Dendritic Cell Priming Research Articles

Priming of dendritic cells by DNA-containing extracellular vesicles from activated T cells through antigen-driven contacts

Interaction of T cell with antigen-bearing dendritic cells (DC) results in T cell activation, but whether this interaction has physiological consequences on DC function is largely unexplored. Here we show that when antigen-bearing DCs contact T cells, DCs initiate anti-pathogenic programs. Signals of this interaction are transmitted from the T cell to the DC, through extracellular vesicles (EV) that contain genomic and mitochondrial DNA, to induce antiviral responses via the cGAS/STING cytosolic DNA-sensing pathway and expression of IRF3-dependent interferon regulated genes. Moreover, EV-treated DCs are more resistant to subsequent viral infections. In summary, our results show that T cells prime DCs through the transfer of exosomal DNA, supporting a specific role for antigen-dependent contacts in conferring protection to DCs against pathogen infection. The reciprocal communication between innate and adaptive immune cells thus allow efficacious responses to unknown threats.

Vaccine immunotherapy with ARNAX induces tumor-specific memory T cells and durable anti-tumor immunity in mouse models.

Immunological checkpoint blockade therapies benefit a limited population of cancer patients. We have previously shown that vaccine immunotherapy with Toll‐like receptor (TLR)3‐adjuvant and tumor antigen overcomes anti‐programmed death ligand‐1 (PD‐L1) resistance in mouse tumor models. In the present study, 4 different ovalbumin (OVA)‐expressing tumor cell lines were implanted into syngeneic mice and subjected to anti‐tumor immunotherapy using ARNAX and whole OVA protein. ARNAX is a TLR3‐specific agonist that does not activate the mitochondrial antiviral‐signaling protein (MAVS) pathway, and thus does not induce systemic inflammation. Dendritic cell priming and proliferative CTL were induced by ARNAX + OVA, but complete remission was achieved only in a PD‐L1‐low cell line of EG7. Addition of anti‐PD‐L1 antibody to the ARNAX + OVA therapy brought complete remission to another PD‐L1‐high subline of EG7. Tumor shrinkage but not remission was observed in MO5 in that regimen. We analyzed tumor cells and tumor‐infiltrating immune cells to identify factors associated with successful ARNAX vaccine therapy. Tumors that responded to ARNAX therapy expressed high levels of MHC class I and low levels of PD‐L1. The tumor‐infiltrating immune cells in ARNAX‐susceptible tumors contained fewer immunosuppressive myeloid cells with low PD‐L1 expression. Combination with anti‐PD‐L1 antibody functioned not only within tumor sites but also within lymphoid tissues, augmenting the therapeutic efficacy of the ARNAX vaccine. Notably, ARNAX therapy induced memory CD8+ T cells and rejection of reimplanted tumors. Thus, ARNAX vaccine + anti‐PD‐L1 therapy enabled permanent remission against some tumors that stably present antigens.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent <em>In Vitro</em> Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes.

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Type I Interferon-Independent Dendritic Cell Priming and Antitumor T Cell Activation Induced by a Mycoplasma fermentans Lipopeptide.

Mycoplasma fermentans-derived diacylated lipoprotein M161Ag (MALP404) is recognized by human/mouse toll-like receptor (TLR) 2/TLR6. Short proteolytic products including macrophage-activating lipopeptide 2 (MALP2) have been utilized as antitumor immune-enhancing adjuvants. We have chemically synthesized a short form of MALP2 named MALP2s (S-[2,3-bis(palmitoyloxy)propyl]-CGNNDE). MALP2 and MALP2s provoke natural killer (NK) cell activation in vitro but only poorly induce tumor regression using in vivo mouse models loading NK-sensitive tumors. Here, we identified the functional mechanism of MALP2s on dendritic cell (DC)-priming and cytotoxic T lymphocyte (CTL)-dependent tumor eradication using CTL-sensitive tumor-implant models EG7 and B16-OVA. Programmed death ligand-1 (PD-L1) blockade therapy in combination with MALP2s + ovalbumin (OVA) showed a significant additive effect on tumor growth suppression. MALP2s increased co-stimulators CD80/86 and CD40, which were totally MyD88-dependent, with no participation of toll-IL-1R homology domain-containing adaptor molecule-1 or type I interferon signaling in DC priming. MALP2s + OVA consequently augmented proliferation of OVA-specific CTLs in the spleen and at tumor sites. Chemokines and cytolytic factors were upregulated in the tumor. Strikingly, longer duration and reinvigoration of CTLs in spleen and tumors were accomplished by the addition of MALP2s + OVA to α-PD-L1 antibody (Ab) therapy compared to α-PD-L1 Ab monotherapy. Then, tumors regressed better in the MALP2s/OVA combination than in the α-PD-L1 Ab monotherapy. Hence, MALP2s/tumor-associated antigens combined with α-PD-L1 Ab is a good therapeutic strategy in some mouse models. Unfortunately, numerous patients are still resistant to PD-1/PD-L1 blockade, and good DC-priming adjuvants are desired. Cytokine toxicity by MALP2s remains to be settled, which should be improved by chemical modification in future studies.

