Healthy Peripheral Blood Mononuclear Cells Research Articles

Benzoxazole Derivative K313 Induces Cell Cycle Arrest, Apoptosis and Autophagy Blockage and Suppresses mTOR/p70S6K Pathway in Nalm-6 and Daudi Cells.

Benzoxazole derivative K313 has previously been reported to possess anti-inflammatory effects in lipopolysaccharide-induced RAW264.7 macrophages. To date, there have been no related reports on the anticancer effects of K313. In this study, we found that K313 reduced the viability of human B-cell leukemia (Nalm-6) and lymphoma (Daudi) cells in a dose-dependent manner without affecting healthy peripheral blood mononuclear cells (PBMCs) and induced moderate cell cycle arrest at the G0/G1 phase. Meanwhile, K313 mediated cell apoptosis, which was accompanied by the activation of caspase-9, caspase-3, and poly ADP-ribose polymerase (PARP). Furthermore, cells treated with K313 showed a significant decrease in mitochondrial membrane potential (MMP), which may have been caused by the caspase-8-mediated cleavage of Bid, as detected by Western blot analysis. We also found that K313 led to the downregulation of p-p70S6K protein, which plays an important role in cell survival and cell cycle progression. In addition, treatment of these cells with K313 blocked autophagic flux, as reflected in the accumulation of LC3-II and p62 protein levels in a dose- and time-dependent manner. In conclusion, K313 decreases cell viability without affecting normal healthy PBMCs, induces cell cycle arrest and apoptosis, reduces p-p70S6K protein levels, and mediates strong autophagy inhibition. Therefore, K313 and its derivatives could be developed as potential anticancer drugs or autophagy blockers in the future.

Chloroquine and Rapamycin Augment Interleukin-37 Expression via the LC3, ERK, and AP-1 Axis in the Presence of Lipopolysaccharides

IL-37 is a cytokine that plays critical protective roles in many metabolic inflammatory diseases, and its therapeutic potential has been confirmed by exogenous IL-37 administration. However, its regulatory mechanisms remain unclear. U937 cells were treated with autophagy-modifying reagents (3-MA, chloroquine, and rapamycin) with or without LPS stimulation. Thereafter, IL-37 expression and autophagic markers (Beclin1, P62/SQSTM1, and LC3) were determined. For regulatory signal pathways, phosphorylated proteins of NF-κB (p65 and IκBα), AP-1 (c-Fos/c-Jun), and MAPK signal pathways (Erk1/2 and p38 MAPK) were quantified, and the agonists and antagonists of MAPK and NF-κB pathways were also used. Healthy human peripheral blood mononuclear cells were treated similarly to confirm our results. Four rhesus monkeys were also administered chloroquine to evaluate IL-37 induction in vivo and its bioactivity on CD4 proliferation and activation. IL-37 was upregulated by rapamycin and chloroquine in both U937 cells and human PBMCs in the presence of LPS. IL-37 was preferentially induced in autophagic cells associated with LC3 conversion. AP-1 and p65 binding motifs could be deduced in the sequence of the IL-37 promoter. Inductive IL-37 expression was accompanied with increased phosphorylated Erk1/2 and AP-1 and could be completely abolished by an Erk1/2 inhibitor or augmented by Erk1/2 agonists. In monkeys, chloroquine increased IL-37 expression, which was inversely correlated with CD4 proliferation and phosphorylated STAT3. IL-37 levels were induced by rapamycin and chloroquine through the LC3, Erk1/2, and NF-κB/AP-1 pathways. Functional IL-37 could also be induced in vivo.

Removal of Human Leukemic Cells from Peripheral Blood Mononuclear Cells by Cell Recognition Chromatography with Size Matched Particle Imprints.

We report a cell recognition chromatography approach for blood cancer cell separation from healthy peripheral blood mononuclear cells (PBMCs) based on size-matched functionalized particle imprints. Negative imprints were prepared from layers of 15 μm polymeric microbeads closely matching the size of cultured human leukemic cells (HL60). We replicated these imprints on a large scale with UV curable polyurethane resin using nanoimprinting lithography. The imprints were functionalized with branched polyethylene imine (bPEI) and passivated by Poloxamer 407 to promote a weak attraction toward cells. When a matching cell fits into an imprint cavity, its contact area with the imprint is maximized, which amplifies the attraction and the binding selectivity. We tested these imprints specificity for depleting myeloblasts from a mixture with healthy human PBMCs in a cell recognition chromatography setup hosting the imprint. The mixture of fixed HL60/PBMCs ratio was circulated over the imprint and at each step the selectivity toward HL60 was assessed by flow cytometry. The role of the imprint length, flow rate, channel depth, and the bPEI coating concentration were examined. The results show that HL60 cells, closely matching the imprint cavities, get trapped on the imprint, while the smaller PBMCs are carried away by the drag force of the flow. Lower flow rates, longer imprints, and interim channel depth favor HL60 specific retention. The bPEI concentration higher than 1 wt % on the imprint made it less selective toward the HL60 because of indiscriminate attraction with all cells. Particle imprint based cell recognition chromatography was able to achieve selective myeloblast depletion from initial 11.7% HL60 (88.3% PBMC) to less than 1.3% HL60 for 3 h of circulation. The cell recognition chromatography with size-matched microbead imprints can be employed as an efficient cell separation technique and potentially lead to alternative therapies for myeloblasts removal from peripheral blood of patients with acute myeloid leukemia.

