Human Monocyte-derived Immature Dendritic Cells Research Articles

Abstract 1363: Blocking inhibitory receptor ILT3 by an antibody with enhanced Fc function promoted adaptive immunity against hematopoietic malignancy independent of ADCC

Abstract Immunoglobulin-like transcript 3 (ILT3) is an inhibitory receptor that is expressed by mature monocytes, dendritic cells (DC), plasma blasts, and the malignant cells arising thereof including acute myeloid leukemia (AML), B cell lymphoma and multiple myeloma (MM). We developed an anti-ILT3 blocking monoclonal antibody (mAb) 17G8 to test the hypothesis that blockade of ILT3 on hematopoietic malignant cells might drive anti-cancer responses through several mechanisms such as cell migration, ADCC, and T cell driven immune responses. Blockade of ILT3 using 17G8 without Fc effector function impaired AML cell line THP1 dissemination into distant sites in vivo and using 17G8 with enhanced Fc function through afucosylation further reduced the tumor burden. This effect of afucosylated 17G8 appears to be mediated by a mechanism other than ADCC, since afucosylated 17G8 showed modest tumor inhibition in ICR mice carrying ILT3 positive AML and MM xenografts. In immune competent mice carrying murine syngeneic AML cell C1498 expressing human ILT3, 17G8 also showed modest anti-tumor effects as a single agent, however, when combined with anti-PD1, afucosylated 17G8 increased CD8+ effector memory T cells, reduced proportion of cells with an exhausted phenotype and significantly improved survival when compared to different controls. Since T cells don’t express ILT3, the effects of 17G8 on T cells are hypothesized to be mediated by blocking ILT3 expressed on myeloid cells. The expression of ILT3 on immature human monocyte-derived DC (moDC) is higher than on mature moDC. Blocking ILT3 with afucosylated 17G8 on immature moDC potentiated maturation of moDC when compared to the controls in vitro. Surprisingly, afucosylated 17G8 stimulated IFNγ production from moDC during maturation. The enhanced DC function induced by afucosylated 17G8 was subsequently found to be translated into enhanced T cell function in an allogeneic mixed lymphocyte reaction (allo-MLR) assay, in which afucosylated 17G8 significantly increased IFNγ production when combined with anti-PD1. The activation of effector T cells was also monitored through a cytotoxicity assay by mixing THP-1 cells with human PBMC in culture. Afucosylated 17G8 induced strong cytotoxicity on THP-1 cells with minimal effects on normal ILT3 positive cells such as monocytes or moDC, suggesting that blockade of ILT3 with afucosylated 17G8 induced more potent T cell mediated allogeneic cytotoxicity than innate cell mediated ADCC killing. In summary, ILT3 is a highly selective M4/M5 AML target. Developing a blocking antibody with enhanced Fc function could add an additional therapeutic strategy of inducing host anti-tumor immunity to current AML treatments which induce direct tumor killing, such as chemotherapy, targeted therapy, and antibody drug conjugate. Citation Format: Shiming Ye, Dong Zhang, Min-Zu Wu, Harini Raghu, Diane Cohen, Mally Romero, Siu-Sze Tan, Donghee Choi, Jonathan Hickson, John Engelhardt, Alex Shoemaker. Blocking inhibitory receptor ILT3 by an antibody with enhanced Fc function promoted adaptive immunity against hematopoietic malignancy independent of ADCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1363.

Podocalyxin Expressed in Antigen Presenting Cells Promotes Interaction With T Cells and Alters Centrosome Translocation to the Contact Site.

