Generation Of DCs Research Articles

Nanoparticle-Based Drug Delivery Systems for Induction of Tolerance and Treatment of Autoimmune Diseases.

Autoimmune disease is a chronic inflammatory disease caused by disorders of immune regulation. Antigen-specific immunotherapy has the potential to inhibit the autoreactivity of inflammatory T cells and induce antigen-specific immune suppression without impairing normal immune function, offering an ideal strategy for autoimmune disease treatment. Tolerogenic dendritic cells (Tol DCs) with immunoregulatory functions play important roles in inducing immune tolerance. However, the effective generation of tolerogenic DCs in vivo remains a great challenge. The application of nanoparticle-based drug delivery systems in autoimmune disease treatment can increase the efficiency of inducing antigen-specific tolerance in vivo. In this review, we discuss multiple nanoparticles, with a focus on their potential in treatment of autoimmune diseases. We also discuss how the physical properties of nanoparticles influence their therapeutic efficacy.

Development of a serum-free induction medium for the induction of human CD40+CD209+ dendritic cells from CD14+ monocytes

BackgroundDendritic cells (DCs) are a promising cell type for cell immunotherapy due to their unique functions. The generation of monocyte-derived DCs for clinical immunotherapy requires the use of well-defined components and culture conditions to follow clinical regulations. Therefore, the aim of this study was to develop a serum-free medium for the induction of monocyte-derived DCs. MethodsIn this study, monocytes were isolated from cord blood. First, three different concentration compositions of cytokines (GM-CSF and IL-4) were tested. Then, effective serum substitutes were selected to optimize the compositions. Herein, a serum-free DC (SF-DC) induction medium was proposed to further induce large numbers of functional DCs from monocytes. After culturing, the characteristics and functions of induced DCs were confirmed by surface markers, morphology, TNF-α-stimulated maturation, mixed lymphocyte reaction, endocytosis ability and cytokine secretion. Significant findingsThe results showed that the serum-free induced DCs expressed DC-related surface markers and exhibited endocytosis ability. Importantly, the serum-free induced DCs secreted higher amounts of IL-1β, IL-6, IL-12p70 and TNF and showed a better ability to stimulate T cell proliferation than those in serum-containing medium. Taken together, SF-DC induction medium was successfully developed and can provide a promising source of DCs for future cell immunotherapy.

Potent adjuvant effect elicited for tumor immunotherapy by a liposome conjugated pH-sensitive polymer and dendritic cell-targeting Toll-like-receptor ligand

The generation of DCs with augmented functions is a strategy for obtaining satisfactory clinical outcomes in tumor immunotherapy. We developed a novel synthetic adjuvant comprising a liposome conjugated with a DC-targeting Toll-like-receptor ligand and a pH-sensitive polymer for augmenting cross-presentation. In an in vitro study using mouse DCs, these liposomes were selectively incorporated into DCs, significantly enhanced DC function and activated immune responses to present an epitope of the incorporated antigen on the major histocompatibility complex class I molecules. Immunization of mice with liposomes encapsulating a tumor antigen significantly enhanced antigen-specific cytotoxicity. In tumor-bearing mice, vaccination with liposomes encapsulating a tumor antigen elicited complete tumor remission. Furthermore, vaccination significantly enhanced cytotoxicity, targeting not only the vaccinated antigen but also the other antigens of the tumor cell. These results indicate that liposomes are an ideal adjuvant to develop DCs with considerably high potential to elicit antigen-specific immune responses; they are a promising tool for cancer therapy with neoantigen vaccination.

Methods to assess DC-dependent priming of T cell responses by dying cells.

