Soluble Ligand Research Articles

Wnt5a influences the directional displacement of ovarian cancer cells

Objectives: The noncanonical Wnt ligand Wnt5a is found in high concentrations in ovarian cancer ascites. This microenvironmental Wnt5a has recently been shown to potentiate ovarian cancer metastasis. We sought to identify whether Wnt5a modulates ovarian cancer cell motility. Methods: We engineered and validated a microfluidic device that establishes stable gradients of soluble ligands. After a Wnt5a gradient was established, ovarian cancer cells (ES2 or OVCAR5) were introduced and allowed to adhere to a 2-dimensional fibronectin substrate. Real time imaging was carried out over 7 hours and cellular migration was analyzed with respect to speed, displacement, and directionality. In a subset of experiments, ROR2, a noncanonical Wnt5a receptor involved in planar cell polarity (PCP) pathways, was depleted with siRNA to assess contribution of ROR2-dependent PCP to Wnt5a mediated behaviors. Results: In the presence of Wnt5a, ES2 and OVCAR5 ovarian cancer cells demonstrated an increase in both mean and instantaneous velocity of migration when compared to absence of ligand (P Conclusions: Wnt5a promotes the motility of ovarian cancer cells by influencing both directionality of motion as well as degree of displacement. Furthermore, the Wnt5a receptor ROR2 was shown to be critical in mediating these behaviors, suggesting a role for planar cell polarity pathways in ovarian cancer metastasis. As Wnt5a is produced at high levels by peritoneal mesothelial cells and omental adipose tissue in the metastatic microenvironment of ovarian cancer, these data suggest a potential mechanism by which ovarian cancer cells ‘home’ to their metastatic niche. ROR2 represents an attractive target for disrupting this tumor cell/microenvironment interaction and represents a possible new therapeutic target.

Soluble FAS Ligand Enhances Suboptimal CD40L/IL-21-Mediated Human Memory B Cell Differentiation into Antibody-Secreting Cells.

Differentiation of Ag-specific B cells into class-switched, high-affinity, Ab-secreting cells provides protection against invading pathogens but is undesired when Abs target self-tissues in autoimmunity, beneficial non-self-blood transfusion products, or therapeutic proteins. Essential T cell factors have been uncovered that regulate T cell-dependent B cell differentiation. We performed a screen using a secreted protein library to identify novel factors that promote this process and may be used to combat undesired Ab formation. We tested the differentiating capacity of 756 secreted proteins on human naive or memory B cell differentiation in a setting with suboptimal T cell help in vitro (suboptimal CD40L and IL-21). High-throughput flow cytometry screening and validation revealed that type I IFNs and soluble FAS ligand (sFASL) induce plasmablast differentiation in memory B cells. Furthermore, sFASL induces robust secretion of IgG1 and IgG4 Abs, indicative of functional plasma cell differentiation. Our data suggest a mechanistic connection between elevated sFASL levels and the induction of autoreactive Abs, providing a potential therapeutic target in autoimmunity. Indeed, the modulators identified in this secretome screen are associated with systemic lupus erythematosus and may also be relevant in other autoimmune diseases and allergy.

Laboratory Approaches to Test the Function of Antiphospholipid Antibodies

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder caused by the presence of aPLs (antiphospholipid antibodies, i.e., anti-β2-glycoprotein I and anti-cardiolipin). Everyday practice in terms of laboratory diagnostics of APS includes determination of aPLs and well-known functional assays assessing for lupus anticoagulant (LA), in turn using various tests. According to recent guidelines, the recommended method for LA identification or exclusion is based on the Russell Viper Venom test and a sensitive activated partial thromboplastin time assay. Despite the fact that LA can be quantified in laboratory practice in this way, LA is still used as a binary parameter that is just one of the risk factors of thrombosis in APS. As of today, there are no other functional assays to routinely assess the risk of thrombosis in APS. It is well-known that APS patients display a wide range of clinical outcomes although they may express very similar laboratory findings. One way to solve this dilemma, could be if antibodies could be further delineated using more advanced functional tests. Therefore, we review the diagnostic approaches to test the function of aPLs. We further discuss how thrombin generation assays, and rotational thromboelastometry tests can be influenced by LA, and how experimental methods, such as flow cytometric platelet activation, surface plasmon resonance, or nano differential scanning fluorimetry can bring us closer to the puzzling interaction of aPLs with platelets as well as with their soluble protein ligand. These novel approaches may eventually enable better characterization of aPL, and also provide a better linkage to APS pathophysiology.