Casticin: A Promising Candidate to Develop a Stem Cell Targeted Strategy in AML Treatment

Purpose: Acute myeloid leukemia is the most common form of acute leukemia with genetic and epigenetic heterogeneity. Although current therapeutic agents provide successful remission, 5-year survival rates are still low. Insufficiency of targeting leukemia stem cells is considered as the main obstacle that causes drug resistance and relapse. Phytochemicals remain a promising source for targeted drug research. Studies showed that Casticin has antiproliferative effects on leukemic cells, but its effects on leukemic stem cells are still unclear. In this study, we aimed to investigate the antiproliferative capacity of Casticin on acute myeloid leukemia stem-like KG1a cells and its relatively mature parental KG1 cells in comparison with healthy peripheral blood mononuclear cells PBMC . Method: The antiproliferative effects of Casticin on cells and IC50 values were determined by MTT test. The effects of Casticin on caspase 3/7 activity, apoptosis and necrosis in cells were evaluated by flow cytometry and TUNEL assays. Results: 2 μM Casticin treatment for 24 h was increased apoptotic cell death and caspase 3/7 activation in KG1 27.2%; 17.30%; p

KIF15 Expression in Tumor-associated Monocytes Is a Prognostic Biomarker in Hepatocellular Carcinoma.

Kinesin family member 15 (KIF15) participates in the transport of macromolecules in essential cellular processes. In this study we evaluated the clinical relevance of KIF15 expression in hepatocellular carcinoma (HCC). Association between KIF15 expression and clinical outcomes in HCC patients was analyzed using three independent cohorts. Localization of KIF15 expression was assessed by immunohistochemical analysis. Co-culture experiments were performed using healthy donor peripheral blood mononuclear cells (PBMC) and HCC cell lines. Immunohistochemical analysis showed that KIF15 was mainly expressed in inflammatory monocytes around cancer cells. Multivariate analysis indicated high KIF15 expression was an independent poor prognostic factor for survival. HCC cells with high expression of minichromosome maintenance protein 2 (MCM2) were located close to KIF15-expressing inflammatory monocytes. The proliferation ability of HCC cells was increased by co-culture with PBMC. High KIF15 expression in inflammatory monocytes in tumor tissues may serve as a prognostic marker for poor outcome in HCC.

Health impact of the commercially cultivated mushroom Agaricus bisporus and wild-growing mushroom Ganoderma resinaceum - a comparative overview

The health promoting effects of hot water extracts obtained from fruiting bodies of the commercially cultivated mushroom Agaricus bisporus (AbHW) and the wild-growing mushroom Ganoderma resinaceum (GrHW) originating from northern Serbia are presented in this research. These abilities were compared in vitro by the prevention of lipid peroxidation (LPx) in a linoleic acid model system, inhibition of the angiotension converting I enzyme (ACE) that could help in the maintenance of a normal blood pressure level and strengthening the ability of the central cholinergic neuron by inhibiting the activity of acetylcholinesterase (AChE). Cytotoxic activities were observed towards selected human malignant (HeLa and K562) cell lines and normal- -human peripheral blood mononuclear cells (PBMC). GrHW contains higher phenolics (5.9 g (100 g)-1), inhibition of LPx (EC50 = 1.07 mg mL-1), ACE (IC50 = 0.54 mg mL-1) and AChE (IC50 = 0.37 mg mL-1), and exhibited a significant selectivity in the antitumour action against HeLa (IC50 = 0.14 mg mL-1) and K562 (IC50 = 0.11 mg mL-1) cells. AbHW contained higher total protein (6.4 g (100 g)-1), carbohydrate (75.4 g (100 g)-1) and ?-glucan (55.1 g (100 g)-1) contents and induced significant proliferation of healthy PBMC from 152?116 % in the concentration range of 0.047?0.187 mg mL-1. The difference in the biological activity of the extracts provides guidance on their use as functional food.

