Healthy Human Peripheral Blood Research Articles

Human decidua basalis mesenchymal stem/stromal cells enhance anticancer properties of human natural killer cells, in vitro.

Mesenchymal stem cells/stromal cells from the Decidua Basalis of the human placenta (DBMSCs) express wide range of effector molecules that modulate the functions of their target cells. These properties make them potential candidate for use in cellular therapy. In this study, we have investigated the consequences of interaction between DBMSCs and natural killer (NK) cells for both cell types. DBMSCs were cultured with IL-2-activated and resting non-activated NK cells isolated from healthy human peripheral blood and various functional assays were performed including, NK cell proliferation and cytolytic activities. Flow cytometry and microscopic studies were performed to examine the expression of NK cell receptors that mediate these cytolytic activities against DBMSCs. Moreover, the mechanism underlying these effects was also investigated. Our findings revealed that, co-culture of DBMSCs and NK cells resulted in inhibition of proliferation of resting NK cells, while proliferation of IL-2 activated NK cells was increased. Contrarily, treatment of DBMSC's with comparatively high numbers of IL-2 activated NK cells, resulted in their lysis, whereas treatment with low numbers resulted in reduction in their proliferation. Cytolytic activity of NK cells against DBMSCs was mediated by several activating NK cell receptors. In spite of the expression of HLA class I molecules by DBMSCs, they were still lysed by NK cells, excluding their involvement in cytolytic activity. In addition, preconditioning NK cells by DBMSCs, enhanced their ability to suppress tumor cell proliferation and in severe cases resulted in their partial lysis. Lysis and decrease of tumor cell proliferation is associated with increased expression of important molecules involved in anticancer activities. We conclude that DBMSCs exhibit dualfunctions on NK cells that enhance their anticancer therapeutic potential.

The β2-adrenergic biased agonist nebivolol inhibits the development of Th17 and the response of memory Th17 cells in an NF-κB-dependent manner.

Adrenergic receptors regulate metabolic, cardiovascular, and immunological functions in response to the sympathetic nervous system. The effect of β2-adrenergic receptor (AR) as a high expression receptor on different subpopulations of T cells is complex and varies depending on the type of ligand and context. While traditional β2-AR agonists generally suppress T cells, they potentially enhance IL-17A production by Th17 cells. The effects of pharmacological drugs that count as biased agonists of AR like nebivolol are not completely understood. We investigated the impact of nebivolol on human memory CD4+ T (Th1, Th2, Th17) cells and polarized naive Th17 cells, highlighting its potential for IL-17A suppression via a non-canonical β2-AR cell signaling pathway. The effects of nebivolol were tested on healthy human peripheral blood mononuclear cells, purified memory Th cells, and polarized naive Th17 cells activated with anti-CD3/anti-CD28/anti-CD2 ImmunoCult reagent. IFN-γ, IL-4, and IL-17A, which are primarily derived from Th1, Th2, and Th17 cells, respectively, were quantified by ELISA and flow cytometry. IL-10 was measured by ELISA. Gene expression of RORC, ADRB1, ADRB2, and ADRB3 was evaluated by qPCR. The ADRB2 gene was knocked out in memory Th cells using CRISPR/Cas9. Protein expression of phosphorylated serine133-CREB and phosphorylated NF-κB p65 was assessed by Western blot. Proliferation was assessed by fluorescent dye loading and flow cytometry. Nebivolol treatment decreased IL-17A and IFN-γ secretion by activated memory Th cells and elevated IL-4 levels. Nebivolol reduced the proportion of IL-17A+ Th cells and downregulated RORC expression. Unlike the β2-AR agonist terbutaline, nebivolol inhibited the shift of naive CD4+ T cells toward the Th17 phenotype. IL-10 and the proliferation index remained unchanged. Nebivolol-treated β2-knockout memory Th cells showed significant inhibition of β2-AR-mediated signaling, evidenced by the absence of IL-17A suppression compared to controls. Phosphorylation of the NF-κB p65 subunit was inhibited by nebivolol, but CREB phosphorylation was not changed, suggesting a selective transcriptional control. The findings demonstrate that nebivolol acts through a β2-AR-mediated signaling pathway, as a distinctive anti-inflammatory agent capable of selectively shifting Th17 cells and suppressing the phosphorylation of NF-κB. This highlights nebivolol's potential for therapeutic interventions in chronic autoimmune conditions with elevated IL-17A levels.

Establishment of a humanized mouse model of HIV-1 infection and quantification of integrated HIV proviral DNA in vivo

In recent years, virological, pathological, and immunological studies need to be carried out for the emerging anti-human immunodeficiency virus (HIV) therapies such as gene therapy, broadly neutralizing antibodies, and the derived chimeric antigen receptor (CAR)-T immunotherapy, which necessitates suitable, simple, and inexpensive small-animal models and methods for accurate quantification of the viral genome in the HIV-1 infected. In our research, the HIV-∆ENV-Jurkat-EGFP-mCherry cell line was engineered through the infection with a dual-labelled HIV pseudovirus. A nested quantitative PCR (nested-qPCR) method with the cellular genome as the integrated standard was established for the quantification of HIV proviral copies. We administered intravenous injections of healthy human peripheral blood mononuclear cell (PBMC) into NOD/Prkdcscid/IL2rgnull (NPG) mice. To verify engraftment kinetics, we analyzed the percentages of hCD45+, hCD3+, hCD4+, and hCD8+ cells in the peripheral blood of hu-PBMC-NPG mice. To evaluate HIV-1 infection in hu-PBMC-NPG mice, we inoculated these mice with HIV NL4-3-NanoLuc by intraperitoneal (IP) injection. We then monitored the luciferase expression by the small animal imaging system and measured the viral load in the spleen by qPCR. The infiltration of human PBMCs in mice was detected 3-5 weeks after intravenous injection, and the percentage of hCD45 in humanized mouse PBMCs were more than 25% five weeks after IP inoculation. The expression of the virus-associated luciferase protein was detected by luciferase imaging 27 days post infection. Moreover, the viral total DNA, RNA, and proviral DNA copies reached 18 000 copies/106 cells, 15 000 copies/μg RNA, and 15 000 copies/106 cells, respectively, in the mouse spleen. Taken together, we reported a convenient method for building a simple humanized mouse model of HuPBMC-NPG/severe combined immunodeficiency (SCID) by intravenous injection with hu-PBMCs without advanced surgical skills and irradiation. Furthermore, we established a convenient method for the efficient determination of proviral DNA to assess HIV replication in vivo, viral reservoir sizes, and efficacy of novel anti-HIV therapies including CAR-T immunotherapy and gene therapy.