Triangle of cytokine storm, central nervous system involvement, and viral infection in COVID-19: the role of sFasL and neuropilin-1.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is identified as the cause of coronavirus disease 2019 (COVID-19), and is often linked to extreme inflammatory responses by over activation of neutrophil extracellular traps (NETs), cytokine storm, and sepsis. These are robust causes for multi-organ damage. In particular, potential routes of SARS-CoV2 entry, such as angiotensin-converting enzyme 2 (ACE2), have been linked to central nervous system (CNS) involvement. CNS has been recognized as one of the most susceptible compartments to cytokine storm, which can be affected by neuropilin-1 (NRP-1). ACE2 is widely-recognized as a SARS-CoV2 entry pathway; However, NRP-1 has been recently introduced as a novel path of viral entry. Apoptosis of cells invaded by this virus involves Fas receptor-Fas ligand (FasL) signaling; moreover, Fas receptor may function as a controller of inflammation. Furthermore, NRP-1 may influence FasL and modulate cytokine profile. The neuroimmunological insult by SARS-CoV2 infection may be inhibited by therapeutic approaches targeting soluble Fas ligand (sFasL), cytokine storm elements, or related viral entry pathways. In the current review, we explain pivotal players behind the activation of cytokine storm that are associated with vast CNS injury. We also hypothesize that sFasL may affect neuroinflammatory processes and trigger the cytokine storm in COVID-19.

Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2.

To date, no study has demonstrated that soluble Fas ligand (sFasL)-mediated inflammation is regulated via interaction with Fas in vivo. We found that FasL interacts specifically with tumor necrosis factor receptor superfamily (TNFRSF)10B, also known as death receptor (DR)5. Autoantibody-induced arthritis (AIA) was attenuated in FasL (Faslgld/gld)- and soluble FasL (FaslΔs/Δs)-deficient mice, but not in Fas (Faslpr/lpr and Fas-/-)- or membrane FasL (FaslΔm/Δm)-deficient mice, suggesting sFasL promotes inflammation by binding to a Fas-independent receptor. Affinity purification mass spectrometry analysis using human (h) fibroblast-like synovial cells (FLSCs) identified DR5 as one of several proteins that could be the elusive Fas-independent FasL receptor. Subsequent cellular and biochemical analyses revealed that DR5 interacted specifically with recombinant FasL-Fc protein, although the strength of this interaction was approximately 60-fold lower than the affinity between TRAIL and DR5. A microarray assay using joint tissues from mice with arthritis implied that the chemokine CX3CL1 may play an important downstream role of the interaction. The interaction enhanced Cx3cl1 transcription and increased sCX3CL1 production in FLSCs, possibly in an NF-κB-dependent manner. Moreover, the sFasL-DR5 interaction-mediated CX3CL1-CX3CR1 axis initiated and amplified inflammation by enhancing inflammatory cell influx and aggravating inflammation via secondary chemokine production. Blockade of FasL or CX3CR1 attenuated AIA. Therefore, the sFasL-DR5 interaction promotes inflammation and is a potential therapeutic target.

Abstract LB123: A high value pharmacological platform dedicated to the real time study of stimulatory immune checkpoint signaling pathways

Abstract Immune checkpoint (ICP) co-receptors play a key role in the fine modulation of the immune response. While inhibitory ICP co-receptors are mainly characterized in cancer by the exhaustion of the immune response, stimulatory ICP co-receptors are clearly involved in an anarchic activation of the immune system leading to autoimmune diseases. But in another hand, this subtype of co-receptor shows a great asset in cancer therapy by increasing anti-tumoral response. All together, these co-receptors show a great interest for the development of innovative therapies in immunology or immuno-oncology. Nowadays, treatments targeting these co-receptors are currently tested in clinical trials or approved for use in man, especially antibodies targeting both inhibitory and stimulatory ICP. Small molecule-based strategy is tested in early drug discovery and in early clinical trials. In order to screen and assess the potency and efficiency of these therapies, robust and reliable cell-based assays are urgently needed. At Domain Therapeutics, using our powerful proprietary BRET technology: bioSens-AllTM, we successfully develop and validate a cell-based assay platform to assess immune checkpoint pharmacology. The activation or blockade of the signaling of checkpoint co-receptors are monitored in real time and in living cells. BRET assays dedicated to stimulatory ICPs: 4-1BB/4-1BB Ligand and OX-40/OX-40 Ligand were designed. BRET assay development is inspired by the natural recruitment and proximity of specific cytoplasmic partners very close to their co-receptors in an activated pathway. Indeed, the first assay design is based on the co-receptor fused with rGFP and its cytoplasmic partner fused with a rLucII. In the second assay design, the co-receptor is no longer fused, but is co-transfected with a membrane anchor fused with the rGFP and co-transfected with the cytoplasmic partner fused with rLucII. So, in an activated system (soluble ligand, antibody or in co-culture system) a modulation of BRET can be observed. For ICP assays dedicated to 4-1BB, BRET signal is triggered using soluble ligand, agonist antibody, or co-culture where TRAF1 is the biosensor fused with rLucII. Further validation was obtained for OX-40/OX-40 Ligand. Those BRET assays complement once previously developed for inhibitory ICPs axis: PD-1/PD-Ligand 1 or PD-Ligand 2 and CTLA-4/CD80-CD86. The ICP Platform set up here shows a good accuracy and robustness and represents a strong and reliable technology for drug discovery dedicated to ICPs. Our spatio-temporal cell-based functional assays can support broad drug programs, including: High Throughput Functional Screening, Lead Optimization and Bioanalytical QC lot Release. Citation Format: Alice GENTIL DIT MAURIN, Christel FRANCHET, Stephan SCHANN, Xavier LEROY. A high value pharmacological platform dedicated to the real time study of stimulatory immune checkpoint signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB123.