Comparative immunophenotyping of Saccharomyces cerevisiae and Candida spp. strains from Crohn’s disease patients and their interactions with the gut microbiome

Investigation of the fungal communities in animal models of Inflammatory Bowel Diseases (IBD) showed a controversial role of Saccharomyces cerevisiae and Candida spp. In health and disease. These conflicting observations could be ascribed to immunogenic differences among co-specific strains. To assess the relevance of intra-strains differences on yeast immunogenicity and impact on the microbiota, we screened S. cerevisiae and Candida spp. Strains isolated from fecal samples of IBD patients. We compared the cytokine profiles, obtained upon stimulation of Peripheral Blood Mononuclear Cells (PBMCs) and Dendritic Cells with different yeast strains, and evaluated the relationship between strain’s cell wall sugar amount and immune response. Moreover, the gut microbiota composition was explored in relation to fungal isolation from fecal samples by metabarcoding analysis. The comparison of cytokine profiles showed strain dependent rather than species-dependent differences in immune responses. Differences in immunogenicity correlated with the cell wall composition of S. cerevisiae intestinal strains. Stimulation of human healthy PBMCs with different strains showed a pro-inflammatory IL-6 response counterbalanced by IL-10 production. Interestingly, Crohn’s (CD) patients responded differently to “self” and “non-self” strains, eliciting pure Th1 or Th17 cytokine patterns. The differences observed in vitro were recapitulated in vivo, where different strains contributed in dramatically different ways to local epithelial activity and to the inflammation of wild type and Interleukin-deficient mice. Furthermore, we observed that the gut microbiota profiles significantly differentiated according to the presence of Saccharomyces or Candida spp. or the absence of fungal isolates in fecal samples. Our results show the importance to deepen metagenomics and immunophenotyping analyses to the strain level, to elucidate the role of fungal and bacterial communities in health and disease.

Ex vivo immunological evaluation of stable mixed chimeric patients after matched related donor allogeneic transplantation in sickle cell disease

Background aimsAllogeneic hematopoietic stem cell transplantation is curative for sickle cell disease, and the use of matched related donors, non-myeloablative conditioning and sirolimus immunosuppression results in stable mixed chimerism without graft-versus-host disease (GVHD). However, the time to terminate sirolimus while maintaining mixed chimerism is unclear. MethodsIn this study, we developed a two-way mixed lymphocyte reaction (MLR) to evaluate ex vivo immunoreaction in mixed chimeric patients. ResultsIn co-culture of peripheral blood mononuclear cells (PBMCs) from two healthy controls (without irradiation), we detected proliferation at various ratios of PBMC mixtures (1:9 to 9:1) as well as various concentrations of sirolimus, suggesting that two-way MLR is applicable to patients (having >10% chimerism) undergoing sirolimus treatment. In two-way MLR using PBMCs (including donor and recipient cells) from mixed chimeric patients (n = 28), greater ex vivo proliferation was observed <6 months compared with >6 months post-transplant and healthy control PBMC monoculture. Robust ex vivo proliferation was observed in a patient with acute GVHD, and persistent ex vivo proliferation (until 2 years) was observed in a patient with decreasing donor chimerism. ConclusionsIn summary, we demonstrated that in two-way MLR, ex vivo immunoreaction decreases to low levels ~6 months post-transplant. These findings suggest a rationale to continue immunosuppression for 6 months.

Chimeric Antigen Receptor T Cells Targeting NKG2D-Ligands Show Robust Efficacy Against Acute Myeloid Leukemia and T-Cell Acute Lymphoblastic Leukemia

