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Background: Dendritic cells (DCs) are vital for initiating antigen-specific T cell-dependent antitumor immune responses. However, emerging evidence reveals that local DCs within the tumor microenvironment (TME) are tolerized to facilitate immune evasion. Reprogramming of DCs is recognized as a promising strategy for tumor immunotherapy. Leukocyte immunoglobulin-like receptor B4 (LILRB4) is an inhibitory receptor belonging to the LILR family which is mainly expressed on normal myeloid cells and myeloid-derived malignant cells. It is upregulated on tolerogenic DCs (tolDCs), which exhibit low expression levels of costimulatory molecules and resistance to DC maturation. Targeting LILRB4 to reprogram tolDCs has been reported to be a promising strategy for cancer treatment. Here we report the preclinical development of an LILRB4 antagonist antibody, ATG-034. Method: The protein-based and cell-based binding affinity of ATG-034 were measured using SPR, ELISA and FACS analysis. Fc receptor (FcR) stimulation assay was used to evaluate the ability of ATG-034 to block the interaction of LILRB4 with its ligand fibronectin. ATG-034-mediated enhancement of antigen presentation and co-stimulation ability of DCs was determined by FACS analysis for the change of surface expression of HLA-DR, HLA-ABC, CD86, and CD206. The mixed lymphocyte reaction (MLR) assay was used to assess the immunomodulatory potential of ATG-034. The in vivo antitumor efficacy of ATG-034 was evaluated in a radiation therapy-resistant murine Lewis lung carcinoma (LLC) syngeneic model. Results: ATG-034 binds to LILRB4 protein with a single-digit nM affinity. It potently reversed the fibronectin-mediated inhibition of FcR-driven TNF-α cytokine production. TolDCs were reprogrammed by ATG-034 to immunogenic DCs with significant upregulation of the expression of HLA-DR, HLA-ABC, CD86 and downregulation of the expression of CD206. Furthermore, ATG-034 remarkably reinvigorated tolDCs for priming T cell activation with noticeable upregulation of the expression of CD25 and the secretion of IFN-γ, enhancing antitumor immunity. In addition, 10 mg/kg ATG-034 significantly inhibited the LLC tumor growth in vivo with a TGI of 40.28%, while a benchmark antibody only demonstrated a 24.43% TGI. Conclusion: Our data show that ATG-034 reprograms tolDC, enhancing anti-tumor immunity, and demonstrates potent in vivo anti-tumor efficacy. Therefore, ATG-034 may be a promising strategy for the treatment of cancer. Citation Format: Ao Sun, Enlin Zheng, Huili Cao, Mengshi Cao, Suya Bai, Peng Chen, Linjie Tian, Jay Mei, Bo Shan, Bing Hou. ATG-034, an LILRB4 antagonist antibody, reinvigorates dendritic cells and prevents tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6384.