CIB1 Regulates Hemostasis through Integrin Outside-in Signaling

On circulating platelets, integrin αIIbβ3, the fibrinogen receptor, is in a low affinity state unable to bind soluble ligands. Agonist-dependent inside-out signaling is necessary for activation of the integrin αIIbβ3 in order to bind ligands such as fibrinogen (Fg). Ligand binding induces a cascade of signaling that culminates in platelet aggregation, leading to hemostatic plug formation and clot retraction. Although it is well known that signaling through integrin αIIbβ3 is necessary, the mechanism of clot retraction is not well understood. Previously, we have identified a calcium- and integrin-binding protein 1 (CIB1) that specifically interacts with integrin αIIbβ3. One study implicated CIB1 as an endogenous inhibitor of integrin αIIbβ3 activation, while another study suggested CIB1 to be an activator of inside-out signaling. Using a novel technique to inhibit interaction of CIB1 with integrin in vivo, we have shown that CIB1 regulates outside-in signaling through integrin αIIbβ3. Definitive proof for the role of CIB1 in platelets remained absent, however. Here, using Cib1 null platelets, we show for the first time that CIB1 is a key regulator of hemostasis. Genetic ablation of CIB1 had no effect on agonist-induced platelet aggregation, excluding the role for CIB1 in inside-out signaling. However, deletion of the Cib1 gene significantly increased mouse bleeding times. More than 50% of the mice also showed a rebleeding phenotype, suggesting a defect in clot stability. Incomplete and significantly delayed clot retraction was observed in Cib1 null platelets compared to wildtype littermates. The absence of CIB1 also affects the ability of platelets to spread on immobilized Fg. Platelet adhesion and subsequent filopodia formation was not affected by the absence of CIB1. Platelet spreading can be restored by addition of exogenous ADP, suggesting that the defect is not in the ability of the platelet to spread. These in vivo and in vitro results clearly show that CIB1 regulates hemostasis by supporting transition of platelets from filopodial stage to fully spread morphology during outside-in signaling through integrin αIIbβ3.

The Application of ‘Drug-Reversible’ CAR-T Cells Directed Against Recipient Hematopoietic Cells As a Selective Conditioning Strategy for Stem Cell Transplantation