WU-NK-101 (W-NK), a Memory-like (ML) NK Cell, Intrinsically Overcomes Factors Restricting Adoptive Cell Therapy (ACT) in Acute Myeloid Leukemia (AML)

ML-NK cells are generated from conventional (c) NK cells activated with cytokines IL-12/15/18 which results in enhanced fitness, in vivo persistence, and function against tumor targets. While effective in early phase clinical trials, a 1 donor to 1 recipient cell product hinders commercial development as a cellular therapeutic. Here, W-NK is a cytokine-reprogrammed, expanded, cryopreserved ML-NK product derived from healthy human peripheral blood (PBMC) cNK cells, with scalable manufacturability. W-NKs harness the benefits of ML-NKs, and further overcome challenges previously reported for adoptive cell therapy (ACT) in AML: antigen escape, limited tumor trafficking/infiltration, and restricted function in tumor microenvironment (TME). W-NK characterization was performed by scRNA-seq (10X Genomics) and multidimensional flow and mass cytometry.Functionality was evaluated by in vitro cytotoxicity assays against AML cell lines (THP-1, and HL-60) and bioenergetics assays (Seahorse XF), under different conditions: conventional (N) media (pH 7, glucose 11 mM), or two models for TME (TME-aligned media - pH 5.9, glucose 6 mM, immune suppressive agents, e.g., PGE2, TGFb1, and malignant ascites), and in hypoxia (0.1% O 2; AVATAR). In vivo efficacy was evaluated in NSG mice bearing THP-1 AML tumors. Proteomic data was acquired by tandem-mass spectrometry (Sciex Zeno TOF 7600). Cell trafficking was measured by fluorescent-labeled W-NK in NSG mice. W-NK cells are transcriptionally distinct from cNKs. Sc-RNA-seq analysis revealed important transcriptional and compositional differences compared to cNK cells, including novel NK clusters expressing metabolic ( GLUL), proliferation-associated ( Ki67) and IL-15-inducible genes ( TXNIP) known to enhance NK responses. Additionally, W-NKs express higher levels of activating receptors ( i.e. 2B4, DNAM-1, NKG2D, NKp30, etc.), cytotoxic effector proteins ( i.e. granzyme B), and lower levels of inhibitory receptors leading to increased potency in vitro against HL-60 cells (mEC50 1.7 and 5.2; p= 0.032; W-NK vs. cNK respectively) and demonstrated a dose dependent reduction of tumor burden in THP-1 xenografts ( p=0.015). W-NK also express high levels of CXCR4 (81.6% positive, MFI 5552), contributing to improved bone marrow (BM) homing (16.5 ± 2.4%) compared to data previously reported for IL-2-expanded NK cells (3%; Sato et al.CCR. 2020). Once in the TME, unlike cNK and T-cells, W-NK cytotoxic function is preserved (TME-media vs. N-media-; W-NK: 96.5% vs. 88.2%, p=0.45; cNK: 44.1% vs.13.5%, p=0.009; T-cell: 53.2 % vs. 25.2%, p=0.041, respectively). This is potentially due to W-NK resistance to hypoxia, and improved metabolic fitness/flexibility. W-NK cytotoxic activity was also preserved in hypoxic conditions (90% vs. 89.5%, p=0.2) compared to cNK (24% vs. 6.9%, p=0.02; normoxia vs. hypoxia). Metabolically, W-NK express higher levels of cell surface nutrient transporters, implying better adaptation to adverse TMEs ( i.e. upregulation of amino acids, lactate, and pyruvate). This might enable utilization of diverse macronutrients, activation of metabolic pathways, and optimal ATP manufacturing, and is supported by upregulation of REACTOME pathways capturing amino-acid and lipid metabolism, as well as mitochondrial function. Lastly, W-NK did not attack cells from healthy human tissues including PBMCs, indicating that W-NK are not indiscriminate killers. This observation would predict low myelosuppression and a favorable safety profile. In summary, W-NK are effective against AML cells, robustly home to the BM, overcome hypoxia, immunosuppression, and nutritional scarcity to survive/function within the leukemia TME. Moreover, they are not restricted to a single antigen target thereby mitigating antigen escape. These features, which overcome challenges for ACT, herald the promise of NK cell therapy and augur positively for patients being treated with W-NK in a Phase 1 study in R/R AML (NCT# 05470140).