BackgroundCollagen and C1q in the extracellular matrix (ECM), are the predominant ligands for LAIR-1, an inhibitory receptor expressed on the cell surface of several immune cell subsets that inhibits immune activation and migration. LAIR-2, a soluble homolog of LAIR-1, competes for binding to collagen and C1q and serves as a natural decoy to promote immune function under normal conditions. Dysregulation of the ECM and increased expression of collagen and C1q within the tumor microenvironment (TME) plays a critical role in promoting tumor progression. NC410 was engineered to overcome this highly immunosuppressive environment by blocking LAIR-1 function, reversing immune suppression, and inducing ECM remodeling to promote immune cell infiltration within the TME.MethodsThis is a first in human, phase 1/2, open-label, single-armed dose-escalation study to determine the safety, tolerability, dose-limiting toxicity (DLT), maximum tolerated dose (MTD), recommended phase 2 dose, preliminary efficacy and to explore pharmacodynamic biomarkers of NC410 (figure 1). Key eligibility criteria include subjects with advanced or metastatic solid tumors with measurable disease based on RECIST v1.1.ResultsAs of 07/22/2021, a total of 16 patients have been enrolled, treated, and completed the DLT period. NC410 (up to 60 mg), was well tolerated, with no safety concerns, infusion-related toxicities, or DLT reported. No anti-drug antibody (ADA) was detected post-NC410 up to 60 mg treatment. As expected, the C1Q level decreased immediately after the NC410 infusion and was replenished after two hours. Evaluation from samples to date available up to cycle 5 suggests that there was no reduction in the baseline C1Q level with subsequent dosing (figure 2). LAIR-2 levels continued to increase in a dose-dependent fashion post-NC410 dosing and marginal increase pre-dose (figure 3). Interestingly, we observed an increase in soluble LAIR-1 over time in a similar pattern to LAIR-2 (figure 4). Furthermore, immunophenotyping of patient whole blood suggests a trend towards an increase in CD4+ and CD8+ T cells including LAIR-1+ T cells in cohort 4, although overall expression levels of LAIR-1 did not appear to increase (ongoing analysis). Cytokines, chemokines, and collagen degradation products (CDP) will be evaluated as potential pharmacodynamic biomarkers as we continue through higher dose cohorts.Abstract 487 Figure 1NC410: study schemaAbstract 487 Figure 2C1Q levels show immediate reduction after NC410 dosing with no accumulated depletionAbstract 487 Figure 3LAIR-2 levels show an increase in dose-dependent fashion after NC410 dosing with marginal increase pre-doseAbstract 487 Figure 4Soluble LAIR-1 levels show a similar pattern to LAIR-2 levelsConclusionsPreliminary evaluation of NC410 in subjects with advanced or metastatic solid tumors appears to be safe and well-tolerated with evidence of immune modulation consistent with predictive PK/PD modeling in a Phase 1/2 open-label study. Further evaluation will be done with increasing doses to confirm these initial findings.Trial RegistrationNCT04408599Ethics ApprovalThis study has been approved by the IRB of all the participating institutions, and all participants have given informed consent before taking part in the study.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Background: CD73 is a cell surface enzyme which is highly expressed in the tumor microenvironment (TME). Degradation of adenosine monophosphate (AMP) into adenosine by CD73 results in the generation of an immunosuppressed and pro-angiogenic niche within the TME that promotes the onset and progression of cancer. Targeting CD73 has resulted in favourable anti-tumor effects in preclinical models and in clinic. One of the key mechanisms of therapeutic activity by CD73 inhibition is restoring the normal function of effector T cells from adenosine inhibition. ATG-037 is a highly potent and selective oral small molecule inhibitor of CD73. This study compared the ability of ATG-037 and clinical CD73 antibodies in reverting immunosuppression in higher-AMP environments in vitro. Methods: The activity of ATG-037 and CD73 antibodies (Hu101-28, MEDI9447) in inhibiting enzyme function of cell surface CD73 was evaluated by measuring the ATP-dependent oxidation of luciferin, which is inhibited by AMP, using Cell Titer Glo assay. Reversal of AMP/adenosine-mediated immune suppression of human T cells by CD73 inhibitors was determined by measuring T cell function in the presence of different concentrations of exogenous AMP. Markers for T cell proliferation, activation and cytotoxicity were assessed by flow cytometry, and the cytokine levels were measured by ELISA. Results: ATG-037, Hu101-28 and MEDI9447 inhibited the enzyme activity of cell surface-expressed CD73 on human A375 cells under 100 µM AMP with a half-maximal inhibitory concentration (IC50) of 0.36 nM,20.94 nM and 3.46 nM, respectively. ATG-037 demonstrated complete inhibition of CD73 activity, whereas MEDI9447 did not reach complete CD73 inhibition. T cell proliferation and activation induced by CD3/CD28 dynabeads were suppressed by 100 µM of extracellular AMP and the suppression was relieved by the addition of ATG-037 with half-maximal effective concentration (EC50) of 21.6 nM for CD8+ T cell proliferation, 27 nM for T cell activation, 2 nM for granzyme B expression, and 6.5 nM for T cell IFN-γ production. While Hu101-28 and MEDI9447 fail to restore the T cell function. In a mixed lymphocyte reaction assay, ATG-037, but not Hu101-28 or MEDI9447 reversed AMP-dependent T cell suppression. In addition, ATG-037 rescued immune cell activation in high AMP (500 µM, 1000 µM) environments. Conclusion: ATG-037 demonstrated potent and complete CD73 enzyme inhibition and stronger ability in restoring T cell function from high-level AMP mediated suppression, compared with clinical anti-CD73 antibodies. These data implicate the potential therapeutic advantages of small molecule CD73 inhibitors over blocking antibodies. Citation Format: Gang Bian, Jiahui Hua, Linjie Tian, Kevin Lynch, Jay Mei, Bo Shan, Bing Hou. ATG-037, a highly potent small molecule CD73 inhibitor has superior activity of reversing immunosuppression in higher-AMP environments compared with anti-CD73 antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2576.

Background: Antibody-drug conjugates (ADCs) have become promising antitumor agents in recent years. ADCs are comprised of a monoclonal antibody, targeting antigens expressed at higher levels on tumor cells than on normal cells, cross-linked to small molecule payloads with cytotoxic activity. Human Claudin 18.2 (CLDN18.2) is overexpressed in a large proportion of gastric and pancreatic cancers, with restricted normal tissue expression. The monoclonal antibody targeting CLDN18.2 (IMAB362) has demonstrated promising clinical benefit in combination with chemotherapies for gastric cancer patients. However, the same antibody shows suboptimal efficacy in patients with low CLDN18.2 expression. Here we report the potent in vivo efficacy of ATG-022, a CLDN18.2 ADC, in multiple gastric cancer patient-derived xenograft (PDX) models, including those with low CLDN18.2 expression. Methods: The binding affinity of ATG-022 with CLDN18.2 was detected by SPR and FACs analysis. The in vitro 50% inhibition concentration (IC50) of ATG-022 was determined in CLDN18.2 positive cell lines using CellTiter-Glo luminescent cell viability assay. In vivo efficacy of ATG-022 was evaluated in a series of gastric cancer PDX models with different expression level of CLDN18.2 The expression level of CLDN18.2 was determined using IHC staining. ATG-022 was dosed twice at 1 mg/kg, 3 mg/kg or 10 mg/kg every two weeks. Results: ATG-022 binds to CLDN18.2 protein with sub-nM affinity. It induced potent in vitro cytotoxicity in CHOK1 cells overexpressing CLDN18.2, with an IC50 of 5-7nM. Bystander killing by ATG-022 was observed. ATG-022 demonstrated potent in vivo antitumor efficacy in a gastric cancer PDX model with high CLDN18.2 expression. Intravenous dosing of 1 mg/kg, 3 mg/kg, and 10 mg/kg ATG-022 induced 35.41%, 71.56% tumor growth inhibition (%TGI) and tumor regression respectively. In a PDX model with low CLDN18.2 expression, 3 mg/kg ATG-022 or 10 mg/kg clinical benchmark CLDN18.2-ADC did not inhibit tumor growth, while 10 mg/kg ATG-022 induced tumor regression. Conclusions: ATG-022 demonstrated potent in vitro and in vivo antitumor effects, with in vivo efficacy observed in CLDN18.2-low expression PDX models, suggesting a promising therapeutic strategy for gastric cancer patients with a broad range of CLDN18.2 expression levels. Citation Format: Peng Chen, Yun Liu, Min Deng, Linjie Tian, Kevin Lynch, Bo Shan, Jay Mei, Bing Hou. ATG-022, an antibody-drug conjugate targeting Claudin 18.2, demonstrated potent in vivo efficacy in gastric cancer patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1143.