The transplant of autologous or allogeneic hematopoietic stem cells (HSCs) has the proven ability to treat a wide array of malignant and non-malignant hematological diseases. The preparative regimen, however, routinely entails aggressive and genotoxic treatment with total body irradiation and/or chemotherapy, which brings severe and even life-threatening complications that limit its broader application. Previous experimental studies have established that depletion of recipient HSCs is an essential requirement of these conditioning regimens in allowing successful engraftment of the composite donor HSCs. Animal and clinical studies have also indicated that alloreactive anti-HSC donor T cells additionally facilitate stem cell engraftment, but this is often accompanied by the risks of GvHD. This has prompted the consideration of alternative conditioning methods for the depletion of HSCs with less toxic side-effects, such as anti-c-kit and anti-CD45 antibody-directed treatments. In this way, more precise HSC targeting may also be achieved by the application of short-lived, genetically engineered chimeric antigen receptor (CAR)-T cells for stem cell transplantation conditioning.We developed a novel and controllable CAR-T approach for recipient HSC targeting via genetic modification using the non-viral piggyBac™ (PB) transposon system. As opposed to viral vector delivery systems, the relatively large carrying capacity of PB allows the stable introduction of at least three separate genes encoded within the same tri-cistronic transgene cassette. This includes a second-generation CAR that targets either human c-kit (CD117) or prominin-1 (CD133), markers known to be antigenically expressed on the surface of HSCs. In addition, a drug resistance element serves as a selection gene that, in combination with a non-genotoxic drug, provides an effective method of CAR-T cell purification during manufacture. Importantly, a small molecule drug-inducible safety switch gene is also included to facilitate rapid in vivo clearance of the CAR-T cells after depletion of recipient HSCs and prior to donor HSC transplant. Lastly, as a result of the manufacturing process, the majority of the CAR-T cells express chemokine receptors such as CXCR4 that can allow more selective trafficking to the bone marrow (BM) for eradication of resident HSCs.To select a lead candidate from a panel of anti-HSC CAR constructs, CD3/CD28 stimulated T cells from human peripheral blood were first electroporated with mRNA encoding each of the CAR candidates directed against either c-kit or CD133. CAR surface expression was confirmed in transfected T cells by flow cytometry. In vitro functional assays were performed by co-culturing mRNA-transfected CAR-T cells with mouse or human cell lines (EML-C1, TF-1 and K562), expressing either c-kit or CD133, as well as mouse and human primary BM cells. Lead CAR candidates were identified from their specific activation of the CAR-T cells through degranulation according to CD107a expression and secretion of IFNγ. Furthermore, those CARs were also capable of selectively depleting c-kit or CD133 positive cells. Interestingly, some mRNA-transfected CAR-T cells retained effector activity against target c-kit+ TF-1 cells even in the presence of its soluble ligand, stem cell factor. Next, lead CAR candidates were co-expressed with the selection and drug-inducible safety switch genes in the same tri-cistronic transgene and then stably delivered to T cells using PB. The manufacturing process yielded CAR-T cells that were mainly of the T memory stem cell (Tscm) phenotype, as determined by positive expression of CD62L and CD45RA, and also expressed high levels of the CXCR4 chemokine receptor. Similar to the mRNA-transfected CAR-T cells, these stably-transposed cells were capable of extensive effector capabilities including specific depletion of c-kit or CD133 expressing target cells.Future studies will evaluate PB-produced lead anti-HSC CAR-T cells in immune-deficient NSG mice with pre-established xenogeneic human hematopoietic chimerism, along with standard busulfan or radiation conditioning controls. This approach constitutes a novel targeted biological therapy that is envisaged to lead the way towards minimally toxic transplant regimens for depletion of endogenous HSCs in the BM and to procure their replacement with engrafted allogeneic or gene-corrected stem cells. DisclosuresTimberlake:Poseida Therapeutics, Inc.: Employment. Richter:Poseida Therapeutics, Inc.: Employment. Wang:Poseida Therapeutics, Inc.: Employment. Cam:Poseida Therapeutics, Inc.: Employment. Tan:Poseida Therapeutics, Inc.: Employment. Ostertag:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Shedlock:Poseida Therapeutics, Inc.: Employment. Down:Poseida Therapeutics, Inc.: Employment.

Evaluation of a FLT3 Bite® for Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a disease with high unmet medical need. Standard induction chemotherapy can lead to complete remission in patients with a favorable genetic risk profile; however, about half of patients are ineligible for this intensive regimen. In those patients with a non-favorable genetic risk profile, the preferred treatment strategy is allogeneic stem cell transfer, but the majority of patients are ineligible due to age and other comorbidities.FLT3 is a receptor tyrosine kinase that induces cell growth in myeloid progenitor cells. Activating mutations in FLT3 have been identified in approximately 30% of AML patients. Expression of mutant FLT3 and overexpression of wild type FLT3 are associated with poor prognosis in AML, leading to clinical investigation of targeted FLT3 inhibitors, including midostaurin which was recently approved for patients with newly diagnosed FLT3-mutant AML. These inhibitors are likely to be effective only in certain patient subpopulations and may ultimately give rise to acquired resistance. To drive cures in AML, effective therapies for broad patient populations that can eliminate residual disease are needed.Bispecific T cell engaging antibody constructs (BiTE®) are a class of immunotherapy agents which redirect T lymphocytes to a specific antigen expressed on tumor cells. Blinatumomab, a BiTE® containing a single chain variable fragment (scFv) that binds CD19 on B lymphocytes and an scFv that binds CD3 on T lymphocytes, has shown clinical efficacy for treating CD19-positive hematological malignancies such as ALL. To extend the serum half-life, next generation BiTE® antibody constructs have been modified to contain Fc-moieties, allowing for weekly dosing.To evaluate FLT3 as a BiTE® target in AML, we assessed expression of FLT3 on primary AML patient blasts using flow cytometry. Over 83% of AML patients (n = 192) were FLT3-positive (MFI ratio > 1.5) with similar expression in bulk and leukemic stem cells. A half-life extended FLT3 BiTE® (FLT3 HLE BITE®) was generated and evaluated in vitro as well as in vivo in a mouse xenograft tumor model and non-human primates (NHP). FLT3 HLE BiTE® had subnanomolar binding affinity for human FLT3 and nanomolar binding affinity for human CD3 and NHP FLT3 and CD3. In cytotoxic assays targeting human AML cell lines, FLT3 HLE BiTE® exhibited picomolar efficacy using as effector cells human pan T cells in a 10:1 E:T ratio (range: 2 to 10 pM) or NHP PBMCs in a 5:1 E:T ratio (range: 30 to 80 pM). In mice injected with FLT3-positive MOLM13 cells and human CD3-positive T cells, FLT3 HLE BiTE® induced robust anti-tumor activity and extended survival ≥ 2-fold at all doses (n = 10 per group, p ≤ 0.0015). Following a single 5 µg/kg dose in NHP (n=2), FLT3 HLE BiTE® exhibited a reproducible pharmacokinetic profile with a 66 hour half-life. In this study, we observed hallmarks of BiTE® activation such as induction of IL-6 and MCP-1 and indication of a pharmacodynamic effect through increased soluble FLT3 ligand. Following repeat-dosing with FLT3 HLE BiTE®, digital droplet PCR was used to measure FLT3 transcript in NHP blood and bone marrow. Robust depletion of FLT3+ cells was observed in both NHP blood (≥ 95% reduction) and bone marrow (≥ 68% reduction) at all doses (5 µg/kg, 25 µg/kg, 75 µg/kg, n = 3/group). Taken together, FLT3 HLE BiTE® has potent in vitro and in vivo activity and may provide therapeutic benefit to AML patients. DisclosuresGoldstein:Amgen: Employment. Henn:Amgen Research (Munich) GmbH: Employment, Equity Ownership. Archibeque:Amgen: Employment. Frank:Amgen Inc.: Employment, Equity Ownership. Balazs:Amgen Inc.: Employment, Equity Ownership. Dahlhoff:Amgen Research (Munich): Employment, Equity Ownership. Raum:Amgen Research (Munich): Employment. Li:Amgen, Inc.: Employment. Wahl:Amgen Research (Munich): Employment. Rock:Amgen, Inc.: Employment, Equity Ownership. Thomas:Amgen Research (Munich): Employment. Karbowski:Amgen, Inc.: Employment. Krupka:Amgen Research (Munich): Research Funding; Amgen, Inc: Research Funding. Subklewe:Amgen Research (Munich): Research Funding; Amgen, Inc.: Research Funding. Coxon:Amgen, Inc.: Employment. Chapman-Arvedson:Amgen, Inc.: Employment, Equity Ownership.