Adjuvant immunotherapy for cancer: both dendritic cell-priming and check-point inhibitor blockade are required for immunotherapy.

The immune system eliminates advanced cancer when treated with programmed cell death protein-1 (PD-1) or its ligand (PD-L1) blockade, but PD-1 therapy is effective in only ∼20% of patients with solid cancer. The PD-1 antibody mainly acts on the effector phase of cytotoxic T lymphocytes (CTLs) in tumors but induces no activation of the priming phase of antigen-presenting dendritic cells (DCs). It is reasonable that both DC-priming and PD-1/L1 blocking are mandatory for efficient CTL-mediated tumor cytolysis. For DC-priming, a therapeutic vaccine containing Toll-like receptor (TLR) agonists, namely a priming adjuvant, is a good candidate; however, a means for DC-targeting by TLR adjuvant therapy remains to be developed. TLR adjuvants usually harbor cytokine toxicity, which is a substantial barrier against drug approval. Here, we discuss the functional properties of current TLR adjuvants for cancer immunotherapy and introduce a TLR3-specific adjuvant (ARNAX) that barely induces cytokinemia in mouse models.

PP5.9 - Novel dual role of dendritic cells in priming de novo CTL responses while inhibiting memory CTL responses to HIV-1 through the PD-L1 pathway

PP5.9 - Novel dual role of dendritic cells in priming de novo CTL responses while inhibiting memory CTL responses to HIV-1 through the PD-L1 pathway

Bone Marrow Dendritic Cells Derived Microvesicles for Combinational Immunochemotherapy against Tumor

AbstractVarious types of cell can change the cytoskeleton and shed microvesicles (MVs) with biomimic properties as parent cells in response to stimuli. To take use of the drug package capability of MVs and the potent antigen presentation property of dendritic cells (DCs), DC‐derived antigenic MVs are constructed by priming DCs with tumor‐derived MVs and then encapsulating a chemotherapeutic drug during MVs shedding. This kind of MVs exhibit significant inhibition on melanoma tumor growth and metastasis. The MV‐encapsulated chemotherapeutics can induce direct cytotoxicity and immunogenic cell death in tumor cells. Moreover, a robust antitumor immunity is induced in both, the tumor‐draining lymph node and the tumor microenvironment as the infiltration and activation of T lymphocytes increases. This kind of MVs is further explored in a hepatic ascites model with remarkable prolonged overall survival of mice. More importantly, the MVs can extend the survival of 60% mice more than 150 d without ascites even after rechallenging the tumor twice. This study demonstrates that antigenic MVs with chemotherapeutics possess great potential in cancer immunochemotherapy.

