Activation Of Human Dendritic Cells Research Articles

MARCH-I: A negative regulator of dendritic cell maturation

The expression of costimulatory molecules such as MHC-II, CD86 and CD83 on dendritic cells (DCs) are strongly regulated during cellular activation. Ubiquitination of some of these markers by the E3 ubiquitin ligase MARCH-I affects the maturation state of DCs and subsequently modulates immune responses. The effects of MARCH-I gene overexpression on the functional activity of human DCs is not well understood.Here, we investigate how MARCH-I, regulates maturation of DCs. We now provide evidence that MARCH-I transduced DCs secrete high levels of IL10 despite low secretion of IL 6 and IL 12 in response to LPS stimulation. They are weak stimulators of T lymphocyte cells but skewed T cell polarization toward T regulatory subset.These results exhibit that reduced expression of surface costimulatory molecules suppresses DC activation. It can be concluded that overexpression of MARCH-I gene in DCs leads to the production of tolerogenic DC.

Sulforaphane Reduces the Chronic Inflammatory Immune Response of Human Dendritic Cells.

Sulforaphane (SFN) is an isothiocyanate of vegetable origin with potent antioxidant and immunomodulatory properties. The characterization of its pleiotropic activity in human dendritic cells (DCs) is poorly summarized. The aim of this work was to study the immunomodulatory power of SFN in response to an inflammatory microenvironment on human monocyte-derived DCs (moDCs). We studied the immunological response induced by SFN. Apoptosis and autophagy assays were performed using flow cytometry on moDCs and a cancer cell line (THP-1). These included moDC maturation, lymphocyte proliferation and cytokine production under different experimental conditions. We investigated whether these results were associated with an inflammatory microenvironment induced by lipopolysaccharides (LPSs). Our results demonstrated that SFN could interact with moDCs, significantly reducing the autophagy process and enhancing apoptosis similarly to cancer cell line THP-1 cells in a chronic inflammatory microenvironment. Under chronic inflammation, SFN modulated the phenotypical characteristics of moDCs, reducing the expression of all markers (CD80, CD83, CD86, HLA-DR and PD-L1). SFN significantly reduced the Th2 proliferative response, with a decrease in the IL-9 and IL-13 levels. Although we did not observe any changes in the regulatory proliferative response, we noted an increase in the IL-10 levels. These findings demonstrate that SFN exerts protective effects against LPS-induced inflammation via the modulation of moDCs/T cells towards a regulatory profile. SFN may be a potential candidate for the treatment of pathologies with an inflammatory profile.

Lipoteichoic acid of Streptococcus gordonii as a negative regulator of human dendritic cell activation.

Streptococcus gordonii, an opportunistic Gram-positive bacterium, causes an infective endocarditis that could be fatal to human health. Dendritic cells (DCs) are known to be involved in disease progression and immune responses in S. gordonii infection. Since lipoteichoic acid (LTA) is a representative virulence factor of S. gordonii, we here investigated its role in the activation of human DCs stimulated with LTA-deficient (ΔltaS) S. gordonii or S. gordonii LTA. DCs were differentiated from human blood-derived monocytes in the presence of GM-CSF and IL-4 for 6 days. DCs treated with heat-killed ΔltaS S. gordonii (ΔltaS HKSG) showed relatively higher binding and phagocytic activities than those treated with heat-killed wild-type S. gordonii (wild-type HKSG). Furthermore, ΔltaS HKSG was superior to wild-type HKSG in inducing phenotypic maturation markers including CD80, CD83, CD86, PD-L1, and PD-L2, antigen-presenting molecule MHC class II, and proinflammatory cytokines such as TNF-α and IL-6. Concomitantly, DCs treated with the ΔltaS HKSG induced better T cell activities, including proliferation and activation marker (CD25) expression, than those treated with the wild-type. LTA, but not lipoproteins, isolated from S. gordonii weakly activated TLR2 and barely affected the expression of phenotypic maturation markers or cytokines in DCs. Collectively, these results demonstrated that LTA is not a major immuno-stimulating agent of S. gordonii but rather it interferes with bacteria-induced DC maturation, suggesting its potential role in immune evasion.