Background: RAS proteins play a vital role in cellular proliferation, differentiation, and survival, through their association and crosstalk with multiple pathways, the best characterized of which are the Ras/Raf/Mek/Erk and PI3K/PTEN/Akt/mTOR pathways. Activating mutations in KRAS are a hallmark of cancer. Clinical targeting of KRAS G12C mutation has demonstrated profound overall response rate (ORR) with relatively short progression-free survival (PFS). Some potential reasons for short PFS are MAPK pathway re-activation through wild type RAS and activation of PI3K/mTOR pathway, which lead to the bypass and acquired resistance to KRAS G12C inhibitors. The combination of KRAS G12C inhibitor with mTOR or ERK1/2 inhibitor might overcome drug resistance and prolong PFS. This study tested the antitumor effects induced by the combination of ATG-012, a KRAS G12C inhibitor, with SHP2 inhibitor (ET0038), ERK 1/2 kinase inhibitor (ATG-017), mTORC1/2 kinase inhibitor (ATG-008) or XPO1 inhibitor (selinexor), in preclinical tumor models. Methods: The in vivo combinations of the drugs were tested in NCI-H358 (non-small cell lung cancer) and Mia-Paca-2 (pancreatic cancer) CDX mouse model. The tumor bearing mouse were treated with vehicle control, ATG-012 (3 to 100 mg/kg, QD), ET0038 (5mg/kg, QD), selinexor (10mg/kg, MWF), ATG-017 (25mg/kg, QD), ATG-008 (10mg/kg, QD) or the combination for 18 to 27 days. Tumor size was measured twice a week and tumor growth inhibition (TGI) was evaluated compared with vehicle control group. Results: In the NCI-H358 CDX in vivo study, mono therapy with ATG-012 (10mg/kg), selinexor, or ATG-017 induced tumor growth inhibition (TGI) of 44.29%,73.71% and 80.85%, respectively on day 8 after grouping. The combination of ATG-012 and selinexor showed 84.96% TGI, while ATG-012 plus ATG-017 showed 97.13% TGI. The combinations induced continual tumor regression from day 9 to day 21.In the Mia-PaCa-2 CDX in vivo study, the mono therapy with ATG-012 (3 mg/kg) showed 65.6% TGI on day 27 after grouping, while single agent ET0038 or ATG-008 treatment induced 84.6% and 75.4% TGI, respectively. The combination of ATG-012 and ET0038 showed 119.1% TGI; and ATG-012 -ATG-008 combination showed 92.3% TGI. Conclusions: Strong in vivo synergism has been observed for the combination of a Kras (G12C) inhibitor (ATG-012) with a SHP2 inhibitor, ERK 1/2 inhibitor, mTORC1/2 inhibitor or XPO1 inhibitor, suggesting promising clinical therapeutic strategies for cancer patients carrying the KRAS G12C mutation. Citation Format: Jian Wang, Peng Chen, Ya Kong, Hui Yuwen, Bin Jiang, Linjie Tian, Bing Hou, Jay Mei, Bo Shan. Synergistic effects of the combination of Kras (G12C) with SHP2, ERK 1/2, mTORC1/2 or XPO1 inhibition for the treatment of Kras (G12C) mutated cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2679.

Background Abnormalities in the extracellular matrix of tumor microenvironments support tumor progression, lead to immune dysfunction, and provide a target for cancer therapeutics. Collagens are a primary component of the extracellular matrix. Abnormal levels of collagen and of the collagen-domain containing complement component 1q (C1q) in tumor microenvironments has been proposed to disrupt anti-tumor immunity. LAIR-1 is an adhesion molecule and inhibitory receptor expressed on the cell surface of several immune cell subsets. LAIR-1 binding to collagen-like domains present in collagens and C1q inhibit immune cell function. LAIR-2 is a soluble homolog of LAIR-1 that binds to and outcompetes LAIR-1 binding to collagens and C1q and serves as a natural decoy to promote immune function. Methods Taking advantage of a natural decoy system, we designed a protein biologic, NC410, composed of LAIR-2 fused with a functional IgG1 Fc domain to target collagen-rich tumors and promote immune activation, infiltration and effector function. Results NC410 has increased avidity due to Fc mediated dimerization, and blocks LAIR-1 interactions with ligands, and LAIR-1 signaling. In vivo administration of NC410 in humanized tumor models reduced tumor growth in a dose dependent fashion. NC410 increased the numbers of infiltrating human CD8+ and CD4+ T cells in the tumor, which is associated with increased levels of chemokines in the local tumor environment. Effector function was also enhanced, as denoted by increased levels of IFN-gamma and Granzyme B in the local tumor environment. In addition, NC410 increased specific collagen degradative products in the serum of humanized tumor-bearing mice, suggesting NC410 may promote tumor microenvironment remodeling and immune accessibility to further promote anti-tumor immunity. Conclusions These data support NC410 as a novel therapeutic for targeting collagen-rich tumors and enabling normalization of the tumor-immune microenvironment. FIH studies have recently been initiated with NC410.

Collagens are a primary component of the extracellular matrix and are functional ligands for the inhibitory immune receptor leukocyte associated immunoglobulin-like receptor-1 (LAIR-1). Leukocyte associated immunoglobulin-like receptor-2 (LAIR-2) is a secreted protein that can act as a decoy receptor by binding collagen with higher affinity than LAIR-1. We propose that collagens promote immune evasion by interacting with LAIR-1 and that LAIR-2 could release LAIR-1 mediated immune suppression. Analysis of public datasets shows high LAIR-2 expression being associated with a favorable outcome in certain tumors. We designed a dimeric LAIR-2 with a functional IgG1 Fc tail, NC410, and showed that NC410 reduces tumor growth and increases T cell expansion and effector function in humanized tumor models. Immunohistochemical analysis of human tumors shows that NC410 binds to collagen-rich areas where LAIR-1+immune cells are localized. Our findings show that NC410 might be a powerful new strategy for cancer immunotherapy for immune-excluded tumors.

