisingly, CD8⁺ T cells, but not NK1.1⁺ cells, are required in the effector phase of the antitumour immune response. The depletion of CD4⁺ T cells during immunization phase did not affect the anti-tumour activity. In addition, T cells from surviving-immunized animals secreted IFNγ when were co-cultured with IFNα-treated B16 melanoma cells or DCs pulsed with melanoma extract. Paradoxically, in spite of the glycolipidic nature of this antigen, these findings demonstrate the direct involvement of the cellular immune response in the anti-tumour protection induced by a ganglioside-based vaccine. Grant support: Center of Molecular Immunology, Elea Laboratories and Recombio.     

Chemically engineered glycan-modified cancer vaccines to mobilize skin dendritic cells

Dendritic cell (DC)–targeting vaccines show great promise in increasing antitumor immunity. Glycan-engineered vaccines facilitate both DC targeting and increased uptake by DCs for processing and presentation to CD4⁺ and CD8⁺ T cells to induce tumor-specific T-cell responses. However, the complexity of various DC subsets in skin tissues, expressing different glycan-binding receptors that can mediate vaccine uptake or drainage of vaccines via lymphatics directly to the lymph node–resident DCs, complicates the success of vaccines. Moreover, the influx of inflammatory immune cells to the site of vaccination, such as monocytes that differentiate to DCs and coexpress glycan-binding receptors, may contribute to the strength of DC-targeting glycovaccines for future clinical use.     

Human CD141+ dendritic cells generated from adult peripheral blood monocytes

Human CD141⁺ dendritic cells (DCs), specialized for cross-presentation, have been extensively studied in the development of DC-based therapy against cancer. A series of attempts was made to generate CD141⁺ DCs from cord blood CD34⁺ hematopoietic progenitors to overcome the practical limitation of in vivo rareness. In the present study, we identified a culture system that generates high CD141⁺ DCs. After culture of CD14⁺ monocytes in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 for 8 days, CD141 was detected on cells that adhered to the bottom of the culture plate. The attached cells exhibited typical features of immature monocyte-derived DCs (moDCs), except for higher CD86 expression, more dendrites and higher granularity compared with those that did not attach. With 3 additional days of culture, increased CD141 expression on the cells was retained along with adhesion ability and partial expression of CLEC9A, a c-type lectin receptor. Furthermore, the cells exhibited effective uptake of dead cells. Interestingly, the attached moDCs differently responded to polyinosinic:polycytidylic acid (poly I:C) stimulation as well as a mixed lymphocyte reaction. Collectively, our findings show that human CD141⁺ DCs can be sufficiently generated from peripheral blood CD14⁺ monocytes, potentiating further investigation into generation of higher yields of cross-priming human DCs in vitro.     

Bacterial product cantastim derived from Pseudomonas aeruginosa induces migration and maturation of dendritic cells

Dendritic cells (DCs) play a pivotal role in linking innate and adaptive immunity. Migration to the lymph nodes and maturation of DCs are crucial steps in the initiation of specific immune responses. The bacterial product CANTASTIM (CS) is a purified extract of Pseudomonas aeruginosa that induces non-specific protection against bacterial infection, enhances macrophage effector functions and modulates cytokines production. In this study, we used a mouse skin explant culture model and human monocyte-derived DCs to study the effect of CS on the migration and maturation of DCs, respectively. We noticed a significant increase in the number of DCs which migrated from the skin explants when CS was added to the culture medium. Also, CS was able to induce the expression of maturation-associated marker CD83 on human monocyte-derived DCs. DC-based tumor vaccines represent a promising approach for cancer immunotherapy and the migration rate and maturation state of DCs are important parameters for their clinical effectiveness. CS may be an attractive candidate to be tested for the production of DC-based vaccine.     