Dendritic cells have been widely investigated in cancer immunotherapy clinical trials for the last two decades mainly due to their robust ability to elicit an adaptive anticancer immune response of the cellular and humoral types. Immature DCs can be easily loaded with desired antigens. However, to become efficient antigen-presenting cells, DCs must first undergo a process of maturation. Protocols for the generation of DCs for use in cancer immunotherapy, including the generation of a large number of immature DCs for antigen pulsing and the selection of a well-defined immunostimulatory agent to achieve complete and reproducible maturation, which is a crucial step for further stimulation of T cell activation, must carefully consider the characteristics of DC physiology. In this report, we provided a detailed protocol for DC generation, pulsation and activation with the subsequent induction of T cell-specific immune responses.

Induction of tolerogenic dendritic cells by activated TGF-\u03b2/Akt/Smad2 signaling in RIG-I-deficient stemness-high human liver cancer cells

BackgroundDendritic cells (DCs) alter their role from being immunostimulatory to immunosuppressive at advanced stages of tumor progression, but the influence of cancer stem cells (CSCs) and their secreted factors on generation and phenotypic change of DCs is unknown. Retinoic acid-inducible gene I (RIG-I) plays a role in regulation of other cellular processes including leukemic stemness besides its antiviral function.MethodsShort hairpin RNA-mediated gene silencing was employed to generate stable RIG-I-knocked-down human hepatocellular carcinoma (HCC) cell lines. Expression levels of genes and proteins in spheres of those HCC cells were determined by quantitative real-time PCR and Western bot, respectively. Levels of secreted cytokines were measured by ELISA. The surface molecule expression levels of DCs were analyzed using flow cytometry. The ability of DCs to induce proliferation of T cells was assessed by a mixed lymphocyte reaction (MLR) assay.ResultsRIG-I-knocked-down HCC cells showed upregulated expression of stem cell marker genes, enhanced secretion of factors suppressing in vitro generation of DCs into the conditioned medium (CM), and induction of a phenotype of tumor-infiltrating DCs (TIDCs) with low levels of DC markers in their tumors in nude mice. Those DCs and TIDCs showed reduced MLR, indicating RIG-I deficiency-induced immunotolerance. The RIG-I-deficient HCC cells secreted more TGF-β1 than did reference cells. The tumors formed after injection of RIG-I-deficient HCC cells had higher TGF-β1 contents than did tumors derived from control cells. DC generation and MLR suppressed by the CM of RIG-I-deficient HCC cells were restored by an anti-TGF-β1 antibody. TGF-β1-induced phosphorylation of Smad2 and Akt was enhanced in RIG-I-deficient HCC spheres, knockdown of AKT gene expression abolishing the augmentation of TGF-β1-induced Smad2 phosphorylation. Akt and p-Akt were co-immunoprecipitated with Smad2 in cytoplasmic proteins of RIG-I-deficient spheres but not in those of control spheres, the amounts of co-immunoprecipitated Akt and p-Akt being increased by TGF-β stimulation.ConclusionsOur results demonstrate that RIG-I deficiency in HCC cells induced their stemness, enhanced secretion and signaling of TGF-β1, tolerogenic TIDCs and less generation of DCs, and the results suggest involvement of TGF-β1 in those RIG-I deficiency-induced tolerogenic changes and involvement of CSCs in DC-mediated immunotolerance.

Epitope-based ligation of ICAM-1: Therapeutic target for protection against the development of rheumatoid arthritis

Identification of a particular epitope on the domain 2 of human ICAM-1 led us to focus on its role in the treatment of rheumatoid arthritis (RA). Key observations from our previous xenotransplantation research included the generation of tolerogenic DCs, antigen-specific T-cell tolerance, and reduced production of inflammatory cytokines. The critically important point is the fact that it works initially on DC maturation. Ligation of this epitope with a recognizing antibody, MD-3, was also able to create a tolerogenic environment in RA in a manner sililar to that created by xenotransplantation. In this study, we noted that the disease progression, in terms of arthritis score and histopathology of joints, was significantly less severe in the MD-3-treated group than in the vehicle-treated group. Defective production of IL-6 and reduced proliferation of collagen-specific T cells were most remarkable laboratory findings. This type of ligation has a greater advantage over other types of therapeutics, in a sense that simple injection of this antibody inhibits antigen-specific T cell response. Due to the possibility of viral infection in this process, we regularly monitored cytomegalovirus reactivation status without detection of any viral gene replication. We are hoping that remarkable specializations that this interaction has, would be a promising target for therapeutic antibody in RA.