CAR T cell approaches to effectively target AML and T-ALL without off-tumor effects on healthy myeloid or T cell compartments respectively are an unmet medical need. NKG2D-ligands are a promising target given their absence on healthy cells and surface expression in a wide range of malignancies. NKG2D-ligand expression in T-ALL has not previously been interrogated, and reports regarding the prevalence and density of NKG2D-ligand expression in AML vary. A first-in-human clinical trial of CART cells targeting NKG2D ligands in patients with AML demonstrated safety and feasibility (Baumeister et al, Cancer Immunology Research 2019). However, at low CAR T cell doses without lymphodepletion clinical efficacy was limited and in this selective patient cohort, NKG2D-ligand expression was only detectable at low levels. To broadly assess the level of NKG2D-ligand expression in AML and T-ALL and evaluate how this correlates with leukemia susceptibility to CAR T cells targeting NKG2D-ligands, we employed NKG2D-fusion protein-based flow-cytometry and RNA sequencing. CAR T cells bearing the full-length native NKG2D-receptor coupled to the CD3ζ cytoplasmic domain were generated using γ-retroviral transduction and then evaluated in functional assays. We show that NKG2D-ligands are expressed in T-ALL cell lines and primary T-ALL and confirm that NKG2D-ligands are frequently expressed at the surface and RNA-level in primary AML, albeit at relatively low levels. We demonstrate that high-expressing AML and T-ALL targets are exquisitely susceptible to in vitro activity of CAR T cells targeting NKG2D-ligands as evidenced by CD107a degranulation, cytotoxicity and production of IFN-γ and TNF-α. Importantly, we show that even low-level ligand expression in primary leukemia targets results in robust NKG2D-CAR activity. Using mass-cytometry, we reveal a distinct phenotypic profile of NKG2D-CAR T cells following incubation with ligand-positive leukemia targets when compared to controls. This includes selective upregulation of ICOS, 41BB, PD-1, LAG-3 and OX40. Given clinical precedent of pharmacologic HDAC inhibition in AML, we next assessed the impact of preincubation with Valproic Acid (VPA) on NKG2D-ligand expression in AML and whether this enhances susceptibility to NKG2D-CART cells in functional assays. We demonstrate that NKG2D-ligand expression can be selectively enhanced in low-expressing AML cell lines and primary AML blasts when AML cells are pre-incubated with 1mM VPA for 24 hours. Such pharmacologic ligand induction significantly augments NKG2D-CAR anti-leukemic activity in functional assays without affecting healthy Peripheral Blood Mononuclear Cells or CAR T cells. Our findings demonstrate that NKG2D-ligands are viable targets for CAR T cell therapy in AML and T-ALL despite low-level cell-surface expression and provide the rationale for the combination of HDAC-inhibitors with NKG2D-CAR T cell therapy as a potential strategy to achieve clinical NKG2D-CAR T cell efficacy in AML. Figure: A) Specific fluorescence intensity of NKG2D-ligand surface expression in Cell lines and Primary Leukemia cells are displayed in comparison B) NKG2D-CAR T cells (CAR) degranulate and produce proinflammatory cytokines during co-culture with AML and T-ALL lines (representative flow plot, left panel), but not to negative controls, whereas T cells transduced with vector lacking the CAR-transgene (Empty) do not. C) NKG2D-CAR T cells but not Empty control T cells display striking killing activity against AML and T-ALL cell lines. D) Preincubation of primary AML blasts with 1mM VPA for 24 hours enhances NKG2D-ligand expression and significantly augments NKG2D-CAR T cell activity, shown here as IFN-γ production during co-culture with 3 independent primary AML samples. Figure Disclosures Dranoff: Novartis: Employment. Ritz:Equillium: Research Funding; Merck: Research Funding; Kite Pharma: Research Funding; Aleta Biotherapeutics: Consultancy; Celgene: Consultancy; Avrobio: Consultancy; LifeVault Bio: Consultancy; Draper Labs: Consultancy; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy.

Overcoming the Supportive Stroma-Induced Proliferation in Waldenstrom's Macroglobulinemia By Selective Inhibition of the FGF/FGF-Receptor Axis

The human fibroblast growth factor receptor (FGF-R) family plays an essential role in a wide range of cellular processes, such as cell growth, proliferation, differentiation, migration and survival. It has been reported that FGF-Rs are expressed in hematopoietic cells; and FGF/FGFR signaling deregulation is largely involved in hematologic malignancies, including Waldenström macroglobulinemia (WM). WM is still an incurable disease, and patients succumb due to disease progression. Therefore, novel therapeutics designed to specifically target deregulated signaling pathways in WM are required. We aimed to investigate the role of FGF/FGF-R system in FGF-dependent WM cell lines by using an anti-pan FGF trap molecule (NSC12), responsible for FGF/FGF-R blocking. We first interrogated the GSE9656 dataset in order to confirm the expression of FGFs and FGF-Rs in WM cells, demonstrating an enrichment of several FGF- and FGF-R-isoforms in primary WM patients' derived tumor cells compared to the normal cellular counterpart (P&lt;0.05); and demonstrated the ability of NSC12 to inhibit FGF-secretion within the conditioned media of NCS12-treated WM cells, as shown by ELISA. Wide-transcriptome profiling of NSC12-treated WM cells (BCWM.1; MWCL1) revealed a significant inhibition of Myc-target related genes, coupled with silencing of genes involved in cell cycle progression, cell proliferation, PI3K-AKT-mTOR signaling, oxidative phosphorylation (Hallmark; FDR&lt;0.25; P&lt;0.05). This prompted us to evaluate the anti-tumor functional sequelae exerted by NSC12 in WM cells: NSC12 induced significant inhibition of WM cell growth (BCWM1 and WMCL1) in a dose-dependent fashion (0.1-10μM; IC50 ~3μM), even in the presence of bone marrow microenvironment. In addition, a significant effect was also observed in primary tumor cells from WM patients; while no effect was observed on healthy donor-derived peripheral blood mononuclear cells. The growth inhibitory effect was associated with induction of apoptotic cell death, caspase activation and PARP cleavage, as demonstrated by flow cytometry and western blot, respectively. Moreover, we also observed a NSC12 dose-dependent increase of mitochondrial reactive oxigen species (ROS), at protein level. Cell cycle analysis revealed a reduction of the S-phase and increase of G0/G1 phase. Mechanistically, NSC12 targeted WM cells by inhibiting MAPK, JAK/STAT3 and PI3K-Akt pathways known to be FGFRs-activated signaling cascades. Importantly, the same effect was maintained in WM cells even in the presence of the supporting BM microenvironment. Functional studies demonstrated the ability of NSC12 to impair the adhesion of both cell lines to the supportive primary bone marrow stromal cells, in vitro. NCS12-dependnet anti-WM activity was also tested in combination with bortezomib, carfilzomib, everolimus and ibrutinib: the combinatory treatment (48h) resulted in a more significant dose-dependent inhibition of WM cell survival and proliferation (P&lt;0.05), thus suggesting the rational for combining FGF-blockade with proteasome-, mTOR-, or BTK-inhibitors. In vivo studies are being performed, in order to further corroborate the anti-WM activity of NSC12 using WM animal models. Disclosures Ronca: Associazione Italiana per la Ricerca sul Canctro (AIRC): Research Funding. Rossi:Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead: 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; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Roccaro:AstraZeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; European Hematology Association: Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Transcan2-ERANET: Research Funding; AstraZeneca: Research Funding; European Hematology Association: Research Funding; Transcan2-ERANET: Research Funding.

