Blocking Antibody Research Articles

Enhanced Interleukin 6 Trans-Signaling Modulates Disease Process in Amyotrophic Lateral Sclerosis Mouse Models.

Background/Objectives: Charcot first described ALS in 1869, but the specific mechanisms that mediate the disease pathology are still not clear. Intense research efforts have provided insight into unique neuroanatomical regions, specific neuronal populations and genetic associations for ALS and other neurodegenerative diseases; however, the experimental results also suggest a convergence of these events to common toxic pathways. We propose that common toxic pathways can be therapeutically targeted, and this intervention will be effective in slowing progression and improving patient quality of life. Here, we focus on understanding the role of IL6 trans-signaling in ALS disease processes. Methods: We leveraged unique mouse models of IL6 trans-signaling that we developed that recapitulate the production of active sIL6R in a genotypic and quantitative fashion observed in humans. Given that the SOD1 transgenic mouse is one of the most highly studied and characterized models of ALS, we bred SOD1G93A mice with IL6R trans-signaling mice to determine how enhanced trans-signaling influenced symptom onset and pathological processes, including neuromuscular junction (NMJ) denervation, glial activation and motoneuron (MN) survival. Results: The results indicate that in animals with enhanced trans-signaling, symptom onset and pathological processes were accelerated, suggesting a role in disease modification. Administration of an IL6R functional blocking antibody failed to alter accelerated symptom onset and disease progression. Conclusions: Future work to investigate the site-specific influence of enhanced IL6 trans-signaling and the tissue-specific bioavailability of potential therapeutics will be necessary to identify targets for precise therapeutic interventions that may limit disease progression in the 60% of ALS patients who inherit the common Il6R Asp358Ala variant.

NcSWP8, a New Spore Wall Protein, Interacts with Polar Tube Proteins in the Parasitic Microsporidia Vairimorpha (Nosema) ceranae.

Vairimorpha (Nosema) ceranae is a pathogen that affects Apis mellifera and Apis ceranae Fabricius, capable of spreading within and between honeybee colonies. The spore wall of microsporidia is the initial structure to contact the host cell directly, which may play a crucial role in the infection process. Currently, several spore wall proteins have been identified in microsporidia, but only two spore wall proteins from V. ceranae have been characterized. Here, we report the expression and identification of a novel spore wall protein, NcSWP8, with a molecular mass of 21.37 kDa in V. ceranae. Subcellular localization analysis revealed that NcSWP8 was localized on the spore wall of V. ceranae. Co-immunoprecipitation and Far-Western blotting experiments demonstrated that NcSWP8 could stably interact with polar tube proteins, NcPTP2 and NcPTP3. The antibody blocking assay significantly decreased their infection rate, indicating that NcSWP8 played a significant role in the process of V. ceranae infection. These results together suggested that NcSWP8 was a new spore wall protein localized to the spore wall and interacted with the polar tube proteins, playing a crucial role in supporting the formation of the spore wall and potentially affecting the process of infection of V. ceranae.

Simultaneous Blockade of CD209 and CD209L by Monoclonal Antibody Does Not Provide Sufficient Protection Against Multiple Viral Infections InVivo.

Many virus species, including Ebola virus, Marburg virus, SARS-CoV-2, dengue virus (DENV) and Zika virus (ZIKV), exploit CD209 and CD209L as alternative or attachment receptors for viral cis- or trans-infection. Thus, CD209 and CD209L may be critical targets for the development of therapeutic monoclonal blocking antibody drugs to disrupt the infection process caused by multiple viruses. Here, we produced a human chimeric monoclonal blocking antibody that simultaneously blocks CD209 and CD209L, namely 7-H7-B1. We show that 7-H7-B1 effectively blocks multiple pseudotyped or live viral infections invitro, including SARS-CoV, SARS-CoV-2, Ebola virus, Marburg virus, ZIKV and DENV infections. However, the 7-H7-B1 mAb does not provide favourable protection against Zaire Ebola virus or ZIKV infection in hCD209 knock-in mice invivo. Thus, our findings indicate that although CD209 and CD209L are critical for multiple viral infections invitro, they may play only a partial role in viral infections invivo.

AAVR Expression is Essential for AAV Vector Transduction in Sensory Hair Cells.