Collagens are a primary component of the extracellular matrix and are functional ligands for the inhibitory immune receptor leukocyte-associated immunoglobulin-like receptor (LAIR)-1. LAIR-2 is a secreted protein that can act as a decoy receptor by binding collagen with higher affinity than LAIR-1. We propose that collagens promote immune evasion by interacting with LAIR-1 expressed on immune cells, and that LAIR-2 releases LAIR-1-mediated immune suppression. Analysis of public human datasets shows that collagens, LAIR-1 and LAIR-2 have unique and overlapping associations with survival in certain tumors. We designed a dimeric LAIR-2 with a functional IgG1 Fc tail, NC410, and showed that NC410 increases human T cell expansion and effector function in vivo in a mouse xenogeneic-graft versus-host disease model. In humanized mouse tumor models, NC410 reduces tumor growth that is dependent on T cells. Immunohistochemical analysis of human tumors shows that NC410 binds to collagen-rich areas where LAIR-1+ immune cells are localized. Our findings show that NC410 might be a novel strategy for cancer immunotherapy for immune-excluded tumors.

Extensive research has led to recent approval of novel therapies such as mylotarg, venetoclax, glasdegib and CC486, and small molecule inhibitors against actionable mutations such as ivosidenib (IDH1), enasidenib (IDH2), gliteritinib and midostaurin (FLT3) in AML. However, the mainstay of treatment in AML remains unchanged since the 1970s. There is a significant unmet need for AML patients that fail to respond to or relapse after standard-of-care (SOC) treatments including allogeneic stem cell transplantation and targeting actionable mutations. In addition, a large fraction of SOC patients invariably relapse due to persistence of chemotherapy-resistant leukemia stem cells (LSCs) or immune evasion. Therefore, identification of unique therapies that preferentially target elusive LSCs and promote immune responses to AML to prevent relapse are highly sought after. Unlike, targeting acute lymphoblastic leukemia (ALL) with CD19 or CD22 with various modalities, when developing AML therapies, it is of paramount importance to differentiate LSCs from hematopoietic stem cells (HSCs) to lessen or abolish unavoidable cytopenias. Leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) is an immune checkpoint receptor on T cells and myeloid cells that delimits immune cell activation through binding to endogenous collagen ligands. In addition, LAIR-1 is universally expressed on AML blasts and may sustain AML survival signals. We demonstrated using multi-color flow cytometry that LAIR-1 is highly expressed in AML blasts (n=9 of 9) and that LAIR-1 expression in LSCs (markers: CD34 +CD38 -CD90 -CD45RA +/- or CD34 -CD117 +CD244 +/-) is high compared with negligible expression of LAIR-1 in HSCs (markers: CD34 +CD38 -CD90 +CD99 -) (n=3) (Figure 1). Based on these findings, we hypothesized that a LAIR-1 monoclonal antibody (mAb) would disrupt LAIR-1 mediated survival signaling and preferentially target LAIR-1 expressing AML LSCs and blast cells but not HSCs. To test this, we developed a novel LAIR-1 targeting mAb with a functional human IgG 1 isotype that blocks LAIR-1 binding to its ligands (including collagens, complement component C1q, MBL and SP-D) To characterize the anti-leukemic effect of the LAIR-1 mAb we performed an in vitro antibody dependent cell cytotoxicity (ADCC) assay with LAIR-1 expressing AML cells (MOLT4 and MV-4-11). Compared with isotype control, the LAIR-1 mAb significantly increased leukemia cell death (MV411 = 17% above isotype, and MOLT4 = 29.24% above isotype at 15 µg/ml), suggesting that the LAIR-1 mAb confers ADCC activity against LAIR1 + AML cells (Figure 2). To elucidate if the LAIR-1 mAb has a direct signaling effect on LAIR-1 + AML cells, a colony forming unit assay using primary AML cells was carried out. Interestingly, the LAIR-1 mAb inhibited colony formation by AML CD34 + cells (40-60% decreased compared with isotype control, N=4), but not normal CD34 + cells. These data suggests that our LAIR-1 mAb stimulated LAIR-1 signaling that inhibits LSC self-renewal. We then tested the in vivo anti-leukemia effect of the mAb in cell line derived xenograft (CDX) models (immune deficient mice transplanted with MV-4-11 expressing luciferase). In vivo bioluminescence imaging indicated that the LAIR-1 mAb significantly inhibited in vivo AML growth (91% reduction of total flux)(Figure 3). A significant increase in cell death was observed in the presence of the mAb in the blood (47%), spleen (89.4%) and bone marrow (27.6%). Similar to the anti-leukemic effect in CDX AML models, the LAIR-1 mAb significantly suppressed in vivo growth of AML patient derived xenografts (5 different primary AML donors) (10-90% human CD33 + AML cells in isotype control treatment vs 0.5-5% CD33 + AML cells in anti-LAIR-1 treatment, N=3) (Figure 4), while minimally impacting normal immune cells. Taken together, our studies suggest that the LAIR-1 mAb we generated is a novel AML immunomedicine that preferentially eradicates AML LSCs and blasts while preserving healthy HSCs through disruption of AML survival signals and clearance of AML through ADCP and ADCC. Additional studies are currently evaluating if this novel LAIR-1 mAb has other mechanisms of action that contribute to overall in vivo activity, including reduction of AML niche implantation, regulation of bone marrow homing and regulation of anti-tumor immunity. Figure 1 Figure 1. Disclosures Tian: NextCure: Ended employment in the past 24 months. Paucarmayta: NextCure: Current Employment. Lovewell: NextCure: Current Employment. Maloveste: NextCure: Current Employment. Copeland: NextCure: Current Employment. O'Neill: NextCure: Current Employment. Patel: NextCure: Current Employment. Liu: NextCure: Current Employment, Current holder of stock options in a privately-held company. Myint: NextCure: Current Employment, Current holder of stock options in a privately-held company. Langermann: NextCure: Current Employment, Current holder of stock options in a privately-held company. Flies: NextCure: Current Employment, Current holder of stock options in a privately-held company. Kim: Nextcure: Research Funding.

