Pro-inflammatory Dendritic Cells Research Articles

Protists protecting food tolerance

Celiac disease (CeD) is an immune disorder characterized by gluten intolerance that can be unleashed by enteric viral infections in mice. However, Sanchez-Medina et al. recently identified a murine commensal protist, Tritrichomonas arnold, that protects against reovirus-induced intolerance to dietary protein by counteracting virus-induced epithelial stress and proinflammatory dendritic cell (DC) activation.

Vibrio cholerae Porin OmpU Activates Dendritic Cells via TLR2 and the NLRP3 Inflammasome.

OmpU is one of the major porins of Vibrio cholerae, a Gram-negative human pathogen. Previously, we showed that OmpU stimulates host monocytes and macrophages and induces the production of proinflammatory mediators via activation of the Toll-like receptor 1/2 (TLR1/2)-MyD88-dependent pathways. In the present study, we show that OmpU activates murine dendritic cells (DCs) via activation of the TLR2-mediated pathway and the NLRP3 inflammasome, leading to the production of proinflammatory cytokines and DC maturation. Our data reveal that although TLR2 plays an important role in providing both priming and the activation signal for the NLRP3 inflammasome in OmpU-activated DCs, OmpU is capable of activating the NLRP3 inflammasome, even in the absence of TLR2, if a priming signal is given. Furthermore, we show that the OmpU-mediated interleukin-1β (IL-1β) production in DCs depends on calcium flux and mitochondrial reactive oxygen species (mitoROS) generation. Interestingly, both OmpU translocation to the mitochondria of DCs as well as calcium signaling contribute to mitoROS production and prompt NLRP3 inflammasome activation. We also demonstrate that OmpU induces downstream signaling via activation of phosphoinositide-3-kinase (PI3K)-AKT, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and transcription factor NF-κB. Furthermore, our data reveal that OmpU-mediated activation of TLR2 induces signaling via PKC, MAPKs p38 and extracellular signal-regulated kinase (ERK), and transcription factor NF-κB; however, PI3K and MAPK Jun N-terminal protein kinase (JNK) are activated in TLR2 independent manner.

Opposing Roles of DCs and iNKT Cells in the Induction of Foxp3 Expression by MLN CD25+CD4+ T Cells during IFNγ-Driven Colitis

We have previously shown that a deficiency of CD1d-restricted invariant natural killer T (iNKT) cells exacerbates dextran sulfate sodium (DSS)-induced colitis in Yeti mice that exhibit IFNγ-mediated hyper-inflammation. Although iNKT cell-deficiency resulted in reduced Foxp3 expression by mesenteric lymph node (MLN) CD4+ T cells in DSS-treated Yeti mice, the cellular mechanisms that regulate Foxp3 expression by CD25+CD4+ T cells during intestinal inflammation remain unclear. We found that Foxp3-CD25+CD4+ T cells expressing Th1 and Th17 phenotypic hallmarks preferentially expanded in the MLNs of DSS-treated Yeti/CD1d knockout (KO) mice. Moreover, adoptive transfer of Yeti iNKT cells into iNKT cell-deficient Jα18 KO mice effectively suppressed the expansion of MLN Foxp3-CD25+CD4+ T cells during DSS-induced colitis. Interestingly, MLN dendritic cells (DCs) purified from DSS-treated Yeti/CD1d KO mice promoted the differentiation of naive CD4+ T cells into Foxp3-CD25+CD4+ T cells rather than regulatory T (Treg) cells, indicating that MLN DCs might mediate Foxp3+CD25+CD4+ T cell expansion in iNKT cell-sufficient Yeti mice. Furthermore, we showed that Foxp3-CD25+CD4+ T cells were pathogenic in DSS-treated Yeti/CD1d KO mice. Our result suggests that pro-inflammatory DCs and CD1d-restricted iNKT cells play opposing roles in Foxp3 expression by MLN CD25+CD4+ T cells during IFNγ-mediated intestinal inflammation, with potential therapeutic implications.