Abstract 2993: STING signaling in breast tumor microenvironment modulates immune checkpoint blockade efficacy in the neu-N mouse model of breast cancer

Abstract Background: The presence of tumor infiltrating T cells (TILs) is associated with improved clinical outcomes in multiple tumor types and is also necessary for response to immune checkpoint blockade. While T cell responses to some tumors occur spontaneously, the majority of cancers are not naturally recognized by the immune system. The lack of response is attributed primarily to insufficient T cell infiltration into the tumor microenvironment (TME). Activating the STING pathway represents one strategy for increasing infiltration of T cells into the TME. This induces interferon-β (IFN-β) production, leading to dendritic cell (DC) activation and priming of tumor antigen specific CD8+ T cells that mediate tumor regression. The intratumoral injection (IT) of ML-RR-S2-CDA (ADU-S100), a synthetic cyclic dinucleotide STING agonist, has antitumor efficacy in several aggressive mouse tumor models, including B16 melanoma, CT26 colon carcinoma, Panc02 pancreatic carcinoma, and 4T1 triple negative breast cancer. However, the impact of antigen-specific tolerance on tumor regression associated with STING-activation remains poorly understood. Therefore, we evaluated the efficacy of IT ADU-S100 in both non-tolerant parental FVB/N and the immune tolerant neu/N transgenic mice bearing established HER-2+ breast tumors. Methods: First we first evaluated the impact of IT ADU-S100 on tumor regression, survival, innate sensing, and priming of HER-2 specific CD8+ T cells in both tumor-bearing non-tolerant FVB/N control and tolerant neu/N mice. Then we determined whether modulating the most highly expressed immune checkpoints on tumor infiltrating CD8+ T cells enhanced intratumoral STING activation with ADU-S100 in the tolerant neu/N mouse model of HER-2+ breast cancer. Results: ADU S-100 induced HER-2-specific CD8+ T cell priming and durable tumor clearance in 100% of non-tolerant, FVB/N mice. In contrast, ADU S-100 failed to sufficiently prime HER-2-specific CD8+ T cells in tolerant neu/N mice, delaying tumor growth and clearing tumors in only 10% of mice. No differences in IFN-β production, DC priming, or HER-2-specific CD8+ T cell trafficking were detected between FVB/N and neu/N mice. However, activation and expansion of HER-2-specific CD8+ T cells was defective in neu/N mice. Immune cell infiltrates of untreated tumor-bearing neu/N mice expressed high levels of PD1 and OX40 on CD8+ T cells, and high levels of PD-L1 on both myeloid and tumor cells. Modulating PD-L1 and OX40 signaling combined with IT ADU S-100 enhanced HER-2-specific CD8+ T cell activity, clearing tumors from 40% of neu/N mice. Conclusions: Intratumoral STING activation synergizes with PD1 pathway-blockade and OX40 receptor stimulation to overcome immune tolerance and prime tumor antigen-specific CD8+ T cell responses that mediate effective tumor regression. Citation Format: Jeremy B. Foote, Marlene Kok, James M. Leatherman, Todd D. Armstrong, Bridget Marcinkowski, David B. Kanne, Elizabeth M. Jaffee, Thomas W. Dubensky, Leisha A. Emens. STING signaling in breast tumor microenvironment modulates immune checkpoint blockade efficacy in the neu-N mouse model of breast cancer [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 2993. doi:10.1158/1538-7445.AM2017-2993

Nimotuzumab Induces NK Cell Activation, Cytotoxicity, Dendritic Cell Maturation and Expansion of EGFR-Specific T Cells in Head and Neck Cancer Patients.

Survival benefit and long-term duration of clinical response have been seen using the epidermal growth factor receptor (EGFR)-targeted monoclonal antibody (mAb) nimotuzumab. Blocking EGFR signaling may not be the only mechanism of action underlying its efficacy. As an IgG1 isotype mAb, nimotuzumab’s capacity of killing tumor cells by antibody dependent cellular cytotoxicity (ADCC) and to induce an immune response in cancer patients have not been studied. ADCC-induced by nimotuzumab was determined using a 51Cr release assay. The in vitro effect of nimotuzumab on natural killer (NK) cell activation and dendritic cell (DC) maturation and the in vivo frequency of circulating regulatory T cells (Tregs) and NK cells were assessed by flow cytometry. Cytokine levels in supernatants were determined by ELISA. ELISpot was carried out to quantify EGFR-specific T cells in nimotuzumab-treated head and neck cancer (HNSCC) patients. Nimotuzumab was able to kill EGFR+ tumor cells by NK cell-mediated ADCC. Nimotuzumab-activated NK cells promoted DC maturation and EGFR-specific CD8+ T cell priming. Interestingly, nimotuzumab led to upregulation of some immune checkpoint molecules on NK cells (TIM-3) and DC (PD-L1), to a lower extent than another EGFR mAb, cetuximab. Furthermore, circulating EGFR-specific T cells were identified in nimotuzumab-treated HNSCC patients. Notably, nimotuzumab combined with cisplatin-based chemotherapy and radiation increased the frequency of peripheral CD4+CD39+FOXP3+Tregs which otherwise were decreased to baseline values when nimotuzumab was used as monotherapy. The frequency of circulating NK cells remained constant during treatment. Nimotuzumab-induced, NK cell-mediated DC priming led to induction of anti-EGFR specific T cells in HNSCC patients. The association between EGFR-specific T cells and patient clinical benefit with nimotuzumab treatment should be investigated.