Human Monocyte-Derived Dendritic Cells Are the Pharmacological Target of the Immunosuppressant Flavonoid Silibinin.

Silibinin, a natural polyphenolic flavonoid, is known to possess anti-inflammatory, anticancer, antioxidant, and immunomodulatory properties. However, the effects of Silibinin on the maturation and immunostimulatory functions of human dendritic cells (DC) remain to be elucidated. In this study, we have attempted to ascertain whether Silibinin influences the maturation, cytokine production, and antigen-presenting capacity of human monocyte-derived DC. We show that Silibinin significantly suppresses the upregulation of costimulatory and MHC molecules in LPS-stimulated mature DC and inhibits lipopolysaccharide (LPS)-induced interleukin (IL)-12, IL-23, and TNF-α production. Furthermore, Silibinin impairs the proliferation response of the allogenic memory CD4 T lymphocytes elicited by LPS-matured DC and their Th1/Th17 profile. These findings demonstrate that Silibinin displays immunosuppressive activity by inhibiting the maturation and activation of human DC and support its potential application of adjuvant therapy in the treatment of autoimmune diseases.

EP08.01-036 Low-dose EGFR-TKIs Directly Induce Maturation and Functional Activity of Human Dendritic Cells in an EGFR-independent manner

Recently, several epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have been administered concurrently with the anti-PD-1 antibody pembrolizumab in patients with EGFR mutated non-small cell lung cancer (NSCLC) in clinical trials. EGFR-TKIs with immune checkpoint inhibitors (ICIs) does have preclinical rationale in tumor-bearing syngeneic mouse models. However, the direct effect of EGFR-TKIs on APC (antigen presenting cells) such as DCs (dendritic cells) remains elusive.

Triiodothyronine-stimulated dendritic cell vaccination boosts antitumor immunity against murine colon cancer

Given the ability of dendritic cells (DCs) to modulate other immune players, their manipulation holds great potential for inducing efficient antitumor immunity. However, DC vaccine manufacturing deserve optimization since tumor cell cargo and DC functional state induced by maturation signals influence their in vivo immunogenic potential. We reported that triiodothyronine (T3) stimulates mice DCs’ maturation and their ability to promote pro-inflammatory and cytotoxic T-cell responses. This study aimed to evaluate the efficacy of T3-conditioned DC vaccination in a murine model of colon carcinoma, deciphering the molecular players involved; and to examine the effects of T3 on the maturation and activation of human DCs (huDCs). Bone marrow-derived DCs were exposed to T3 and MC38 cancer cells that underwent cell death (MC38-Apo/Nec) by UVB irradiation. Our results showed that MC38-Apo/Nec cells are efficiently uptaken by DCs and that T3 upregulates CD86 expression with increased production of the pro-inflammatory cytokines IL-12, IL-6 and TGF-β. In a colon cancer model, vaccination with T3-stimulated and tumor antigen-loaded DCs inhibited tumor growth in wild type mice, an effect that was eliminated in IL-17-deficient animals. Notably, secretion of high levels of IFN-γ and IL-17, induction of Th1, Th17 and tumor-specific cytotoxic T lymphocytes characterized the antitumor response upon vaccination. Moreover, our initial findings demonstrated a significant increase in CD86 expression and IL-12 production by huDCs induced by T3. Overall, these results reinforce the adjuvant properties of T3-conditioned DCs to potentiate T-cell-mediated antitumor immunity and support promising advances in the translation process to human oncotherapy.