425.6: Recognition of Donor MHCI Disrupts Recipient Reparative Macrophage Differentiation

Introduction: After tissue injury, infiltrating monocytes differentiate into CD86hiLy6chi pro-inflammatory and CD206+CD301+ reparative macrophages (MΦs) that complete repair. MΦs differentiation is coordinated by tissue injury signals, often referred to as damage-associated molecular patterns (DAMPs), and local cytokines. For example, after heart transplant (HTx) IL-33 is released from graft fibroblasts and acts as a reparative/regulatory DAMP that metabolically reprograms MΦs to differentiate into reparative subsets that protect against chronic HTx rejection. Transplanting allogeneic materials is a novel situation where innate allorecognition of mismatched MHCI may disrupt normal MΦ responses to damaged tissues. For instance, paired immunoglobulin-like receptor (PIR)-A3 on C57BL/6 (B6) monocytes recognition of BALB/c H2-Dd triggers their differentiation into pro-inflammatory dendritic cells that promotes HTx rejection. Monocyte PIR-B binding to self MHCI negatively regulates PIR-A signals. How PIRs shapes DAMP-mediated MΦ differentiation after Tx is not understood, so we hypothesize that in addition to DAMPs, recognition of self versus allogeneic MHCI by PIRs modulates reparative or inflammatory MΦ differentiation. Methods: Monocyte-derived MΦ differentiation in response to donor materials was tested by administration of 20x106 irradiated BALB/c allogeneic (H-2d; Allo) or (B6) syngeneic (H-2b; Syn) splenocytes intraperitoneally alone or with recombinant IL-33. Recipients of donor cellular materials were B6 wildtype (WT), Rag2-/-γc-/-, Pira-/- and Pirb-/- mice. The precise impact of H-2Dd (Allo) or H-2Db (Syn) recombinant MHCI on MΦ differentiation was determined using B6 WT and Pira-/- bone marrow-derived MΦs (BMDMs). Flow cytometry was utilized to assess MΦ differentiation. Results: Administration of Syn cells predominantly stimulated monocyte differentiation into CD206+CD301+ reparative MΦs. Allo cells instead drove monocyte differentiation to pro-inflammatory Ly6chiCD86hi MΦs. A similar outcome was observed in Rag2-/-γc-/- recipients, suggesting these changes are not driven by adaptive immunity. Incubation of BMDMs with recombinant allo MHCI, but not syn MHCI, triggered inflammatory Ly6chiCD86hi MΦs in both WT and Pira-/- BMDMs. Co-administration of allo cells with recombinant IL-33 supported differentiation into CD206+CD301+ reparative MΦs. Interestingly, Pirb-/- mice display reduced C206+CD301+ MΦs, which correlated with decreased IL-33 receptor expression. Conclusions: These data indicate allorecognition by monocytes or MΦs disrupts the typical generation of reparative MΦs in response to injury. We show that monocyte-derived MΦs PIR-B recognition of self MHCI may trigger expression of the receptor needed for IL-33-mediated reparative MΦ differentiation. These new data provide important insights into how innate allorecognition and repair pathways intersect to direct the differentiation of reparative or inflammatory MΦs that shape HTx outcomes. #R01AR073527 (HRT) #5T32AI074490-14 (JPW).

Abstract 601: IO-108, A fully human therapeutic antibody blocking the myeloid checkpoint LILRB2/ILT4, promotes innate and adaptive anti-cancer immunity in preclinical studies

Abstract Tumor-associated myeloid cells inhibit anti-cancer immune responses systemically and in the tumor microenvironment (TME), thereby limiting the efficacy of T cell checkpoint inhibitors. However, the plasticity of myeloid cells may enable therapeutic intervention. The immune inhibitory receptor LILRB2 (also known as ILT4) is expressed primarily by myeloid cells (monocytes, macrophages, dendritic cells and neutrophils), and has emerged as a key myeloid checkpoint contributing to the tolerogenic activity of myeloid cells associated with cancer. LILRB2 has several ligands (classical and non-classical MHC-I, ANGPTL2/5, SEMA4A and CD1) and most of these are known to contribute to immune suppression in the TME. Using computational biology approaches, we found that high LILRB2 expression is associated with macrophage infiltration in many solid tumor types from TCGA. Therefore, LILRB2 is a compelling target to overcome immune suppressive activity of cancer-associated myeloid cells. IO-108 is a fully human IgG4 therapeutic antibody that binds to LILRB2 with high affinity and specificity. IO-108 binds to all myeloid cells in the solid TME and periphery. In vitro studies support that blockade of LILRB2 interaction with its various ligands is the anticipated mechanism of action of IO-108. The LILRB2 antagonist activity of IO-108 produces the desired pro-inflammatory (re)polarization of myeloid cells. IO-108 treatment results in increased pro-inflammatory responses and an enhanced antigen-presenting cell (APC) phenotype to multiple stimuli (e.g., T cell activators, STING and TLR agonists) in ex vivo assays. In addition, IO-108 precludes the anti-inflammatory myeloid cell phenotype resulting from “tumor conditioning” and promotes the differentiation of monocytes and immature dendritic cells (DC) into pro-inflammatory DC. IO-108 enhances the effect of PD-1 blocking antibodies in allogeneic mixed leukocyte reactions of CD4+ T cells and macrophages. Moreover, IO-108 polarized primary myeloid cells isolated from solid tumor patient blood and ovarian cancer-associated ascites towards a proinflammatory phenotype and attenuated their suppressive effect on autologous T cell proliferation and production of proinflammatory cytokines. In vivo, IO-108 inhibits tumor growth in mouse models, which is associated with immune cell activation. Importantly, IO-108 presents a favorable pharmacokinetic and safety profile in preclinical models. Collectively, the preclinical characterization of IO-108 enabled a comprehensive clinical biomarker plan and lends rationale to the clinical study (NCT05054348) of IO-108 as a novel immunotherapy for multiple solid tumor types, including those relapsed/refractory to standard of care therapies. Citation Format: Jing-Tyan Ma, Xiaoye Liu, Ryan Stafford, Krista McCutcheon, Heyu Chen, Tao Huang, Hongyu Tian, Kyu Hong, ChengCheng Zhang, Luke Chung, An Song, Paul Woodard, XCharlene Liao, MariaJose Costa. IO-108, A fully human therapeutic antibody blocking the myeloid checkpoint LILRB2/ILT4, promotes innate and adaptive anti-cancer immunity in preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 601.