Tolerogenic dendritic cells are efficiently generated using minocycline and dexamethasone

Tolerogenic dendritic cells (tDCs) represent a promising tool for cellular therapy against autoimmune diseases, allergies, and transplantation rejection. Numerous pharmacological agents are known to induce tDC generation. Minocycline, which has long been used as a broad-spectrum antibiotic, was recently shown to significantly increase the generation of DCs with regulatory properties. Here, we examined the effect of the combination of minocycline with dexamethasone, rapamycin, vitamin D3, and interleukin (IL)-10, which are all known inducers of tDC generation. The highest number of tDCs was generated when minocycline and dexamethasone were used together with granulocyte colony-stimulating factor (GM-SCF) and IL-4. The tolerogenicity of the minocycline/dexamethasone-conditioned tDCs was much better than or at least equal to those of the tDCs generated with either one of these agents, as assessed through in vitro phenotypic and functional assays. In addition, pretreatment with MOG35-55 peptide-pulsed minocycline/dexamethasone-conditioned tDCs significantly ameliorated the clinical signs of experimental autoimmune encephalitis induced by MOG peptide injection in a murine model. These results confirmed that tDCs with potent tolerogenic properties could be efficiently generated by the combined use of minocycline and dexamethasone, along with GM-CSF and IL-4. Our results would help in the development of ex vivo tDC-based immunotherapies.

Abstract 3060: Role of curcumin in targeting cancer-associated fibroblasts and modulation of tumor microenvironment in dendritic cell-based immunotherapy

Abstract INTRODUCTION: Cancer associated fibroblasts (CAFs), main component of tumor microenvironment (TME), modulate the recruitment and functions of tumor-associated immune cells by secreting various growth hormones, miRNAs and cytokines; thus having an important role in generation of immunosuppressive TME which is yet to be elucidated. Curcumin is known to have various properties including capability to modulate numerous target proteins including transcription factors, receptors, kinases, cytokines, enzymes and growth factors. Thus, aim of the study was to evaluate the effect of miRNAs and cytokines released by lung cancer patients’ derived CAFs and to assess immunomodulatory potential of curcumin on DC maturation by targeting these CAFs through modulating their TME. METHODOLOGY: CAFs were cultured from lung cancer patient derived tumor tissue biopsy and characterised using CAF-specific markers. Further, immature DCs (imDCs) were differentiated in presence of rGM-CSF and rIL-4 for 5-6 days. These imDCs were cultured in the presence of conditioned media derived from CAFs (CAFs-CM) as well as NFs (Normal Fibroblasts, NFs-CM) at day 6 for 48 hours to evaluate the effect of CAFs on DC maturation. Mature DCs (mDCs) were characterized by the presence of maturation markers CD80, CD83, CD86 and CTLA4 using qRT-PCR. Moreover, expression of miR-221, miR-222, miR-155, miR-142-3p and miR-146a was assessed to evaluate the role of epigenetic regulators on DC maturation. Cytokine profiling of CAFs-CM as well as CAFs-CM treated with curcumin was conducted. RESULTS: α-SMA+Vimentin+ cells were considered as CAFs. A significant upregulation of CD80, CD83 and CD86 was observed when cultured in the presence of NFs-CM while a remarkable downregulation of these markers was found when cultured in CAFs-CM. CTLA-4 was down regulated in NFs-CM as compared to CAFs-CM, suggesting the role of CAFs in generation of regulatory DCs. Amongst all miRNAs, miR-146a was shown to be up regulated dramatically in CAF-DCs (DCs cultured in CAFs-CM) as well as in CAFs-CM, suggesting the immunosuppressive role of miR-146a. Further, an increased expression of miR-146a was positively correlated with increased expression of anti-inflammatory cytokines like IL-6, IL-10, TGF-β and decreased expression of TNF-α (pro-inflammatory) in CM derived from CAF-DCs. Moreover, curcumin had the potential to convert regulatory DCs facilitated by CAFs into mDCs, which were characterized by high expression of co-stimulatory molecules, low expression of CTLA4, lower levels of immune suppressive cytokines production, lower levels of miR-146a. CONCLUSION: These findings provide insight into understanding the immunomodulatory role of curcumin in targeting CAFs and modulating the tumor microenvironment, thus enhancing antitumor immune response in DC based therapy. Citation Format: Sheefa Mirza, Nayan Jain, Rakesh Rawal. Role of curcumin in targeting cancer-associated fibroblasts and modulation of tumor microenvironment in dendritic cell-based immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3060. doi:10.1158/1538-7445.AM2017-3060