Based on the structural advantages of trough beam and corrugated steel web, considering the capacity, durability and rapid construction of composite structure, a new type of composite structure beam is proposed in this paper, namely Composite Trough Beam with Corrugated Steel Web wrapped with Steel Plate (CTBCS). A scale experimental CTBCS was designed and fabricated to study the bending properties of failure mode, load-displacement curve and section strain distribution of CTBCS. In addition, the calculation method of bending capacity of CTBCS was proposed, and the effects of the thickness of steel plate, the diameter of steel bar in upper flange concrete slab and the height to span ratio on the bending properties of CTBCS were studied. The study in this paper provides reference values for the experimental scheme formulation of composite trough beam, and lays a foundation for the design and application of CTBCS.

Proinflammatory cytokine overproduction and excessive cell death, coupled with impaired clearance of apoptotic cells, have been implicated as causes of failure to resolve gut inflammation in inflammatory bowel diseases. Here we have found that dendritic cells expressing the apoptotic cell–recognizing receptor CD300f play a crucial role in regulating gut inflammatory responses in a murine model of colonic inflammation. CD300f-deficient mice failed to resolve dextran sulfate sodium–induced colonic inflammation as a result of defects in dendritic cell function that were associated with abnormal accumulation of apoptotic cells in the gut. CD300f-deficient dendritic cells displayed hyperactive phagocytosis of apoptotic cells, which stimulated excessive TNF-α secretion predominantly from dendritic cells. This, in turn, induced secondary IFN-γ overproduction by colonic T cells, leading to prolonged gut inflammation. Our data highlight a previously unappreciated role for dendritic cells in controlling gut homeostasis and show that CD300f-dependent regulation of apoptotic cell uptake is essential for suppressing overactive dendritic cell–mediated inflammatory responses, thereby controlling the development of chronic gut inflammation.

In this study, three full-scale orthotropic steel plates set with single U rib, double U ribs and three U ribs were fabricated based on the mechanical characteristics of Changhong bridge in Ningbo. Several displacement and strain measuring points were selected as the analysis indexes, and two loading modes of axial and biased loading were designed. The buckling shape, out of plane displacement and strain variation law of the components under different loading conditions were analyzed. The DIC method was used to analyze the buckling and force transfer mechanism of the component in the loading process. Moreover, the finite element simulation and parameter analysis were carried out under eight component parameters by using ANSYS software. Through the experimental and parameter analysis in this paper, the instability mode, change of out of plane displacement, strain distribution and force transfer mechanism of orthotropic steel plates under different loading modes were obtained, which can provide reference value for the stability study of orthotropic steel plate and orthotropic steel box girder.

Receptor CD300b is implicated in regulating the immune response to bacterial infection by an unknown mechanism. Here, we identified CD300b as a lipopolysaccharide (LPS)-binding receptor and determined the mechanism underlying CD300b augmentation of septic shock. In vivo depletion and adoptive transfer studies identified CD300b-expressing macrophages as the key cell type augmenting sepsis. We showed that CD300b, and its adaptor DAP12, associated with Toll-like receptor 4 (TLR4) upon LPS binding, thereby enhancing TLR4-adaptor MyD88- and TRIF-dependent signaling that resulted in an elevated pro-inflammatory cytokine storm. LPS engagement of the CD300b-TLR4 complex led to the recruitment and activation of spleen tyrosine kinase (Syk) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). This resulted in an inhibition of the ERK1/2 protein kinase- and NF-κB transcription factor-mediated signaling pathways, which subsequently led to a reduced interleukin-10 (IL-10) production. Collectively, our data describe a mechanism of TLR4 signaling regulated by CD300b in myeloid cells in response to LPS.

Efficient phagocytosis of apoptotic cells (efferocytosis) is essential for immune homeostasis. Phospholipids exposed on the surface of apoptotic cells, such as phosphatidylserine, supply important "eat-me" signals. Liposomes are lipid bilayer vesicles that can be generated from one or several types of phospholipids of interest. Thus, these vesicles offer versatility, flexibility, and, importantly, a three-dimensional structure for studying the interaction between lipids and their receptors as well as the lipid-receptor interaction-mediated signaling events controlling efferocytosis by cells like professional phagocytes. Here, we describe methods to prepare liposomes, perform liposome-based lipid-receptor binding assays, use liposomes to block efferocytosis, and utilize liposome-coated beads as apoptotic cell surrogates for phagocytosis. © 2018 by John Wiley & Sons, Inc.

Homeostasis requires the immunologically silent clearance of apoptotic cells before they become pro-inflammatory necrotic cells. CD300f (CLM-1) is a phosphatidylserine receptor known to positively regulate efferocytosis by macrophages, and CD300f gene-deficient mice are predisposed to develop a lupus-like disease. Here we show that, in contrast to CD300f function in macrophages, its expression inhibits efferocytosis by DC, and its deficiency leads to enhanced antigen processing and T-cell priming by these DC. The consequences are the expansion of memory T cells and increased ANA levels in aged CD300f-deficient mice, which predispose CD300f-deficient mice to develop an overt autoimmune disease when exposed to an overload of apoptotic cells, or an exacerbated autoimmunity when combined with FcγRIIB deficiency. Thus, our data demonstrates that CD300f helps to maintain immune homeostasis by promoting macrophage clearance of self-antigens, while conversely inhibiting DC uptake and presentation of self-antigens.