Reduced pro-inflammatory dendritic cell phenotypes are a potential indicator of successful peanut oral immunotherapy.

Dendritic cells are important mediators in the early presentation of antigen and regulation of the differentiation of T cells. Peanut oral immunotherapy (POIT) results in desensitization in most peanut allergic individuals (responders), but not in others due to allergic reactions (non-responders). Delineation of early immunologic changes contributing to desensitization would help clarify the POIT mechanism of action. We analyzed dendritic cells in 15 pediatric subjects (5–12 years) undergoing a phase 1 single-center POIT study. We examined dendritic cells at baseline, 6-, 12-, 18- and 24-weeks after initiation of POIT and responders of therapy were compared to non-responders and healthy controls. The distribution frequency of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) from peripheral blood samples were measured in vitro. A general linear mixed model was used, and included fixed effects for cohort (responder, non-responder, or healthy control), time (0-, 6-, 12-, 18-, and 24-weeks), and the cohort-time interaction term. P-values were adjusted for multiple hypothesis testing using Tukey’s method. We observed that POIT responders had reduced TNFa producing myeloid dendritic cells (mDCs) compared to non-responders. Additionally, non-responders had increased OX40L expressing mDCs at 18-weeks compared to responders. In conclusion, our findings suggest that a reduced pro-inflammatory phenotype in DCs could potentially serve as a predictor of early outcome and success of POIT desensitization.

Profiling the myeloid compartment of PBMC in active tuberculosis reveals substantial changes in CD14+ cells and upregulation of CD16 in pro-inflammatory dendritic cells

Abstract Tuberculosis (TB) is the second leading infectious killer after COVID-19 worldwide. Gene expression analysis of whole blood and peripheral blood mononuclear cells (PBMC) from TB infected versus uninfected donors suggests that myeloid cells contribute to the immune signature of TB infection. In this study, we carried out the largest cellular and molecular profiling of the circulating myeloid compartment in the context of Mycobacterium tuberculosis (Mtb) infection to date. Using flow cytometry and RNA sequencing, we isolated and interrogated the transcriptomic profile of myeloid cell subsets isolated from PBMC in a cohort of active TB (ATB) patients with paired sampling at diagnosis and mid-treatment, as well as from Mtb sensitized (IGRA+) and unsensitized (IGRA−) healthy individuals. We identified an increased frequency of CD14+CD16− and CD14+CD16+ myeloid cells in ATB at diagnosis with upregulated expression of interferon signaling genes that significantly overlapped with previously reported blood TB signatures. In CD14+CD16+ cells, there was an additional increased expression of MHC-II related genes, which could be traced down to a subset of pro-inflammatory dendritic cells (DCs), namely CD14+CD163+ DC3. This cell population significantly upregulated CD16 in ATB at diagnosis, thus displaying a CD14+CD16+ phenotype, similarly to intermediate monocytes. This result also suggests CD16 might play an important role in inflammatory DC function in ATB. Thus, our study demonstrates quantitative and qualitative changes in CD14+ myeloid cells that are contributing to blood TB signatures. Additionally, it reveals phenotypic overlaps between subsets of monocytes and DCs in human blood that may hold disease relevance. Supported by a grant from NIH (U19 AI118626)