Monoclonal anti-dsDNA antibody 2C10 escorts DNA to intracellular DNA sensors in normal mononuclear cells and stimulates secretion of multiple cytokines implicated in lupus pathogenesis.

SummaryThere have been many studies on the mechanisms of internalization of DNA–anti‐DNA immune complexes by cells, including the one used for rheumatoid factor‐expressing mouse B cells. In parallel, studies on the role of intracellular DNA sensors in the pathogenesis of systemic lupus erythematosus (SLE) have been conducted, including the one using a mouse model lacking one of the sensors. These and other data have established a framework for understanding the pathogenic role of anti‐DNA antibodies, but studies on normal cells are limited. Here, we used the monoclonal anti‐dsDNA antibody 2C10, 2‐kbp dsDNA and healthy human peripheral blood mononuclear cells (PBMCs) to test whether and how 2C10 and/or DNA cause pathology in normal cells. We found that on culture with PBMCs, 2C10 preferentially entered monocytes and that DNA enhanced this internalization. In contrast, DNA alone was not significantly internalized by monocytes, but 2C10 facilitated its internalization. This was suppressed by cytochalasin D, but not by methyl‐β‐cyclodextrin, chloroquine or an Fc blocker, suggesting the involvement of macropinocytosis in this process. Internalization of 2C10 and DNA together resulted in production of interferon (IFN)‐α, IFN‐γ, tumor necrosis factor (TNF)‐α, monocyte chemoattractant protein‐1 (MCP‐1), interleukin (IL)‐1β, IL‐6, IL‐10 and IL‐33 by PBMCs. Cytokine production was suppressed by chloroquine and shikonin, but not by RU.521, suggesting dependence on activation of the Toll‐like receptor (TLR)‐9 and absent in melanoma 2 (AIM‐2) pathways. These results established a simple model to demonstrate that anti‐DNA antibodies can cause dysregulation of cytokine network mimicking systemic lupus erythematosus in culture of normal PBMCs, and emphasize again the importance of maintaining anti‐DNA antibodies at low levels by treatment.

Effect of β-D-Mannuronic Acid (M2000) on Oxidative Stress Enzymes' Gene Using Healthy Donor Peripheral Blood Mononuclear Cells for Evaluating the Anti-Aging Property.

This research aimed to study the anti-aging and anti-inflammatory effects of low and high doses of the β-D-mannuronic (M2000) on gene expression of enzymes involved in oxidative stress (including SOD2, GST, GPX1, CAT, iNOS, and MPO) in peripheral blood mononuclear cells (PBMCs) of healthy donors under in vitro conditions. The PBMCs were separated and the RNAs were then extracted and the cDNAs synthesized, and expression levels of the mentioned genes were detected by qRT-PCR. Our results indicated that the high dose of this drug could significantly reduce the expression level of the SOD2 gene compared to the lipopolysaccharide (LPS) group (p < 0.0001). Moreover, it was found that the high dose of this drug could significantly decrease the expression level of the GST gene compared to the LPS group (p < 0.0001). However, no significant reductions were observed in expression levels of the CAT and GPX1 genes compared to the LPS group. Furthermore, our data revealed that the level of iNOS and MPO gene expression was significantly reduced, in both doses of M2000, respectively, compared to the LPS group (p < 0.0001). This research showed that M2000 as a novel NSAID with immunosuppressive properties could modify oxidative stress through lowering expression levels of the SOD2, GST, iNOS, and MPO genes compared to the healthy expression levels, with a probable reduction of the risk of developing inflammatory diseases related to age and aging.