GATA2 regulates dendritic cell differentiation

AbstractDendritic cells (DCs) are critical immune response regulators; however, the mechanism of DC differentiation is not fully understood. Heterozygous germ line GATA2 mutations induce GATA2-deficiency syndrome, characterized by monocytopenia, a predisposition to myelodysplasia/acute myeloid leukemia, and a profoundly reduced DC population, which is associated with increased susceptibility to viral infections, impaired phagocytosis, and decreased cytokine production. To define the role of GATA2 in DC differentiation and function, we studied Gata2 conditional knockout and haploinsufficient mice. Gata2 conditional deficiency significantly reduced the DC count, whereas Gata2 haploinsufficiency did not affect this population. GATA2 was required for the in vitro generation of DCs from Lin−Sca-1+Kit+ cells, common myeloid-restricted progenitors, and common dendritic cell precursors, but not common lymphoid-restricted progenitors or granulocyte-macrophage progenitors, suggesting that GATA2 functions in the myeloid pathway of DC differentiation. Moreover, expression profiling demonstrated reduced expression of myeloid-related genes, including mafb, and increased expression of T-lymphocyte–related genes, including Gata3 and Tcf7, in Gata2-deficient DC progenitors. In addition, GATA2 was found to bind an enhancer element 190-kb downstream region of Gata3, and a reporter assay exhibited significantly reduced luciferase activity after adding this enhancer region to the Gata3 promoter, which was recovered by GATA sequence deletion within Gata3 +190. These results suggest that GATA2 plays an important role in cell-fate specification toward the myeloid vs T-lymphocyte lineage by regulating lineage-specific transcription factors in DC progenitors, thereby contributing to DC differentiation.

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.

Flt3 Ligand Is Essential for Survival and Protective Immune Responses during Toxoplasmosis.

Dendritic cells (DCs) are critical for resistance to Toxoplasma gondii, and infection with this pathogen leads to increased numbers of DCs at local sites of parasite replication and in secondary lymphoid organs, but the factors that regulate this expansion are poorly understood. The cytokine Flt3 ligand (Flt3L) is critical for the generation and maintenance of DCs, and Flt3L(-/-) mice were found to be highly susceptible to acute toxoplasmosis. This phenotype correlated with decreased production of IL-12 and IFN-γ, as well as impaired NK cell responses. Surprisingly, despite low basal numbers of DCs, Flt3L(-/-) mice infected with T. gondii displayed an expansion of CD8α(+) and CD11b(lo)CD8α(-) DCs. Infection also induced an expansion of parasite-specific CD4(+) and CD8(+) T cells in Flt3L(-/-) mice; however, these cells were reduced in number and displayed impaired ability to produce IFN-γ relative to wild-type controls. Exogenous IL-12 treatment partially restored NK and T cell responses in Flt3L(-/-) mice, as well as acute resistance; however, these mice eventually succumbed to toxoplasmic encephalitis, despite the presence of large numbers of DCs and T cells in the brain. These results highlight the importance of Flt3L for resistance to toxoplasmosis and demonstrate the existence of Flt3L-independent pathways that can mediate infection-induced expansion of DCs and T cell priming.