Podocalyxin (PODXL), a cell surface sialomucin expressed in diverse types of normal and malignant cells, mediates cellular adhesion to extracellular matrix and cell-to-cell interaction. A previous study reported the expression of PODXL protein on monocytes undergoing macrophage differentiation, yet the expression of this molecule in other antigen presenting cells (APCs) and its function in the immune system still remain undetermined. In this study, we report that PODXL is expressed in human monocyte-derived immature dendritic cells at both the mRNA and protein levels. Following dendritric cells maturation using pro-inflammatory stimuli, PODXL expression level decreased substantially. Furthermore, we found that PODXL expression is positively regulated by IL-4 through MEK/ERK and JAK3/STAT6 signaling pathways. Our results revealed a polarized distribution of PODXL during the interaction of APCs with CD4+ T cells, partially colocalizing with F-actin. Notably, PODXL overexpression in APCs promoted their interaction with CD4+ T cells and CD8+ T cells and decreased the expression of MHC-I, MHC-II, and the costimulatory molecule CD86. In addition, PODXL reduced the translocation of CD4+ T-cell centrosome toward the APC-contact site. These findings suggest a regulatory role for PODXL expressed by APCs in immune responses, thus representing a potential target for therapeutic blockade in infection and cancer.

Antiviral effects of deoxynojirimycin (DNJ)-based iminosugars in dengue virus-infected primary dendritic cells

Dendritic cells (DCs) are important targets for dengue virus (DENV) infection and play a significant role in the early immune response. Antiviral effects of iminosugars against DENV in primary cells have been demonstrated previously in monocyte-derived macrophages (MDMΦs). Given the important role played by DCs in innate immune defense against DENV, the antiviral effects of three deoxynojirimycin (DNJ) derivatives (NN-DNJ, EOO-DNJ and 2THO-DNJ) and a deoxygalactonojirimycin (DGJ) negative control were evaluated in DENV-infected primary human monocyte-derived immature DCs (imDCs). DNJ- but not DGJ-derivatives elicited antiviral activity in DENV-infected imDCs, similar to that observed in MDMΦs. The DNJ-derivatives inhibited DENV secretion in a dose-dependent manner. Endoplasmic reticulum (ER) α-glucosidase I inhibition by DNJ-derived iminosugars, at concentrations of 3.16 μM, correlated with a reduction in the specific infectivity of virions that were still secreted, as well as a reduction in DENV-induced tumour necrosis factor alpha secretion. This suggests iminosugar-mediated ER α-glucosidase I inhibition may give rise to further benefits during DENV infection, beyond the reduction in viral secretion associated with ER α-glucosidase II inhibition.

PEGylated single-walled carbon nanotubes as co-adjuvants enhance expression of maturation markers in monocyte-derived dendritic cells.

Aim: This study investigated the application of phospholipid-PEGylated single-walled carbon nanotubes (PL-PEG-SWCNTs) as a safe co-adjuvant for the commercial recombinant hepatitis B virus vaccine to enhance induction of monocyte-derived dendritic cells (MDDCs) differentiation and activation in vitro as an immune response initiator cell to prompt a long-term immune response after a single dose injection. Methods: Immature MDDCs were exposed to PL-PEG-SWCNTs alone and in combination with hepatitis B vaccine. Results & conclusion: Study results confirm the enhanced expression of maturation markers in human immature MDDCs after PL-PEG-SWCNT exposure. The results suggest that PL-PEG-SWCNT is an efficient co-adjuvant for the commercial recombinant hepatitis B virus vaccine to enhance dendritic cell response stimulation in vitro.

Human monocyte-derived dendritic cells as an in vitro alternative model cell to evaluate the immunotoxicity of 2, 4-Dinitrochlorobenzene

Improvements in science and technology have led to the increasing threats of new chemicals to the public health. It is crucial to evaluate the toxicity, especially immunotoxicology. Dendritic cells (DCs) are believed to be more favorable choices in immunotoxicity evaluations. To obtain and evaluate the value of human monocyte-derived immature DCs (imDCs) in vitro applications in immunotoxicology, compared the results in vitro. DCs were obtained from enriched leukocytes of peripheral blood by using magnetic cell sorting and cytokine (rhGM-CSF + rhIL-4) co-induction. imDCs function in vitro and the surface antigens changes both in imDCs and THP-1 after 24 h of 2,4-dinitrochlorobenzene (DNCB) exposure were determined. The results were compared with those of DNCB-induced rats. The feasibility of imDCs applications in immunotoxicology was evaluated. In vivo, the splenic nodules, lymphocytes, and CD103+DC surface antigen expression were altered in the spleen of DNCB-induced rats. Moreover, DNCB exposure increased CD8+ T cell numbers both in peripheral blood and in the spleen of DNCB-induced rats. In vitro, DNCB exposure reduced the antigen uptake capacity and enhanced the T cell proliferative capacity of imDCs. The results are consistent with in vivo, but superior to that of the THP-1. Our results suggest that human monocyte-derived DCs may have potential applications as an attractive in vitro alternative cell model to evaluate the sensitization of DNCB.