Activation of Human Dendritic Cells by Nanoparticles Carrying CTL Epitopes of Non-Structural Proteins of Hepatitis C Virus

Activation of Human Dendritic Cells by Nanoparticles Carrying CTL Epitopes of Non-Structural Proteins of Hepatitis C Virus

Glioma-targeted delivery of exosome-encapsulated antisense oligonucleotides using neural stem cells

Tropism of neural stem cells (NSCs) to hypoxic tumor areas provides an opportunity for the drug delivery. Here, we demonstrate that NSCs effectively transport antisense oligonucleotides (ASOs) targeting oncogenic and tolerogenic signal transducer and activator of transcription 3 (STAT3) protein into glioma microenvironment. To enable spontaneous, scavenger receptor-mediated endocytosis by NSCs, we used previously described CpG-STAT3ASO conjugates. Following uptake and endosomal escape, CpG-STAT3ASO colocalized with CD63+ vesicles and later with CD63+CD81+ exosomes. Over 3 days, NSCs secreted exosomes loaded up to 80% with CpG-STAT3ASO. Compared to native NSC exosomes, the CpG-STAT3ASO-loaded exosomes potently stimulated immune activity of human dendritic cells or mouse macrophages, inducing nuclear factor κB (NF-κB) signaling and interleukin-12 (IL-12) production. Using orthotopic GL261 tumors, we confirmed that NSC-mediated delivery improved oligonucleotide transfer from a distant injection site into the glioma microenvironment versus naked oligonucleotides. Correspondingly, the NSC-delivered CpG-STAT3ASO enhanced activation of glioma-associated microglia. Finally, we demonstrated that NSC-mediated CpG-STAT3ASO delivery resulted in enhanced antitumor effects against GL261 glioma in mice. Peritumoral injections of 5 × 105 NSCs loaded ex vivo with CpG-STAT3ASO inhibited subcutaneous tumor growth more effectively than the equivalent amount of oligonucleotide alone. Based on these results, we anticipate that NSCs and NSC-derived exosomes will provide a clinically relevant strategy to improve delivery and safety of oligonucleotide therapeutics for glioma treatment.

Mycobacterium tuberculosis Rv3628 is an effective adjuvant via activation of dendritic cells for cancer immunotherapy

Tumor antigens (Ags) are weakly immunogenic and elicit inadequate immune responses, thus induction of antigen-specific immune activation via the maturation of dendritic cells (DCs) is a strategy used for cancer immunotherapy. In this study, we examined the effect of Rv3628 from Mycobacterium tuberculosis (Mtb) on activation of DCs and anti-tumor immunity in vivo. Intravenous injection of mice with Rv3628 promoted DC activation of spleen and lymph nodes. More importantly, Rv3628 also induced activation of DCs and enhanced Ag presentation in tumor-bearing mice. In mice bearing ovalbumin (OVA)-expressing tumors, combination treatment with Rv3628 and OVA peptide promoted OVA-specific T cell activation and accumulation of interferon (IFN)-γ and tumor necrosis factor (TNF)-α-producing OT-I and OT-II cells in tumor-draining lymph nodes. Moreover, three different tumor Ags in three different tumor models showed enhanced anti-tumor activity with Rv3628 as adjuvant, including inhibition of growth of OVA-expressing B16 melanoma, CT26 carcinoma, and B16 melanoma tumors, and a synergistic effect with anti-programmed cell death protein 1 (PD-1) antibody treatment. Additionally, potential application against human tumors was indicated by similar activation of human peripheral blood DCs by Rv3628. Taken together, these data demonstrate that Rv3628 could be an effective adjuvant in tumor immunotherapy via enhanced capacity of DC activation and Ag presentation.

Gut microbiota-mediated secondary bile acids regulate dendritic cells to attenuate autoimmune uveitis through TGR5 signaling.

Gut microbiota-mediated secondary bile acids (BAs) play an important role in energy balance and host metabolism via G protein-coupled receptors and/or nuclear receptors. Emerging evidence suggests that BAs are important for maintaining innate immune responses via these receptors. However, the effect of BAs on autoimmune uveitis is still unknown. Here, we demonstrate decreased microbiota-related secondary BA concentration in feces and serum of animals with experimental autoimmune uveitis (EAU). Restoration of the gut BAs pool attenuates severity of EAU in association with inhibition of nuclear factor κB (NF-κB)-related pro-inflammatory cytokines in dendritic cells (DCs). TGR5 deficiency partially reverses the inhibitory effect of deoxycholic acid (DCA) on DCs. TGR5 signaling also inhibits NF-κB activation via the cyclic AMP (cAMP)-protein kinase A (PKA) pathway in DCs. Additionally, both DCA and TGR5 agonists inhibit human monocyte-derived DC activation. Taken together, our results suggest that BA metabolism plays an important role in adaptive immune responses and might be a therapeutic target in autoimmune uveitis.