A STING Agonist Given with OX40 Receptor and PD-L1 Modulators Primes Immunity and Reduces Tumor Growth in Tolerized Mice.

Stimulator of interferon genes (STING) signaling induces IFNβ production by intratumoral dendritic cells (DC), driving T-cell priming and recruitment into the tumor microenvironment (TME). We examined to what extent preexisting antigen-specific tolerance influenced the efficacy of in situ delivery of a potent STING-activating cyclic dinucleotide (CDN), ADU S-100, against established HER-2+ breast tumors. ADU S-100 induced HER-2-specific CD8+ T-cell priming and durable tumor clearance in 100% of nontolerant parental FVB/N mice. In contrast, ADU S-100 did not sufficiently prime HER-2-specific CD8+ T cells in tolerant neu/N mice, resulting in only delayed tumor growth and tumor clearance in 10% of the mice. No differences in IFNβ production, DC priming, or HER-2-specific CD8+ T-cell trafficking were detected between FVB/N and neu/N mice. However, activation and expansion of HER-2-specific CD8+ T cells were defective in neu/N mice. Immune cell infiltrates of untreated tumor-bearing neu/N mice expressed high numbers of PD1 and OX40 receptors on their CD8+ T cells, and PD-L1 was highly expressed on both myeloid and tumor cells. Modulating PD-L1 and OX40 receptor signaling combined with intratumoral ADU S-100 administration enhanced HER-2-specific CD8+ T-cell activity, clearing tumors in 40% of neu/N mice. Thus, intratumoral STING agonists could potently prime tumor antigen-specific CD8+ T-cell responses, and adding PD-L1 blockade and OX40 receptor activation can overcome antigen-enforced immune tolerance to induce tumor regression. Cancer Immunol Res; 5(6); 468-79. ©2017 AACR.

CD137 Stimulation Enhances Cetuximab-Induced Natural Killer: Dendritic Cell Priming of Antitumor T-Cell Immunity in Patients with Head and Neck Cancer.

Cetuximab, an EGFR-specific antibody (mAb), modestly improves clinical outcome in patients with head and neck cancer (HNC). Cetuximab mediates natural killer (NK) cell:dendritic cell (DC) cross-talk by cross-linking FcγRIIIa, which is important for inducing antitumor cellular immunity. Cetuximab-activated NK cells upregulate the costimulatory receptor CD137 (4-1BB), which, when triggered by agonistic mAb urelumab, might enhance NK-cell functions, to promote T-cell-based immunity. CD137 expression on tumor-infiltrating lymphocytes was evaluated in a prospective cetuximab neoadjuvant trial, and CD137 stimulation was evaluated in a phase Ib trial, in combining agonistic urelumab with cetuximab. Flow cytometry and cytokine release assays using NK cells and DC were used in vitro, testing the addition of urelumab to cetuximab-activated NK, DC, and cross presentation to T cells. CD137 agonist mAb urelumab enhanced cetuximab-activated NK-cell survival, DC maturation, and tumor antigen cross-presentation. Urelumab boosted DC maturation markers, CD86 and HLA DR, and antigen-processing machinery (APM) components TAP1/2, leading to increased tumor antigen cross-presentation. In neoadjuvant cetuximab-treated patients with HNC, upregulation of CD137 by intratumoral, cetuximab-activated NK cells correlated with FcγRIIIa V/F polymorphism and predicted clinical response. Moreover, immune biomarker modulation was observed in an open label, phase Ib clinical trial, of patients with HNC treated with cetuximab plus urelumab. These results suggest a beneficial effect of combination immunotherapy using cetuximab and CD137 agonist in HNC. Clin Cancer Res; 23(3); 707-16. ©2016 AACR.