Adeno-associated virus (AAV) vectors are a leading platform for gene therapy. Recently, AAV-mediated gene therapy in the inner ear has progressed from laboratory use to clinical trials, but the lower transduction rates in outer hair cells (OHCs) in the organ of Corti and in vestibular hair cells in adult mice still pose a challenge. OHCs are particularly vulnerable to inner ear insults. In this study, we demonstratedthat expression of a key AAV receptor (AAVR, Kiaa0319l, or Au040320) in OHCs and vestibular hair cells decreases significantly in mature mice and AAV particles directly interact with AAVR by forming complexes. Consequently, antibody blockage of AAVR significantly inhibits AAV transduction in sensory hair cells in cochlear explants. Moreover, use of AAVR knockout mice confirms inhibition of AAV transduction in sensory hair cells in vivo. Finally, conditional overexpression of AAVR in sensory hair cells of adult mice successfully restores AAV transduction efficiency in OHCs and vestibular hair cells. In conclusion, this strong evidence that AAVR is essential for AAV transduction in sensory hair cells will help to increase the efficacy of future gene therapy in inner ear.

PD1 BLOCKADE IMPROVES SURVIVAL AND CD8+ CYTOTOXIC CAPACITY, WITHOUT INCREASING INFLAMMATION, DURING NORMAL MICROBIAL EXPERIENCE IN OLD MICE

Abstract The elderly and those with chronic diseases have a 2 to 10-fold increased risk of hospitalization and mortality following infection as compared with healthy adults. A significant contributor to disease severity is the aging of the immune system, including an expansion of exhausted T cells. These cells simultaneously promote excessive levels of inflammation and display impaired T cell activation and function. How exhausted T cells from aged individuals respond to infection and whether acute restoration of T cell activation further exacerbates the inflammatory environment or generates functional T cells is unclear. Previously, we used a mouse model of normal microbial experience (NME), where old specific pathogen-free (SPF) mice show 100% mortality following exposure to multiple microbes ordinarily found in pet store mice. In the present study, we show that when exposed to NME, old SPF mice have increased expression of multiple inflammatory pathways and exhibit elevated frequencies of a heterogenous exhausted CD8+ T cell pool that expresses differing combinations of the inhibitory receptor programmed cell death protein 1 (PD1), TOX, and CXCR5. Pre-treatment or intervention with an anti-PD1 monoclonal blocking antibody during the exposure of old SPF mice to NME significantly improves both antibody production and survival, without altering the acute inflammatory response. Anti-PD1 checkpoint blockade-mediated survival is dependent on CD8+, but not CD4+ T cells. Correspondingly, CD8+ PD1+ T cells from old mice given anti-PD1 monoclonal antibody have increased granzyme B production. These data reveal a new approach for reducing vulnerability to infections in the elderly by targeting CD8+ T cell exhaustion through PD1 checkpoint blockade immunotherapy.

Genetically Engineered T Cells and Recombinant Antibodies to Target Intracellular Neoantigens: Current Status and Future Directions

Recombinant antibodies and, more recently, T cell receptor (TCR)-engineered T cell therapies represent two immunological strategies that have come to the forefront of clinical interest for targeting intracellular neoantigens in benign and malignant diseases. T cell-based therapies targeting neoantigens use T cells expressing a recombinant complete TCR (TCR-T cell), a chimeric antigen receptor (CAR) with the variable domains of a neoepitope-reactive TCR as a binding domain (TCR-CAR-T cell) or a TCR-like antibody as a binding domain (TCR-like CAR-T cell). Furthermore, the synthetic T cell receptor and antigen receptor (STAR) and heterodimeric TCR-like CAR (T-CAR) are designed as a double-chain TCRαβ-based receptor with variable regions of immunoglobulin heavy and light chains (VH and VL) fused to TCR-Cα and TCR-Cβ, respectively, resulting in TCR signaling. In contrast to the use of recombinant T cells, anti-neopeptide MHC (pMHC) antibodies and intrabodies neutralizing intracellular neoantigens can be more easily applied to cancer patients. However, different limitations should be considered, such as the loss of neoantigens, the modification of antigen peptide presentation, tumor heterogenicity, and the immunosuppressive activity of the tumor environment. The simultaneous application of immune checkpoint blocking antibodies and of CRISPR/Cas9-based genome editing tools to engineer different recombinant T cells with enhanced therapeutic functions could make T cell therapies more efficient and could pave the way for its routine clinical application.

