Antigen-presenting Dendritic Cells Research Articles

Interview with the Guest Editor – Ana-Maria Lennon-Duménil

ABSTRACT Ana-Maria Lennon-Duménil was born in Chile and grew up in France before returning to South America to study biology at the University of Chile. For her PhD, she joined the laboratory of Marc Fellous at Institut Pasteur, Paris, France. Subsequently, Ana-Maria did her postdoctoral work with Hidde Ploegh at Harvard Medical School, Boston, USA on MHC class II antigen presentation. In 2004, she established her own research group at the Department of Immunity and Cancer at Institut Curie in Paris. Ana-Maria has been an elected EMBO member since 2018 and was awarded an INSERM national research award in the same year. Her research focuses on the coordination of cell migration and antigen presentation in dendritic cells and the contribution of cell polarity to the establishment of the immune synapse in B-lymphocytes. Ana-Maria is the Guest Editor for the 2020 Cell Biology of the Immune System Special Issue in Journal of Cell Science.

Cyto-Immuno-Therapy for Cancer: A Pathway Elicited by Tumor-Targeted, Cytotoxic Drug-Packaged Bacterially Derived Nanocells

Immunotherapy has emerged as a powerful new chapter in the fight against cancer. However, it has yet to reach its full potential due in part to the complexity of the cancer immune response. We demonstrate that tumor-targeting EDV nanocells function as an immunotherapeutic by delivering a cytotoxin in conjunction with activation of the immune system. These nanocells polarize M1 macrophages and activate NK cells concurrently producing a Th1 cytokine response resulting in potent antitumor function. Dendritic cell maturation and antigen presentation follows, which generates tumor-specific CD8+ Tcells, conferring prolonged tumor remission. The combination of cytotoxin delivery and activation of innate and adaptive antitumor immune responses results in a potent cyto-immunotherapeutic with potential in clinical oncology.

The Variable Genomic NK Cell Receptor Locus Is a Key Determinant of CD4+ T Cell Responses During Viral Infection.

Increasing evidence points to a key role for NK cells in controlling adaptive immune responses. In studies examining the role of CD1d on CD4+ T cell responses, we found that a line of CD1d-deficient mice on the C57BL/6J background had a homozygous 129 locus on chromosome 6 containing the entire NK cell gene cluster. Mice possessing this locus (C57BL/6.NKC129) displayed a >10-fold reduction in antigen-specific CD4+ T cell responses after intracranial infection with lymphocytic choriomeningitis virus (LCMV). Neither parental strain displayed defects in viral-specific CD4+ T cell responses. Interestingly, following infection, increased numbers of NK cells accumulated in the lymph nodes of C57BL/6.NKC129 mice and displayed enhanced in vivo functionality. Moreover, depletion of NK cells with anti-asialo-GM-1 antibody in C57BL/6.NKC129 mice resulted in a >20-fold increase in viral-specific CD4+ T cell responses. Mechanistically, we found that dendritic cell antigen presentation and early type I IFN production were significantly decreased in C57BL/6.NKC129 mice, but were restored in perforin-deficient C57BL/6.NKC129 mice or following NK depletion. Together, these data reveal that the variable genomic regions containing the activating/inhibitory NK cell receptors are key determinants of antigen-specific CD4+ T cell responses, controlling type I IFN production and the antigen-presenting capacity of dendritic cells.

A Potential Herbal Adjuvant Combined With a Peptide-Based Vaccine Acts Against HPV-Related Tumors Through Enhancing Effector and Memory T-Cell Immune Responses