Adoptively Infused Memory-like Natural Killer Cells Impact Adaptive Immune Responses in Patients with Acute Myeloid Leukemia

Natural killer (NK) therapies have been challenging owing to antigen escape, restricted trafficking, and limited tumor infiltration, translating into modest clinical benefit. We have shown that memory-like (ML)-NK cells acquire an in vivo differentiated phenotype distinct from conventional NK cells and are well-tolerated without cytokine release syndrome, GVHD, or neurotoxicity. WU-NK-101 (W-NK) is an allogeneic cytokine-reprogrammed, expanded, cryopreserved ML-NK cell product derived from healthy human peripheral blood mononuclear cells, with scalable manufacturability, that is currently in clinical development in relapsed/refractory (R/R) AML (NCT# 05470140). We conducted a multi-modal analysis of the immune tumor microenvironment (TME) in 13 patients with R/R AML receiving ML-NK cells (NCT#01898793) with the aim to determine whether TME modifications induced by ML-NK cells correlate with clinical benefit. NK cells were purified from leukapheresate, and activated with IL-12, IL-15, and IL-18 for 12-16 hours. Patients were lymphodepleted with fludarabine and cyclophosphamide. NK cells were infused in escalating doses: 0.5 × 10 6/kg, 1.0 × 10 6/kg, and all NK cells from a single leukapheresis, capped at 10 × 10 6/kg. Response was defined as <5% BM blasts on d+28. BM trephines for immune gene expression profiling (nCounter IO360®) and spatial proteomics (GeoMx™ DSP; n=53 immuno-oncology [IO] targets; NanoString Technologies, Seattle) were collected at baseline (BL) and on treatment (OT). Immune infiltration, NK and T cell number from BM samples was quantified manually on immunofluorescent imaging. Oncomine™ TCR Pan-Clonality assay (Thermo Fisher) was used to profile TCRb and g repertoires by NGS. The Shannon index was used to describe TCR repertoire diversity, and clonal evenness was used as a measure of the similarity of clone sizes. BM infiltration with CD8 + effectors, enhanced glycolysis and cell cycling was higher in BM samples from responders (CR) to ML-NK cells. By contrast, OXPHOS RNA scores and gene signatures of immune senescence and terminal T-cell exhaustion were higher in non-responders (NR). We employed machine learning to construct an 8-gene predictor of response which encompassed T/NK markers, such as CD7, and IFN regulatory factors and TLRs. This score was significantly higher at baseline in CR and positively correlated with response duration. Principal component analysis (PCA) of spatially resolved IO proteins separated CR from NR, particularly in OT specimens, where T-cell markers CD3/CD8 and inhibitory receptors CTLA4/LAG3 were among the top contributing features. CD34, type I IFN signaling molecule STING1 and PARP1 were significantly higher at baseline compared with OT, with OT showing higher expression of cytotoxicity markers and inhibitory receptors (e.g., Tim-3 and CD276). This observation strongly suggests modulation of the immune TME after the infusion of ML-NK cells. Response to ML-NK cells was associated with coordinated up-regulation of T cell, NK cell and B cell protein markers. To further substantiate the co-localization of T cells and NK cells in the BM FFPE sections (Fig. A), and its correlation with clinical efficacy we annotated the 93 ROIs in our spatial proteomics dataset based on a median split of CD3 and CD56 protein barcodes. ROIs from CR were largely CD3 highCD56 high, both at BL and OT. Conversely, ROIs from BM sections of NR to ML-NK cells had a predominantly CD3 lowCD56 low phenotype. Quantitative analysis of T-cell infiltration post ML-MK dosing showed a significant association with response, 0.9 (PD) vs. 5.3 (CR)-fold-increase ( P=0.01). Interestingly, data demonstrate selected intra-BM clonal expansion as evidenced by decreased TCR diversity (mean ± SEM, 4.6±0.3 vs. 3.3±0.4, pre vs. post; P=0.013)and increased clonality (Fig. B). Thus, pre-existing T-cell responses appear to be co-opted in the AML TME of CR, suggesting the potential for durable effectiveness of ML-NK cells. Multi-modal analyses of WU-NK-101 highlight unique transcriptional and functional states, which could underpin in vivo potency and persistence. WU-NK-101 are currently being evaluated in a phase 1 study of R/R AML (#NCT05470140).

WU-NK-101 (W-NK), a Memory-like (ML) NK Cell, Naturally Overcomes Tumor Microenvironment (TME) Metabolic Challenges, Retaining Anti-Tumor Potency

Cellular engineering revolutionized adoptive cell therapy (ACT) through improved antigen recognition and cellular activation. Cancer cells are metabolically active and extensively consume nutrients from the TME, rendering it nutrient-deprived, acidic, and hypoxic, and resulting in intratumoral metabolic demands which negatively impact ACT antitumor activity. This is central to T cells, however, some of these challenges can be overcome through engineering, an ideal solution would be to have a cellular product that is inherently adept at overcoming these challenges. W-NK is a cytokine-reprogrammed, expanded, cryopreserved, ML-NK cell product derived from healthy human peripheral blood mononuclear cells; it comes with naturally effective potency towards tumor cell killing, and importantly can overcome metabolic challenges present in the TME. We aim at deciphering what metabolic features make W-NK particularly potent in adverse TME. W-NK characterization of metabolic fitness/adaptability was with transcriptomics (sc-RNA-seq; 10X Genomics) flow cytometry, bioenergetics (Seahorse Real-Time Cell Metabolic Analysis; Agilent), and proteomic (Sciex Zeno TOF 7600 tandem-mass spectrometry). The aforementioned assays evaluated the effect of differing media/TME on W-NK: 1) conventional (N) media (pH 7, glucose 11 mM); 2) TME-aligned media (pH 5.9, glucose 6 mM, immune suppressive agents, e.g., PGE2, TGFb1); and 3) ascites derived from patients with malignancy. Media composition was analyzed biochemically using clinical assays (Core Lab Clinical Studies at Washington University, St. Louis, USA), and by immune secretome profiling (Nomic Bio, Quebec, CA). At baseline, W-NK cells have a unique metabolic phenotype, with higher expression of cell surface nutrient transporters, e.g., GLUT1, CD98, ASCT2, MCT1 and PiT1, compared to conventional NK cells (cNK); additionally, W-NK metabolism was consistent with aerobic (“Warburg”) glycolysis, with ~80% of ATP coming from glycolysis vs. ~30% for cNK. In TME/ascites media, W-NK surface nutrient receptor expression adapted to meet metabolic demand. For example, transporters for amino acids, lactate and pyruvate are upregulated in these hypoglycemic medias. This coincided with a 50% shift in ATP manufacturing from glycolysis to mitochondria, suggesting that nutrients bypassed glycolysis to enter tricarboxylic acid cycle and oxidative phosphorylation. Proteomic analyses of W-NK cells cultured with either TME-aligned media or ascites supported this hypothesis, with up-regulation of metabolic pathways (ShinyGO) capturing amino-acid and lipid metabolism, and mitochondrial function. Overall, W-NK retained cytotoxic function in TME and ascites, as well as in native TME-aligned 3D assays from primary surgical tumor samples. Conversely, cNKs did not adapt to TME media, whether in nutrient transporter expression or ATP manufacturing, and demonstrated reduced survival and function, including cytotoxicity. W-NK has enhanced metabolic fitness, flexibility, and plasticity. This enables W-NK to utilize diverse macronutrients, engage different metabolic pathways, and maintain optimal ATP manufacturing. Overall, W-NK inherently survives and maintains function in the TME, a limiting factor for immune cell-based ACT. These data herald the promise of NK cell therapy; a Phase 1 clinical study of W-NK in acute myeloid leukemia is currently open and enrolling patients (NCT# 05470140).