Differential regulation of CD103 (αE integrin) expression in human dendritic cells by retinoic acid and Toll-like receptor ligands.

CD103 (αE integrin) is an important dendritic cell (DC) marker that characterizes functionally distinct DC subsets in mice and humans. However, the mechanism by which CD103 expression is regulated in human DCs and the role of CD103 for DC function are not very well understood. Here, we show that retinoic acid (RA) treatment of human monocyte-derived DCs (MoDCs) increased the ability of the DCs to synthesize RA and induced MoDC expression of CD103 and β7 at the mRNA and protein level. In contrast, RA was unable to induce the expression of CD103 in primary human DCs isolated from the gastric mucosa. Inhibition of TGF-β signaling in MoDCs down-regulated RA-induced CD103 expression, indicating that TGF-β-dependent pathways contribute to the induction of CD103. Conversely, when RA-treated MoDCs were stimulated with live Helicobacter pylori, commensal bacteria, LPS, or a TLR2 agonist, the RA-induced up-regulation of CD103 and β7 integrin expression was completely abrogated. To determine whether CD103 expression impacts DC priming of CD4+ T cells, we next investigated the ability of CD103+ and CD103─ DCs to induce mucosal homing and T cell proliferation. Surprisingly, RA treatment of DCs enhanced both α4β7 expression and proliferation in cocultured T cells, but no difference was seen between RA-treated CD103+ and CD103─ DCs. In summary, our data demonstrate that RA, bacterial products, and the tissue environment all contribute to the regulation of CD103 on human DCs and that DC induction of mucosal homing in T cells is RA dependent but not CD103 dependent.

Role of heterogeneous cell population on modulation of dendritic cell phenotype and activation of CD8 T cells for use in cell-based immunotherapies

Dendritic cell (DC)-based immunotherapies have much utility in their ability to prime antigen-specific adaptive immune responses. However, there does not yet exist a consensus standard to how DCs should be primed. In this study, we aimed to determine the role of heterogeneous co-cultures, composed of both CD11c+ (DCs) and CD11c− cells, in combination with monophosphoryl lipid A (MPLA) stimulation on DC phenotype and function. Upon DC priming in different co-culture ratios, we observed reduced expression of MHCII and CD86 and increased antigen uptake among CD11c+ cells in a CD11c− dependent manner. DCs from all culture conditions were induced to mature by MPLA treatment, as determined by secretion of pro-inflammatory cytokines IL-12 and TNF-α. Antigen-specific stimulation of CD4+ T cells was not modulated by co-culture composition, in terms of proliferation nor levels of IFN-γ. However, the presence of CD11c− cells enhanced cross-presentation to CD8+ T cells compared to purified CD11c+ cells, resulting in increased cell proliferation along with higher IFN-γ production. These findings demonstrate the impact of cell populations present during DC priming, and point to the use of heterogeneous cultures of DCs and innate immune cells to enhance cell-mediated immunity.

HSP70L1-mediated intracellular priming of dendritic cell vaccination induces more potent CTL response against cancer.

Heat-shock protein (HSP)-based immunotherapy is established on its adjuvant effects when applied via an extracellular approach to pulse and activate dendritic cells (DCs). Our previous studies indicate that DCs pulsed with recombinant fusion proteins of antigenic fragment and HSP70-like protein 1 (HSP70L1) are potent in stimulating antigen-specific Th1 responses. We herein evaluated the cytotoxic T cell (CTL) response by an intracellular approach of priming DCs with transfection of recombinant adenovirus-expressing the fusion gene of the 576-699 fragment of carcinoembryonic antigen (CEA) and HSP70L1. As compared with DCs pulsed with extracellular fusion protein, the DCs transfected with recombinant adenovirus expressing the fusion gene displayed equivalent mature phenotypes but less inflammatory appearance. However, the transfected DCs were superior to the pulsed DCs in inducing CEA-specific CTLs. Consistently, immunization of HLA-A2.1/H-2Kb transgene mice with the transfected DCs could induce more quantities of HLA-A2.1-restricted CEA-specific CTLs, protecting nude mice more significantly from human CEA-expressing colon tumor challenge when adoptively transferred. Mechanistic investigation indicated that intracellular expression of the fusion protein empowered the transfected DCs by activation of STAT1 possibly via inducing IFN-β and ERK pathways. Therefore, the more potent ability to induce anti-CEA CTL responses enables the DCs, which transfected with recombinant adenovirus expressing the fusion gene of antigenic CEA fragment and Th1 adjuvant, as an alternative promising approach for the immunotherapy of CEA-positive tumors.