Viral infection is associated with many types of tumorigenesis, including human papillomavirus (HPV)-induced cervical cancer. The induction of a specific T-cell response against virus-infected cells is desired to develop an efficient therapeutic approach for virus-associated cancer. Chinese herbal medicine (CHM) has a long history in the treatment of cancer patients in Asian countries. Hedyotis diffusa Willd (Bai Hua She She Cao, BHSSC) is frequently used clinically and has been shown to inhibit tumor growth in vitro. However, in vivo data demonstrating the antitumor efficacy of BHSSC are still lacking. We showed that BHSSC induces murine and human antigen-presenting cell (APC) activation via the MAPK signaling pathway and enhances antigen presentation in bone marrow-derived dendritic cells (BMDCs) in vitro. Furthermore, we identified that treatment with BHSSC leads to improved specific effector and memory T-cell responses in vivo. Variant peptide-based vaccines combined with BHSSC improved antitumor activity in preventive, therapeutic, and recurrent HPV-related tumor models. Furthermore, we showed that rutin, one of the ingredients in BHSSC, induces a strong specific immune response against HPV-related tumors in vivo. In summary, we demonstrated that BHSSC extract and its active compound, rutin, can be used as adjuvants in peptide-based vaccines to increase immunogenicity and to bypass the requirement of a conditional adjuvant.

The lactate receptor GPR81 promotes breast cancer growth via a paracrine mechanism involving antigen-presenting cells in the tumor microenvironment

GPR81 is a G-protein-coupled receptor for lactate, which is upregulated in breast cancer and plays an autocrine role to promote tumor growth by tumor cell-derived lactate. Here we asked whether lactate has any paracrine role via activation of GPR81 in cells present in tumor microenvironment to help tumor growth. First, we showed that deletion of Gpr81 suppresses breast cancer growth in a constitutive breast cancer mouse model (MMTV-PyMT-Tg). We then used a syngeneic transplant model by monitoring tumor growth from a mouse breast cancer cell line (AT-3, Gpr81-negative) implanted in mammary fat pad of wild-type mice and Gpr81-null mice. Tumor growth was suppressed in Gpr81-null mice compared with wild-type mice. There were more tumor-infiltrating T cells and MHCIIhi-immune cells in tumors from Gpr81-null mice compared with tumors from wild-type mice. RNA-seq analysis of tumors indicated involvement of immune cells and antigen presentation in Gpr81-dependent tumor growth. Antigen-presenting dendritic cells expressed Gpr81 and activation of this receptor by lactate suppressed cell-surface presentation of MHCII. Activation of Gpr81 in dendritic cells was associated with decreased cAMP, IL-6 and IL-12. These findings suggest that tumor cell-derived lactate activates GPR81 in dendritic cells and prevents presentation of tumor-specific antigens to other immune cells. This paracrine mechanism is complementary to the recently discovered autocrine mechanism in which lactate induces PD-L1 in tumor cells via activation of GPR81 in tumor cells, thus providing an effective means for tumor cells to evade immune system. As such, blockade of GPR81 signaling could boost cancer immunotherapy.

Defects of CTLA-4 Are Associated with Regulatory T Cells in Myasthenia Gravis Implicated by Intravenous Immunoglobulin Therapy.

Myasthenia gravis (MG) is a CD4+ T cell-dependent autoimmune disease resulting from aberrant immune response mediated by circulating autoantibodies at the neuromuscular junction. Intravenous immunoglobulin (IVIg) is an expensive and commonly used immunotherapeutic approach to treat patients with MG. The mechanisms of actions involved in IVIg treatment, however, remain to be investigated. In an effort to examine the roles of various subsets of CD4+ T cells in the periphery blood of MG and uncover the mechanisms that contribute to the therapeutical effects of IVIg, we first demonstrated that a subset of CD4+ T cells, CTLA-4-expressing regulatory T (Treg) cells, were underrepresented and functionally defective in MG patients. The dynamic profiling during the IVIg therapy course further revealed an inverse relationship between the frequency of CTLA-4+ Treg and the quantitative MG (QMG) score that represents disease severity. Our mechanistic studies indicated that IVIg expands CTLA-4-Treg cells via modulating antigen-presenting dendritic cells (DCs). To determine the molecular defects of CTLA-4 in abnormities of Treg in MG patients, we demonstrated hypermethylation at -658 and -793 CpGs of CTLA-4 promoter in MG Tregs. Interestingly, IVIg therapy significantly reduced the methylation level at these two sites in MG patients. Overall, our study may suggest a role of CTLA-4 in functionally defected Treg cells in MG and its actions involved in IVIg therapy.

Sugar-Nanocapsules Imprinted with Microbial Molecular Patterns for mRNA Vaccination.