Soluble PD-1 Ligands Regulate T-Cell Function in Waldenstrom's Macroglobulinemia

While immune checkpoint molecules contribute to the malignant development of several cancers, including certain types of lymphomas, their potential significance in malignant progression of Waldenstrom's Macroglobulinemia (WM), a low-grade B-cell lymphoma, has not fully been described. The main characteristic of WM is infiltration of lymphoplasmacytic cells in the bone marrow (BM) and increased monoclonal IgM production by malignant cells within BM microenvironment. While next generation sequencing analysis has identified mutations in the MYD88 gene as the driver of WM development, studies by our group have also highlighted a role for the BM microenvironment in pathophysiology of WM and shown that elevated levels of certain cytokines in the BM, such as IL-6 and IL-21, promote both cell proliferation and IgM secretion in WM.In this study, we investigated the impact of cytokines on the immune checkpoint receptor Programmed Death-1 (PD-1) and its ligands PD-L1 and PD-L2 and evaluated how these molecules contribute to malignant progression in WM.Using RT-PCR analysis we have shown that treatment of WM cell lines with IL-21 and IL-6 significantly induce PD-L1 and PD-L2 gene expression, implying that elevated levels of these cytokines within the BM microenvironment could be associated with increased PD-L1 and PD-L2 expression by malignant cells in WM patients. We therefore performed RT-PCR analysis on cells isolated from patients' BM and confirmed increased PD-L1 and PD-L2 expression by both WM and BM cells when compared normal BM cells. In contrast, flow cytometry analysis showed minimal expression of these ligands on the surface of the WM cells, with no significant difference between WM and normal BM cells, indicating that there might be a regulatory mechanism controlling surface expression of these ligands. Further analysis using ELISA and Western Blotting showed that PD-L1 and PD-L2 are found as secreted forms in both BM plasma and peripheral blood serum of the WM patients and their level is significantly higher in WM as compared to normal subjects. Interestingly, in vitro treatment of the WM cell lines by IL-21 and IL-6 increased secreted PD-L1 and PD-L2 in the culture media of the cells, providing an additional line of evidence for elevated PD-L1 and PD-L2 levels in WM patients. Subsequent analysis showed that the secreted form of the PD-L1 and PD-L2 binds to T-cells and induces signals that reduce T-cell proliferation and cell cycle progression, the effects that are associated with a reduction in p-ERK and cyclin A levels.In summary, our data identifies that secreted form of PD-1 ligands is elevated in WM and play an important role in regulating T-cell function. Given the capability of secreted form to be bioactive at distant sites, soluble PD-1 ligands may have potential to promote disease progression. DisclosuresAnsell:Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Affimed: Research Funding; Merck: Research Funding; Celldex: Research Funding.