Switch of NAD Salvage to de novo Biosynthesis Sustains SIRT1-RelB-Dependent Inflammatory Tolerance.

A typical inflammatory response sequentially progresses from pro-inflammatory, immune suppressive to inflammatory repairing phases. Although the physiological inflammatory response resolves in time, severe acute inflammation usually sustains immune tolerance and leads to high mortality, yet the underlying mechanism is not completely understood. Here, using the leukemia-derived THP-1 human monocytes, healthy and septic human peripheral blood mononuclear cells (PBMC), we report that endotoxin dose-dependent switch of nicotinamide adenine dinucleotide (NAD) biosynthesis pathways sustain immune tolerant status. Low dose endotoxin triggered nicotinamide phosphoribosyltransferase (NAMPT)-dependent NAD salvage activity to adapt pro-inflammation. In contrast, high dose endotoxin drove a shift of NAD synthesis pathway from early NAMPT-dependent NAD salvage to late indoleamine 2,3-dioxygenase-1 (IDO1)-dependent NAD de novo biosynthesis, leading to persistent immune suppression. This is resulted from the IDO1-dependent expansion of nuclear NAD pool and nuclear NAD-dependent prolongation of sirtuin1 (SIRT1)-directed epigenetics of immune tolerance. Inhibition of IDO1 activity predominantly decreased nuclear NAD level, which promoted sequential dissociations of immunosuppressive SIRT1 and RelB from the promoter of pro-inflammatory TNF-α gene and broke endotoxin tolerance. Thus, NAMPT-NAD-SIRT1 axis adapts pro-inflammation, but IDO1-NAD-SIRT1-RelB axis sustains endotoxin tolerance during acute inflammatory response. Remarkably, in contrast to the prevention of sepsis death of animal model by IDO1 inhibition before sepsis initiation, we demonstrated that the combination therapy of IDO1 inhibition by 1-methyl-D-tryptophan (1-MT) and tryptophan supplementation rather than 1-MT administration alone after sepsis onset rescued sepsis animals, highlighting the translational significance of tryptophan restoration in IDO1 targeting therapy of severe inflammatory diseases like sepsis.

Mitochondrial Function in Peripheral Blood Mononuclear Cells (PBMC) Is Enhanced, Together with Increased Reactive Oxygen Species, in Severe Asthmatic Patients in Exacerbation.

Asthma is a chronic inflammatory lung syndrome with an increasing prevalence and a rare but significant risk of death. Its pathophysiology is complex, and therefore we investigated at the systemic level a potential implication of oxidative stress and of peripheral blood mononuclear cells’ (PBMC) mitochondrial function. Twenty severe asthmatic patients with severe exacerbation (GINA 4–5) and 20 healthy volunteers participated at the study. Mitochondrial respiratory chain complexes activities using different substrates and reactive oxygen species (ROS) production were determined in both groups by high-resolution respirometry and electronic paramagnetic resonance, respectively. Healthy PBMC were also incubated with a pool of plasma of severe asthmatics or healthy controls. Mitochondrial respiratory chain complexes activity (+52.45%, p = 0.015 for VADP) and ROS production (+34.3%, p = 0.02) were increased in asthmatic patients. Increased ROS did not originate mainly from mitochondria. Plasma of severe asthmatics significantly increased healthy PBMC mitochondrial dioxygen consumption (+56.8%, p = 0.031). In conclusion, such asthma endotype, characterized by increased PMBCs mitochondrial oxidative capacity and ROS production likely related to a plasma constituent, may reflect activation of the immune system. Further studies are needed to determine whether increased PBMC mitochondrial respiration might have protective effects, opening thus new therapeutic approaches.

P2.04-44 Combined Immune Checkpoint Blockade in Mesothelioma: In Vivo Investigation of in Vitro Data

Malignant pleural mesothelioma (MPM) is an aggressive cancer that is causally associated with asbestos exposure. Due to its aggressive nature and despite the effectiveness of conventional anti-cancer treatment, the prognosis of patients diagnosed with MPM remains dismal, highlighting the urgent need for new therapeutic strategies. Our group and others have recently demonstrated PD-1 and PD-L1 expression in MPM patients, providing rationale to evaluate their suitability as immunotherapeutic targets in MPM. Tree human cell lines representative for the epithelioid and sarcomatoid subtypes of MPM were placed in allogeneic co-cultures with healthy donor peripheral blood mononuclear cells. The co-cultures were treated with the following immune checkpoint blocking antibodies: anti PD-1 (Nivolumab®, BMS) or anti PD-L1 (Durvalumab®, AstraZeneca) in combination with anti TIM-3 or anti LAG-3. Supernatant was collected and enzyme-linked immunosorbent assays and multiplex electrochemo-luminescence were used to look at the secretion of 7 cytokines, being IFNγ, IL-2/5/6/10, IL-1band TNF-α, as well as the enzyme granzyme B. Significant differences were found for the secretion of IFNγ, granzyme B, IL-2, IL-5 and IL-10. Though the differences were not always significant for the 3 MPM cell lines, the same trends were observed among them. Interestingly, highest concentrations of the aforementioned cytokines were all noticed for monotherapy treatment with anti PD-1, anti PD-L1 or or their combination with anti TIM-3. In vivo investigation of PD-1, PD-L1 and TIM-3 blockade, alone or in combination is required for validation of our in vitro results and is currently ongoing. Our data show that treatment with anti PD-1, anti PD-L1 or their respective combination with anti TIM-3 resulted in the highest secretion of cytokines and granzyme B, suggesting that these treatments stimulate the antitumor response the most. Results of our in vivo validation are awaited in order to confirm our in vitro findings.