IL-4 receptor (R) α, the common receptor chain for IL-4 and IL-13, is a critical component in IL-4- and IL-13-mediated signaling and subsequent effector functions such as those observed in type 2 inflammatory responses. Nonetheless, the existence of intrinsic pathways capable of amplifying IL-4Rα-induced responses remains unknown. In this study, we identified the myeloid-associated Ig receptor CD300f as an IL-4-induced molecule in macrophages. Subsequent analyses demonstrated that CD300f was colocalized and physically associated with IL-4Rα. Using Cd300f(-/-) cells and receptor cross-linking experiments, we established that CD300f amplified IL-4Rα-induced responses by augmenting IL-4/IL-13-induced signaling, mediator release, and priming. Consistently, IL-4- and aeroallergen-treated Cd300f(-/-) mice displayed decreased IgE production, chemokine expression, and inflammatory cell recruitment. Impaired responses in Cd300f(-/-) mice were not due to the inability to generate a proper Th2 response, because IL-4/IL-13 levels were markedly increased in allergen-challenged Cd300f(-/-) mice, a finding that is consistent with decreased cytokine consumption. Finally, CD300f expression was increased in monocytes and eosinophils obtained from allergic rhinitis patients. Collectively, our data highlight a previously unidentified role for CD300f in IL-4Rα-induced immune cell responses. These data provide new insights into the molecular mechanisms governing IL-4Rα-induced responses, and may provide new therapeutic tools to target IL-4 in allergy and asthma.

Autism spectrum disorder (ASD) has complex neurodevelopmental impairments and origins that are linked to both genetic and environmental factors. Hence, there is an urgency to establish animal models with ASD-like characteristics to understand the underlying mechanisms of ASD. Prenatal exposure to valproic acid (VPA) has been shown to cause ASD-like symptoms in humans, rats, and recently zebrafish. The present study investigated the use of VPA exposure to create an ASD model in zebrafish that was verified through observation of ASD-like phenotypes in brain development and behavioral changes in embryonic and larval zebrafish. Our findings revealed that treating zebrafish embryos with VPA starting at 8 hours post fertilization (hpf) resulted in significant: increase in the ASD macrocephalic phenotype; hyperactivity of embryo/larvae movement behaviors; and increases of ASD-like larval social behaviors. Further analysis showed increases in cell proliferation, the proportion of mature newborn neurons, and neural stem cell proliferation in the brain region, which may contribute to the brain overgrowth and macrocephaly observed following VPA exposure. Our study demonstrated that VPA exposure can generate ASD-like phenotypes and behaviors, showing the validity of zebrafish as an alternative model for ASD and underlying mechanism research.

Steel Bridge has been regarded as one of the most popular choices in engineering. Many bridges with large-span and attractive appearance emerge unceasingly due to the use of steel. A new structure of steel ring-web beam (SRWB) was presented, which has the similar appearance with castellated steel beam. SRWB whose top and bottom slabs are connected to circular steel rings by welding or bolting uses circular steel rings to form the web. First, the structure stress characteristics of SRWB were studied. To derive the equivalent flexural stiffness and equivalent shear stiffness for SRWB, the unit stiffness method based on the theory of Timoshenko Beam was proposed. Furthermore, the deflection formula of SRWB was derived, which was validated by finite element analysis (FEA) method. The detailed design of SRWB was completed for studying bending behavior of the structure through two scale model tests. The failure mode of the first specimen under four-point loading, reflecting the inadequacy of the design, was the local instability on the edge of top slab. The failure mode of the second specimen under three-point loading was due to the local buckling of the top slab. The loading and deformation progress of the structure were obtained through two bending tests. The yield loads, strains and test phenomena were also captured through two bending tests. The FEA models of two specimens whose results are compared with the tests were realized. The stress distribution of FEA models were analyzed in order to provide reference for further improvement of design. It can be concluded that SRWB with the rational design has good bending performance.

FcR specific for pentameric IgM (FCMR) is expressed at high levels by B cells. Although circulating IgM has profound effects on responses to pathogens, autoimmunity, and B cell homeostasis, the biologic consequences of its binding to FCMR are poorly understood. We interrogated FCMR contributions to B cell function by studying mice that lack FCMR. FCMR transcripts are expressed at different levels by various B cell subsets. FCMR-deficient mice have reduced numbers of developing B cells, splenic follicular and peritoneal B-2 cells, but increased levels of peritoneal B-1a cells and autoantibodies. After immunization, germinal center B cell and plasma cell numbers are increased. FCMR-deficient B cells are sensitive to apoptosis induced by BCR ligation. Our studies demonstrate that FCMR is required for B cell differentiation and homeostasis, the prevention of autoreactive B cells, and responsiveness to antigenic challenge.

Reportedly, CD300f negatively regulates interactions between dendritic and T cells and acts as an anti-inflammatory molecule in a multiple sclerosis mouse model. We found that a CD300f/Fc chimeric protein specifically binds to apoptotic/dead splenocytes and to apoptotic cells from starved or irradiated lymphocytic cell lines, an observation extended to insect cells. CD300f also binds PMA/ionomycin-activated splenocytes and Ag-stimulated T cells, an interaction inhibited by Annexin V. By ELISA, cosedimentation, and surface plasmon resonance using phospholipid-containing liposomes, we show that CD300f preferentially binds phosphatidylserine and requires a metal ion. Exogenous expression of CD300f in cell lines results in enhanced phagocytosis of apoptotic cells. We conclude that expression of CD300f conveys additional capacity to recognize phosphatidylserine to myeloid cells. The result of this recognition may vary with the overall qualitative and quantitative receptor content, as well as signaling capacity of the expressing effector cell, but enhanced phagocytosis is one measurable outcome.