A Functional Assay to Determine the Capacity of Oncolytic Viruses to Induce Immunogenic Tumor Cell Death.

Oncolytic immunotherapy efficacy relies partially on the induction of immunogenic tumor cell death following infection with oncolytic viruses (OV) to induce an antitumor immune response. Here, we describe a method to determine if an OV is able to induce such an immunogenic tumor cell death. This method consists in testing whether tumor cells lysed by an OV are able to induce the maturation of human monocyte-derived immature dendritic cells (Mo-iDC).

Vector uncoating limits adeno-associated viral vector-mediated transduction of human dendritic cells and vector immunogenicity

AAV vectors poorly transduce Dendritic cells (DC), a feature invoked to explain AAV’s low immunogenicity. However, the reason for this non-permissiveness remained elusive. Here, we performed an in-depth analysis using human monocyte-derived immature DC (iDC) as model. iDC internalized AAV vectors of various serotypes, but even the most efficient serotype failed to transduce iDC above background. Since AAV vectors reached the cell nucleus, we hypothesized that AAV’s intracellular processing occurs suboptimal. On this basis, we screened an AAV peptide display library for capsid variants more suitable for DC transduction and identified the I/VSS family which transduced DC with efficiencies of up to 38%. This property correlated with an improved vector uncoating. To determine the consequence of this novel feature for AAV’s in vivo performance, we engineered one of the lead candidates to express a cytoplasmic form of ovalbumin, a highly immunogenic model antigen, and assayed transduction efficiency as well as immunogenicity. The capsid variant clearly outperformed the parental serotype in muscle transduction and in inducing antigen-specific humoral and T cell responses as well as anti-capsid CD8+ T cells. Hence, vector uncoating represents a major barrier hampering AAV vector-mediated transduction of DC and impacts on its use as vaccine platform.

The Vacuolar Pathway of Long Peptide Cross-Presentation Can Be TAP Dependent.

The intracellular pathway of cross-presentation, which allows MHC class I-restricted presentation of peptides derived from exogenous Ags, remains poorly defined and may vary with the nature of the exogenous Ag and the type of APC. It can be cytosolic, characterized by proteasome and TAP dependency, or vacuolar, usually believed to be proteasome and TAP independent. Cross-presentation is particularly effective with long synthetic peptides, and we previously reported that the HLA-A2-restricted cross-presentation of a long peptide derived from melanoma Ag gp100 by human monocyte-derived immature dendritic cells occurred in a vacuolar pathway, making use of newly synthesized HLA-A2 molecules that follow a nonclassical secretion route. In this article, we show that the HLA-A1-restricted cross-presentation of a long peptide derived from tumor Ag MAGE-A3 by human monocyte-derived immature dendritic cells also follows a vacuolar pathway. However, as opposed to the HLA-A2-restricted peptide, cross-presentation of the HLA-A1-restricted peptide is TAP dependent. We show that this paradoxical TAP-dependency is indirect and reflects the need for TAP to load HLA-A1 molecules with peptides in the endoplasmic reticulum, to allow them to escape the endoplasmic reticulum and reach the vacuole, where peptide exchange with the cross-presented peptide likely occurs. Our results confirm and extend the involvement of the vacuolar pathway in the cross-presentation of long peptides, and indicate that TAP-dependency can no longer be used as a key criterion to distinguish the cytosolic from the vacuolar pathway of cross-presentation. They also stress the existence of an alternative secretory route for MHC class I, which will be worthy of further studies.

Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response.