PGE1-Containing Protocols Generate Mature (Leukemia-Derived) Dendritic Cells Directly from Leukemic Whole Blood.

Dendritic cells (DCs) and leukemia-derived DC (DCleu) are potent stimulators of various immunoreactive cells and they play a pivotal role in the (re-) activation of the immune system. As a potential treatment tool for patients with acute myeloid leukemia, we developed and analyzed two new PGE1-containing protocols (Pici-PGE1, Kit M) to generate DC/DCleu ex vivo from leukemic peripheral blood mononuclear cells (PBMCs) or directly from leukemic whole blood (WB) to simulate physiological conditions. Pici-PGE1 generated significantly higher amounts of DCs from leukemic and healthy PBMCs when compared to control and comparable amounts as the already established protocol Pici-PGE2. The proportions of sufficient DC-generation were even higher after DC/DCleu-generation with Pici-PGE1. With Kits, it was possible to generate DCs and DCleu directly from leukemic and healthy WB without induction of blast proliferation. The average amounts of generated DCs and DCleu-subgroups were comparable with all Kits. The PGE1 containing Kit M generated significantly higher amounts of mature DCs when compared to the PGE2-containing Kit K and increased the anti-leukemic-activity. In summary PGE1-containing protocols were suitable for generating DC/DCleu from PBMCs as well as from WB, which reliably (re-) activated immunoreactive cells, improved the overall ex vivo anti-leukemic activity, and influenced cytokine-release-profiles.

Cystic Fibrosis Plasma Blunts the Immune Response to Bacterial Infection.

Cystic fibrosis (CF) is caused by mutations of the gene encoding the CF transmembrane conductance regulator. It remains unclear whether the abnormal immune response in CF involves extrinsic signals released from the external or internal environment. We sought to characterize the peripheral immune signatures in CF and its association with clinical phenotypes. Healthy peripheral blood mononuclear cells (PBMCs) were cultured with plasma from CF probands (CFPs) or healthy control subjects (HCs) followed by nCounter gene and microRNA (miRNA) profiling. A discovery cohort of 12 CFPs and 12 HCs and a validation cohort of 103 CFPs and 31 HCs (our previous microarray data [GSE71799]) were analyzed to characterize the composition of cultured immune cells and establish a miRNA‒mRNA network. Cell compositions and miRNA profiles were associated with clinical characteristics of the cohorts. Significantly differentially expressed genes and abundance of myeloid cells were downregulated in PMBCs after culture with CF plasma (P < 0.05). Top-ranked miRNAs that increased in response to CF plasma (adjusted P < 0.05) included miR-155 and miR-146a, which target many immune-related genes, such as IL-8. Pseudomonas aeruginosa infection was negatively associated with abundance of monocytes and the presence of those regulatory miRNAs. Extrinsic signals in plasma from patients with CF led to monocyte inactivation and miRNA upregulation in PBMCs. An improved understanding of the immune effects of extrinsic factors in CF holds great promise for integrating immunomodulatory cell therapies into current treatment strategies in CF.

ADAM17-Mediated Reduction in CD14++CD16+ Monocytes ex vivo and Reduction in Intermediate Monocytes With Immune Paresis in Acute Pancreatitis and Acute Alcoholic Hepatitis.