CD6 regulates CD4 T follicular helper cell differentiation and humoral immunity during murine coronavirus infection.

CD6 monoclonal blocking antibodies are being therapeutically administered to inhibit T cell activation in autoimmune disorders. However, the multifaceted nature of CD6 allows for multiple and even opposing functions under different circumstances of T cell activation. We therefore sought to characterize how CD6 regulates T cell activation in the context of viral infections using an in vivo murine coronavirus model. In contrast to its role in autoimmunity, but consistent with its function in the presence of superantigens, we found that CD6 deficiency enhances CD4 T cell activation and CD4 T cell help to germinal center-dependent antiviral humoral responses. Finally, we provide evidence that CD6 regulates transcription of its intracellular binding partner UBASH3a, which suppresses T cell receptor (TCR) signaling and consequently T cell activation. These findings highlight the context-dependent flexibility of CD6 in regulating in vivo adaptive immune responses, which may be targeted to enhance antiviral immunity.

Therapeutic agents for Alzheimer’s disease: a critical appraisal

Alzheimer’s disease (AD) is the most common form of dementia. Mutations in genes and precursors of β amyloid (Aβ) are found in the familial form of the disease. This led to the evaluation of seven monoclonal antibodies against Aβ in subjects with AD, two of which were approved for use by the FDA. They caused only a small improvement in cognitive function, probably because they were given to those with much more prevalent sporadic forms of dementia. They also have potentially serious adverse effects. Oxidative stress and elevated pro-inflammatory cytokines are present in all subjects with AD and are well correlated with the degree of memory impairment. Drugs that affect these processes include TNFα blocking antibodies and MAPK p38 inhibitors that reduce cognitive impairment when given for other inflammatory conditions. However, their adverse effects and inability to penetrate the brain preclude their use for dementia. Rosiglitazone is used to treat diabetes, a risk factor for AD, but failed in a clinical trial because it was given to subjects that already had dementia. Ladostigil reduces oxidative stress and suppresses the release of pro-inflammatory cytokines from activated microglia without blocking their effects. Chronic oral administration to aging rats prevented the decline in memory and suppressed overexpression of genes adversely affecting synaptic function in relevant brain regions. In a phase 2 trial, ladostigil reduced the decline in short-term memory and in whole brain and hippocampal volumes in human subjects with mild cognitive impairment and had no more adverse effects than placebo.

Results of SIGNAL‐AD, a randomized, phase 1b/2 trial to evaluate safety and efficacy of targeting reactive astrocytes with pepinemab, SEMA4D blocking antibody, in people with mild Alzheimer’s dementia

Results of SIGNAL‐AD, a randomized, phase 1b/2 trial to evaluate safety and efficacy of targeting reactive astrocytes with pepinemab, SEMA4D blocking antibody, in people with mild Alzheimer’s dementia

Blocking IL1RAP on cancer-associated fibroblasts in pancreatic ductal adenocarcinoma suppresses IL-1-induced neutrophil recruitment

BackgroundPancreatic ductal adenocarcinoma (PDAC) represents a major clinical challenge due to its tumor microenvironment, which exhibits immune-suppressive properties that facilitate cancer progression, metastasis, and therapy resistance. Interleukin 1 (IL-1) signaling...

High-resolution crystal structure of PD-1 in complex with retifanlimab, the FDA-approved immune checkpoint blocking antibody for treating Merkel cell carcinoma

Retifanlimab is a humanized monoclonal antibody that specifically targets programmed cell death protein 1 (PD-1), an essential immune checkpoint that modulates T-cell immune responses. Several anti-PD-1 antibodies have been market-approved, marking a significant advancement in the treatment of diverse tumor types by restoring the T-cell immune response. Recently, the US FDA approved retifanlimab for treating metastatic or recurrent locally advanced Merkel cell carcinoma. We present the crystal structure of PD-1 in complex with the retifanlimab Fab at a resolution of 1.54 Å to elucidate the structural basis for the mechanism of action of this antibody. This work clarifies the detailed interactions and conformational alterations that occur upon antibody binding. The epitope of retifanlimab partially overlaps with the ligand binding site, and its binding induced unique conformations of the flexible loops within PD-1, including BC, C'D, and FG loops, thereby optimizing interactions with the antibody. A thorough analysis of its interaction with PD-1 and other FDA-approved anti-PD-1 antibodies may provide valuable insights into the rational design of enhanced therapies to regulate immune responses in cancer treatment.