Intrinsic differences in the mechanisms of Tie2 binding to angiopoietins exploited by directed evolution to create an Ang2-selective ligand trap

Angiopoietins Ang1 and Ang2 are secreted ligands for the endothelial receptor tyrosine kinase Tie2 essential for vascular development and maintenance. Ang1 acts as an agonist to maintain normal vessel function, whereas Ang2 acts as a Tie2 antagonist. Ang2 is increased in macular edema, sepsis, and other conditions, in which it blocks Ang1-mediated signaling, causing vascular dysfunction and contributing to disease pathology. Therefore, Ang2 is an attractive therapeutic target. Previously, we reported a Tie2 ectodomain variant that selectively binds Ang2 and acts as soluble ligand trap to sequester Ang2; however, the mechanism of Ang2-binding selectivity is unknown. In the present study, we used directed protein evolution to enhance Ang2-binding affinity of this Tie2 ectodomain trap. We examined contributions of individual residues in the ligand-binding interface of Tie2 to Ang1 and Ang2 binding. Surprisingly, different combinations of Tie2 residues were found to bind each ligand, with hydrophobic residues binding both ligands and polar residues contributing selectively to either Ang1 or Ang2 binding. Our analysis also identified a single Tie2 residue, His168, with a pivotal role in both Ang1 and Ang2 binding, enabling competition between binding ligands. In summary, this study reports an enhanced-affinity Ang2-selective ligand trap with potential for therapeutic development and reveals the mechanism behind its selectivity. It also provides the first analysis of contributions of individual Tie2 residues to Ang1 and Ang2 binding and identifies selectivity-determining residues that could be targeted in the future design of small molecule and other inhibitors of Ang2 for the treatment of vascular dysfunction.

The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region.

We have previously reported that the 26-amino acid N-terminus stalk region of soluble Fas ligand (sFasL), which is separate from its binding site, is required for its biological function. Here we investigate the mechanisms that link the structure of the sFasL stalk region with its function. Using site-directed mutagenesis we cloned a mutant form of sFasL in which all the charged amino acids of the stalk region were changed to neutral alanines (mut-sFasL). We used the Fas-sensitive Jurkat T-cell line and mouse and human alveolar epithelial cells to test the bioactivity of sFasL complexes, using caspase-3 activity and Annexin-V externalization as readouts. Finally, we tested the effects of mut-sFasL on lipopolysaccharide-induced lung injury in mice. We found that mutation of all the 8 charged amino acids of the stalk region into the non-charged amino acid alanine (mut-sFasL) resulted in reduced apoptotic activity compared to wild type sFasL (WT-sFasL). The mut-sFasL attenuated WT-sFasL function on the Fas-sensitive human T-cell line Jurkat and on primary human small airway epithelial cells. The inhibitory mechanism was associated with the formation of complexes of mut-sFasL with the WT protein. Intratracheal administration of the mut-sFasL to mice 24 hours after intratracheal Escherichia coli lipopolysaccharide resulted in attenuation of the inflammatory response 24 hours later. Therefore, the stalk region of sFasL has a critical role on bioactivity, and changes in the structure of the stalk region can result in mutant variants that interfere with the wild type protein function in vitro and in vivo.

Airway monocyte modulation relates to tumour necrosis factor dysregulation in neutrophilic asthma.

BackgroundDysregulation of tumour necrosis factor-α (TNF-α) signalling is implicated in neutrophilic asthma. TNF-α signalling involves membrane-bound and soluble ligand (TNF-α) and receptors (TNFRs); however, little is known about how these proteins are altered in asthma. We hypothesised that intercompartment-, immune cell- and/or asthma inflammatory phenotype-dependent regulation could relate to TNF dysregulation in neutrophilic asthma.MethodsMeasurements were made in 45 adults with asthma (36 non-neutrophilic, 9 neutrophilic) and 8 non-asthma controls. Soluble TNF-α, TNF receptor 1 (TNFR1) and TNFR2 were quantified in plasma and sputum supernatant by ELISA, and membrane-bound TNF-α/TNFR1/TNFR2 measured on eosinophils, neutrophils, monocytes, and macrophages in blood and sputum by flow cytometry. Marker expression was compared between inflammatory phenotypes and compartments, and relationship of membrane-bound and soluble TNF markers and immune cell numbers tested by correlation.ResultsSoluble sputum TNFR1 and TNFR2 were increased in neutrophilic versus non-neutrophilic asthma (p=0.010 and p=0.029). Membrane-bound TNF-α expression was upregulated on sputum versus blood monocytes, while TNFR1 and TNFR2 levels were reduced on airway versus blood monocytes and neutrophils. Soluble TNFR1 and TNFR2 in sputum significantly correlated with the number of airway monocytes (p=0.016, r=0.358 and p=0.029, r=0.327).ConclusionOur results imply that increased sputum soluble TNF receptor levels observed in neutrophilic asthma relate to the increased recruitment of monocytes and neutrophils into the airways and their subsequent receptor shedding. Monocytes also increase TNF-α ligand expression in the airways. These results suggest an important contribution of airway monocytes to the altered inflammatory milieu in neutrophilic asthma.