Paired Immunoglobulin-Like Receptors Impact The Differentiation of Reparative Macrophages Following Allogeneic Challenge

Abstract Monocytes differentiate into CD206+CD301+ reparative macrophages (MΦs) that orchestrate tissue repair in response to damage signals and cytokines. Yet, transplanting allogeneic materials produces a novel situation where innate allorecognition of mismatched MHCI may disrupt normal MΦ responses. B6 monocyte paired immunoglobulin-like receptor (PIR)-A3 recognizes BALB/c H2-Dd to generate pro-inflammatory dendritic cells that promote kidney and heart transplant rejection. Conversely, monocyte PIR-B binding to self MHCI negatively regulates PIR-A signals. How PIRs shape MΦ differentiation after Tx was not understood, so we tested the hypothesis that recognition of self-versus-allogeneic MHCI by PIRs divergently modulates MΦ differentiation. Monocyte-derived MΦ differentiation in wild type (WT), Rag2−/−γc−/−, Pira−/− and Pirb−/− B6 mice was assessed by flow cytometry 3 days after exposure to 20×106 radiation-damaged BALB/c allogeneic (allo) or B6 syngeneic (syn) splenocytes administered intraperitoneally. Syn materials predominantly stimulated monocyte differentiation into phenotypically reparative MΦs. Allo cells instead transformed monocytes exclusively into pro-inflammatory Ly6chiCD86hi MΦs. Rag2−/−γc−/− mice behaved similarly. IL-33 released from injured cells metabolically reprograms infiltrating monocytes and MΦs to support their differentiation into CD206+CD301+MΦs. Interestingly, Pirb−/− mice display greatly reduced C206+CD301+MΦs, which correlated with their decreased expression of the IL-33 receptor. Our data provide important insights into how innate allorecognition by PIR-A disrupts the typical generation of reparative MΦs in response to injured self that is supported by PIR-B. Supported by 5T32AI074490-14

Cord blood sphingolipids are associated with atopic dermatitis and wheeze in the first year of life.

Background:Allergen-sensitized pregnant mice have increased plasma levels of the lipids β-glucosylceramides (βGlcCers) that are transplacentally transferred to the fetus, increased subsets of proinflammatory dendritic cells in the fetal liver and pup lung, and increased allergen-induced offspring lung inflammation.Objective:Our aim was to determine whether these preclinical observations extend to a human association of βGlcCers with wheeze and allergic disease in the prospective Wisconsin Infant Study Cohort.Methods:We measured 74 lipids in cord blood plasma by using mass spectrometry detection of sphingolipids, eicosanoids, and docosinoids, as well as an ELISA for 13-hydroxyoctadecadienoic acid. Lipid profiles were determined by unbiased Uniform Manifold Approximation and Projection dimensional reduction machine learning. Lipid profiles and a proinflammatory lipid index were analyzed for association with maternal allergy and childhood outcomes of wheeze, atopic dermatitis, cord blood leukocytes, and total IgE level at age 1 year.Results:Uniform Manifold Approximation and Projection analysis of lipids defined 8 cluster-specific plasma lipid profiles. Cluster 6 had significantly lower levels of plasma βGlcCers and a higher frequency of cord blood plasmacytoid dendritic cells that mediate anti-inflammatory responses, which is consistent with an anti-inflammatory profile. For clusters and for each infant, a proinflammatory lipid index was calculated to reflect the sum of the proinflammatory lipids minus the anti-inflammatory lipids that were significantly different than in cluster 6. The cluster proinflammatory lipid index was associated with cord blood basophil frequency and with wheeze and atopic dermatitis in the first year of life. The infant inflammatory lipid index was associated with increased risk of wheeze in the first year of life.Conclusion:The cord blood proinflammatory lipid index is associated with early-life atopic dermatitis and wheezing.

Adding Fuel to the Fire? A Role of Intraepithelial Lymphocytes in Enteric Immune Responses to SARS-CoV-2 Infection

Adding Fuel to the Fire? A Role of Intraepithelial Lymphocytes in Enteric Immune Responses to SARS-CoV-2 Infection

The miR-424(322)/503 gene cluster regulates pro- versus anti-inflammatory skin DC subset differentiation by modulating TGF-β signaling.