Effects of DKK-3, a Wnt signaling inhibitor, on dendritic cell phenotype and T cell polarization

Wnt signaling plays crucial roles in regulation of a wide range of processes in different cell types including immune cells and, in particular, dendritic cells and T cells. Growing indications point out that Wnt pathway components modulate the both innate and adaptive immune responses through regulating DC functions. We investigated the effects of recombinant DKK-3 protein on the phenotype and biological functions of bone marrow-derived DCs (BM-DCs) as well as T cell polarization. The phenotype and the cytokine production of BM-derived DCs in the presence DKK-3 were analyzed using flow cytometry and ELISA, respectively. Also, capability of DCs to activate T cells was evaluated by CFSE-labeled splenocytes. Regulatory T cell induction, T cell polarization, and cytokine secretion were assessed by flow cytometry and ELISA in splenocytes cultured in the presence of DKK-3. Our results showed that the expression of CD86 and CD40 increased in the DKK-3-treated DC, while the expression of PDL-1 and PDL-2 diminished. Furthermore, the presence of DKK-3 decreased IL-10 and IL-4 production and increased IFN-gamma production by treated DCs.DKK-3. Moreover, DKK-3 shifted naive CD4 T cells towards TH1 cells through up-regulation of T-bet and down-regulation of GATA-3. Our results, therefore, suggest that DKK-3 protein has the ability to promote the generation of Th1-immunostimulatory DCs from its precursors.

In vitro and in vivo studies of the immunomodulatory effect of Echinacea purpurea on dendritic cells.

BackgroundExtracts of Echinacea have been used traditionally for the treatment of diverse types of infections and wounds. They have become very familiar immunostimulant herbal medicine. However, the specific immunomodulatory effect of Echinacea remains to be elucidated.AimIn our study, the effect of Echinacea purpurea extract on the generation of immature DCs from monocytes was described, as well as its effect on DC differentiation. In addition, an in vivo experiment was conducted to investigate whether treatment of mice with extracts derived from E. purpurea has immunomodulatory effect on murine splenic DCs.MethodsImmature DCs were generated by incubating peripheral blood monocytes with cytokine cocktail (GM-CSF + IL-4) and matured by tumor necrosis factor-α (TNF-α). The cells were randomized to 5 groups to investigate E. purpurea effect in different stages. Phenotypic analysis of cell marker CD83-expressed on DCs was performed by flow cytometry. Mice were randomly divided into 3 groups; control, E. purpurea treated and E. purpurea-TNF-α treated group. The murine splenic DCs were isolated and phenotyped for CD83 and CD11c by flow cytometry.ResultsTreatment of monocytes with E. purpurea prior to addition of the maturation factor TNF-α resulted in a significant decrease in the yield of DC expressing CD83. On the other hand, immature DCs generated in the culture in the presence of GM-CSF and IL-4, when treated simultaneously with E. purpurea and TNF-α, exhibited an insignificant change in the yield of CD83-expressing DCs compared with untreated control. The in vivo experiments showed that splenic DCs obtained from mice treated with E. purpurea with or without TNF-α did not exhibit significant changes in CD83 or CD11c compared with those obtained from control mice.ConclusionOur findings suggest that the immunomodulatory mechanisms of E. purpurea impact generation fate of DCs rather than differentiation stages. The results obtained in the in vivo study utilizing murine splenic DCs supported those observed in vitro.