Diversity of Dominant Peripheral T Cell Receptor Clone and Soluble Immune Checkpoint Proteins Associated With Clinical Outcomes Following Immune Checkpoint Inhibitor Treatment in Advanced Cancers.

Dynamic changes of the peripheral T cell receptor (TCR) and soluble receptors and ligands (sRLs) have the potential to be used as biomarkers to monitor the evolution of the immune system in tumor patients undergoing immunotherapy. These functional biomarkers could be used to predict immune response to treatment with immune checkpoint inhibitors (ICIs) and to provide high-value information on the immune function status of cancer patients, thereby helping physicians to make effective clinical decisions. We collected paired pre- and post-treatment peripheral blood samples from 31 solid tumor patients treated with ICIs. TCR and sRL status were investigated using next-generation sequencing and magnetic bead panels. We found that the diversity of the dominant TCR clone at baseline was correlated with durable clinical benefit in patients receiving single-agent treatment. The D50 index, the diversity from the cumulative 50% of the total complementary determinant region 3, was obtained during treatment. A significant difference in progression-free survival was demonstrated between the D50 high and D50 low groups. This result was validated in an independent cohort. A signature including soluble immune checkpoint proteins (sICPs) was identified. Upregulation of the signature during treatment was correlated with durable clinical benefit. All these results indicate that a novel biomarker based on peripheral TCR and sICPs has the potential to be used in prognostic prediction and for rapid determination of therapeutic outcomes in patients treated with immune checkpoint inhibitors.

CLEC-2 Prevents Accumulation and Retention of Inflammatory Macrophages During Murine Peritonitis.

Platelets play a key role in the development, progression and resolution of the inflammatory response during sterile inflammation and infection, although the mechanism is not well understood. Here we show that platelet CLEC-2 reduces tissue inflammation by regulating inflammatory macrophage activation and trafficking from the inflamed tissues. The immune regulatory function of CLEC-2 depends on the expression of its ligand, podoplanin, upregulated on inflammatory macrophages and is independent of platelet activation and secretion. Mechanistically, platelet CLEC-2 and also recombinant CLEC-2-Fc accelerates actin rearrangement and macrophage migration by increasing the expression of podoplanin and CD44, and their interaction with the ERM proteins. During ongoing inflammation, induced by lipopolysaccharide, treatment with rCLEC-2-Fc induces the rapid emigration of peritoneal inflammatory macrophages to mesenteric lymph nodes, thus reducing the accumulation of inflammatory macrophages in the inflamed peritoneum. This is associated with a significant decrease in pro-inflammatory cytokine, TNF-α and an increase in levels of immunosuppressive, IL-10 in the peritoneum. Increased podoplanin expression and actin remodelling favour macrophage migration towards CCL21, a soluble ligand for podoplanin and chemoattractant secreted by lymph node lymphatic endothelial cells. Macrophage efflux to draining lymph nodes induces T cell priming. In conclusion, we show that platelet CLEC-2 reduces the inflammatory phenotype of macrophages and their accumulation, leading to diminished tissue inflammation. These immunomodulatory functions of CLEC-2 are a novel strategy to reduce tissue inflammation and could be therapeutically exploited through rCLEC-2-Fc, to limit the progression to chronic inflammation.

Pro-cachectic factors link experimental and human chronic kidney disease to skeletal muscle wasting programs.

Skeletal muscle wasting is commonly associated with chronic kidney disease (CKD), resulting in increased morbidity and mortality. However, the link between kidney and muscle function remains poorly understood. Here, we took a complementary interorgan approach to investigate skeletal muscle wasting in CKD. We identified increased production and elevated blood levels of soluble pro-cachectic factors, including activin A, directly linking experimental and human CKD to skeletal muscle wasting programs. Single-cell sequencing data identified the expression of activin A in specific kidney cell populations of fibroblasts and cells of the juxtaglomerular apparatus. We propose that persistent and increased kidney production of pro-cachectic factors, combined with a lack of kidney clearance, facilitates a vicious kidney/muscle signaling cycle, leading to exacerbated blood accumulation and, thereby, skeletal muscle wasting. Systemic pharmacological blockade of activin A using soluble activin receptor type IIB ligand trap as well as muscle-specific adeno-associated virus-mediated downregulation of its receptor ACVR2A/B prevented muscle wasting in different mouse models of experimental CKD, suggesting that activin A is a key factor in CKD-induced cachexia. In summary, we uncovered a crosstalk between kidney and muscle and propose modulation of activin signaling as a potential therapeutic strategy for skeletal muscle wasting in CKD.

Context-aware synthetic biology by controller design: Engineering the mammalian cell.