Transforming growth factor β (TGF-β) family ligands are key regulators of dendritic cell (DC) differentiation and activation. Epidermal Langerhans cells (LCs) require TGF-β family signaling for their differentiation, and canonical TGF-β1 signaling secures a non-activated LC state. LCs reportedly control skin inflammation and are replenished from peripheral blood monocytes, which also give rise to pro-inflammatory monocyte-derived DCs (moDCs). By studying mechanisms in inflammation, we previously screened LCs versus moDCs for differentially expressed microRNAs (miRNAs). This revealed that miR-424/503 is the most strongly inversely regulated (moDCs > LCs). We here demonstrate that miR-424/503 is induced during moDC differentiation and promotes moDC differentiation in human and mouse. Inversely, forced repression of miR-424 during moDC differentiation facilitates TGF-β1-dependent LC differentiation. Mechanistically, miR-424/503 deficiency in monocyte/DC precursors leads to the induction of TGF-β1 response genes critical for LC differentiation. Therefore, the miR-424/503 gene cluster plays a decisive role in anti-inflammatory LC versus pro-inflammatory moDC differentiation from monocytes.

Intestinal Host Response to SARS-CoV-2 Infection and COVID-19 Outcomes in Patients With Gastrointestinal Symptoms

Background & AimsGiven that gastrointestinal (GI) symptoms are a prominent extrapulmonary manifestation of COVID-19, we investigated intestinal infection with SARS-CoV-2, its effect on pathogenesis, and clinical significance.MethodsHuman intestinal biopsy tissues were obtained from patients with COVID-19 (n = 19) and uninfected control individuals (n = 10) for microscopic examination, cytometry by time of flight analyses, and RNA sequencing. Additionally, disease severity and mortality were examined in patients with and without GI symptoms in 2 large, independent cohorts of hospitalized patients in the United States (N = 634) and Europe (N = 287) using multivariate logistic regressions.ResultsCOVID-19 case patients and control individuals in the biopsy cohort were comparable for age, sex, rates of hospitalization, and relevant comorbid conditions. SARS-CoV-2 was detected in small intestinal epithelial cells by immunofluorescence staining or electron microscopy in 15 of 17 patients studied. High-dimensional analyses of GI tissues showed low levels of inflammation, including down-regulation of key inflammatory genes including IFNG, CXCL8, CXCL2, and IL1B and reduced frequencies of proinflammatory dendritic cells compared with control individuals. Consistent with these findings, we found a significant reduction in disease severity and mortality in patients presenting with GI symptoms that was independent of sex, age, and comorbid illnesses and despite similar nasopharyngeal SARS-CoV-2 viral loads. Furthermore, there was reduced levels of key inflammatory proteins in circulation in patients with GI symptoms.ConclusionsThese data highlight the absence of a proinflammatory response in the GI tract despite detection of SARS-CoV-2. In parallel, reduced mortality in patients with COVID-19 presenting with GI symptoms was observed. A potential role of the GI tract in attenuating SARS-CoV-2–associated inflammation needs to be further examined.

Dendritic cells sub-sets are associated with inflammatory cytokine production in progressive vitiligo disease.

In autoimmune onset of vitiligo, perilesional area shows inflammatory cells including T cytotoxic, helper cells and macrophages. Dendritic Cells (DCs) regulate immune activities by antigen presentation to T cells or cytokine production. It is evident that pro- and anti-inflammatory DCs are responsible for their respective cytokines release. However, role of DCs in vitiligo is enigmatic. In the present study, we assessed DCs markers (CD11b and CD11c) along with pro- and anti-inflammatory cytokines (IL-17A, IL-10 and IL-12p70) in stable and active vitiligo patients. Our results revealed a significant augmented expression of CD11b+CD11c+ (pro-inflammatory DC) in peripheral blood mononuclear cells (PBMCs) and skin tissues of active vitiligo patients versus control and stable vitiligo group. Unlikely, CD11b+ (anti-inflammatory DC) levels were significantly impeded in active vitiligo group as compared to another two groups. CD11c (T helper 1 stimulating DC) presented no significant alterations in any group. Furthermore, we perceived significantly up-regulated IL-17A (pro-inflammatory cytokine) and down-regulated IL-10 (anti-inflammatory cytokine) expressions in active vitiligo group as compared to control and stable group (in sera, PBMCs and skin tissue). Also, a significant positive correlation was observed between CD11b+CD11c+ and IL-17A; and CD11b+ and IL-10. Contrarily, CD11b+CD11c+ and CD11b+ were negatively correlated with IL-10 and IL-17A, respectively. In conclusion, modulation of pro- and anti-inflammatory DCs in active vitiligo patients may affect cytokines production and thereby, lead to further depigmentation of skin.