Exogenous Addition of Arachidonic Acid to the Culture Media Enhances the Functionality of Dendritic Cells for Their Possible Use in Cancer Immunotherapy

The development of dendritic cell based vaccines is a promising approach in cancer immunotherapy. For their successful use in the clinics, the propagation and functionality of DCs is crucial. We earlier established a two-step method for the large scale generation of DCs from umbilical cord blood derived MNCs/CD34+ cells. This work aims at improving their functionality based on the following observations: in vitro generated DCs can be less efficient in migration and other functional activities due to lower eicosanoid levels. The production of eicosanoids from Arachidonic Acid (AA) can be hampered due to suppression of the enzyme phospholipase A2 by IL-4, an essential cytokine required for the differentiation of DCs. We hypothesized that exogenous addition of AA to the culture media during DC generation may result in DCs with improved functionality. DCs were generated with and without AA. The two DC sets were compared by phenotypic analysis, morphology and functional assays like antigen uptake, MLR, CTL assay and in vitro and in vivo migration. Though there were no differences between the two types of DCs in terms of morphology, phenotype and antigen uptake, AA+ DCs exhibited an enhanced in vitro and in vivo migration, T cell stimulatory capacity, CTL activity and significantly higher transcript levels of COX-2. AA+ DCs also show a favorable Th1 cytokine profile than AA- DCs. Thus addition of AA to the culture media is skewing the DCs towards the secretion of more IL-12 and less of IL-10 along with the restoration of eicosanoids levels in a COX-2 mediated pathway thereby enhancing the functionality of these cells to be used as a potent cellular vaccine. Taken together, these findings will be helpful in the better contriving of DC based vaccines for cancer immunotherapy.

Inhibition of breast cancer resistance protein (ABCG2) in human myeloid dendritic cells induces potent tolerogenic functions during LPS stimulation.

Breast cancer resistance protein (ABCG2), a member of the ATP-binding cassette transporters has been identified as a major determinant of multidrug resistance (MDR) in cancer cells, but ABC transporter inhibition has limited therapeutic value in vivo. In this research, we demonstrated that inhibition of efflux transporters ABCG2 induced the generation of tolerogenic DCs from human peripheral blood myeloid DCs (mDCs). ABCG2 expression was present in mDCs and was further increased by LPS stimulation. Treatment of CD1c+ mDCs with an ABCG2 inhibitor, Ko143, during LPS stimulation caused increased production of IL-10 and decreased production of pro-inflammatory cytokines and decreased expression of CD83 and CD86. Moreover, inhibition of ABCG2 in monocyte-derived DCs (MDDCs) abrogated the up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines in these cells in response to LPS. Furthermore, CD1c+ mDCs stimulated with LPS plus Ko143 inhibited the proliferation of allogeneic and superantigen-specific syngenic CD4+ T cells and promoted expansion of CD25+FOXP3+ regulatory T (Treg) cells in an IL-10-dependent fashion. These tolerogenic effects of ABCG2 inhibition could be abolished by ERK inhibition. Thus, we demonstrated that inhibition of ABCG2 in LPS-stimulated mDCs can potently induce tolerogenic potentials in these cells, providing crucial new information that could lead to development of better strategies to combat MDR cancer.

Hepatitis B virus e antigen (HBeAg) may have a negative effect on dendritic cell generation

Hepatitis B virus (HBV) continues to be a serious worldwide health problem despite the use of protective HBV vaccines and therapeutic regimens against chronic HBV infection. Chronic HBV patients cannot induce sufficient immune responses against the virus. HBV and its antigens are believed to suppress immune responses during chronic infection. Hence, studying the role of HBV in immune suppression is very important for the development of alternative therapeutic strategies for HBV infections.In the present study, we investigated the effect of Hepatitis B virus e antigen (HBeAg) on the generation of bone marrow derived dendritic cells (BMDCs) and the stimulation of plasmacytoid DCs (pDCs). In the presence of HBeAg, the ratio of BMDCs was decreased, but the ratio of CD11b+Ly6G+ immature myeloid cells was increased. The expression of 47 proteins was also changed during HBeAg treatment; however, CpG-induced MHC-II expression on pDCs was not affected. Our results indicate that HBeAg may have a negative effect on the generation of DCs from bone morrow precursors.