Viscum album : from high dilutions to phytotherapy

Background : Besides conventional anticancer therapy, cancer patients use complementary and alternative medicine like the medicinal herb mistletoe (Viscum album L.), used in oral form or injections.
 For this condition Viscum album (VA) is also used in high dilutions, in injections, as in Brazil.
 
 Aim:
 Our aim is to compare results of basic research about these two presentations: herbal medicine and high dilutions and to look for an eventual continuity of action between the molecular form (herbal medicine) and the informative one (high dilutions)
 
 Method:
 About VA in herbal medicine : a lot of references in immunology, in particular those published by the team of Srini Kaveri and Stephan Baumgartner.(1) (2) (3) (4) (5)
 About VA in high dilutions : the work in immunology of Aloiso Cunha de Carvalho and Leoni Bonamin published in a thesis. (6)
 And then to compare those results with our practice as veterinarian on dogs and cats .
 
 Results : 
 VA in herbal medecine: from basic research results, we know that in low concentration, 
 an immunostimulary action is observed while in higher doses it is a cytotoxic activity.
 These two properties are found in our practical use of VA in dogs or cats : stabilization of the size of tumors for the low concentrations, necrosis of tumors and abscess for the high concentrations. 
 VA in high dilutions (from D3 to D30) : from basic research, an immunostimutating effect is observed. More exactly, on a murin model of tumor of Ehrlich, it is observed an improvement of quality of life, a reduction of the tumor immunosuppressive effect, an enlargement of local inflammations (Oedema) for all concentrations, but only 3D reduces tumor growth, without modifying local necrosis and the immunological parameters.
 In our practice, on two cases of osteosarcoma on dogs, we verify those properties using VA both in herbal medicine and in high dilutions. 
 
 Conclusions:
 From these results, both in basic research and in the practice, we should estimate there is probably a continuity of action fom VA, in High Dynamised Dilutions ( no more molecule), to VA in herbal medicine (molecular activity). From immunostimation to cytotoxicity, from information to molecular activity.
 
 Does each Viscum album’s preparation touch
 the same level of the Immune System ? The same cells ? The same receptors ? A lot of questions remain…
 
 
 References 
 
 
 1) Duong Van Huyen JP and al. Variable sensitivity of lymphoblastoid cells to apoptosis induced by Viscum album Qu FrF, a therapeutic preparation of mistletoe lectin. Chemotherapy. (2001)
 
 2) Duong Van Huyen JP and al. Induction of apoptosis of endothelial cells by Viscum album: a role for anti-tumoral properties of mistletoe lectins. Mol Med. (2002)
 
 3)Duong Van Huyen JP and al Comparative study of the sensitivity of lymphoblastoid and transformed monocytic cell lines to the cytotoxic effects of Viscum album extracts of different origin.. Chemotherapy. (2003)
 
 4) Estko M and al. Tumour cell derived effects on monocyte/macrophage polarization and function and modulatory potential of Viscum album lipophilic extract in vitro.
 BMC Complement Altern Med. 2015
 
 
 5) Saha C, and al Differential Effects of Viscum album Preparations on the Maturation and Activation of Human Dendritic Cells and CD4⁺ T Cell Responses.
 Molecules. 2016 Jul 14;21(7)
 
 6) Aloisio Cunha de Carvalho , ATIVIDADE ANTI-NEOPLÁSICA DE Viscum album (L) EM TUMORES EXPERIMENTAIS: REVISÃO CRÍTICA E ESTUDO EXPERIMENTAL EM TUMOR DE EHRLICH, Thesis Sao Paulo

Birch Pollen Induces Toll-Like Receptor 4-Dependent Dendritic Cell Activation Favoring T Cell Responses.