Innate immune cells recognize and respond to pathogen-associated molecular patterns. In particular, polysaccharides found in the microbial cell wall are potent activators of dendritic cells (DCs). Here, we report a new class of nanocapsules, termed sugar-capsules, entirely composed of polysaccharides derived from the microbial cell wall. We show that sugar-capsules with a flexible polysaccharide shell and a hollow core efficiently drain to lymph nodes and activate DCs. In particular, sugar-capsules composed of mannan (Mann-capsule) carrying mRNA (mRNA) promote strong DC activation, mRNA translation, and antigen presentation on DCs. Mann-capsules elicit robust antigen-specific CD4+ and CD8α+ T-cell responses with antitumor efficacy in vivo. The strategy presented in this study is generally applicable for utilizing pathogen-derived molecular patterns for vaccines and immunotherapies.

The Probiotic BB12 Induces MicroRNAs Involved in Antigen Processing and Presentation in Porcine Monocyte-Derived Dendritic Cells.

MicroRNAs (miRNAs) mediate the regulation of gene expression. Several reports indicate that probiotics induce miRNA-mediated immunomodulation at different levels, such as cytokine production and the up-regulation of several markers related to antigen presentation in antigen-presenting cells. The objective of this work was to identify target genes of miRNAs that are involved in the processing and presentation of antigens in monocyte-derived dendritic cells (moDCs) stimulated with the probiotic Bifidobacterium animalis ssp. lactis BB12 (BB12). First, an in silico prediction analysis for a putative miRNA binding site within a given mRNA target was performed using RNAHybrid software with mature sequences of differentially expressed miRNAs retrieved from a Genbank data set that included BB12-stimulated and unstimulated porcine monocytes. From them, 23 genes resulted in targets of 19 miRNAs, highlighting miR-30b-3p, miR-671-5p, and miR-9858-5p, whose targets were costimulatory molecules, and were overexpressed (p < 0.05) in BB12-stimulated moDCs. The analysis of moDCs showed that the percentage of cells expressing SLA-DR+CD80+ decreased significantly (p = 0.0081) in BB12-stimulated moDCs; interleukin (IL)-10 production was unchanged at 6 h but increased after 24 h of culture in the presence of BB12 (p < 0.001). In summary, our results suggest that SLA-DR and CD80 can be down-regulated by miRNAs miR-30b-3p, miR-671-5p, and miR-9858-5p, while miR-671-5p targets IL-10.

Engineering ApoE3-incorporated biomimetic nanoparticle for efficient vaccine delivery to dendritic cells via macropinocytosis to enhance cancer immunotherapy

Efficient delivery of vaccines to dendritic cells (DCs) is critical for inducing sufficient immune response and realizing effective cancer immunotherapy. In the past decade, researchers have spent tremendous effort in delivering vaccines by using nanoparticles. However, most of the present strategies are designed based on receptor-mediated endocytosis to increase nanovaccines uptake by DCs, and underestimate the role of macropinocytosis in taking up exogenous antigen. Here, we proposed that macropinocytosis, an efficient pathway for DCs to internalize extracellular fluid-phase solutes, might be utilized as a highly-effective approach to facilitate nanovaccines uptake in DCs. Accordingly, we designed a biomimetic nanovaccine (R837-αOVA-ApoE3-HNP), composing of a poly-(D, l-lactide-co-glycolide) (PLGA) core to encapsulate adjuvant imiquimod (R837), a phospholipid membrane to load antigen peptide (αOVA), and apolipoprotein E3 (ApoE3), to boost the internalization of antigens into DCs. The nanovaccine exhibited highly efficient cellular uptake into DCs through the macropinocytosis pathway, and significantly promoted DCs maturation and antigen presentation. After subcutaneous injection, the nanovaccine was efficiently drained to lymph nodes. Strong T cell immune responses including the generation of antigen-specific CD8+ T cells, expansion of IFN-γ+ CD8+ T cells and the secretion of IFN-γ+ were observed after the vaccination of R837-αOVA-ApoE3-HNP. It also efficiently inhibited the formation of tumor metastasis in lung as a prevention vaccine, and exerted superior therapeutic efficiency on B16-OVA tumor-bearing mice when in combination with αPD-1 therapy. Overall, our work demonstrated that by utilizing the macropinocytosis pathway, ApoE3-incorporated biomimetic nanoparticle has great potential to function as a feasible, effective, and safe nanovaccine for cancer immunotherapy.