PD-1 antibody interactions with Fc gamma receptors enable PD-1 agonism to inhibit T cell activation – therapeutic implications for autoimmunity

PD-1 has emerged as a central inhibitory checkpoint receptor in maintaining immune homeostasis and as a target in cancer immunotherapies. However, targeting PD-1 for the treatment of autoimmune diseases has been more challenging. We recently showed in a phase 2a trial that PD-1 could be stimulated with the PD-1 agonist antibody peresolimab to treat rheumatoid arthritis. Here, we demonstrate that PD-1 antibodies can elicit agonism and inhibit T cell activation by co-localization of PD-1 with the T cell receptor via Fcγ receptor (FcγR) engagement. Three PD-1 agonist antibodies with different antigen binding domains, including the clinically validated PD-1 blocking antibody pembrolizumab, suppressed T cell activation to a similar degree; this finding suggests that a specific PD-1-binding epitope is not required for PD-1 agonism. We next explored whether antibody-mediated clustering was an important driver of inhibition of T cell activation; however, we found that a monovalent PD-1 antibody was not inferior to a conventional bivalent antibody in its ability to suppress T cell activation. Importantly, we found that affinity to PD-1 correlated positively with inhibition of T cell activation, with higher affinity antibodies exhibiting higher levels of inhibition. Using a series of human Fc mutants with altered affinities to various FcγRs, we dissected the contributions of FcγRs and found that multiple FcγRs rather than a single receptor contribute to agonist activity. Our work reveals an important role for FcγR binding in the activity of PD-1 antibodies, which has implications for optimizing both PD-1 agonist and antagonist antibodies.

Sialylation inhibition improves macrophage mediated tumor cell phagocytosis of breast cancer cells triggered by therapeutic antibodies of different isotypes.

Tumor cell phagocytosis by macrophages is considered a relevant mechanism of action for many therapeutic IgG antibodies. However, tumor cells employ several mechanisms to evade immune recognition, including hypersialylation. Here, we describe how reduction of sialic acid exposure on tumor cells promotes antibody-dependent tumor cell phagocytosis (ADCP) by macrophages. Incubation with the sialyltransferase inhibitor (STi) P-3Fax-Neu5Ac reduced sialylation on two breast cancer cell lines, rendering these cells more susceptible to macrophage mediated phagocytosis by EGFR or HER2 antibodies. This was observed with not only IgG1 and IgG2 antibodies but also IgA2 variants. These results show that inhibiting sialic acid exposure triggers enhanced tumor cell phagocytosis by macrophages irrespective of the antibody isotype and the tumor target antigen. Investigating the underlying mechanisms of enhanced ADCP, we observed reduced binding of soluble sialic acid-binding immunoglobulin-like lectins (Siglec)-7 and Siglec-9 to tumor cells after sialylation inhibition. However, Fc silent blocking antibodies against Siglec-7 or Siglec-9, or their combination, only marginally improved ADCP. Our results further promote the concept of cancer hypersialylation as immune escape mechanism, which could serve as target to improve tumor immunotherapy with monoclonal antibodies.

Monoclonal antibodies that block Roundabout 1 and 2 signaling target pathological ocular neovascularization through myeloid cells.