Dietary Fatty Acids in Postprandial Triglyceride-Rich Lipoproteins Modulate Human Monocyte-Derived Dendritic Cell Maturation and Activation.

Dietary fatty acids have been demonstrated to modulate systemic inflammation and induce the postprandial inflammatory response of circulating immune cells. We hypothesized that postprandial triglyceride-rich lipoproteins (TRLs) may have acute effects on immunometabolic homeostasis by modulating dendritic cells (DCs), sentinels of the immunity that link innate and adaptive immune systems. In healthy volunteers, saturated fatty acid (SFA)-enriched meal raised serum levels of granulocyte/macrophage colony-stimulating factor GM-CSF (SFAs > monounsaturated fatty acids (MUFAs) = polyunsaturated fatty acids (PUFAs)) in the postprandial period. Autologous TRL-SFAs upregulated the gene expression of DC maturation (CD123 and CCR7) and DC pro-inflammatory activation (CD80 and CD86) genes while downregulating tolerogenic genes (PD-L1 and PD-L2) in human monocyte-derived DCs (moDCs). These effects were reversed with oleic acid-enriched TRLs. Moreover, postprandial SFAs raised IL-12p70 levels, while TRL-MUFAs and TRL-PUFAs increased IL-10 levels in serum of healthy volunteers and in the medium of TRL-treated moDCs. In conclusion, postprandial TRLs are metabolic entities with DC-related tolerogenic activity, and this function is linked to the type of dietary fat in the meal. This study shows that the intake of meals enriched in MUFAs from olive oil, when compared with meals enriched in SFAs, prevents the postprandial production and priming of circulating pro-inflammatory DCs, and promotes tolerogenic response in healthy subjects. However, functional assays with moDCs generated in the presence of different fatty acids and T cells could increase the knowledge of postprandial TRLs’ effects on DC differentiation and function.

569P Intratumoral injection of allogeneic pro-inflammatory dendritic cells (ilixadencel) in combination with anti-CTLA-4 treatment induce complete tumour responses and anti-tumour immune memory in a mouse tumour model

569P Intratumoral injection of allogeneic pro-inflammatory dendritic cells (ilixadencel) in combination with anti-CTLA-4 treatment induce complete tumour responses and anti-tumour immune memory in a mouse tumour model

Human CD83-targeted chimeric antigen receptor T cells prevent and treat graft-versus-host disease.

Graft-versus-host disease (GVHD) remains an important cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HCT). For decades, GVHD prophylaxis has included calcineurin inhibitors, despite their incomplete efficacy and impairment of graft-versus-leukemia (GVL). Distinct from pharmacologic immune suppression, we have developed what we believe is a novel, human CD83-targeted chimeric antigen receptor (CAR) T cell for GVHD prevention. CD83 is expressed on allo-activated conventional CD4+ T cells (Tconvs) and proinflammatory dendritic cells (DCs), which are both implicated in GVHD pathogenesis. Human CD83 CAR T cells eradicate pathogenic CD83+ target cells, substantially increase the ratio of regulatory T cells (Tregs) to allo-activated Tconvs, and provide durable prevention of xenogeneic GVHD. CD83 CAR T cells are also capable of treating xenogeneic GVHD. We show that human acute myeloid leukemia (AML) expresses CD83 and that myeloid leukemia cell lines are readily killed by CD83 CAR T cells. Human CD83 CAR T cells are a promising cell-based approach to preventing 2 critical complications of allo-HCT - GVHD and relapse. Thus, the use of human CD83 CAR T cells for GVHD prevention and treatment, as well as for targeting CD83+ AML, warrants clinical investigation.

Regulation of Intestinal Epithelial Thymic Stromal Lymphopoietin Expression by Retinoic Acid Receptor Alpha