Cytokine-Based Log-Scale Expansion of Functional Murine Dendritic Cells

Background

Limitations of the clinical efficacy of dendritic cell (DC)-based immunotherapy, as well as difficulties in their industrial production, are largely related to the limited number of autologous DCs from each patient. We here established a possible breakthrough, a simple and cytokine-based culture method to realize a log-scale order of functional murine DCs (>1,000-fold), which cells were used as a model before moving to human studies.

Methodology/Principal Findings

Floating cultivation of lineage-negative hematopoietic progenitors from bone marrow in an optimized cytokine cocktail (FLT3-L, IL-3, IL-6, and SCF) led to a stable log-scale proliferation of these cells, and a subsequent differentiation study using IL-4/GM-CSF revealed that 3-weeks of expansion was optimal to produce CD11b⁺/CD11c⁺ DC-like cells. The expanded DCs had typical features of conventional myeloid DCs in vitro and in vivo, including identical efficacy as tumor vaccines.

Conclusions/Significance

The concept of DC expansion should make a significant contribution to the progress of DC-based immunotherapy.     

Effect of dendritic cell–based immunotherapy on hepatocellular carcinoma: A systematic review and meta-analysis

Background aims

Dendritic cell (DC)-based immunotherapy has recently been reported frequently in the treatment of hepatocellular carcinoma (HCC); however, its efficacy remains controversial. In this study, we aimed to evaluate the clinical efficacy of DC-based immunotherapy on HCC by conducting a systematic review and meta-analysis.

Specific microtubule-depolymerizing agents augment efficacy of dendritic cell-based cancer vaccines

Background

Damage-associated molecular patterns (DAMPs) are associated with immunogenic cell death and have the ability to enhance maturation and antigen presentation of dendritic cells (DCs). Specific microtubule-depolymerizing agents (MDAs) such as colchicine have been shown to confer anti-cancer activity and also trigger activation of DCs.

Methods

In this study, we evaluated the ability of three MDAs (colchicine and two 2-phenyl-4-quinolone analogues) to induce immunogenic cell death in test tumor cells, activate DCs, and augment T-cell proliferation activity. These MDAs were further evaluated for use as an adjuvant in a tumor cell lysate-pulsed DC vaccine.

Results

The three test phytochemicals considerably increased the expression of DAMPs including HSP70, HSP90 and HMGB1, but had no effect on expression of calreticulin (CRT). DC vaccines pulsed with MDA-treated tumor cell lysates had a significant effect on tumor growth, showed cytotoxic T-lymphocyte activity against tumors, and increased the survival rate of test mice. In vivo antibody depletion experiments suggested that CD8⁺ and NK cells, but not CD4⁺ cells, were the main effector cells responsible for the observed anti-tumor activity. In addition, culture of DCs with GM-CSF and IL-4 during the pulsing and stimulation period significantly increased the production of IL-12 and decreased production of IL-10. MDAs also induced phenotypic maturation of DCs and augmented CD4⁺ and CD8⁺ T-cell proliferation when co-cultured with DCs.

Conclusions

Specific MDAs including the clinical drug, colchicine, can induce immunogenic cell death in tumor cells, and DCs pulsed with MDA-treated tumor cell lysates (TCLs) can generate potent anti-tumor immunity in mice. This approach may warrant future clinical evaluation as a cancer vaccine.     

Tumor-specific CD4+ T cells are activated by "cross-dressed" dendritic cells presenting peptide-MHC class II complexes acquired from cell-based cancer vaccines