Inflammatory Role of TLR-MyD88 Signaling in Multiple Sclerosis.

Multiple sclerosis (MS) is a neuro-autoimmune and neurodegenerative disorder leading to chronic inflammation, demyelination, axonal, and neuronal loss in the central nervous system (CNS). Despite intense research efforts, the pathogenesis of MS still remains unclear. Toll-like receptors (TLRs) are a family of type I transmembrane receptors that play a crucial role in the innate immune response. Myeloid differentiation factor 88 (MyD88) is the adaptor of major TLRs. It has been widely considered that the TLR-MyD88 signaling pathway plays an important role in the occurrence and development of autoimmune disease. Data have revealed that the TLR-MyD88 signaling may be involved in the pathogenesis of MS and experimental autoimmune encephalomyelitis (EAE), an animal model for MS, by regulating the antigen presentation of dendritic cells, the integrity of blood-brain barrier (BBB), and the activation of T cells and B cells. Here, we summarize the role of TLRs and MyD88 in MS and discuss the possible therapies that are based on these molecules.

Macropinocytosis drives T cell growth by sustaining the activation of mTORC1

Macropinocytosis is an evolutionarily-conserved, large-scale, fluid-phase form of endocytosis that has been ascribed different functions including antigen presentation in macrophages and dendritic cells, regulation of receptor density in neurons, and regulation of tumor growth under nutrient-limiting conditions. However, whether macropinocytosis regulates the expansion of non-transformed mammalian cells is unknown. Here we show that primary mouse and human T cells engage in macropinocytosis that increases in magnitude upon T cell activation to support T cell growth even under amino acid (AA) replete conditions. Mechanistically, macropinocytosis in T cells provides access of extracellular AA to an endolysosomal compartment to sustain activation of the mechanistic target of rapamycin complex 1 (mTORC1) that promotes T cell growth. Our results thus implicate a function of macropinocytosis in mammalian cell growth beyond Ras-transformed tumor cells via sustained mTORC1 activation.

FAT10 localises in dendritic cell aggresome-like induced structures and contributes to their disassembly.

Dendritic cell (DC) aggresome-like induced structures (DALIS) are protein aggregates of polyubiquitylated proteins that form transiently during DC maturation. DALIS scatter randomly throughout the cytosol and serve as antigen storage sites synchronising DC maturation and antigen presentation. Maturation of DCs is accompanied by the induction of the ubiquitin-like modifier FAT10 (also known as UBD), which localises to aggresomes, structures that are similar to DALIS. FAT10 is conjugated to substrate proteins and serves as a signal for their rapid and irreversible degradation by the 26S proteasome similar to, yet independently of ubiquitin, thereby contributing to antigen presentation. Here, we have investigated whether FAT10 is involved in the formation and turnover of DALIS, and whether proteins accumulating in DALIS can be modified through conjunction to FAT10 (FAT10ylated). We found that FAT10 localises to DALIS in maturing DCs and that this localisation occurs independently of its conjugation to substrates. Additionally, we investigated the DALIS turnover in FAT10-deficient and -proficient DCs, and observed FAT10-mediated disassembly of DALIS. Thus, we report further evidence that FAT10 is involved in antigen processing, which may provide a functional rationale as to why FAT10 is selectively induced upon DC maturation.