We have previously targeted the retinoic acid receptor alpha (RARα) isoform in murine intestinal epithelial cells (IECs) to explore the role of epithelial intrinsic retinoid signaling in intestinal immune homeostasis. We noted a significant decrease in the CD11c+ and CD103+ dendritic cell (DC) subpopulations in the intestines of intestinal‐epithelial specific RARα‐deficient (RARαvillin) mice. In the present study, we identified Thymic Stromal Lymphopoietin (TSLP) as a contributing factor that influences intrinsic RARα signaling within IECs as this epithelial‐derived cytokine preferentially stimulates CD103+ DCs to a more tolerogenic phenotype in the GI tract, induces Tregs and drives TH2 polarization. TSLP also influences immune homeostasis by modulating the activation of myeloid cells, inhibition of proinflammatory DCs, and production of IL‐12. Notably, TSLP levels are decreased in Crohn’s disease (CD) where inflammation is driven by IL‐12.We hypothesize that RA intrinsic signaling within IECs modulates TSLP expression via activation of RARα. We sought to generate a RARα−/− murine intestinal epithelial cell line utilizing the CRISPR/CAS9 system to examine the effects of RARα ablation. Second, we aim to establish RARα’s role in regulating intestinal epithelial TSLP expression in both the RARαvillin mice and the CRISPR‐generated RARα−/− cell line.Exon 5 in the open reading frame of the RARα gene in Mode‐K murine IECs was targeted using CRISPR/CAS9. CRISPR cleavage and knockdown of RARα activity was confirmed via surveyor/luciferase assays. The cell lines were phenotyped with regards to their morphology, proliferative ability, and viability. Next, we examined TSLP mRNA expression in the colons of RARαvillin mice and the CRISPR RARα−/− knockout cell line via qRT‐PCR under baseline and stimulated conditions. Wild‐type (WT) and RARα−/− cells were stimulated with RARα‐selective agonist, BMS753, and muramyl dipeptide (MDP), a NOD2‐selective agonist.TSLP expression in surface colonic epithelium was elevated 2.3‐fold in RARαvillin mice compared to WT litter mates. TSLP expression was elevated 10‐fold in the Mode‐K RARα−/− cells versus parent cells at baseline. BMS753 stimulation resulted in a 15‐fold increase in TSLP expression in the RARα−/− cells compared to baseline. When stimulated with MDP, a NOD2‐selective agonist and known stimulus of TSLP production, TSLP expression was elevated 60‐fold in the RARα−/− cells, but significantly impaired compared to the WT cells (400‐fold), suggesting a different regulatory mechanism under stimulated conditions.Identifying a link between dietary factors, such as RA, its receptors and the TSLP molecular pathway can facilitate our understanding of how environmental components contribute to the pathogenesis of inflammatory bowel disease. TSLP expression is controlled by RARα in colonic IECs where it may act as a repressor of TSLP promoter transactivation under baseline conditions. This suggests an important role for RA on myeloid and T cell function via effects on intestinal epithelial TSLP expression.Support or Funding InformationUniversity of Calgary, Department of Medicine and Crohn's Colitis Canada

Recombinant fusion proteins for targeting dendritic cell subsets in therapeutic cancer vaccine.

Dendritic cells (DCs) are professional antigen-presenting cells, which are optimal for the priming of a T cell response against pathogens and tumors. Therefore, many efforts are made to develop therapeutic cancer vaccines which preferentially target the antigen to DC subsets. To this aim, we developed two types of recombinant fusion proteins, which favor antigen delivery to pro-inflammatory DCs as well as the crosstalk between specialized subpopulations of DCs. The first approach combines peptide/CpG vaccination with the recruitment of iNKT cells to the tumor site via CD1d-antitumor scFv fusion proteins. The second approach is targeting the tumor antigen to cross-presenting Xcr1+ DCs via a fusion protein made of Xcl1 fused to a synthetic long peptide followed by an IgG1 Fc fragment. Both strategies allow a potent tumor-specific CD8 T cell response associated with tumor regression or tumor growth delay depending on the model. In the case of iNKT cell activation, the strategy relies on a strong IL-12 release by splenic DCs, while in the second case, the T cell response is strictly dependent on the presence of Xcr1+ cross-presenting DCs.

Sarcocystis fusiformis whole cyst antigen activates pro-inflammatory dendritic cells.

Sarcocystis fusiformis is a coccidian tissue parasite that causes infection in buffalo in countries such an Egypt, China, Iraq and Iran, resulting in significant economic losses to the agricultural industry annually. There is a lack of studies examining host-parasite interactions at the level of the immune response and the present study investigates the interaction between S. fusiformis whole cyst antigens (SFWCA) and dendritic cells (DCs), cells critical to the activation of adaptive immunity. In this study bone marrow derived DCs (BMDCs) were phenotyped following treatment with SFWCA by measuring cell viability, cytokine secretion, and cell surface marker expression. While SFWCA exhibited cytotoxic effects on BMDCs at higher concentrations, lower concentrations of SFWCA activated pro-inflammatory DCs that significantly secreted interleukin (IL)-12p40, tumor necrosis factor alpha, IL-6 and IL-10. These cells also displayed enhanced expression of TLR4, CD80, CD86 and MHC II on their surface, which is indicative of full DCs maturation. Moreover, SFWCA significantly attenuated the capacity of BMDCs to suppress Th2 associated cytokines, notably IL-5 and IL-13, while simultaneously exhibiting no effects on the secretion of interferon (IFN)-γ, IL-2, IL-17, and IL-10. In conclusion, this is the first study to provide fundamental insight into the activation of DCs by SFWCA, providing us with some awareness into the interaction of the Sarcosystis parasite with its host. The pro-inflammatory inducing ability of this antigen is in keeping with studies performed in other protozoan parasites and therefore understanding these interactions is important in the development of future therapeutic strategies.