The human IL22RA2 gene co-produces three protein isoforms in dendritic cells [IL-22 binding protein isoform-1 (IL-22BPi1), IL-22BPi2, and IL-22BPi3]. Two of these, IL-22BPi2 and IL-22BPi3, are capable of neutralizing the biological activity of IL-22. The function of IL-22BPi1, which differs from IL-22BPi2 through an in-frame 32-amino acid insertion provided by an alternatively spliced exon, remains unknown. Using transfected human cell lines, we demonstrate that IL-22BPi1 is secreted detectably, but at much lower levels than IL-22BPi2, and unlike IL-22BPi2 and IL-22BPi3, is largely retained in the endoplasmic reticulum (ER). As opposed to IL-22BPi2 and IL-22BPi3, IL-22BPi1 is incapable of neutralizing or binding to IL-22 measured in bioassay or assembly-induced IL-22 co-folding assay. We performed interactome analysis to disclose the mechanism underlying the poor secretion of IL-22BPi1 and identified GRP78, GRP94, GRP170, and calnexin as main interactors. Structure-function analysis revealed that, like IL-22BPi2, IL-22BPi1 binds to the substrate-binding domain of GRP78 as well as to the middle domain of GRP94. Ectopic expression of wild-type GRP78 enhanced, and ATPase-defective GRP94 mutant decreased, secretion of both IL-22BPi1 and IL-22BPi2, while neither of both affected IL-22BPi3 secretion. Thus, IL-22BPi1 and IL-22BPi2 are bona fide clients of the ER chaperones GRP78 and GRP94. However, only IL-22BPi1 activates an unfolded protein response (UPR) resulting in increased protein levels of GRP78 and GRP94. Cloning of the IL22RA2 alternatively spliced exon into an unrelated cytokine, IL-2, bestowed similar characteristics on the resulting protein. We also found that CD14++/CD16+ intermediate monocytes produced a higher level of IL22RA2 mRNA than classical and non-classical monocytes, but this difference disappeared in immature dendritic cells (moDC) derived thereof. Upon silencing of IL22RA2 expression in moDC, GRP78 levels were significantly reduced, suggesting that native IL22RA2 expression naturally contributes to upregulating GRP78 levels in these cells. The IL22RA2 alternatively spliced exon was reported to be recruited through a single mutation in the proto-splice site of a Long Terminal Repeat retrotransposon sequence in the ape lineage. Our work suggests that positive selection of IL-22BPi1 was not driven by IL-22 antagonism as in the case of IL-22BPi2 and IL-22BPi3, but by capacity for induction of an UPR response.

The Bacterial Toxin CNF1 Induces Activation and Maturation of Human Monocyte-Derived Dendritic Cells.

Cytotoxic necrotizing factor 1 (CNF1) is a bacterial protein toxin primarily expressed by pathogenic Escherichia coli strains, causing extraintestinal infections. The toxin is believed to enhance the invasiveness of E. coli by modulating the activity of Rho GTPases in host cells, but it has interestingly also been shown to promote inflammation, stimulate host immunity and function as a potent immunoadjuvant. The mechanisms underlying the immunostimulatory properties of CNF1 are, however, poorly characterized, and little is known about the direct effects of the toxin on immune cells. Here, we show that CNF1 induces expression of maturation markers on human immature monocyte-derived dendritic cells (moDCs) without compromising cell viability. Consistent with the phenotypic maturation, CNF1 further triggered secretion of proinflammatory cytokines and increased the capacity of moDCs to stimulate proliferation of allogenic naïve CD4+ T cells. A catalytically inactive form of the toxin did not induce moDC maturation, indicating that the enzymatic activity of CNF1 triggers immature moDCs to undergo phenotypic and functional maturation. As the maturation of dendritic cells plays a central role in initiating inflammation and activating the adaptive immune response, the present findings shed new light on the immunostimulatory properties of CNF1 and may explain why the toxin functions as an immunoadjuvant.

Cytotoxicity and Intracellular Uptake of Fluorescent BSA-AuNCs from Human Monocyte-Derived Immature Dendritic Cells

Cytotoxicity and Intracellular Uptake of Fluorescent BSA-AuNCs from Human Monocyte-Derived Immature Dendritic Cells

Valproic Acid and Lithium Meditate Anti-Inflammatory Effects by Differentially Modulating Dendritic Cell Differentiation and Function.