IgG from patients with mild or severe COVID‑19 reduces the frequency and modulates the function of peripheral mucosal-associated invariant T cells in PBMCs from healthy individuals.

Lower levels of peripheral mucosal-associated invariant T (MAIT) cells have been observed in the peripheral blood of patients with severe coronavirus disease 2019 (COVID-19). Following on from previous research into the effect of the IgG repertoire on human lymphocytes, the present study aimed to evaluate if immunoglobulin G (IgG) antibodies obtained from patients with mild or severe COVID-19 contribute to these effects on MAIT cells. Culture experiments were performed using healthy human peripheral blood mononuclear cells (PBMCs) and different repertoires of IgG obtained from patients with COVID-19 as a mild or severe disease and compared with mock, healthy control or therapeutic IgG conditions. The results indicate that the IgG repertoire induced during the development of mild and severe COVID-19 has, per se, the in vitro potential to reduce the frequency of MAIT cells and the production of IFN-γ by the MAIT cell population in PBMCs from healthy individuals. In conclusion, the results of the present study indicate that IgG in patients with severe COVID-19 may participate in the reduction of peripheral MAIT cell frequency and hinder the antiviral activity of these cells.

Reconditioned monocytes are immunomodulatory and regulate inflammatory environment in sepsis

Sepsis is caused by dysregulated immune response to severe infection and hyper inflammation plays a central role in worsening the disease. The immunomodulatory properties of mesenchymal stem cells (MSCs) have been evaluated as a therapeutic candidate for sepsis. Reconditioned monocytes (RM), generated from healthy human peripheral blood mononuclear cells (PBMCs) exhibit both macrophage and MSCs-like properties. RM were administered at different stages of sepsis in a mouse model. It reduced serum levels of IL6, MCP-1, IL-10, improved hypothermia, increased survival, and recovery from 0 to 66% when combined with antibiotics in the mouse model. The reduced human leucocyte antigen DR molecules expression on RM enables their co-culture with PBMCs of sepsis patients which resulted in reduced ROS production, and up-regulated TGF-β while down-regulating IL6, IL8, and IL-10 in-vitro. RM are potentially immunomodulatory, enhance survival in sepsis mouse model and modulate inflammatory behaviour of sepsis patient’s PBMCs.

Production of an interleukin-10 blocking antibody by genetically engineered macrophages increases cancer cell death in human gastrointestinal tumor slice cultures.

Although it can promote effector T-cell function, the summative effect of interleukin-10 (IL-10) in the tumor microenvironment (TME) appears to be suppressive; therefore, blocking this critical regulatory cytokine has therapeutic potential to enhance antitumor immune function. As macrophages efficiently localize to the TME, we hypothesized that they could be used as a delivery vehicle for drugs designed to block this pathway. To test our hypothesis, we created and evaluated genetically engineered macrophages (GEMs) that produce an IL-10-blocking antibody (αIL-10). Healthy donor human peripheral blood mononuclear cells were differentiated and transduced with a novel lentivirus (LV) encoding BT-063, a humanized αIL-10 antibody. The efficacy of αIL-10 GEMs was assessed in human gastrointestinal tumor slice culture models developed from resected specimens of pancreatic ductal adenocarcinoma primary tumors and colorectal cancer liver metastases. LV transduction led to sustained production of BT-063 by αIL-10 GEMs for at least 21 days. Transduction did not alter GEM phenotype as evaluated by flow cytometry, but αIL-10 GEMs produced measurable quantities of BT-063 in the TME that was associated with an ~5-fold higher rate of tumor cell apoptosis than control.

Effect of Sodium Hypochlorite 0.05% on MMP-9 Extracellular Release in Chronic Wounds.