Intestinal mucus acts is a maturation factor for dendritic cells

BackgroundGastrointestinal (GI) epithelial cells are covered by a thick layer of mucus which acts as a barrier, shielding the host epithelium from the luminal contents. Secreted mucus is expelled from goblet cells scattered throughout the GI tract, while adherent mucins are found on all epithelial cells. Mucus consists of highly glycosylated mucin proteins, primarily MUC2. Beneath the mucus and epithelial layer lies the lamina propria which contains multiple immune cell types. Intestinal dendritic cells (DCs) are professional antigen presenting cells capable of extending projections through the epithelium to sample antigen from the mucus layer and intestinal lumen. In this way, DCs are capable of interacting with antigens and metabolites from the intestinal microbiome. DCs act as sentinels in the GI tract by capturing and processing antigen, undergoing maturation, and regulating immune responses via production of cytokines. Previous studies have demonstrated that DCs are capable of extending processes through the mucus layer or acquiring antigen from antigen‐presenting goblet cells. However, little is known about the effect of mucus recognition by DCs and how this affects interaction with the microbiome. We hypothesize that intestinal mucin glycans stimulate monocytes toward DC lineages, thereby acting on innate immunity and immune/microbiota cross talk.MethodsTo assess the role of mucus in DC maturation and cytokine production the human monocyte cell line THP‐1 were matured to DC by stimulation with IL‐4 and GM‐CSF. Alternatively THP‐1 cells were stimulated with human stool mucus, mucus producing human cell line HT29‐MTX secreted mucus, pig stomach mucus or O‐linked mucus glycans. Mucus‐stimulated THP‐1 cells or IL‐4, GM‐CSF DCs were subjected to bacterial lipopolysaccharide (100 ng/ml LPS) for 3 hrs. Cells were analyzed by flow cytometry for mature DC markers CD11c and HLA and supernatants were examined for TNF production by ELISA. Human blood monocytes were isolated from whole blood and processed in the same manner.ResultsFlow cytometry data demonstrated that THP‐1 cells and human blood monocytes could be stimulated towards a DC‐like phenotype with IL‐4, GM‐CSF based on increased expression of CD11c, CD83 and HLA. Introduction of LPS resulted in an enhanced secretion of TNF in DCs compared to unstimulated monocytes or THP‐1 cells (8‐fold higher TNF). Interestingly, the presence of stool, pig stomach or HT29‐MTX cell‐derived mucus produced an increase in the CD83 hi CD11chi and CD11chiHLAhi DC populations from unstimulated THP‐1 cells. This increased DC population was also observed in gross morphology as mucus stimulated THP‐1s presented with increased diameter and projections. The ability of DCs to perform bacterial uptake in the presence of mucus was assessed using fluorescent carboxyfluorescein diacetate succinimidyl ester (CFDA‐SE) labeled bacteria. Mucus did not alter TNF production in unstimulated or LPS‐treated THP‐1 or in unstimulated or LPS‐treated DCs. However human and HT29‐MTX mucus did increase the CD11chiHLAhi population among mucus and LPS‐treated cells.ConclusionsTogether, this data indicates that intestinal mucus may act as a DC maturation factor and works to prime DC for interaction with members of the gut microbiota. This action of mucins has far‐reaching implications for immune interactions with the host and the intestinal microbiomeSupport or Funding InformationT32 DK007664

Tolerogenic effect of mouse fibroblasts on dendritic cells.