Glucocorticoids in Graves’ orbitopathy: mechanisms of action and clinical application

Background:Graves’ orbitopathy (GO) is the most frequent extrathyroidal manifestation of the autoimmune Graves’ disease. GO significantly impacts quality of life and has a psycho-social morbidity. Inflammation and swelling of the orbital tissue often leads to proptosis, diplopia, and decrease of visual acuity. Due to the inflammatory background of the disease, glucocorticoids (GC) have been used as a first-line treatment for decades.Methods:PubMed and MeSH database were searched for original articles, clinical trials, reviews, and meta-analyses published between 1 January 2000 and 31 March 2020 and pertaining to both the mechanism of action and immunological effects of GC as well as to the treatment of GO by GC. The publications were evaluated according to their setting and study design.Results:GC act through genomic (trans-activation and trans-repression) and rapid non-genomic mechanisms. GC in general, and the intravenous (IV) administration of GC in particular, markedly decrease the activity and number of the most potent antigen-presenting dendritic cells. According to the internationally acknowledged European Thyroid Association Guidelines for the management of GO, weekly IVGC application over 12 weeks is recommended as first-line treatment for patients with active and severe GO. The daily and cumulative dose should be tailored according to clinical severity, for example, 4.5 g of IV methylprednisolone for the inflammatory component versus 7.5 g in the presence of diplopia and severe proptosis. Fast and significant improvements in orbital symptoms and signs are noted in 65–70% of patients. Long-term experience over decades, and worldwide availability at low cost, underline the clinical and therapeutic relevance of GC. Adverse events are rarely severe, dose-dependent, and usually reversible, hence easy to handle by medical investigators. Oral GC application on a daily basis is characterized by high bioavailability but reduced efficacy and increased toxicity.Conclusion:IVGC still represents the standard of care in active/severe GO. Innovative biologicals, like monoclonal antibodies targeting the thyrotropin/Insulin-like growth factor-1 receptors or pro-inflammatory cytokines (e.g., Interleukin-6) should be compared with standard GC treatment with respect to short- and long-term efficacy, safety, costs, and global availability.

Immune Characterization in Aneurysmal Subarachnoid Hemorrhage Reveals Distinct Monocytic Activation and Chemokine Patterns.

The pathophysiology of delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH) is incompletely understood. Intrathecal activation of inflammatory immune cells is suspected to play a major role for the induction of DCI. The aim of this study is to identify immune cell subsets and mediators involved in the pathogenesis of DCI. We prospectively collected blood and CSF from 25 patients with aSAH at early and late time points. We performed multicolor flow cytometry of peripheral blood and CSF, analyzing immune cell activation and pro-inflammatory cyto- and chemokines. In addition to the primary immune analysis, we retrospectively analyzed immune cell dynamics in the CSF of all our SAH patients. Our results show an increased monocyte infiltration secondary to aneurysm rupture in patients with DCI. Infiltrating monocytes are defined by a non-classical (CD14dim CD16+) phenotype at early stages. The infiltration is most likely triggered by the intrathecal immune activation. Here, high levels of pro-inflammatory chemokines, such as CXCL1, CXCL9, CXCL10, and CXCL11, are detected. The intrathecal cellular activation profile of monocytes was defined by upregulation of CD163 and CD86 on monocytes and a presumable later differentiation into antigen-presenting plasmacytoid dendritic cells (pDCs) and hemosiderophages. Peripheral immune activation was reflected by CD69 upregulation on T cells. Analysis of DCI prevalence, Hunt and Hess grade, and clinical outcome correlated with the degree of immune activation. We demonstrate that monocytes and T cells are activated intrathecally after aSAH and mediate a local inflammatory response which is presumably driven by chemokines. Our data shows that the distinct pattern of immune activation correlates with the prevalence of DCI, indicating a pathophysiological connection to the incidence of vasospasm.

Type I IFN ineffectively activates neonatal dendritic cells limiting respiratory antiviral T-cell responses.

Insufficient T cell responses contribute to the increased burden of viral respiratory disease in infancy. Neonatal dendritic cells (DCs) often provide defective activation of pathogen-specific T cells through mechanisms that are incompletely understood, which hinders vaccine design for this vulnerable age group. Enhancing our characterization of neonatal DC sub-specialization and function is therefore critical to developing their potential for immunomodulation of T cell responses. In this study, we engineered respiratory syncytial virus (RSV) to express a model protein, ovalbumin, to track antigen-presenting DCs in vivo. We found that murine neonatal conventional DC1s (cDC1s) efficiently migrated and presented RSV-derived antigen, challenging the paradigm that neonatal DCs are globally immature. In a key observation, however, we discovered that during infection neonatal cDC1s presenting viral antigen were unable to upregulate costimulatory molecules in response to type I interferons (IFN-I), contributing to poor antiviral T cell responses. Importantly, we showed that the deficient response to IFN-I was also exhibited by human neonatal cDC1s, independent of infection. These findings reveal a functionally distinct response to IFN-I by neonatal cDC1s that may leave young infants susceptible to viral infections, and provide a new target for exploration, in light of failed efforts to design neonatal RSV vaccines.