Human CD83 Targeted Chimeric Antigen Receptor T Cell for the Prevention of Graft Versus Host Disease and Treatment of Myeloid Leukemia

Distinct from pharmacologic immunosuppression, we designed a programmed cytolytic effector T cell that prevents graft versus host disease (GVHD). CD83 is expressed on allo-activated conventional T cells (Tconv) and pro-inflammatory dendritic cells (DCs), which are implicated in GVHD pathogenesis. Therefore we developed a novel human CD83 targeted chimeric antigen receptor (CAR) T cell for GVHD prophylaxis. Here we demonstrate that human CD83 CAR T cells eradicate cell mediators of GVHD, significantly increase the ratio of regulatory T cells (Treg) to allo-activated Tconv, and provide lasting protection from xenogeneic GVHD. Further, we show human, acute myeloid leukemia (AML) expresses CD83 and can be targeted by CD83 CAR T cells. A 2nd generation CD83 CAR was generated with CD3ζ and 41BB costimulatory domain that was retrovirally transduced in human T cells to generate CD83 CAR T cells. The CD83 CAR construct exhibited a high degree of transduction efficiency of about 60%. The CD83 CAR T cells demonstrated robust IFN-γ and IL-2 production, killing, and proliferation when cultured with CD83+ target cells. To test whether human CD83 CAR T cells reduce alloreactivity in vitro, we investigated their suppressive function in allogeneic mixed leukocyte reactions (alloMLR). CD83 CAR T cells were added to 5-day alloMLRs consisting of autologous T cells and allogeneic monocyte-derived DCs at ratios ranging from 3:1 to 1:10. The CD83 CAR T cells potently reduced alloreactive T cell proliferation compared to mock transduced and CD19 CAR T cells. We identified that CD83 is differentially expressed on alloreactive Tconv, compared to Tregs. Moreover, the CD83 CAR T cell efficiently depletes CD83+ Tconv and proinflammatory DCs with 48 hours of engagement. To test the efficacy of human CD83 CAR T cells in vivo, we used an established xenogeneic GVHD model, where mice were inoculated with human PBMCs (25x106) and autologous CD83 CAR (1-10x106) or mock transduced T cells. The CD83 CAR T cells were well tolerated by the mice, and significantly improved survival compared to mock transduced T cells (Figure 1A). Mice treated with CD83 CAR T cells exhibited negligible GVHD target organ damage at day +21 (Figure 1B). Mice inoculated with CD83 CAR T cells demonstrated significantly fewer CD1c+, CD83+ DCs (1.7x106 v 6.2x105, P=0.002), CD4+, CD83+ T cells (4.8x103 v 5.8x102, P=0.005), and pathogenic Th1 cells (3.1x105 v 1.1x102, P=0.005) at day +21, compared to mice treated with mock transduced T cells. Moreover, the ratio of Treg to alloreactive Tconv (CD25+ non-Treg) was significantly increased among mice treated with CD83 CAR T cells (78 v 346, P=0.02), compared to mice injected with mock transduced T cells. Further, CD83 appears to be a promising candidate to target myeloid malignancies. We observed CD83 expression on malignant myeloid K562, Thp-1, U937, and MOLM-13 cells. Moreover, the CD83 CAR T cells effectively killed AML cell lines. Many AML antigens are expressed on progenitor stem cells. Thus, we evaluated for stem cell killing in human colony forming unit (CFU) assays, which demonstrated negligible on-target, off-tumor toxicity. Therefore, the human CD83 CAR T cell is an innovative cell-based approach to prevent GVHD, while providing direct anti-tumor activity against myeloid malignancies. Figure Disclosures Blazar: Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Fate Therapeutics, Inc.: Research Funding; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Davila:Atara: Research Funding; Celgene: Research Funding; Precision Biosciences: Consultancy; Bellicum: Consultancy; GlaxoSmithKline: Consultancy; Adaptive: Consultancy; Anixa: Consultancy; Novartis: Research Funding.