In chronic wounds, high concentrations of matrix metalloproteinases (MMPs) can cause excessive proteolysis and slow wound healing. Consequently, restoring a proper MMP balance can help reduce the risk of a chronic wound. An antiseptic solution containing 0.05% sodium hypochlorite (Amukine Med 0.05%, Angelini S.p.A.; hereafter termed NaClO solution) is available on the market. The NaClO solution was proven effective and safe in managing infected skin wounds. To further characterize its activity, this study evaluated the in vitro activity of the NaClO solution on the monocyte release of MMPs. Human monocytic THP-1 (ATCC® TIB-202™) cell lines were differentiated into macrophages and treated with different concentrations of NaClO (from 0.05% to 5 × 10-7%). In addition, the THP-1 cell line was stimulated with wound fluid (WF) from patients with active venous leg ulcers in the inflammatory phase. The effect of NaClO (0.025-0.0062%) was also evaluated on healthy human peripheral blood serum samples. The effects of treatments on the gelatinolytic activity of MMP-9 were evaluated by gelatin zymography. The effects on MMPs release were evaluated through the Pro™ Human MMP 9-plex Assay. An exploratory scratch wound healing assay was also performed. The NaClO solution reduced the gelatinolytic activity of MMP-9 and its activated form. The downregulation of MMP-9 gelatinolytic activity was also observed in peripheral blood serum. The MMPs profile showed a reduction in MMP-1 release (p < 0.05) and a slight reduction of the release of MMP-9 and MMP-12 after the treatment with LPS and the NaClO solution. A slight improvement in wound healing was observed after macrophage activation and treatment with the NaClO solution. The results obtained suggest a possible ability of the NaClO solution to modulate the proteolytic pathways in the wound microenvironment, further characterizing its activity and use in clinical practice during wound care.

Lipid Droplet Protein PLIN1 Regulates Inflammatory Polarity in Human Macrophages and is Involved in Atherosclerotic Plaque Development by Promoting Stable Lipid Storage.

Perilipins (PLINs), peripheral lipid droplet (LD) proteins, play important roles in lipid accumulation and maturation in adipocytes. The relationship between PLIN family proteins and macrophage polarization in atherosclerosis has not been elucidated. The experiments used tissues from human arteries of 65 patients who had undergone a carotid endarterectomy, and cultured macrophages generated from healthy human peripheral blood mononuclear cells. Plaque immunohistochemistry demonstrated co-expression of PLIN1 and PLIN2 in both symptomatic (n=31) and asymptomatic patients (n=34). PLIN2 mRNA expression increased 3.38-fold in the symptomatic group compared with those from asymptomatic. PLIN1 was not expressed on small LDs at a shorter incubation but was on large LDs at longer incubation with oxidized LDL and VLDL, while PLIN2 was observed after 24 h and increased with a longer incubation in cultured M1 macrophage. In M2 macrophages, PLIN1 was seen as early as 24 h following incubation with VLDL, and LD size increased with longer incubation. PLIN1 overexpression increased the size of LDs in M1 macrophages, even after a short incubation, and reduced the RNA expression of TNFA, MMP2, ABCA1, and ABCG1 versus the M1 control. Conversely, silencing of PLIN1 in M2 macrophages had the opposite effects on LD size and RNA expression. There was a relationship between macrophage polarity, cytosolic LD size, and PLIN1/PLIN2 expression levels. PLIN2 was mainly expressed in arterial plaques in symptomatic stroke patients, and associated with the inflammatory phenotype of human macrophages, while PLIN1 expression is closely associated with plaque stability and the anti-inflammatory phenotype.

Hepatocyte growth factor-mediated apoptosis mechanisms of cytotoxic CD8+ T cells in normal and cirrhotic livers

Intrahepatic stem/progenitor cells and cytotoxic CD8+ T cells (CD8+ T cells) in the cirrhotic liver undergo apoptosis, which potentially facilitates progression to cancer. Here, we report that hepatocyte growth factor (HGF) signaling plays an important role in promoting normal and damaged liver CD8+ T cell Fas-mediated apoptosis through its only receptor, c-Met. In addition to binding with HGF, c-Met also binds to Fas to form a complex. Using a diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis mouse model, immunostaining, and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining, we found that HGF secretion was significantly higher at 10 weeks post-DEN, the liver cirrhotic phase (LCP), than at 3 weeks post-DEN, the liver fibrotic phase (LFP). Correspondingly, differences in CD8+ T cell proliferation and apoptosis were noted between the two phases. Interestingly, staining and TUNEL assays revealed lower smooth muscle actin (α-SMA)+ cell apoptosis, a marker for hepatic stellate cells (HSCs), in the LFP group than in the LCP group, which suggested a beneficial correlation among HGF, CD8+ T cells and HSCs in improving the fibrotic load during damaged liver repair. In cultures, when met different concentrations of recombinant HGF (rHGF), phytohemagglutinin (PHA)-stimulated naive mouse splenic CD8+ T cells (pn-msCD8+ T cells) responded differently; as increases in rHGF increased were associated with decreases in the clonal numbers of pn-msCD8+ T cells, and when the rHGF dose was greater than 200 ng/mL, the clonal numbers significantly decreased. In the presence of 400 ng/mL rHGF, the death-inducing signaling complex (DISC) can be directly activated in both nsCD8+ T cells and healthy human peripheral blood CD8+ T cells (hp-CD8+ T cells), as indicated by recruitment of FADD and caspase-8 because DISC forms via the recruitment of FADD and caspase-8, among others. These findings suggest that Fas-mediated apoptosis, may also indicate a regulatory role of HGF signaling in hepatic homeostasis.

Interleukin-37 alleviates myocardial injury induced by coxsackievirus B3 via inhibiting neutrophil extracellular traps formation.