In this study, cell microarray technology is used to identify novel human genes associated with CRE pathway activation. By reverse transfection, expression plasmids containing full-length cDNAs were cotransfected with the reporter plasmid pCRE-d2EGFP to monitor the activation of the CRE pathway via enhanced green fluorescence protein (EGFP) expression. Of the 575 predominantly novel genes screened, 22 exhibited relatively higher EGFP fluorescence compared with a negative control. After a functional validation with a dual luciferase reporter system that included both cis- and trans-luciferase assays, 4 of the 22 genes (RNF41, C8orf32, C6orf208, and MEIS3P1) were confirmed as CRE-pathway activators. Western blot analysis revealed that RNF41 can promote CREB phosphorylation. These results demonstrate the successful combination of cell microarray technology with this reporting system and the potential of this tool to characterize functions of novel genes in a highly parallel format.

Engulfment of apoptotic cells is predominantly executed by phagocytes via the recognition of “eat me” signals like phosphatidylserine (PS). Various PS-specific receptors exist on phagocytes, including Tyro3, Axl, and MerTK receptor tyrosine kinases (TAMs), T-cell immunoglobulin and mucin domain containing 1 and 4 (TIM1/4), and the newly identified CD300 family. The aim of the present auto-commentary is to highlight recent findings regarding the Cd300lf and Cd300lb receptors and their emerging roles in the development of autoimmune disease.

Apoptotic cell (AC) clearance is essential for immune homeostasis. Here we show that mouse CD300f (CLM-1) recognizes outer membrane-exposed phosphatidylserine, and regulates the phagocytosis of ACs. CD300f accumulates in phagocytic cups at AC contact sites. Phosphorylation within CD300f cytoplasmic tail tyrosine-based motifs initiates signals that positively or negatively regulate AC phagocytosis. Y276 phosphorylation is necessary for enhanced CD300f-mediated phagocytosis through the recruitment of the p85α regulatory subunit of phosphatidylinositol-3-kinase (PI3K). CD300f-PI3K association leads to activation of downstream Rac/Cdc42 GTPase and mediates changes of F-actin that drive AC engulfment. Importantly, primary macrophages from CD300f-deficient mice have impaired phagocytosis of ACs. The biological consequence of CD300f deficiency is predisposition to autoimmune disease development, as FcγRIIB-deficient mice develop a systemic lupus erythematosus-like disease at a markedly accelerated rate if CD300f is absent. In this report we identify the mechanism and role of CD300f in AC phagocytosis and maintenance of immune homeostasis.

Malaria infection triggers vigorous host immune responses; however, the parasite ligands, host receptors, and the signaling pathways responsible for these reactions remain unknown or controversial. Malaria parasites primarily reside within RBCs, thereby hiding themselves from direct contact and recognition by host immune cells. Host responses to malaria infection are very different from those elicited by bacterial and viral infections and the host receptors recognizing parasite ligands have been elusive. Here we investigated mouse genome-wide transcriptional responses to infections with two strains of Plasmodium yoelii (N67 and N67C) and discovered differences in innate response pathways corresponding to strain-specific disease phenotypes. Using in vitro RNAi-based gene knockdown and KO mice, we demonstrated that a strong type I IFN (IFN-I) response triggered by RNA polymerase III and melanoma differentiation-associated protein 5, not Toll-like receptors (TLRs), binding of parasite DNA/RNA contributed to a decline of parasitemia in N67-infected mice. We showed that conventional dendritic cells were the major sources of early IFN-I, and that surface expression of phosphatidylserine on infected RBCs might promote their phagocytic uptake, leading to the release of parasite ligands and the IFN-I response in N67 infection. In contrast, an elevated inflammatory response mediated by CD14/TLR and p38 signaling played a role in disease severity and early host death in N67C-infected mice. In addition to identifying cytosolic DNA/RNA sensors and signaling pathways previously unrecognized in malaria infection, our study demonstrates the importance of parasite genetic backgrounds in malaria pathology and provides important information for studying human malaria pathogenesis.

Phagocytic clearance of apoptotic cells (efferocytosis) is critical for resolution of inflammation and prevention of chronic inflammatory disorders, including inflammatory bowel diseases, and inflammation-associated cancer. CD300f (CLM-1), a cell surface receptor that recognizes phosphatidylserine exposed on apoptotic cells, has been shown to promote efferocytosis by macrophages. In the present study, we found that CD300f plays a critical role in controlling the severity and duration of dextran sulfate sodium (DSS)-induced colitis, and CD300f-expressing macrophages and dendritic cells (DC) were identified as key players controlling disease pathogenesis. Compared to Cd300f+/+ mice, Cd300f-/- mice showed increased susceptibility to DSS-induced colitis and impaired resolution of inflammation. In Cd300f-/- mice, macrophage-mediated efferocytosis was reduced, resulting in apoptotic cell accumulation in the gut mucosa. In contrast, Cd300f-/- DC engulfed apoptotic cells more efficiently than Cd300f+/+ DC. CD300f-deficient DC transfer exacerbated DSS-induced inflammation and delayed its resolution, whereas CD300f-expressing macrophage transfer attenuated DSS-induced inflammation. Hyperactive efferocytosis by CD300f-deficient gut DC resulted in excessive tumor necrosis factor-á (TNF-á) secretion, which induced secondary interferon-ã (IFN-ã) over-production, both of which impaired the resolution of colitis. TNF-á neutralization resulted in decreased levels of gut pro-inflammatory cytokines and significant disease amelioration. Collectively, these results suggest that CD300f is important for preventing chronic inflammation by enhancing macrophage efferocytosis and inhibiting TNF-á production induced by DC efferocytosis. From these findings, it is conceivable that increasing macrophage-mediated efferocytosis and suppressing DC-mediated efferocytosis through upregulation of CD300f expression would provide a new therapeutic strategy for inflammatory bowel disease patients. Citation Format: Ha-Na Lee, Linjie Tian, Jacquice Davis, Mariam Quinones, Yasmine Belkaid, Konrad Krzewski, John E. Coligan. CD300f-regulated efferocytosis by dendritic cells and macrophages controls the initiation and resolution of experimental colitis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4881.