Seasonal exposure to birch pollen (BP) is a major cause of pollinosis. The specific role of Toll-like receptor 4 (TLR4) in BP-induced allergic inflammation and the identification of key factors in birch pollen extracts (BPE) initiating this process remain to be explored. This study aimed to examine (i) the importance of TLR4 for dendritic cell (DC) activation by BPE, (ii) the extent of the contribution of BPE-derived lipopolysaccharide (LPS) and other potential TLR4 adjuvant(s) in BPE, and (iii) the relevance of the TLR4-dependent activation of BPE-stimulated DCs in the initiation of an adaptive immune response. In vitro, activation of murine bone marrow-derived DCs (BMDCs) and human monocyte-derived DCs by BPE or the equivalent LPS (nLPS) was analyzed by flow cytometry. Polymyxin B (PMB), a TLR4 antagonist and TLR4-deficient BMDCs were used to investigate the TLR4 signaling in DC activation. The immunostimulatory activity of BPE was compared to protein-/lipid-depleted BPE-fractions. In co-cultures of BPE-pulsed BMDCs and Bet v 1-specific hybridoma T cells, the influence of the TLR4-dependent DC activation on T cell activation was analyzed. In vivo immunization of IL-4 reporter mice was conducted to study BPE-induced Th2 polarization upon PMB pre-treatment. Murine and human DC activation induced by either BPE or nLPS was inhibited by the TLR4 antagonist or by PMB, and abrogated in TLR4-deficient BMDCs compared to wild-type BMDCs. The lipid-free but not the protein-free fraction showed a reduced capacity to activate the TLR4 signaling and murine DCs. In human DCs, nLPS only partially reproduced the BPE-induced activation intensity. BPE-primed BMDCs efficiently stimulated T cell activation, which was repressed by the TLR4 antagonist or PMB, and the addition of nLPS to Bet v 1 did not reproduce the effect of BPE. In vivo, immunization with BPE induced a significant Th2 polarization, whereas administration of BPE pre-incubated with PMB showed a decreased tendency. These findings suggest that TLR4 is a major pathway by which BPE triggers DC activation that is involved in the initiation of adaptive immune responses. Further characterization of these BP-derived TLR4 adjuvants could provide new candidates for therapeutic strategies targeting specific mechanisms in BP-induced allergic inflammation.

A Dendritic Cells-Targeting Nano-Vaccine by Coupling Polylactic-Co-Glycolic Acid-Encapsulated Allergen with Mannan Induces Regulatory T Cells

Background: The efficacy of allergen-specific immunotherapy (AIT) is mainly depended on the tolerogenic immune responses elicited. Properly conjugated nano-vaccine has the advantages of both specific targeting and continuous and on-demand release of allergen. Objectives: The aim of this study is to investigate the effects of a dendritic cells (DCs)-targeting nano-vaccine for AIT. Methods: The nano-vaccine was produced by coupling polylactic-co-glycolic acid (PLGA)-encapsulated ovalbumin (OVA) with mannan. Allergen capture, human monocytes-derived DCs (hMoDCs) activation, and T cells responses were assessed by flow cytometry, confocal microscopy, quantitative real-time PCR, ELISA, and Cytometric Bead Array. Balb/c mice were immunized with the nano-vaccines, and the immune responses were analyzed. Results: OVA-PLGA nanoparticle (NP) displayed favorable safety profile. OVA-mannan-PLGA NP was captured more efficiently by hMoDCs than OVA-PLGA NP, which was mediated mainly through DC-specific intercellular adhesion molecule 3-grabbing nonintegrin. A tolerogenic phenotype of hMoDCs was induced by OVA-mannan-PLGA NP, but not OVA-PLGA NP, and increased number of regulatory T (Treg) cells was generated subsequently in in vitro coculture. Immunization of Balb/c mice with OVA-mannan-PLGA NP resulted in lower serum level of OVA-specific immunoglobulins and less production of pro-inflammatory cytokines in splenocytes culture than the mice immunized with OVA-PLAG NP, PLGA NP, or OVA, while the number of splenic Treg cells was higher in OVA-mannan-PLGA group than in other groups. Moreover, preimmunization with OVA-mannan-PLGA NP significantly inhibited the Th2 immune response induced by OVA sensitization. Conclusions: The biocompatible PLGA-encapsulated OVA coupling with mannan has augmented ability for tolerance induction and could be developed as a novel vaccine for AIT.