Evaluation of myeloid cell suppression related to cancer cell released products (TUM4P.913)

Abstract Dendritic cells (DCs) and macrophages may recognize tumor antigens and initiate an antitumoral response before and after the formation of tumor mass. However, the secretion of cytokines and factors that inhibit myeloid cells is a mechanism used by tumor cells to escape from immune attack. Our aim was to understand how tumor cells modulate the generation of murine macrophages and human DCs. For this, we collected bone marrow cells from C57Bl/6 mice and differentiated with M-CSF in the presence of 3LL (Lewis lung carcinoma) or MN/MCA (fibrosarcoma) supernatants. Some cells were activated with IFN-γ and LPS to obtain M1 macrophages or IL-4 to M2. Data showed increased expression of arginase-1 mRNA in macrophages differentiated with MN/MCA supernatant and it happened both in M1 and M2. About 20% of cells proliferate in the presence of supernatants, even in the absence of M-CSF and they express higher level of CD16. Moreover, we differentiated human DCs from monocytes with IL-4 and GM-CSF in the presence of K562 (chronic myeloid leukemia) supernatants. TNF-α was added to activate the cells. It was observed higher percentage of cells expressing CD80 when the differentiation was done with K562 supernatant. Higher secretion of IL-10 and lower production of IL-12 were found in cells differentiated with K562 supernatant and later activated. Therefore, the development and function of macrophages and DCs are affected by tumor products and both cells present some suppressive features.

Targeting DNGR‐1 (CLEC9A) with antibody/MUC1 peptide conjugates as a vaccine for carcinomas

DCs are the most potent APCs and are the focus of many immunotherapeutic approaches for the treatment of cancer, although most of these approaches require the ex vivo generation and pulsing of DCs. We have targeted a subset of DCs in vivo using an Ab to DNGR-1, a C-type lectin dedicated to the cross-presentation of Ag expressed by subsets of DCs. HLA-A2 epitopes from the tumour-associated Ag, MUC1, were coupled to the anti-DNGR-1 Ab, and their efficacy in generating a Th1-cell response and inhibiting tumour growth was evaluated in a clinically relevant double transgenic mouse model expressing human MUC1 and A2K/b. Using this strategy, we demonstrate that an effective immune response to MUC1 can be generated, which results in a significant delay in the growth of MUC1-expressing tumours in both prophylactic and therapeutic settings. In addition, we also show, using PBMCs isolated from healthy volunteer blood, that target an MUC1 HLA-A2 epitope to human DNGR-1 in vitro can induce an MUC1-specific CD8+-T-cell response, which confirms the relevance of our in vivo murine results in the human setting.

Transcriptional control of dendritic cell differentiation.

Dendritic cells (DCs) are professional antigen presenting cells involved critically not only in provoking innate immune responses but also in establishing adaptive immune responses. Dendritic cells are heterogenous and divided into several subsets, including plasmactyoid DCs (pDCs) and several types of conventional DCs (cDCs), which show subset-specific functions. Plasmactyoid DCs are featured by their ability to produce large amounts of type I interferons (IFNs) in response to nucleic acid sensors, TLR7 and TLR9 and involved in anti-viral immunity and pathogenesis of certain autoimmune disorders such as psoriasis. Conventional DCs include the DC subsets with high crosspresentation activity, which contributes to anti-viral and anti-tumor immunity. These subsets are generated from hematopoietic stem cells (HSCs) via several intermediate progenitors and the development is regulated by the transcriptional mechanisms in which subset-specific transcription factors play major roles. We have recently found that an Ets family transcription factor, SPI-B, which is abundantly expressed in pDCs among DC subsets, plays critical roles in functions and late stage development of pDCs. SPI-B functions in cooperation with other transcription factors, especially, interferon regulatory factor (IRF) family members. Here we review the transcription factor-based molecular mechanisms for generation and functions of DCs, mainly by focusing on the roles of SPI-B and its relatives.

Large scale (GMP) production and first clinical experience with a new generation of fast DCs transfected with mRNA hTERT and survivin

Large scale (GMP) production and first clinical experience with a new generation of fast DCs transfected with mRNA hTERT and survivin