Roundabout (ROBO) 1 and 2 are transmembrane receptors that bind secreted SLIT ligands through their extracellular domains (ECDs) and signal through their cytoplasmic domains to modulate the cytoskeleton and regulate cell migration, adhesion, and proliferation. SLIT-ROBO signaling regulates pathological ocular neovascularization, which is a major cause of vision loss worldwide, but pharmacological tools to prevent SLIT-ROBO signaling are lacking. Here, we developed human monoclonal antibodies (mAbs) against the ROBO1 and ROBO2 ECDs. One antibody that inhibited in vitro SLIT2 signaling through ROBO1 and ROBO2 (anti-ROBO1/2) also reduced ocular neovascularization in oxygen-induced retinopathy (OIR) and laser-induced corneal neovascularization (CNV) mouse models in vivo. Single-cell RNA sequencing of OIR retinas revealed that antibody treatment affected several cell types relevant to physiological and pathological angiogenesis, including endothelial cells, pericytes, and a heterogeneous population of myeloid cells. mAb treatment improved blood-retina barrier integrity and prevented pathological pericyte activation in OIR. SLIT-ROBO signaling inhibition prevented pathological activation of myeloid cells and increased neuroprotective myeloid populations normally seen in the developing retina. Microglia/infiltrating macrophage-specific ablation of Robo1 and Robo2 or knockout of the downstream effector phosphatidylinositol 3-kinase (Pik3cg) encoding PI3Kγ in both OIR and CNV models phenocopied anti-ROBO1/2 treatment, further demonstrating the key role of myeloid cells as drivers of ocular neovascular diseases. ROBO1/2 blocking antibodies may thus provide a promising strategy to combat inflammation in blinding eye diseases.

Abstract B012: Understanding the role of VISTA in regulating T cell function and the therapeutic potential of blocking VISTA in triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is the most aggressive and highly metastatic breast cancer subtype with limited effective treatment options. Immunotherapy with immune checkpoint inhibitors (ICIs) targeting checkpoint molecules have been widely used and have shown therapeutic efficacy in treating multiple malignancies in the last decade. In TNBC, ICI targeting PD-1, alone or in combination with chemotherapy has been approved for the treatment of patients with both early and advanced stage of the disease. Although, there has been significant clinical benefit achieved from anti-PD-1 and chemotherapy, treatment efficacy is often limited to a subgroup of patients. The limited response to ICIs has been attributed to the increased infiltration of immunosuppressive cells (including MDSCs, neutrophils and tumor-associated macrophages), more importantly, the upregulation of alternative immune checkpoint receptors/ligands that may further suppress cytotoxic T cells (CD8+ T cells) in the tumor microenvironment (TME). Therefore, it has been suggested that targeting other immune checkpoint molecules may be more effective in bolstering anti-tumor immune response. V- domain Ig suppressor of T-cell activation (VISTA) is one of the immune checkpoint molecules attracting increasing attention in the context of anti-tumor immunity due to its broad expression across various cancer types and increased expression upon development of immune checkpoint resistance. However, the role of VISTA in regulating the anti-tumor immune response and its therapeutic potential in TNBC remains unclear. In our study, we observed that VISTA is expressed in TNBC patients, predominantly in neutrophils and macrophages. In the Py230 murine breast cancer model, VISTA expression increased upon paclitaxel (PTX), anti-PD-L1 and paclitaxel combined with anti-PD-L1 treatment compared to the control group, suggesting its potential role as a resistance mechanism in sustaining or further inhibiting T cells. In the Py230 model, VISTA blockade increased the infiltration and activation of CD8+ T cells, as evidenced by the higher number of CD8+ T cells, increased CD69+CD8+ T cells, and Granzyme B production, indicating that VISTA blockade improved CD8+ T cells mediated anti-tumor immunity. To functionally test the role of VISTA, we stimulated CD8+ T cells and co-cultured them with naïve macrophages or tumor educated macrophages in the presence or absence of VISTA blocking antibody. Tumor educated macrophages showed increased immunosuppressive activity which was abrogated with VISTA blockade. Moreover, we found that the immunosuppressive effect of macrophages on T cell activation needs direct cell-cell interaction between VISTA on macrophages and T cells. In conclusion, this study shows the mechanism of action of VISTA in the regulation of T cells mediated anti-tumor immune response and provides the rationale for targeting VISTA in TNBC. Citation Format: Maidinaimu Abudula, Yuliana Astuti, Meirion Raymant Raymant, Vijay Sharma, Michael Schmid, Ainhoa Mielgo. Understanding the role of VISTA in regulating T cell function and the therapeutic potential of blocking VISTA in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B012.

Tackling immunosuppression by Neisseria gonorrhoeae to facilitate vaccine design.