Constitutively Activated DAP12 Induces Functional Anti-Tumor Activation and Maturation of Human Monocyte-Derived DC.

Dendritic cells (DCs) are professional antigen presenting cells with a great capacity for cross-presentation of exogenous antigens from which robust anti-tumor immune responses ensue. However, this function is not always available and requires DCs to first be primed to induce their maturation. In particular, in the field of DC vaccine design, currently available methodologies have been limited in eliciting a sustained anti-tumor immune response. Mechanistically, part of the maturation response is influenced by the presence of stimulatory receptors relying on ITAM-containing activating adaptor molecules like DAP12, that modulates their function. We hypothesize that activating DAP12 in DC could force their maturation and enhance their potential anti-tumor activity for therapeutic intervention. For this purpose, we developed constitutively active DAP12 mutants that can promote activation of monocyte-derived DC. Here we demonstrate its ability to induce the maturation and activation of monocyte-derived DCs which enhances migration, and T cell stimulation in vitro using primary human cells. Moreover, constitutively active DAP12 stimulates a strong immune response in a murine melanoma model leading to a reduction of tumor burden. This provides proof-of-concept for investigating the pre-activation of antigen presenting cells to enhance the effectiveness of anti-tumor immunotherapies.

Cytotoxic activity of human dendritic cells induces RIPK1-dependent cell death

Dendritic cells (DCs), as potent phagocytes engulf dead cells and present peptide fragments of tumor antigens or pathogens derived from infected cells to naïve CD8+ T-lymphocytes. Dendritic cells can also induce apoptosis in target cells, thus getting an opportunity to sample their microenvironment. Here, we present that the supernatants of LPS- or CL075-activated DCs induced cell death in different cell lines, but during the differentiation to mature DCs, they lost their cytotoxic potential. Dexamethasone-pre-treated tolerogenic DCs induced less intensive death indicating that the tissue microenvironment can downregulate DC-mediated killing. Exploring the signaling of DC-induced cell death, we observed that the supernatant of activated DCs induced TNF-dependent cell death, since TNF antagonist blocked the cytotoxic activity of DCs, contrary to inhibitors of Fas and TRAIL receptors. We identified that the DC-induced killing is at least partially a RIPK1-dependent process, as RIPK1 positive target cells were more susceptible to DC-induced cell death than their RIPK1 deficient counterparts. Moreover, both the elevated phosphorylation of RIPK1 and the increase in RIPK1-caspase-8 interaction in target cells suggest that RIPK1-mediated signals contribute to DC supernatant-induced cell death. We also proved that the cytotoxic activity of DC-derived supernatant induced apoptosis in the target cells and not necroptosis, as it was completely abrogated with the pan caspase inhibitor (Z-VAD), while the necroptosis inhibitor (Nec-1) had no effect.Our work revealed that the supernatant of activated DCs induces the apoptosis of target cells in a RIPK1-dependent manner. This phenomenon could be relevant for the initiation of cross-presentation and may broaden the plethora of cytotoxic mechanisms acting against tumor cells.

The FcγRIIa-Syk Axis Controls Human Dendritic Cell Activation and T Cell Response Induced by Infliximab Aggregates.