Valproic acid (VPA), with inhibition activity mainly toward histone deacetylase (HDAC) and Glycogen Synthase Kinase (GSK)-3, and lithium, with inhibition activity mainly toward GSK-3, are both prescribed in clinical as mood-stabilizers and anticonvulsants for the control of bipolar disorder. This study aims to compare the immuno-modulation activities of VPA and lithium, especially on the differentiation and functions of dendritic cells (DC). Our data show that treatment with VPA or lithium effectively alleviated the severity of collagen-induced arthritis triggered by LPS in mice. Both agents reduced the serum level of IL-6 and IL-10 after LPS challenge in mice. VPA and lithium both induce significant down-regulation of group I CD1 expression and secretion of IL-6 during differentiation of human monocyte-derived immature DC, while they differ in the induction of CD83 and CD86 expression, secretion of IL-8, IL-10, and TNF-α. Upon stimulation of immature DC with LPS, VPA, and lithium both reduced the secretion of IL-6 and TNF-α. However, only lithium significantly increased the production of IL-10, while VPA increased the production of IL-8 but substantially reduce the secretion of IL-10 and IL-23. Treatment with VPA resulted in a reduced capacity of LPS-stimulated DC to promote the differentiation of T helper 17 cells that are critical in the promotion of inflammatory responses. Taken together, our results suggest that VPA and lithium may differentially modulate inflammation through regulating the capacity of DC to mediate distinct T cell responses, and they may provide a complementary immunomodulatory effects for the treatment of inflammation-related diseases. J. Cell. Physiol. 232: 1176-1186, 2017. © 2016 Wiley Periodicals, Inc.

Distinct expression of interferon-induced protein with tetratricopeptide repeats (IFIT) 1/2/3 and other antiviral genes between subsets of dendritic cells induced by dengue virus 2 infection.

Dengue virus (DENV) infection is an emerging public health hazard threatening inhabitants of the tropics and sub-tropics. Dendritic cells (DCs) are one of the major targets of DENV and the initiators of the innate immune response against the virus. However, current invitro research on the DENV-DC interaction is hampered by the low availability of exvivo DCs and donor variation. In the current study, we attempted to develop a novel invitro DC model using immature DCs derived from the myeloid leukaemia cell line MUTZ-3 (IMDCs) to investigate the DENV-DC interaction. The IMDCs morphologically and phenotypically resembled human immature monocyte-derived dendritic cells (IMMoDCs). However, the permissiveness of IMDCs to DENV2 was lower than that of IMMoDCs. RT-PCR arrays showed that a group of type I interferon (IFN) -inducible genes, especially IFIT1, IFITM1, and IFI27, were significantly up-regulated in IMMoDCs but not in IMDCs after DENV2 infection. Further investigation revealed that IFIT genes were spontaneously expressed at both transcriptional and protein levels in the naive IMDCs but not in the naive IMMoDCs. It is possible that the poor permissiveness of IMDCs to DENV2 was a result of the high basal levels of IFIT proteins. We conclude that the IMDC model, although less permissive to DENV2, is a useful platform for studying the suppression mechanism of DENV2 and we expand the knowledge of cellular factors that modulate DENV2 infection in the human body.

Actin Dynamics Regulates Dendritic Cell-Mediated Transfer of HIV-1 to T Cells

Whereas human dendritic cells (DCs) are largely resistant to productive infection with HIV-1, they have a unique ability to take up the virus and transmit it efficiently to T lymphocytes through a process of trans-infection or trans-enhancement. To elucidate the molecular and cell biological mechanism for trans-enhancement, we performed an shRNA screen of several hundred genes involved in organelle and membrane trafficking in immature human monocyte-derived dendritic cells (MDDCs). We identified TSPAN7 and DNM2, which control actin nucleation and stabilization, as having important and distinct roles in limiting HIV-1 endocytosis and in maintaining virus particles on dendrites, which is required for efficient transfer to T lymphocytes. Further characterization of this process may provide insights not only into the role of DCs in transmission and dissemination of HIV-1 but also more broadly into mechanisms controlling capture and internalization of pathogens.