Gonorrhoea, caused by Neisseria gonorrhoeae, is a common sexually transmitted infection. Increasing multi-drug resistance and the impact of asymptomatic infections on sexual and reproductive health underline the need for an effective gonococcal vaccine. Outer membrane vesicles (OMVs) from Neisseria meningitidis induce modest cross-protection against gonococcal infection. However, the presence of proteins in OMVs derived from N. gonorrhoeae that manipulate immune responses could hamper their success as a vaccine. Here we modified two key immunomodulatory proteins of the gonococcus; RmpM, which can elicit 'blocking antibodies', and PorB, an outer membrane porin which contributes to immunosuppression. As meningococcal PorB has adjuvant properties, we replaced gonococcal PorB with a meningococcal PorB. Immunisation with OMVs from N. gonorrhoeae lacking rmpM and expressing meningococcal porB elicited higher antibody titres against model antigens in mice compared to OMVs with native PorB. Further, a gonococcal protein microarray revealed stronger IgG antibody responses to a more diverse range of antigens in the Nm PorB OMV immunised group. Finally, meningococcal PorB OMVs resulted in a Th1-skewed response, exemplified by increased serum IgG2a antibody responses and increased IFNɣ production by splenocytes from immunised mice. In summary, we demonstrate that the replacement of PorB in gonococcal OMVs enhances immune responses and offers a strategy for gonococcal vaccine development.

Positive feedback loop PU.1-IL9 in Th9 promotes rheumatoid arthritis development

ObjectivesT helper 9 (Th9) cells are recognised for their characteristic expression of the transcription factor PU.1 and production of interleukin-9 (IL-9), which has been implicated in various autoimmune diseases. However,...

Abstract 4119062: A KLF2-BMPER-Smad1/5 checkpoint regulates high fluid shear stress-mediated artery remodeling

Background: Vascular remodeling to match arterial diameter to tissue metabolic requirements commonly fails in ischemic disease. Endothelial cell (EC) sensing of elevated fluid shear stress (FSS) from blood flow induces vessel outward remodeling to restore physiological FSS, but mechanisms are poorly understood. The Smad1/5 pathway, which is maximally activated at physiological FSS and suppressed at higher flow, opposes activation of Akt, suggesting that inhibiting Smad1/5 may be required for outward remodeling. Methods: In vitro flow studies used ECs in a parallel plate flow chamber. In vivo mouse studies used a carotid-jugular fistula model to induce high flow outward remodeling in the carotid artery, and femoral artery ligation to examine recovery from ischemia and arteriogenesis in the hindlimb. Results: Suppression of Smad1/5 at high FSS is mediated KLF2-dependent induction of the BMP pathway inhibitor BMPER, which suppresses Smad1/5 and de-inhibits Akt. In a mouse arteriovenous fistula (AVF) model, high FSS induces arterial outward remodeling coincident with elevated BMPER expression and Smad1/5 inactivation. Endothelial BMPER deletion impaired blood flow recovery and vascular remodeling in the AVF and a hindlimb ischemia (HLI) model, with the latter reversed by BMP9/10 blocking antibodies (bAbs). In both STZ-induced type 1 and HFD-induced type 2 diabetic mice that show poor recovery from HLI, BMP9/10 bAbs improved outcomes. Conclusions: Suppression of Smad1/5 through a KLF2-BMPER pathway is required for high FSS-mediated outward remodeling. Mimicking this pathway with BMP9/10 antibodies improves vascular remodeling in diabetic mice, suggesting a potential new therapeutic approach for ischemic disease.

Abstract 4118360: Lipocalin10 is Critical for Macrophage Efferocytosis to Repair Myocardial Ischemia/Reperfusion Injury