To investigate whether interleukin-37 (IL-37) could directly inhibit the formation of neutrophil extracellular traps (NETs) in the early stage of acute viral myocarditis (VMC) and its potential mechanisms of action. Acute VMC was induced by intraperitoneally injecting coxsackievirus B3 (CVB3)(103 TCID50) in mice on day 0. Mice were injected with AAV9-IL-37 or AAV9-NC through the caudal vein 1week before intraperitoneal administration of CVB3. DNASE1 (50ug per mouse) was administered on days 0 to 7 to investigate the role of NETs formation during acute VMC. The severity of myocardial inflammation was evaluated by observing the general condition of mice and detecting cardiac histopathology. Moreover, neutrophils isolated from healthy human peripheral blood were stimulated by phorbol myristate acetate (PMA 100nM) and treated with IL-37 (0.1ng/ml) or BAY11-7082(2.5uM) in vitro. The production of NETs was detected by immunofluorescence labeled MPO and DNA. The expression of related proteins (IκBα, P-IκBα, NFκb, P-NFκb) was detected by Western blot. The results showed that, like DNASE1, IL-37 alleviates the symptoms in acute VMC induced by CVB3, reduces inflammatory cell infiltration, improves cardiac function, and inhibits the formation of NETs in the myocardium. Besides, both IL-37 and DNASE1 could effectively inhibit the activation of NFκb /IκBα. In the isolated peripheral blood neutrophils, the inhibitory effect of IL-37 on the formation of NETs and the activation of NFκb /IκBα was further confirmed. IL-37 has a protective effect on VMC by reducing the infiltration of inflammatory cells and inhibiting the formation of NETs at an early phase.

Lyt-200, a Humanized Anti-Galectin-9 Antibody, Exhibits Preclinical Efficacy in Models of Hematological Malignancies

Patients with relapse and refractory (R/R) hematological malignancies typically have 5-year overall survival rates of less than 50% when receiving chimeric antigen receptor (CAR) T-cell therapy and less than 30% after receiving hematopoietic stem cell transplantation (HSCT). Human leukemia cells utilize a variety of biochemical mechanisms, which allow them to escape host immune surveillance. These molecular pathways cause impairment of the anti-cancer activities of immune cells, which could attack and kill leukemia cells. These dismal outcomes for patients with R/R disease highlight the need for novel treatment regimens when current therapeutic options are exhausted. Galectins are s-type lectins that promote diverse biological processes including adhesion, signaling, and immunosuppression. In acute myeloid leukemia (AML), galectin-9 (GAL-9) maintains the leukemia initiating cell (LIC) population and plays an important role in treatment resistance. We recently tested LYT-200, a fully humanized IgG4 αGAL-9 monoclonal antibody, developed by PureTech Health, which has so far been well tolerated with no dose limiting toxicities in a Phase I clinical trial for solid tumors (NCT04666688). We tested LYT-200 in vitro and in vivo across multiple hematological malignancies. Flow cytometry was used to assess the surface expression of GAL-9 on human B-ALL, AML, T-cell acute lymphoblastic leukemia (T-ALL), and diffuse large B-cell lymphoma (DLBCL) cell lines relative to receptor T-cell immunoglobulin and mucin domain-containing protein-3 (TIM-3) and Programmed cell death protein-1 (PD-1) expression. Compared to healthy human peripheral blood mononuclear cells (PBMCs), where GAL-9 surface expression was low or absent depending on the immune cell, GAL-9 was highly expressed on the surface of all human blood cancer cell lines tested (>100-fold increase). Notably, GAL-9 expression was comparable to PD-1 surface expression and higher than that of TIM-3 on AML, B-ALL, T-ALL, and DLBCL cells. Treating all cell lines with LYT-200 relative to control immunoglobulin resulted in extensive cell death, which occurred after 48 and 72 hours of treatment. Furthermore, a side-by-side comparison with αTIM-3 antibody treatment at equivalent concentrations, revealed that LYT-200 treatment was significantly more effective at killing blood cancer cells at low doses of 10-100 ng/mL in vitro. The in vitro efficacy of LYT-200 against multiple blood cancer subtypes extended to in vivo protection and survival benefit in murine models of T-ALL and AML, both in immunocompromised and immunocompetent mice. When immunocompromised mice were transplanted with human T-ALL cell lines or patient-derived samples, single-agent treatment with LYT-200 (1.5 mg/kg/weekly) was comparable to survival results obtained with methotrexate (0.75 mg/kg/weekly) administration. Notably, the combination of LYT-200 and methotrexate treatments led to the best survival outcomes in both contexts. Similar results were obtained in xenograft studies of AML, where single-agent LYT-200 (1.5 mg/kg/weekly) treatment was more effective at protecting mice than cytarabine (100 mg/kg/weekly). However, combining both treatments resulted in the best survival outcomes with greater than 80% of mice surviving over 2 months after disease appearance. In all, our results demonstrate that GAL-9 is an important driver of multiple blood cancer subtypes and that targeting GAL-9 with LYT-200 represents a potential novel treatment strategy which warrants additional pre-clinical and clinical testing.

A cytotoxic survey on 2-amino-1H-imidazol based synthetic marine sponge alkaloid analogues.

Here, we describe the synthesis and biologic activity evaluation of 20 novel synthetic marine sponge alkaloid analogues with 2-amino-1H-imidazol (2-AI) core. Cytotoxicity was tested on murine 4T1 breast cancer, A549 human lung cancer, and HL-60 human myeloid leukemia cells by the resazurin assay. A total of 18 of 20 compounds showed cytotoxic effect on the cancer cell lines with different potential. Viability of healthy human fibroblasts and peripheral blood mononuclear cells upon treatment was less hampered compared to cancer cell lines supporting tumor cell specific cytotoxicity of our compounds. The most cytotoxic compounds resulted the following IC50 values 28: 2.91 µM on HL-60 cells, and 29: 3.1 µM on 4T1 cells. The A549 cells were less sensitive to the treatments with IC50 15 µM for both 28 and 29. Flow cytometry demonstrated the apoptotic effect of the most active seven compounds inducing phosphatidylserine exposure and sub-G1 fragmentation of nuclear DNA. Cell cycle arrest was also observed. Four compounds caused depolarization of the mitochondrial membrane potential as an early event of apoptosis. Two lead compounds inhibited tumor growth in vivo in the 4T1 triple negative breast cancer and A549 human lung adenocarcinoma xenograft models. Novel marine sponge alkaloid analogues are demonstrated as potential anticancer agents for further development.