There is controversy about the immunomodulatory effect of fibroblasts on dendritic cells (DCs). To clarify this issue, in this study, we have evaluated different features of fibroblast-primed DCs including their ability to express co-inhibitory and co-stimulatory molecules, pro-inflammatory and anti-inflammatory cytokines and their ability to induce T-cell proliferation. We also examined migratory capacity of DCs to lymphatic tissues and present fibroblast-derived antigens after encountering fibroblasts. The results of our in vitro study showed that both co-inhibitory (programmed death ligand 1 and ligand 2 and B7H4) and co-stimulatory (CD86) molecules were up-regulated when DCs were co-cultured with fibroblasts. In an animal model, we showed that intra- peritoneal injection (IP) of both syngeneic and allogeneic fibroblasts significantly increased both total DC count and expression level of co-inhibitory and co-stimulatory molecules on DCs. Priming of DCs with syngeneic and allogeneic fibroblasts reduced the proliferation of CD4(+) and CD8(+) T cells. Even activation of fibroblast- primed DCs failed to restore their ability to induce T-cell proliferation. Likewise, priming of DCs with fibroblasts blocked the ability of ovalbumin-pulsed DCs to induce proliferation of ovalbumin-specific CD4(+) T cells. Compared with non-activated DCs, fibroblast-primed DCs had significantly higher expression levels of interleukin-10 and indoleamine 2, 3 dioxygenase. Fibroblast-primed DCs had a significantly reduced interleukin-12 expression level compared with that of activated DCs. After priming with fibroblasts, DCs were able to migrate to lymphatic tissues and present fibroblast-derived antigens (ovalbumin). In conclusion, after priming with fibroblasts, DCs gain tolerogenic features. This finding suggests the potential role of fibroblasts in the maintenance of immune tolerance.

Abstract A053: Therapy with a non-blocking monoclonal antibody targeting soluble NKG2D ligand MIC revamps endogenous innate and adaptive anti-tumor responses and eliminates primary and metastatic tumors

Abstract The human tumor-derived soluble NKG2D ligand, the MHC I-chain related molecule (sMIC), is highly immune suppressive in cancer patients and correlates with poor prognosis. However, the therapeutic effect of targeting sMIC has not been determined, due to the limitation that mice do not express homologs of human MIC. With a clinically relevant transgenic mouse models, we aimed to determine the therapeutic efficacy of a non-blocking sMIC-neutralizing anti-MIC mAb antibody. Treatment of the engineered MIC-expressing “humanized” TRAMP/MIC bi-transgenic mice at advanced disease stages with a sMIC-neutralizing non-blocking mAb effectively induced regression of primary tumors and eliminated metastasis without inducing systemic toxicity. The therapeutic effect is conferred by revamping endogenous anti-tumor immune responses, exemplified by restoring NK cell homeostasis and function, enhancing susceptibility of MIC+-tumor cells to NK cell killing, reviving and sustaining antigen-specific CD8 T cell responses, augmenting CD4 T cells to Th1 responses, priming dendritic cells for antigen presentation, and remodeling tumor microenvironment to be more immune reactive. With a transplantable syngeneic sMIC-expressing B16 melanoma model, we also demonstrated that therapy with the sMIC-neutralizing mAb significantly enhanced the anti-tumor responses of adoptively transferred antigen-specific CD8 and CD4 T cells. We conclude that therapy with a sMIC-neutralizing non-blocking anti-MIC mAb can effectuate anti-tumor immune responses against advanced MIC+ tumors. Our study provided strong rationale for translating sMIC-neutralizing therapeutic mAb into clinics, either alone or in combination with current ongoing standard immunotherapies. Citation Format: Jennifer Wu. Therapy with a non-blocking monoclonal antibody targeting soluble NKG2D ligand MIC revamps endogenous innate and adaptive anti-tumor responses and eliminates primary and metastatic tumors. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A053.