Introduction: Efficient clearance of dead/dying cells by macrophages (MΦs) (termed efferocytosis) is critical for timely repairing the injured heart after ischemia/reperfusion (I/R). Prior work by us and others shows that Lipocalin 10 (Lcn10), a secreted protein of lipocalin family, can regulate MΦ polarization and is greatly downregulated in cardiac tissue of patients with heart failure. However, the potential role of Lcn10 in MΦ efferocytosis during cardiac I/R has never been investigated. Methods: Wild-type (WT) and Lcn10-knockout (KO) mice were subjected to 45 minutes of ischemia via the ligation of left anterior descending artery (LAD) followed by reperfusion. Cardiac function was measured by echocardiography. Flow cytometry was utilized to assess MΦ efferocytosis. Bone marrow-derived MΦs (BMDMs) were used to test efferocytosis in vitro and dissect mechanism. Dead cells were prepared by treating red-dye-labelled H9C2 cells with H 2 O 2 (1mM, 2h). Results: After co-culturing BMDMs with dead H9C2 cells for 2h, we observed a 25% reduction of efferocytosis in Lcn10-KO BMDMs, compared to WTs. Using Lcn10-KO mCherry mouse model (cardiomyocyte-specific overexpression of mCherry in Lcn10-KO background), we consistently found that cardiac MΦs in Lcn10-KO mice taken up fewer dead mCherry-myocytes at 4 days post-I/R surgery, leading to an increased accumulation of cardiac dead cells, higher serum levels of troponin I, greater myocardial fibrosis, and dramatically impaired cardiac function, compared to WT mCherry controls. In contrast, pre-treatment of BMDMs with recombinant Lcn10 protein (rLcn10) enhanced efferocytosis by 50%. Mechanistically, RNA-seq analysis revealed that many efferocytosis-related genes were dysregulated ( e.g., downregulation of Id 1, Id 3, Itga 8, Itga v, Cd 44, Cx3cr 1, Msr 1, and upregulation of Sirp α) in Lcn10-KO BMDMs, compared to WTs. Interestingly, most of these dysregulated genes are controlled by transcription factors Id1/3. Further studies showed that rLcn10 interacted with Cd44 and activated the expression of its downstream Id1/3, leading to enhanced MΦ efferocytosis. Importantly, rLcn10-mediated efferocytosis was greatly impaired by pre-treatment of MΦs with either anti-CD44 blocking antibody (KM201, 10µg/ml) or Id1/3 inhibitor (AGX51, 25µM). Conclusion: Our data indicate that Lcn10 is essential for MΦ efficient efferocytosis to repair I/R-induced cardiac damage though affecting the Cd44-Id1/3 signaling and its related MΦ surface receptors.

TMIC-27. PDPN ACTIVATION OF CLEC5A IN THE PERI-NECROTIC NICHE DRIVES TAM POLARIZATION AND TME IMMUNOSUPPRESSION IN GLIOBLASTOMA

Abstract Glioblastoma, IDH-wildtype (GBM, WHO grade 4) is the most common malignant glioma of adults and is characterized by a severely hypoxic and immunosuppressive tumor microenvironment (TME). CLEC5A regulates immune responses in inflammatory and infectious diseases. However, its role in GBM immune regulation remain unclear. Here, we found that CLEC5A has the strongest association with poor clinical outcomes among all immune-related genes in GBM. Tumor-associated macrophages (TAMs) account for the majority of immune cells in the GBM and CLEC5A expression in this population is highest in hypoxic, peri-necrotic zones. Both hypoxia and GBM conditioned media cause increased CLEC5A expression by THP-1 cells, and overexpression of CLEC5A enhanced TAM polarization, migration toward glioma cells, and increased secretion of cytokines that mediate immunosuppression. We show that CLEC5A is a cell surface receptor activated by podoplanin (PDPN) and induces TAM polarization through Syk-JAK-STAT3 signaling. Co-IP assay showed that CLEC5A and PDPN could be binded together using antibodies for CLEC5A, Flag-CLEC5A or PDPN when we cocultured control THP-1 cells and THP-1 cells overexpressing Flag-CLEC5A with primary GBM cells (NU02068). PDPN blocking antibody (α-PDPN) reduced the specific binding between PDPN and CLEC5A, as well as inhibited the TAM-IM polarization. Overexpression of CLEC5A significantly upregulated the expression of p-Syk, Jak2, p-Jak2, STAT3, p-STAT3, CD163, CD206 and PD-L1. Moreover, Bay 61-3606, a selective Syk inhibitor, rescued CLEC5A overexpression-mediated TAM-IM polarization and Syk-JAK-STAT3 activation in THP-1 cells. In vivo, both silencing CLEC5A in TAMs and silencing PDPN in GBM cells, as well as pharmacologically targeting Syk delayed glioma growth, prolonged survival and was associated with diminished immunosuppressive TAMs. Collectively, we found that PDPN-CLEC5A-Syk-JAK-STAT3 signaling causes TAM polarization and enhances TME immunosuppression in GBM and can be targeted to suppress growth.