446-P: Lipid Droplet Protein PLIN1 Regulates Inflammatory Polarity in Human Macrophages and Is Involved in Atherosclerotic Plaque Development by Promoting Stable Lipid Storage

Background: Lipid droplet (LD) proteins play important roles in lipid accumulation and maturation in adipocytes. The relationship among PLIN family proteins and macrophage polarization in atherosclerosis has not been elucidated. Methods: The experiments used human artery tissues from 65 patients who had undergone a carotid endarterectomy. Cultured macrophages generated from healthy human peripheral blood mononuclear cells are utilized as well. Results: Plaque immunohistochemistry demonstrated co-expression of PLIN1 and PLIN2 in both symptomatic (n=31) and asymptomatic patients (n=34) . PLIN2 mRNA expression increased 3.38-fold in the symptomatic group compared with those from asymptomatic. PLIN1 was not expressed on small LDs at a shorter incubation (24 h) but was on large LDs at longer incubation (7 days) with oxidized LDL and VLDL, while PLIN2 was observed after 24 h and increased with a longer incubation in cultured M1 macrophage. In M2 macrophages, PLIN1 was seen as early as 24 h following incubation with VLDL, and LD size increased with longer incubation. PLIN1 overexpression increased the size of LDs in M1 macrophages, even after a short incubation, and reduced the RNA expression of TNFA, MMP2, ABCA1, and ABCG1 versus the M1 control. Conversely, silencing of PLIN1 in M2 macrophages had the contrary effects on LD size and RNA expression. Conclusion: There was a relationship among macrophage polarity, cytosolic LD size, and PLIN1/PLIN2 expression levels. PLIN2 was mainly expressed in arterial plaques in symptomatic stroke patients, and associated with the inflammatory phenotype of human macrophages, while PLIN1 expression is closely associated with plaque stability and the anti-inflammatory phenotype. Disclosure K.Cho: None. H.Miyoshi: Research Support; Abbott Japan Co., Ltd., Boehringer Ingelheim International GmbH, Daiichi Sankyo, Kowa Company, Ltd., LifeScan, Mitsubishi Tanabe Pharma Corporation, Novo Nordisk, Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Holdings Co., Ltd., Speaker's Bureau; Astellas Pharma Inc., Boehringer Ingelheim International GmbH, Eli Lilly and Company, Kowa Company, Ltd., Merck &amp; Co., Inc., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk, Ono Pharmaceutical Co., Ltd., Sanofi K.K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Holdings Co., Ltd. A.Nakamura: Research Support; Kissei Pharmaceutical Co., Ltd., MSD, Nippon Boehringer Ingelheim, Taisho Pharmaceutical Holdings Co., Ltd. T.Atsumi: Consultant; AbbVie Inc., AstraZeneca, MEDICAL &amp; BIOLOGICAL LABORATORIES CO., Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Ono Pharmaceutical Co., Ltd., Pfizer Inc., Research Support; AbbVie Inc., Alexion Pharmaceuticals, Inc., Astellas Pharma Inc., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo, Mitsubishi Tanabe Pharma Corporation, Nippon Boehringer Ingelheim Co. Ltd., Otsuka Pharmaceutical Co., Ltd., Pfizer Inc., Taiho Pharmaceutical Co. Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited, Speaker's Bureau; AbbVie Inc., Astellas Pharma Inc., Bristol-Myers Squibb Company, Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo, Eisai Co., Ltd., Eli Lilly and Company, Kyowa Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Novartis Pharma K.K., Pfizer Inc., Taiho Pharmaceutical Co. Ltd., Takeda Pharmaceutical Company Limited, UCB, Inc.

3,4,5-O-tricaffeoylquinic acid with anti-radiation activity suppresses LPS-induced NLRP3 inflammasome activation via autophagy in THP-1 macrophages

Damage to normal tissues caused by excessive ionizing radiation (IR) exposure is the major side effect of radiotherapy. Several recent studies have shown that IR-induced damage to tissues leads to a systemic immune response and NLRP3 inflammasome activation in immune cells. 3,4,5-O-tricaffeoylquinic acid (tCQA), extracted from the natural plant Azolla imbricata, relieves inflammation and has radioprotective function. Here, we aimed to investigate the inhibitory effect and molecular mechanism of tCQA on IR-induced NLRP3 inflammasome activation. First, the results of ELISA and qPCR assays showed that tCQA has anti-inflammatory effects in THP-1 cell line and healthy human peripheral blood mononuclear cells. Western blotting and ELISA suggested tCQA could inhibit NF-κB/MAPK signaling pathway, NLRP3 expression and the secretion of IL-1β in lipopolysaccharide (LPS)-stimulated THP-1 macrophages. Then, flow cytometry, LDH assay and western blotting demonstrated that tCQA could inhibit LPS- and nigericin-induced Caspase-1 activation and gasdermin D cleavage, thereby suppressing inflammatory cell death. Furthermore, we found that the autophagy inhibitor chloroquine, not the proteasome inhibitor MG132, could counteract the promoting effect of tCQA on NLRP3 degradation and the inhibitory effect on cell death. Western blotting and autophagosome staining results suggested tCQA could significantly enhance LPS-induced autophagic flux in macrophages and ATG5/ATG7 knockdown reverses the inhibitory effect of tCQA on NLRP3 expression and Caspase-1 activation, indicating that tCQA induces NLRP3 degradation via autophagy. Finally, THP-1 macrophages and BALB/c mice were irradiated with 137Cs γ-rays and tCQA could inhibit IR-induced NLRP3 inflammasome activation both in vitro and in vivo. To conclude, tCQA controls inflammation and NLRP3 inflammasome activation in vitro via NF-κB/MAPK signaling pathway and autophagy, meanwhile inhibits IR-induced NLRP3 inflammasome activation in vivo. Overall, our study provides an experimental and theoretical basis for the application of tCQA as a radioprotectant in clinical radiotherapy.