Abstract C053: NP-G2-044, a novel fascin inhibitor, blocks tumor metastasis and increases antitumor immune response

Abstract Filopodia are finger-like plasma membrane protrusions that are formed upon remodeling of the actin cytoskeleton beneath the plasma membrane. Fascin protein is the main actin-bundling protein in filopodia and invadopodia which are fundamental to cell shape and motility events. Human fascin expression is low or absent in normal adult epithelial cells but highly expressed in metastatic tumors and in antigen-presenting dendritic cells within the tumor microenvironment. Elevated levels of fascin are correlated with poor prognosis and short survival due to aggressive phenotypes with increased lymph node and distant metastases. Mouse genetic studies demonstrate that deletion of fascin gene delays tumor development, slows tumor growth, reduces metastatic colonization, and increases overall survival. These mouse genetic studies provide strong evidence for fascin’s role in tumor initiation, tumor progression and tumor metastasis, making it an attractive target for drug development. Chemical libraries were screened to identify small-molecule compounds that specifically inhibit the biochemical function of fascin to bundle actin filaments. The inhibitors were optimized to produce the lead compound, NP-G2-044. Preclinical studies showed that fascin inhibitors increase the overall survival of tumor-bearing mice and reinvigorate anti-tumor immune response in syngeneic mouse models of breast, lung, pancreatic and prostate tumors. Fascin protein levels are increased in conventional dendritic cells (cDCs) in the immunosuppressive tumor microenvironment. The number of intra-tumoral activated cDCs is increased following fascin inhibitor treatment. Moreover, together with anti-PD-1 antibody, fascin inhibition markedly increases the number of Ki67+ proliferating and Granzyme-B+ activated CD8+ killer T-cells in tumor tissue. These data demonstrate that fascin inhibitors simultaneously act on tumor cells to block tumor metastasis and on intratumoral DCs to reinvigorate anti-tumor immune response. We are currently conducting a first-in-human phase 1 trial of NP-G2-044 in patients with advanced or metastatic solid tumor malignancies. Citation Format: Vincent Chung, Frank Tsai, Komal Jhaveri, Yufeng Wang, Daniel D Von Hoff, Edward G Garmey, J. Jillian Zhang, Xin-Yun Huang. NP-G2-044, a novel fascin inhibitor, blocks tumor metastasis and increases antitumor immune response [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C053. doi:10.1158/1535-7163.TARG-19-C053

Rapid Production of Physiologic Dendritic Cells (phDC) for Immunotherapy.

Generation of large numbers of dendritic cells (DC) for research or immunotherapeutic purposes typically involves in vitro conversion of murine bone marrow precursors or humanblood monocytes to DC via cultivation with supraphysiologic concentrations of cytokines such as GM-CSF and IL-4 for up to 7days. Alternatively, our group has recently established a new approach, based on the underlying mechanism of action of a widely used cancer immunotherapy termed Extracorporeal Photochemotherapy (ECP). Our method of rapid and cytokine-free production of therapeutically relevant DC populations,leveragingthe innate physiologic programs likely responsible for DC differentiation from blood monocytes in vivo, potentially offers a novel, inexpensive, and easily accessiblesource of DC for clinical and research uses. This approach involves ex vivo physiologic reprogramming of blood monocytes to immunologically tunable dendritic antigen-presenting cells, which we term "phDC," for physiological DC. To facilitate access and utilization of these new DC populations by the research community, in this chapter, we describethe use of a scaled-down version of the clinical ECP leukocyte-treatment devicetermed the Transimmunization (TI) chamberor plate, suitable for processing both mouse and human samples. We highlight the methodological sequences necessary to isolate mouse or human peripheral blood mononuclear cell (PBMC) from whole blood, and to expose those PBMC to the TI chamber for facilitating monocyte activation and conversion to physiological DC (phDC) through interaction with blood proteins and activated platelets under controlled flow conditions. We thenprovide sample protocols for potential applications of the generated DC, including their use as vaccinating antigen-presenting cells (APC) in murine in vivo antitumor models, and in human ex vivo T-cell stimulation and antigen cross-presentation assays which mimic clinical vaccination. We additionally highlight the technical aspects of loading mouse or human phDC with tumor-associated antigens (TAA) in the form of peptides or apoptotic tumor cells. We provide a simple and clinically relevant means to reprogram blood monocytes into functional APC, potentially replacing the comparatively expensive and clinically disappointing cytokine-derived DC which have previously dominated the dendritic cell landscape.