PLGA microparticle combination therapy induces a tolerogenic phenotype in human monocyte-derived dendritic cells

Event Abstract Back to Event PLGA microparticle combination therapy induces a tolerogenic phenotype in human monocyte-derived dendritic cells Maigan Hulme1, Joshua Stewart1, Jamal Lewis2 and Benjamin Keselowsky1 1 University of Florida, Biomedical Engineering, United States 2 University of California, Biomedical Engineering, United States Introduction: Type 1 diabetes (T1D) is an autoimmune disease characterized by the loss of immune tolerance to insulin-producing pancreatic beta cells, followed by their T cell-mediated destruction. A major therapeutic goal is the induction of lasting T cell tolerance to beta cell antigens without global immunosuppression. Many immunomodulatory agents have been tested in both prevention and reversal of disease in preclinical studies and human clinical trials, yet report no durable efficacy. We developed a novel poly(lactic-co-glycolic) acid (PLGA) based microparticle (MP) formulation for the simultaneous delivery of multiple agents, tailored to induce a tolerogenic phenotype in dendritic cells (DC). This system employs large (~30uM diameter) MPs for sustained local controlled release, or small (~1 uM diameter) MPs that are readily engulfed by phagocytic cells. In studies performed in the non-obese diabetic (NOD) mouse, successful prevention of T1D was achieved with a quaternary MP therapy delivered subcutaneously: large MPs encapsulating TGF-β1 and GM-CSF, and small MPs encapsulating Vitamin D3 and denatured insulin[1]. Features of this prevention included maturation-resistance in dendritic cells and the induction of regulatory T cell populations. Following these encouraging results, we began investigation of the effects of this formulation on human cells in vitro. Methods: Human monocyte-derived immature DCs (moDC) were generated from healthy subjects in the presence of GM-CSF and IL-4 for 7-9 days. Immature DCs were then incubated for 48 hours alone, with TGF-β1 MPs (1 mg/105 DCs) and Vitamin D3 MPs (10 MPs/DC), or with relevant unloaded MP controls at equivalent ratios. DCs were then treated with LPS (1 ug/ml) for 24 hours to assess maturation, or co-cultured with allogeneic dye-labeled T cells at a DC:T cell ratio of 1:4 to assess T cell proliferation in response to alloantigen. Results and Discussion: moDCs treated with factor-loaded MPs failed to upregulate HLA-DR and the costimulatory molecules CD40, CD80, and CD86 in response to LPS, indicative of maturation resistance (Fig. 1). Additionally, DCs exhibited increased expression of the negative regulators PD-L1 and Galectin 9 (Fig. 2). Treated moDCs also suppressed alloantigen-induced T cell proliferation in both memory and naïve populations (Fig. 3). Conclusions: Characterization of human dendritic cell responses to this formulation in vitro is important to clinical translation. These results suggest similar mechanism of action in mouse preclinical studies and human cells in vitro, and provide support for clinical translation for immunotherapeutic application of this vaccine formulation.

Abstract 297: Immunotherapy with a CD40L/4-1BBL double-armed oncolytic adenovirus drives Th1 immunity and control tumor progression in a pancreas cancer model