DNA Methyltransferase 3b Accelerates the Process of Atherosclerosis.

Background DNA methylation plays a key role in establishing cell type–specific gene expression profiles and patterns in atherosclerosis. The underlying mechanism remains unclear. Previous studies have shown that DNA methyltransferase 3b (DNMT3b) may play an important role in atherosclerosis. This study aimed to establish the regulatory role of DNMT3b in the development of atherosclerosis. Methods We constructed a viral vector carrying Dnmt3b shRNA to transduce ApoE−/− mice. Meanwhile, healthy human peripheral blood Treg cells were treated with inhibitor of DNMT3b (AZA and EGCG) or transduced with DNMT3b shRNA. Results It showed that Dnmt3b silencing attenuated atherosclerosis, including decreased lesion size and macrophage content and increased collagen and smooth muscle cells content in ApoE−/− mice. To further investigate the possible mechanisms, combined with previous studies by our group, we showed that Foxp3-TSDR methylation level was significantly reduced Foxp3 expression and peripheral blood Treg levels were significantly increased by Dnmt3b shRNA vector transduction in animals committed to western diet for 12 and 18 weeks. Consistently, inhibition of DNMT3b (AZA and EGCG) decreased the expression levels of DNMT3b, which can increase the expression levels of FOXP3, and increase the levels of TGF-β and IL-10 and decrease the levels of IL-β and IFN-γ. After transduction with DNMT3b shRNA, the effect was more obvious. Conclusions DNMT3b accelerated atherosclerosis, and may be associated with FOXP3 hypermethylation status in regulatory T cells.

Anti-microbial and anti-cancer activity of gold nanoparticles phytofabricated using clerodin enriched clerodendrum ethanolic leaf extract

BackgroundSearch for options that can overcome microorganism’s resistance to traditional drugs, has shifted the focus towards plant-based molecules and extracts. Latest developments in plant-based nanomaterials have further expanded the role of plant extracts since nano particle - plant extract combinations are shown to possess enhanced biological activity and reduced toxicity to human cells. In the present study, facile, one-pot synthesis of nearly monodispersed gold nanoparticles was developed using Clerodin enriched Clerodendrum infortunatum ethanolic leaf extract under ambient conditions. The synthesized gold nanoparticles were evaluated for broad spectrum anti-microbial activity as well as compatibility with human peripheral blood mononuclear cells. MethodsClerodin enriched fraction from C. infortunatum leaves ethanol extract was used to synthesize gold nanoparticle from chloroauric acid. Synthesized gold nanoparticles were characterized and tested for broad spectrum anti-microbial activity against Staphylococcus aureus, Bacillus cereus, Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa using the standard broth dilution method. Cytotoxicity of the gold nanoparticles was evaluated against human monocyte leukemic cells (THP-I) and healthy peripheral blood mononuclear cells. MTT and FACS based Annexin V/FITC-PI assays were used to study its cytotoxic effect. ResultsTEM analysis showed an average diameter of 33 ± 5 nm for the synthesized nanoparticles. EDX validated the presence of elemental Au (98.8%) and confirmed its nano form. Antimicrobial assay of gold nanoparticles showed significant inhibitory effect in all the tested pathogens. Additionally, MTT and Annexin-V/ PI assay demonstrated the cytotoxic activity of the nanoparticles against THP-I cells. Interestingly, nanoparticles did not show cytotoxicity against healthy human peripheral blood mononuclear cells. ConclusionGold nanoparticles generated in an eco-friendlyprocess using Clerodin enriched fractions showed promising broad spectrum antibacterial activity. Simultaneously, they proved themselves to be benign against healthy human cells indicating potential therapeutic applications. Added benefit for the nanoparticles was, they had anti-proliferative effects against human leukemic monocyte cancer (THP-I) cells.

In Vitro Antitumor Activity of Endophytic and Rhizosphere Gram-Positive Bacteria from Ibervillea sonorae (S. Watson) Greene against L5178Y-R Lymphoma Cells.

Plant-associated microorganisms represent a potential source of new antitumor compounds. The aim of the present study was to isolate endophytic and rhizosphere Gram-positive bacteria from Ibervillea sonorae and produce extracts with antitumor activity. Methanol and ethyl acetate extracts were obtained from 28 d bacterial fermentation, after which murine L5178Y-R lymphoma cells growth inhibition was evaluated at concentrations ranging from 15.62 µg/mL to 500 µg/mL by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide reduction colorimetric assay. IC50 and the selectivity index (SI) were calculated and compared with healthy control human peripheral blood mononuclear cells (PBMC). Identification of the isolated strains was performed using the 16S ribosomal gene and by MALDI-TOF MS mass spectrometry. The endophytic and rhizosphere bacterial extracts from strains ISE-B22, ISE-B26, ISE-B27, ISS-A01, ISS-A06, and ISS-A16 showed significant (p < 0.05) L5178Y-R cell growth inhibition, compared with an untreated control. The rhizosphere Micromonospora echinospora isolate ISS-A16 showed the highest (90.48%) percentage of lymphoma cells growth inhibition and SI (19.1) for PBMC, whereas the Bacillus subtilis ISE-B26 isolate caused significant (p < 0.01) growth inhibition (84.32%) and a SI of 5.2. Taken together, results of the present study evidenced antitumor effects by I. sonorae endophytic and rhizosphere bacteria culture extracts. Further research will involve the elucidation of the compounds that exert the antitumor activity and their evaluation in pre-clinical studies.