P70: Construction and studying expression in eukaryotic cells of allel-specific HLA-A∗0201 DNA-vaccine against breast cancer

Background Breast cancer ranks the first among female oncological diseases and ranks the second after lung cancer among causes of cancer related deaths. Use of contemporary anticancer drugs significantly enhanced possibilities of cancer therapy. However, most of medicines against cancer have such disadvantages as toxicity, narrowness of therapeutic action, and emergence of drug resistance. Recently, an approach based on potential of dendritic cells (DCs) to induce formation of cytotoxic T-cell immune response against cancer cells is widely used. Priming of dendritic cells by DNA-constructs encoding immunogenic epitopes of tumor antigens is regarded as a rather promising approach to design cancer vaccines. The aim of this study is to construct, obtain and study expression in eukaryotic cells of recombinant plasmid – candidate DNA-vaccines inducing T-cell immune response against breast cancer. Materials and methods Using the PolyCTLDesigner software, DNA-vaccine construct was made encoding polyepitope T-cell immunogen BC-A ∗ 0201. Gene encoding target immunogen was synthesized and cloned in the composition of plasmid pcDNA3.1 that is eukaryotic expression vector. Accuracy of gene insertion encoding polyepitopeimmunogen was verified using restriction analysis. Obtained target plasmid pBC-A ∗ 0201 – candidate DNA-vaccine was used for evaluation of gene expression in transfected eukaryotic cells 293T and dendritic cells by means of flow cytofluorometry and immunohistochemistry using MAT to p24 epitope-marker inserted into the structure of target plasmid. Dendritic cells were obtained from peripheral blood precursors by 2-h plastic adhesion. The nonadherent cells were removed 2 h later and the adherent monolayer was cultivated in the medium containing growth factors (GM-CSF and IL-4) according to (Naik, 2010; Ganul and Khranovskaya, 2012). Transfection of immature DCs (iDC) was performed by using the reagents purchased from Promokine. Further cultivation of iDCs was carried out in the medium containing TNF- α to obtain fully mature DCs (mDC). Analysis of mDCsurface markers expression was made using MAO (HLA-DR, CD83, CD86) and flow cytofluorometer (BD FACSCalibur). Results Plasmid pBC-A ∗ 0201 encoding epitopes of breast cancer antigens was constructed and produced in preparative amount. Using the method of intracellular staining of product of expression by specific MAO 29F2 to epitope p24, we showed expression of gene BC-A ∗ 0201 in eukaryotic cells 293T and dendritic cells transfected by plasmid pBC-A ∗ 0201. Maturity of dendritic cells was also proved. Conclusion Thus, obtained candidate DNA-vaccine encoding target polyepitopeimmunogen provides synthesis of relevant protein in the eukaryotic cells culture. In future, we plan to study biological activity of DNA-vaccine to evaluate level of induction of cytotoxic response against SKBR3 breast cancer cells.

Nonblocking Monoclonal Antibody Targeting Soluble MIC Revamps Endogenous Innate and Adaptive Antitumor Responses and Eliminates Primary and Metastatic Tumors.

The human tumor-derived soluble MHC I-chain-related molecule (sMIC) is highly immune suppressive in cancer patients and correlates with poor prognosis. However, the therapeutic effect of targeting sMIC has not been determined, due to the limitation that mice do not express homologs of human MIC. This study is to evaluate the therapeutic effect of a monoclonal antibody (mAb) targeting sMIC in a clinically relevant transgenic animal model. We treated the engineered MIC-expressing "humanized" TRAMP/MIC bitransgenic mice at advanced disease stages with a sMIC-neutralizing nonblocking anti-MIC mAb and assessed the therapeutic efficacy and associated mechanisms. A sMIC-neutralizing nonblocking anti-MIC mAb effectively induced regression of primary tumors and eliminated metastasis without inducing systemic toxicity. The therapeutic effect is conferred by revamping endogenous antitumor immune responses, exemplified by restoring natural killer (NK) cell homeostasis and function, enhancing susceptibility of MIC(+)-tumor cells to NK cell killing, reviving and sustaining antigen-specific CD8 T-cell responses, augmenting CD4 T cells to Th1 responses, priming dendritic cells for antigen presentation, and remodeling tumor microenvironment to be more immune reactive. Therapy with a sMIC-neutralizing nonblocking anti-MIC mAb can effectuate antitumor immune responses against advanced MIC(+) tumors. Our study provided strong rationale for translating sMIC-neutralizing therapeutic mAb into clinics, either alone or in combination with current ongoing standard immunotherapies.