Abstract We hypothesized that an immunostimulatory oncolytic virus (LOAd703) is a potent inducer of anti-tumor immunity and tested its biological functions in models of pancreas cancer. LOAd703 is a double-armed oncolytic adenovirus that introduces the simultaneous expression of a trimerized membrane-bound CD40L and 4-1BBL locally in the tumor aiming to induce potent Th1-mediated anti-tumor immunity. By utilizing an oncolytic adenovirus serotype 5/35 for gene transfer, virally induced tumor cell oncolysis will provide a broad release of antigens at the site of immune activation. Transgene expression is driven by a separate promoter to allow for efficient expression in both tumor cells and tumor stroma while virus replication is restricted to tumor cells by E1A delta24 deletion. CD40 ligand is a potent stimulator of myeloid cells including dendritic cells (DCs) that in turn induce robust T cell responses. CD40L can also reduce the levels of myeloid suppressor cells and M2 macrophages as well as enhancing T cell infiltration into tumors. Further, CD40-mediated signaling combined with TLR stimuli (such as adenovirus) induces intense Th1 immunity. 4-1BBL is known to provide expansion and survival signaling to pre-activated T- and NK cells. Pancreatic cancer cell lines and healthy exocrine pancreas cells were transduced with LOAd703 and it efficiently killed tumor cells while normal cells remained intact as evaluated by an MTS assay. CD40L and 4-1BBL were highly expressed by the transduced tumor cells as detected by flow cytometry. Cell lysis by LOAd703 was equally efficient as a similar oncolytic virus without transgenes demonstrating that the double transgene expression did not interfere with viral replication. Repeated (6x) peritumoral injections of LOAd703 in a xenograft Nu/Nu/Panc01 model showed efficient tumor cell growth control and sustained complete responses. The effect could be further enhanced by gemcitabine in both xenograft mice and in a syngeneic immunocompetent C57BL/6/Panc02 model. Transduction of human monocyte-derived immature DCs resulted in a strong CD40L and 4-1BBL expression without lysis of the cells. Instead, the transduced DCs matured as shown by high CD83 and IL12 expression. The addition of 4-1BBL significantly enhanced DC maturation compared to a virus containing only CD40L, or no transgenes, by expressing higher levels of CD70, IL12, TNFa, IFNg and IL21. Further, LOAd703-transduced DCs pulsed with pp65 CMV peptides potently expanded antigen-specific T cells as well as NK cells. In conclusion, LOAd703 is a novel, double-armed immunostimulatory oncolytic gene therapy that initiates robust Th1 immunity, and eradicates pancreatic cancer in experimental models. A clinical trial using LOAd703 for pancreatic cancer is underway. Citation Format: Emma Svensson, Ioanna Milenova, Rafael Moreno, Ramon Alemany, Angelica Loskog. Immunotherapy with a CD40L/4-1BBL double-armed oncolytic adenovirus drives Th1 immunity and control tumor progression in a pancreas cancer model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 297. doi:10.1158/1538-7445.AM2015-297

The association of complement protein C1q with apoptotic cells ingested by human dendritic cells modulates dendritic cell-mediated T cell activation (INC5P.332)

Abstract Humans deficient in complement protein C1q develop systemic lupus erythematosus (SLE) with nearly 100% penetrance, but the molecular mechanisms underlying this association have not yet been uncovered. Our previous studies have shown that human monocyte-derived immature dendritic cells (DC) ingesting C1q-bound autologous apoptotic cells (AC) (C1q-polarized DC) increase secretion of anti-inflammatory cytokines such as IL-10 and reduce pro-inflammatory cytokines such as TNFα. In this study, we demonstrate that C1q-polarized DC express significantly more surface PD-L2 and CD39 and significantly less CD86 relative to DC ingesting AC alone. To elucidate whether C1q-polarized DC functionally modulate the adaptive immune responses, human autologous T cells were incubated with DC or C1q-polarized DC and the proliferation and induction of CD4+ and CD8+ T cell subset was assessed. We discovered that relative to DC ingesting AC in the absence of C1q, C1q-polarized DC decrease autologous Th17 proliferation and induction and Th1 proliferation when the T cells were maintained in IL-7 prior to the MLR. These data imply that C1q-polarized DC can switch T cell function to reduce T effector cell activation and/or suppress the induction of autoimmunity, identifying new potential pathways for therapeutic intervention in SLE and other inflammatory diseases.