MicroRNA-223-3p modulates dendritic cell function and ameliorates experimental autoimmune myocarditis by targeting the NLRP3 inflammasome

Autoimmune myocarditis is a cause of dilated cardiomyopathy and heart failure. MicroRNAs regulate many immune processes, but their role in aberrant inflammation during autoimmune myocarditis remains unclear. In this study, we investigated the role of miR-223-3p in experimental autoimmune myocarditis (EAM). We found that miR-223-3p expression was significantly lower in EAM mice than that in normal mice. miR-223-3p inhibited NLRP3 inflammasome expression, promoting the polarization of dendritic cells (DCs) towards a tolerogenic DC phenotype. miR-223-3p effectively induced regulatory T cell (Treg) generation by inhibiting the function of antigen-presenting DCs. Transfer of miR-223-3p-overexpressing DCs protected mice against the development of EAM. Our findings suggest that miR-223-3p is involved in the induction of the tolerogenic DC phenotype and regulates tolerance in autoimmune myocarditis.

Reduced Antigen Presentation May Contribute to Immunomodulatory Drug Resistance in Multiple Myeloma

Introduction: Maintenance therapy with the immunomodulatory drug (IMiD) lenalidomide improves progression-free survival (PFS) and overall survival among patients with multiple myeloma (MM). It has been suggested that IMiDs enhance immune-mediated anti-tumor responses through increased activation and proliferation of T cells and natural killer (NK) cells as well as inhibition of regulatory T cells. Despite the effectiveness of this therapy, nearly all patients relapse for reasons that remain uncharacterized. We sought to elucidate mechanisms of resistance to lenalidomide maintenance through investigation of mutations in cell-free DNA (cfDNA) and identification of changes in the innate and adaptive immune system by single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs). While MM is a cancer of plasma cells that primarily reside in the bone marrow, we focused on the peripheral blood which offers a comprehensive view of multiple sites of disease including extramedullary locations commonly found at the time of relapse. Methods: Twenty-four patients with MM and no minimal residual disease by 10-color flow cytometry and IGHVnext-generation sequencing (Invivoscribe) after induction therapy with or without an autologous stem cell transplant were selected for analysis. All patients were enrolled in NCT02538198 at Memorial Sloan Kettering Cancer Center. Peripheral blood was collected serially including timepoints prior to lenalidomide maintenance and after disease progression. Plasma and PBMCs were isolated by Ficoll-Paque density gradient centrifugation. cfDNA was extracted from plasma and targeted next-generation sequencing using a 70-gene panel of recurrently mutated genes in myeloma was performed using the QIAseq Targeted DNA kit (QIAGEN). DNA sequencing was also performed using the same gene panel on granulocytes to exclude germline variants. On PBMC samples, 5' gene expression and T and B cell receptor single-cell sequencing were performed using the Chromium Single Cell V(D)J kit (10X Genomics). Analysis of single-cell sequencing was performed on the top 100 principal components and visualized by Uniform Manifold Approximation and Projection (UMAP). A regression-based classifier was trained to automatically assign clustered cells into 26 immune cell phenotypes using lists of genes reported to be uniquely expressed by each cell type. Results: cfDNA sequencing was performed on 12 patients characterized as durable responders (PFS &gt; 57 months) and 12 early progressors (median PFS 25 months). Prior to maintenance therapy, no significant difference was observed in the number of cfDNA mutations between these groups. The number of non-synonymous somatic mutations in cfDNA tended to increase over time and was significantly higher among patients taking lenalidomide for more than 2 years compared to patients at the time of relapsed disease (mean number of mutations was 7 in responders versus 3 in progressors).ZNF292was among the genes most significantly mutated in responders compared to progressors. ZNF292encodes a zinc finger protein involved in RNA binding and has previously been identified as a potential source of tumor-associated antigens in patients with monoclonal gammopathy of undetermined significance. To identify changes in immune cell phenotype and function that associate with disease progression, we performed single-cell RNA sequencing on PBMCs from the 12 progressors before maintenance and at the time of relapse. The median number of cells sequenced per PBMC sample was 3,132. Over 90% of cells could automatically be classified into a specific immune cell type by their gene expression. We observed the frequency of NK cells and plasmablasts were significantly increased at the time progression on lenalidomide while monocytes, dendritic cells, and basophils were decreased. Conclusion: Mechanisms of resistance among patients receiving treatment with IMiDs is not well known. Using targeted sequencing of cfDNA, we identify patients responding to maintenance tended to have a higher mutational load in cfDNA including genes previously reported to encode tumor-associated antigens. Additionally, we show that a decline in antigen-presenting dendritic cells is more common at the time of disease progression. Taken together, these results suggest reduced neoantigen production and a decline in antigen presentation may contribute to IMiD resistance. Disclosures Landgren: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Other: IDMC; Theradex: Other: IDMC; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees. Green:Celgene: Consultancy; Cellectar Biosciences: Research Funding; Seattle Genetics: Research Funding; Juno Therapeutics: Consultancy, Patents &amp; Royalties, Research Funding; GSK: Consultancy.