Cross-linking of the collagen binding receptor leukocyte-associated Ig-like receptor-1 (LAIR-1) in vitro delivers an inhibitory signal that is able to downregulate activation-mediated signals. To study the in vivo function of LAIR-1, we generated LAIR-1−/− mice. They are healthy and fertile and have normal longevity; however, they show certain phenotypic characteristics distinct from wild-type mice, including increased numbers of splenic B, regulatory T, and dendritic cells. As LAIR-1−/− mice age, the splenic T cell population shows a higher frequency of activated and memory T cells. Because LAIR-1+/+ and LAIR-1−/− T cells traffic with equal proficiency to peripheral lymphoid organs, this is not likely due to abnormal T lymphocyte trafficking. LAIR-1−/− mice have lower serum levels of IgG1 and, in response to T-dependent immunization with trinitrophenyl-OVA, switch less efficiently to Ag specific IgG2a and IgG2b, whereas switching to IgG1 is not affected. Several mouse disease models, including experimental autoimmune encephalitis and colitis, were used to examine the effect of LAIR-1 deficiency, and no differences in the responses of LAIR-1−/− and LAIR-1+/+ mice were observed. Taken together, these observations indicate that LAIR-1 plays a role in regulating immune cells and suggest that any adverse effects of its absence may be balanced in vivo by other inhibitory receptors.

CC chemokine receptor 4 (CCR4) is a kind of G-protein-coupled receptor, which plays a pivotal role in allergic inflammation. The interaction between 2-(2-(4-chloro-phenyl)-5-{[(naphthalen-1-ylmethyl)-carbamoyl]-methyl}-4-oxo-thiazolidin-3-yl)-N-(3-morpholin-4-yl-propyl)-acetamide (S009) and the N-terminal extracellular tail (ML40) of CCR4 has been validated to be high affinity by capillary zone electrophoresis (CZE). The S009 is a known CCR4 antagonist. Now, a series of new thiourea derivatives have been synthesized. Compared with positive control S009, they were screened using ML40 as target by CZE to find some new drugs for allergic inflammation diseases. The synthesized compounds XJH-5, XJH-4, XJH-17 and XJH-1 displayed the interaction with ML40, but XJH-9, XJH-10, XJH-11, XJH-12, XJH-13, XJH-14, XJH-3, XJH-8, XJH-6, XJH-7, XJH-15, XJH-16 and XJH-2 did not bind to ML40. Both qualification and quantification characterizations of the binding were determined. The affinity of the four compounds was valued by the binding constant, which was similar with the results of chemotactic experiments. The established CEZ method is capable of sensitive and fast screening for a series of lactam analogs in the drug discovery for allergic inflammation diseases. Keywords: Capillary zone electrophoresis, CCR4 antagonist, 2-(2-(4-chloro-phenyl)-5-{[(naphthalen-1-ylmethyl)-carbamoyl]-methyl}-4-oxo-thiazolidin-3-yl)-N-(3-morpholin-4-yl-propyl)-acetamide, Interactions, Structural modification

Cell death can proceed via at least two distinct pathways, apoptosis and oncosis. Apoptosis is an energy-dependent process characterized morphologically by cell shrinkage, whereas oncosis is defined as a prelethal pathway leading to cell death associated with cellular swelling, organelle swelling, and increased membrane permeability. In this study, we found that overexpression of chromatin modifying protein 6 (CHMP6) induced cell death by a series of experiments, including morphological observation, intracellular ATP determination, caspase-3 activity, and flow cytometry. Typical morphological characteristics consistent with oncosis were observed by transmission electron microscopy. Simultaneously, we obtained some results that indicated apoptosis, but the anti-apoptotic gene Bcl-xL and caspase family inhibitor Z-VAD-FMK had little effect on CHMP6-induced cell death. These results suggest that CHMP6 overexpression can cause cell death, predominantly via oncosis and to a certain extent via apoptosis, and that CHMP6 might be a novel regulator involved in both oncosis and apoptosis.

PDCD5 (human programmed cell death 5) plays a significant role in apoptotic and paraptotic cell deaths. However, it was found that recombinant PDCD5 added exogenously to culture medium could also enhance programmed cell death triggered by certain stimuli. Here we show that PDCD5 has a remarkable role in intercellular transport in various cells (endogenous caveolin-1-positive and -negative cells) through a clathrin-independent endocytic pathway that originates from heparan sulfate proteoglycan binding and lipid rafts. These conclusions are supported by the studies of slow internalization kinetics of PDCD5 endosomes, by the resistance of endosomes to nonionic detergents, by the overexpression of the clathrin dominant negative mutant form, which did not block PDCD5-fluorescein isothiocyanate uptake, and by PDCD5 localization in lipid rafts by immunofluorescence, electron microscopy techniques, and sucrose density centrifugation. This is further supported by the findings that certain drugs that disrupt lipid rafts, compete with cell membrane heparan sulfate proteoglycans, or block the caveolae pathway, impair the PDCD5 internalization process. The translocation activity of PDCD5 may possess physiological significance and be a potential mechanism for its programmed cell death-promoting activity. PDCD5 protein also has the ability to drive the internalization of large protein cargo, depending on the residues 109-115 mapped by deletion mutagenesis, and can introduce the Mdm-2 binding domain of human p53 into living cells to induce cell death in human cancer cells, indicating that PDCD5 may serve as a vehicle and thus have potential in the field of protein delivery to the cells. This is the first evidence of such findings.