Tumor cells that constitutively express MHC class I molecules and are genetically modified to express MHC class II (MHC II) and costimulatory molecules are immunogenic and have therapeutic efficacy against established primary and metastatic cancers in syngeneic mice and activate tumor-specific human CD4+ T lymphocytes. Previous studies have indicated that these MHC II vaccines enhance immunity by directly activating tumor-specific CD4+ T cells during the immunization process. Because dendritic cells (DCs) are considered to be the most efficient APCs, we have now examined the role of DCs in CD4+ T cell activation by the MHC II vaccines. Surprisingly, we find that DCs are essential for MHC II vaccine immunogenicity; however, they mediate their effect through "cross-dressing." Cross-dressing, or peptide-MHC (pMHC) transfer, involves the generation of pMHC complexes within the vaccine cells, and their subsequent transfer to DCs, which then present the intact, unprocessed complexes to CD4+ T lymphocytes. The net result is that DCs are the functional APCs; however, the immunogenic pMHC complexes are generated by the tumor cells. Because MHC II vaccine cells do not express the MHC II accessory molecules invariant chain and DM, they are likely to load additional tumor Ag epitopes onto MHC II molecules and therefore activate a different repertoire of T cells than DCs. These data further the concept that transfer of cellular material to DCs is important in Ag presentation, and they have direct implications for the design of cancer vaccines.     

A polysaccharide extracted from Grifola frondosa enhances the anti-tumor activity of bone marrow-derived dendritic cell-based immunotherapy against murine colon cancer

We previously isolated the novel heteropolysaccharide maitake Z-fraction (MZF) from the maitake mushroom (Grifola frondosa), and demonstrated that MZF significantly inhibited tumor growth by inducing cell-mediated immunity. In this study, we demonstrated that MZF upregulated the expression of CD80, CD86, CD83, and MHC II on bone marrow-derived dendritic cells (DCs) and significantly increased interleukin-12 (IL-12) and tumor necrosis factor-alpha production by DCs in a dose-dependent manner. MZF-treated DCs significantly stimulated both allogeneic and antigen-specific syngenic T cell responses and enhanced antigen-specific interferon-gamma (IFN-γ) production by syngenic CD4⁺ T cells; however, MZF-treated DCs did not affect IL-4 production. Furthermore, the enhancement of IFN-γ production in CD4⁺ T cells, which was induced by MZF-treated DCs, was completely inhibited by the addition of an anti-IL-12 antibody. These results indicate that MZF induced DC maturation and antigen-specific Th1 response by enhancing DC-produced IL-12. We also demonstrated that DCs pulsed with colon-26 tumor lysate in the presence of MZF induced both therapeutic and preventive effects on colon-26 tumor development in BALB/c mice. These results suggest that MZF could be a potential effective adjuvant to enhance immunotherapy using DC-based vaccination.     

Silencing of the glucocorticoid-induced leucine zipper improves the immunogenicity of clinical-grade dendritic cells

BackgroundThe maturation cocktail composed of interleukin (IL)-6, IL-1β, tumor necrosis factor-α and prostaglandin E₂ is considered the “gold standard” for inducing the maturation of dendritic cells (DCs) for use in cancer immunotherapy. Nevertheless, although this maturation cocktail induces increased expression of several activation markers, such as CD83, the co-stimulation molecules CD80, CD86 and CD40 and the chemokine receptor involved in DC homing in lymph nodes CCR7, the DC immune stimulatory function in vivo contrasts with this mature phenotype, and good clinical outcomes in patients with cancer treated with DC-based vaccines remain rare.MethodsPhenotypic characterization of the immunosuppressive status of DCs differentiated from peripheral blood mononuclear cells of healthy volunteers and matured with the “gold standard” cocktail was performed. Glucocorticoid-induced leucine zipper (GILZ) targeting small interfering RNA (siRNA) was electroporated into DCs after maturation to increase their immunogenicity.ResultsThe mature phenotype of DCs treated for 48 h with this cocktail was associated with the expression of several immunosuppressive regulators, including programmed cell death 1 ligand 1 (PD-L1), IL-10 and GILZ. Electroporation is a very efficient and safe way to deliver siRNA into DCs (80% of DCs receive at least one molecule of siRNA). Silencing GILZ in clinical-grade DCs by siRNA leads to a decrease of the PD-L1 expression associated with an increase in their IL-12 secretion and T-cell induction capability.ConclusionsGILZ silencing is a promising approach to achieving complete clinical-grade DC maturation and avoiding the immunosuppressive effects of the maturation cocktail on DCs intended for clinical use.