The development of anti-drug Abs in response to biological products (BP) is a major drawback in the treatment of patients. Factors related to the patient, the treatment, and the product can influence BP immunogenicity. Among these factors, BP aggregates have been suggested to promote immunogenicity by acting as danger signals recognized by dendritic cells (DC) facilitating the establishment of an anti-BP CD4 T cell-dependent adaptive immune response leading to anti-drug Abs production. To date, little is known on the mechanism supporting the effect of aggregates on DCs and consequently on the T cell response. The aim of this work was to identify key signaling pathways involved in BP aggregate DC activation and T cell response. We generated aggregates by submitting infliximab (IFX), an immunogenic anti-TNF-α chimeric Ab, to heat stress. Our results showed that IFX aggregates were able to induce human monocyte-derived DC (moDC) maturation in a concentration-dependent manner. Aggregate-treated moDCs enhanced allogeneic T cell proliferation and IL-5, IL-9, and IL-13 production compared with native Ab-treated moDCs. We then investigated the implication of FcγRIIa and spleen tyrosine kinase (Syk) in DC activation and showed that they were both strongly implicated in moDC maturation induced by IFX aggregates. Indeed, we found that neutralization of FcγRIIa inhibited DC activation, and consequently, Syk inhibition led to a decrease in T cell proliferation and cytokine production in response to IFX aggregates. Taken together, our results bring new insight, to our knowledge, on how protein aggregates could induce DC and T cell activation via the FcγRIIa-Syk signaling pathway.

Phosphate-modif ied CpG oligonucleotides induce in vitro maturation of human myeloid dendritic cells.

Myeloid dendritic cells (DCs) play an important role in the immune response; therefore, the search for compounds that can effectively activate DCs is a needful goal. This study was aimed to investigate the effect of synthetic CpG oligodeoxynucleotides (CpG-ODN) on the maturation and allostimulatory activity of myeloid DCs in comparison with other PAMP and DAMP molecules. For the research, we synthesized known CpG-ODN class C (SD-101 and D-SL03) containing thiophosphate internucleotide groups, and their original phosphate-modified analogues (SD-101M and D- SL03M) with mesylphosphoramide internucleotide groups (M = μ-modification). The effects of CpG-ODN and other activators were evaluated on DCs generated from blood monocytes in the presence of GM-CSF and IFN-α (IFN-DC) or IL-4 (IL4-DC). Evaluation of the intracellular TLR-9 expression showed that both types of DCs (IFN-DC and IL4-DC) contained on average 52 and 80 % of TLR-9-positive cells, respectively. The CpG-ODNs studied enhanced the allostimulatory activity of IFN-DCs, and the effect of μ-modified CpG-ODNs was higher than that of CpG-ODNs with thiophosphate groups. The stimulating effect of CpG-ODN at a dose of 1.0 μg/ml was comparable (for D-SL03, D-SL03M, SD-101) with or exceeded (for SD-101M) the effect of LPS at a dose of 10 μg/ml. At the same time, IFN-DCs were characterized by greater sensitivity to the action of CpG-ODNs than IL4-DCs. The enhancement of DC allostimulatory activity in the presence of CpG-ODNs was associated with the induction of final DC maturation, which was confirmed by a significant decrease in the number of CD14+DC, an increase in mature CD83+DC and a trend towards an increase in CD86+DC. Interestingly, the characteristic ability of LPS to enhance the expression of the co-stimulatory molecule OX40L on DCs was revealed only for the μ-analogue SD-101M. In addition, CpG-ODNs (SD-101 and SD-101M) had a stimulatory effect on IFN-γ production comparable to the action of LPS. The data obtained indicate a stimulating effect of CpG-ODN on the maturation and allostimulatory activity of human myeloid DCs, which is more pronounced for μ-modified analogs.

Regulation of RLR-Mediated Antiviral Responses of Human Dendritic Cells by mTOR.