RBTT-08. A RANDOMIZED PHASE 2 OPEN LABEL STUDY OF NIVOLUMAB PLUS STANDARD DOSE BEVACIZUMAB VERSUS NIVOLUMAB PLUS LOW DOSE BEVACIZUMAB IN RECURRENT GLIOBLASTOMA (GBM)

Abstract INTRODUCTION Initial trials with anti-PD1 in recurrent glioblastoma showed limited efficacy. Vascular endothelial growth factor (VEGF) is a highly upregulated proangiogenic growth factor in GBM that contributes to tumor-associated immunosuppression by inhibition of dendritic cell maturation and antigen presentation, induction of apoptosis of CD8+ T-cells and enhancing Treg activity. Hence, a combination of anti-PD1 and anti-VEGF is promising approach in recurrent GBM. METHOD This is a 90 patient randomized, open-label, phase 2 safety study of Nivolumab and bevacizumab administered according to standard and reduced dosage schedules in patients with first recurrence of GBM. Patients will undergo 1:1 randomization to receive treatment with either Nivolumab (240 mg flat dosing IV every 2 weeks) and bevacizumab administered according to standard (10 mg/kg IV every 2 weeks; Arm A) and reduced (3 mg/kg IV every 2 weeks; Arm B) dosage schedules for recurrent glioblastoma patients. The study will allow patients that require dexamethasone up to 4 mg/day to participate in the study. All subjects will be followed for safety and tolerability, tumor progression and overall survival. Tumor progression or response endpoints will be assessed using the Radiologic Assessment in Neuro-Oncology (RANO) criteria and an exploratory endpoint will evaluate the response endpoints using the Immunotherapy RANO (iRANO). Treatment with study medication will continue until confirmed tumor progression, unacceptable toxicity or death. It is expected that enrollment and follow-up of randomized subjects (45 subjects in each arm) will take approximately 12months. The one-sample log-rank test will be applied to outcomes observed for each arm individually to test the hypothesis that OS has been improved beyond the null 12-month survival rate of 45%. With N=45 patients per arm, a one-sided test provides power=0.80 to detect survival rate of 58% at 12-months following treatment at the 0.10 significance level. The study (NCT03452579) is ongoing and enrolling patients.