Neuropeptide Y induces potent migration of human immature dendritic cells and promotes a T h 2 polarization

Neuropeptide Y (NPY), a major autonomic nervous system and stress mediator, is emerging as an important regulator of inflammation, implicated in autoimmunity, asthma, atherosclerosis, and cancer. Yet the role of NPY in regulating phenotype and functions of dendritic cells (DCs), the professional antigen-presenting cells, remains undefined. Here we investigated whether NPY could induce DCs to migrate, mature, and polarize naive T lymphocytes. We found that NPY induced a dose-dependent migration of human monocyte-derived immature DCs through the engagement of NPY Y1 receptor and the activation of ERK and p38 mitogen-activated protein kinases. NPY promoted DC adhesion to endothelial cells and transendothelial migration. It failed to induce phenotypic DC maturation, whereas it conferred a T helper 2 (Th2) polarizing profile to DCs through the up-regulation of interleukin (IL)-6 and IL-10 production. Thus, during an immune/inflammatory response NPY may exert proinflammatory effects through the recruitment of immature DCs, but it may exert antiinflammatory effects by promoting a Th2 polarization. Locally, at inflammatory sites, cell recruitment could be amplified in conditions of intense acute, chronic, or cold stress. Thus, altered or amplified signaling through the NPY-NPY-Y1 receptor-DC axis may have implications for the development of inflammatory conditions.-Buttari, B., Profumo, E., Domenici, G., Tagliani, A., Ippoliti, F., Bonini, S., Businaro, R., Elenkov, I., Riganò, R. Neuropeptide Y induces potent migration of human immature dendritic cells and promotes a Th2 polarization.

Role of DC-SIGN in Lassa virus entry into human dendritic cells.

The arenavirus Lassa virus (LASV) causes a severe hemorrhagic fever with high mortality in humans. Antigen-presenting cells, in particular dendritic cells (DCs), are early and preferred targets of LASV, and their productive infection contributes to the virus-induced immunosuppression observed in fatal disease. Here, we characterized the role of the C-type lectin DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) in LASV entry into primary human DCs using a chimera of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) expressing the LASV glycoprotein (rLCMV-LASVGP). We found that differentiation of human primary monocytes into DCs enhanced virus attachment and entry, concomitant with the upregulation of DC-SIGN. LASV and rLCMV-LASVGP bound to DC-SIGN via mannose sugars located on the N-terminal GP1 subunit of LASVGP. We provide evidence that DC-SIGN serves as an attachment factor for rLCMV-LASVGP in monocyte-derived immature dendritic cells (MDDC) and can accelerate the capture of free virus. However, in contrast to the phlebovirus Uukuniemi virus (UUKV), which uses DC-SIGN as an authentic entry receptor, productive infection with rLCMV-LASVGP was less dependent on DC-SIGN. In contrast to the DC-SIGN-mediated cell entry of UUKV, entry of rLCMV-LASVGP in MDDC was remarkably slow and depended on actin, indicating the use of different endocytotic pathways. In sum, our data reveal that DC-SIGN can facilitate cell entry of LASV in human MDDC but that its role seems distinct from the function as an authentic entry receptor reported for phleboviruses.

Aryl hydrocarbon receptor contributes to the MEK/ERK-dependent maintenance of the immature state of human dendritic cells

AbstractDendritic cells (DCs) promote tolerance or immunity depending on their maturation state, which is enhanced or accelerated upon MEK-ERK signaling pathway inhibition. We have determined the contribution of MEK-ERK activation to the profile of gene expression of human immature monocyte-derived dendritic cells (MDDCs) and peripheral blood myeloid DCs. ERK inhibition altered the expression of genes that mediate Chemokine (C-C motif) ligand 19 (CCL19)–directed migration (CCR7) and low-density lipoprotein (LDL) binding (CD36, SCARB1, OLR1, CXCL16) by immature DCs. In addition, ERK upregulated CCL2 expression while impairing the expression of DC maturation markers (RUNX3, ITGB7, IDO1). MEK-ERK–regulated genes exhibited an overrepresentation of cognate sequences for the aryl hydrocarbon receptor (AhR) transcription factor, whose transcriptional and DNA-binding activities increased in MDDCs upon exposure to the MEK1/2 inhibitor U0126. Therefore, the MEK-ERK signaling pathway regulates antigen capture, lymph node homing, and acquisition of maturation-associated genes, and its contribution to the maintenance of the immature state of MDDCs and myeloid DCs is partly dependent on the activity of AhR. Since pharmacologic modulation of the MEK-ERK signaling pathway has been proposed as a potential therapeutic strategy for cancer, our findings indicate that ERK inhibitors might influence antitumor responses through regulation of critical DC effector functions.