To detect replicating viruses, dendritic cells (DCs) utilize cytoplasmic retinoic acid inducible gene-(RIG) I-like receptors (RLRs), which play an essential role in the subsequent activation of antiviral immune responses. In this study, we aimed to explore the role of the mammalian target of rapamycin (mTOR) in the regulation of RLR-triggered effector functions of human monocyte-derived DCs (moDCs) and plasmacytoid DCs (pDCs). Our results show that RLR stimulation increased the phosphorylation of the mTOR complex (mTORC) 1 and mTORC2 downstream targets p70S6 kinase and Akt, respectively, and this process was prevented by the mTORC1 inhibitor rapamycin as well as the dual mTORC1/C2 kinase inhibitor AZD8055 in both DC subtypes. Furthermore, inhibition of mTOR in moDCs impaired the RLR stimulation-triggered glycolytic switch, which was reflected by the inhibition of lactate production and downregulation of key glycolytic genes. Blockade of mTOR diminished the ability of RLR-stimulated moDCs and pDCs to secret type I interferons (IFNs) and pro-inflammatory cytokines, while it did not affect the phenotype of DCs. We also found that mTOR blockade decreased the phosphorylation of Tank-binding kinase 1 (TBK1), which mediates RLR-driven cytokine production. In addition, rapamycin abrogated the ability of both DC subtypes to promote the proliferation and differentiation of IFN-y and Granzyme B producing CD8 + T cells. Interestingly, AZD8055 was much weaker in its ability to decrease the T cell proliferation capacity of DCs and was unable to inhibit the DC-triggered production of IFN-y and Granyzme B by CD8 + T cells. Here we demonstrated for the first time that mTOR positively regulates the RLR-mediated antiviral activity of human DCs. Further, we show that only selective inhibition of mTORC1 but not dual mTORC1/C2 blockade suppresses effectively the T cell stimulatory capacity of DCs that should be considered in the development of new generation mTOR inhibitors and in the improvement of DC-based vaccines.

Profiling of Human Circulating Dendritic Cells and Monocyte Subsets Discriminates Between Type and Mucosal Status in Patients With Inflammatory Bowel Disease.

Intestinal dendritic cells (DC) and macrophages drive disease progression in patients with inflammatory bowel disease (IBD). We aimed to characterize the activation and homing profile of human circulating DC and monocyte subsets in healthy control patients (CP) and IBD patients. Eighteen CP and 64 patients with IBD were categorized by diagnoses of Crohn disease (CD) and ulcerative colitis (UC), either endoscopically active (inflamed) or quiescent. Circulating type 1 conventional DC, type 2 conventional DC, plasmacytoid DC, classical monocytes, nonclassical monocytes, and intermediate monocytes were identified by flow cytometry in each individual and characterized for the expression of 18 markers. Association between DC/monocytes and IBD risk was tested by logistic regression. Discriminant canonical analyses were performed to classify the patients in their own endoscopy category considering all markers on each subset. CCRL1, CCR3, and CCR5 expression on circulating type 1 DC; CCRL1 expression on nonclassical monocytes; and CCR9 and β7 expression on classical monocytes allowed us to discriminate among the different study groups. Indeed, the same markers (excluding β7) were also associated with IBD when all DC and monocyte subsets were considered at the same time. Monitoring the phenotype of human circulating DC and monocyte subsets may provide novel tools as biomarkers for disease diagnosis (CD/UC) or mucosal status (inflamed/noninflamed) in the absence of an invasive colonoscopy.

A Structurally Simple Vaccine Candidate Reduces Progression and Dissemination of Triple-Negative Breast Cancer.

SummaryThe Tn antigen is a well-known tumor-associated carbohydrate determinant, often incorporated in glycopeptides to develop cancer vaccines. Herein, four copies of a conformationally constrained mimetic of the antigen TnThr (GalNAc-Thr) were conjugated to the adjuvant CRM197, a protein licensed for human use. The resulting vaccine candidate, mime[4]CRM elicited a robust immune response in a triple-negative breast cancer mouse model, correlated with high frequency of CD4+ T cells and low frequency of M2-type macrophages, which reduces tumor progression and lung metastasis growth. Mime[4]CRM-mediated activation of human dendritic cells is reported, and the proliferation of mime[4]CRM-specific T cells, in cancer tissue and peripheral blood of patients with breast cancer, is demonstrated. The locked conformation of the TnThr mimetic and a proper presentation on the surface of CRM197 may explain the binding of the conjugate to the anti-Tn antibody Tn218 and its efficacy to fight cancer cells in mice.