Impaired immune responses and increased susceptibility to infection characterize acute inflammatory conditions such as pancreatitis and alcoholic hepatitis and are major causes of morbidity and mortality. However, the mechanisms that drive this apparent immune paresis remain poorly understood. Monocytes mediate host responses to damage and pathogens in health and disease, and three subsets of monocytes have been defined based on CD14 and CD16 expression. We sought to determine the changes in monocyte subsets in acute pancreatitis (AP) and acute alcoholic hepatitis (AAH), together with functional consequences and mechanisms that underlie this change. Peripheral blood mononuclear cells (PBMCs) from patients with AP or AAH were compared with healthy controls. Monocyte subsets were defined by HLA-DR, CD14, and CD16 expression. Changes in surface and intracellular protein expression and phosphorylation were determined by flow cytometry. Phenotype and function were assessed following stimulation with lipopolysaccharide (LPS) or other agonists in the presence of specific inhibitors of TNFα and a disintegrin and metalloproteinase 17 (ADAM17). Patients with AP and AAH had reduced CD14++CD16+ intermediate monocytes compared to controls. Reduction of intermediate monocytes was recapitulated ex vivo by stimulating healthy control PBMCs with Toll-like receptor (TLR) agonists LPS, flagellin or polyinosilic:polycytidylic acid (poly I:C). Stimulation caused shedding of CD14 and CD16, which could be reversed using the ADAM17 inhibitor, TMI005 but not direct inhibitors of TNFα, a known ADAM17-target. Culturing PBMCs from healthy controls resulted in expansion of intermediate monocytes, which did not occur when LPS was in the culture medium. Cultured intermediate monocytes showed reduced expression of CX3CR1, CCR2, TLR4, and TLR5. We found reduced migratory responses, intracellular signaling and pro-inflammatory cytokine production, and increased expression of IL-10. Stimulation with TLR agonists results in ADAM17-mediated shedding of phenotypic markers from CD16+ monocytes, leading to apparent “loss” of intermediate monocytes. Reduction in CD14++CD16− monocytes and increased CD14++CD16+ is associated with altered responses in functional assays ex vivo. Patients with AP and AAH had reduced proportions of CD14++CD16+ monocytes and reduced phosphorylation of NFκB and IL-6 production in response to bacterial LPS. Together, these processes may contribute to the susceptibility to infection observed in AP and AAH.

Building a Bridge between Chemotherapy and Immunotherapy in Malignant Pleural Mesothelioma: Investigating the Effect of Chemotherapy on Immune Checkpoint Expression.

In light of the promising results of immune checkpoint blockade (ICPB) in malignant pleural mesothelioma (MPM), we investigated the effect of different chemotherapeutic agents on the expression of immune checkpoints (ICPs) in order to rationally design a good treatment schedule for their combination with ICP blocking antibodies. Cisplatin, oxaliplatin and pemetrexed are interesting chemotherapeutic agents to combine with immunotherapy given their immunomodulatory capacities. We looked into cisplatin and pemetrexed because their combination is used as first-line treatment of MPM. Additionally, the effect of the immunogenic chemotherapeutic agent, oxaliplatin, was also studied. Three different MPM cell lines were used for representation of both epithelioid and sarcomatoid subtypes. The desired inhibitory concentrations of the chemotherapeutic agents were determined with the SRB-assay. Allogeneic co-cultures of MPM cells with healthy donor peripheral blood mononuclear cells (PBMC) were set up to assess the effect of these chemotherapeutic agents on the expression of ICPs (PD-1, LAG-3, TIM-3) and their ligands (PD-L1, PD-L2, galectin-9). Cisplatin might be a promising treatment to combine with ICP blocking antibodies since our MPM cell lines were most susceptible to this stand-alone treatment. We found that the expression of ICPs and their ligands on both MPM cells and PBMC was mostly downregulated or unaltered when treated with chemotherapeutic agents, though no clear trend could be determined.

Horses with equine recurrent uveitis have an activated CD4+ T-cell phenotype that can be modulated by mesenchymal stem cells in vitro.

Equine recurrent uveitis (ERU) is an immune‐mediated disease causing repeated or persistent inflammatory episodes which can lead to blindness. Currently, there is no cure for horses with this disease. Mesenchymal stem cells (MSCs) are effective at reducing immune cell activation in vitro in many species, making them a potential therapeutic option for ERU. The objectives of this study were to define the lymphocyte phenotype of horses with ERU and to determine how MSCs alter T‐cell phenotype in vitro. Whole blood was taken from 7 horses with ERU and 10 healthy horses and peripheral blood mononuclear cells were isolated. The markers CD21, CD3, CD4, and CD8 were used to identify lymphocyte subsets while CD25, CD62L, Foxp3, IFNγ, and IL10 were used to identify T‐cell phenotype. Adipose‐derived MSCs were expanded, irradiated (to control proliferation), and incubated with CD4+ T‐cells from healthy horses, after which lymphocytes were collected and analyzed via flow cytometry. The percentages of T‐cells and B‐cells in horses with ERU were similar to normal horses. However, CD4+ T‐cells from horses with ERU expressed higher amounts of IFNγ indicating a pro‐inflammatory Th1 phenotype. When co‐incubated with MSCs, activated CD4+ T‐cells reduced expression of CD25, CD62L, Foxp3, and IFNγ. MSCs had a lesser ability to decrease activation when cell‐cell contact or prostaglandin signaling was blocked. MSCs continue to show promise as a treatment for ERU as they decreased the CD4+ T‐cell activation phenotype through a combination of cell‐cell contact and prostaglandin signaling.