The rise of systems biology has ushered a new paradigm: the view of the cell as a system that processes environmental inputs to drive phenotypic outputs. Synthetic biology provides a complementary approach, allowing us to program cell behavior through the addition of synthetic genetic devices into the cellular processor. These devices, and the complex genetic circuits they compose, are engineered using a design-prototype-test cycle, allowing for predictable device performance to be achieved in a context-dependent manner. Within mammalian cells, context effects impact device performance at multiple scales, including the genetic, cellular, and extracellular levels. In order for synthetic genetic devices to achieve predictable behaviors, approaches to overcome context dependence are necessary. Here, we describe control systems approaches for achieving context-aware devices that are robust to context effects. We then consider cell fate programing as a case study to explore the potential impact of context-aware devices for regenerative medicine applications.

MO430MASS-SPECTROMETRIC IDENTIFICATION OF POST-TRANSLATIONAL GUANIDINYLATED PROTEINS IN THE CONTEXT OF SYSTEMIC LUPUS ERYTHEMATOSUS

Abstract Background and Aims With continuous identification of post-translational modified isoforms of proteins, it is becoming increasingly clear that post-translational modifications limit or modify the biological functions of native proteins are majorly involved in development of various chronic disease. This is mostly due to technically advanced molecular identification and quantification methods, mainly based on mass spectrometry. Mass spectrometry has become one of the most powerful tools for the identification of lipids. Method In this study, we used sophisticated high-resolution mass-spectrometric methods to analyze the soluble ligand of receptor Notch-3, namely the Y-box protein (YB)-1, in serum from systemic lupus erythematosus (SLE) patients. In addition, kidneys of lupus-prone (MRL.lpr) mice were analyzed by mass-spectrometric imaging techniques to identify the underlying pathomechanisms. Serum YB-1 was isolated by chromatographic methods, afterwards digested by trypsin and analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The kidneys were fixed in paraffin, then kidney sections were deparaffinized, tryptic digested and analyzed by mass-spectrometric imaging techniques. Mass-spectrometry of extracellular YB-1 in SLE patient serum revealed post-translational guanidinylation of two lysine’s within the highly conserved cold shock domain (CSD) of the YB-1 protein (YB-1-2G). Patients with increased disease activity and those with active renal involvement (lupus nephritis, LN) had a higher degree of dual-guanidinylation within the CSD. Of note, at least one of these modifications was present in all analyzed LN patients, whereas single-guanidinylated YB-1 was present in only one and double modification in none of the control individuals. Mass-spectrometric imaging analyses specifically localized YB-1-2G and increases Notch-3 expression in kidney sections from MRL.lpr mice. Results The data from this study clearly demonstrate the high potential of high-resolution mass spectrometric methods as well as mass spectrometric imaging techniques to identify pathomechanisms of diseases like SLE/LN.

Soluble type immune checkpoint regulators using multiplex luminex immunoassay in chronic hepatitis B patients

AimsSoluble immune checkpoint regulators (sICs) were reported to have clinical impact on the diagnosis and progress of various diseases. This study compared the serum levels of 16 sICs in patients...

Microbial Phagocytic Receptors and Their Potential Involvement in Cytokine Induction in Macrophages.

Phagocytosis is an essential process for the uptake of large (>0.5 µm) particulate matter including microbes and dying cells. Specialized cells in the body perform phagocytosis which is enabled by cell surface receptors that recognize and bind target cells. Professional phagocytes play a prominent role in innate immunity and include macrophages, neutrophils and dendritic cells. These cells display a repertoire of phagocytic receptors that engage the target cells directly, or indirectly via opsonins, to mediate binding and internalization of the target into a phagosome. Phagosome maturation then proceeds to cause destruction and recycling of the phagosome contents. Key subsequent events include antigen presentation and cytokine production to alert and recruit cells involved in the adaptive immune response. Bridging the innate and adaptive immunity, macrophages secrete a broad selection of inflammatory mediators to orchestrate the type and magnitude of an inflammatory response. This review will focus on cytokines produced by NF-κB signaling which is activated by extracellular ligands and serves a master regulator of the inflammatory response to microbes. Macrophages secrete pro-inflammatory cytokines including TNFα, IL1β, IL6, IL8 and IL12 which together increases vascular permeability and promotes recruitment of other immune cells. The major anti-inflammatory cytokines produced by macrophages include IL10 and TGFβ which act to suppress inflammatory gene expression in macrophages and other immune cells. Typically, macrophage cytokines are synthesized, trafficked intracellularly and released in response to activation of pattern recognition receptors (PRRs) or inflammasomes. Direct evidence linking the event of phagocytosis to cytokine production in macrophages is lacking. This review will focus on cytokine output after engagement of macrophage phagocytic receptors by particulate microbial targets. Microbial receptors include the PRRs: Toll-like receptors (TLRs), scavenger receptors (SRs), C-type lectin and the opsonic receptors. Our current understanding of how macrophage receptor stimulation impacts cytokine production is largely based on work utilizing soluble ligands that are destined for endocytosis. We will instead focus this review on research examining receptor ligation during uptake of particulate microbes and how this complex internalization process may influence inflammatory cytokine production in macrophages.