Substrate-induced condensation activates plant TIR domain proteins.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR-NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.

Structural insights into the unique pH-responsive characteristics of the anti-TIGIT therapeutic antibody Ociperlimab.

TIGIT is mainly expressed on T cells and is an inhibitory checkpoint receptor that binds to its ligand PVR in the tumor microenvironment. Anti-TIGIT monoclonal antibodies (mAbs) such as Ociperlimab and Tiragolumab block the TIGIT-PVR interaction and are in clinical development. However, the molecular blockade mechanism of these mAbs remains elusive. Here, we report the crystal structures of TIGIT in complex with Ociperlimab_Fab and Tiragolumab_Fab revealing that both mAbs bind TIGIT with a large steric clash with PVR. Furthermore, several critical epitopic residues are identified. Interestingly, the binding affinity of Ociperlimab toward TIGIT increases approximately 17-fold when lowering the pH from 7.4 to 6.0. Our structure shows a strong electrostatic interaction between ASP103HCDR3 and HIS76TIGIT explaining the pH-responsive mechanism of Ociperlimab. In contrast, Tiragolumab does not show an acidic pH-dependent binding enhancement. Our results provide valuable information that could help to improve the efficacy of therapeutic antibodies for cancer treatment.

A conserved isoleucine in the binding pocket of RIG-I controls immune tolerance to mitochondrial RNA.

RIG-I is a cytosolic receptor of viral RNA essential for the immune response to numerous RNA viruses. Accordingly, RIG-I must sensitively detect viral RNA yet tolerate abundant self-RNA species. The basic binding cleft and an aromatic amino acid of the RIG-I C-terminal domain(CTD) mediate high-affinity recognition of 5'triphosphorylated and 5'base-paired RNA(dsRNA). Here, we found that, while 5'unmodified hydroxyl(OH)-dsRNA demonstrated residual activation potential, 5'-monophosphate(5'p)-termini, present on most cellular RNAs, prevented RIG-I activation. Determination of CTD/dsRNA co-crystal structures and mutant activation studies revealed that the evolutionarily conserved I875 within the CTD sterically inhibits 5'p-dsRNA binding. RIG-I(I875A) was activated by both synthetic 5'p-dsRNA and endogenous long dsRNA within the polyA-rich fraction of total cellular RNA. RIG-I(I875A) specifically interacted with long, polyA-bearing, mitochondrial(mt) RNA, and depletion of mtRNA from total RNA abolished its activation. Altogether, our study demonstrates that avoidance of 5'p-RNA recognition is crucial to prevent mtRNA-triggered RIG-I-mediated autoinflammation.

Binding Mechanism of CD47 with SIRPα Variants and Its Antibody: Elucidated by Molecular Dynamics Simulations.

The intricate complex system of the differentiation 47 (CD47) and the signal-regulatory protein alpha (SIRPα) cluster is a crucial target for cancer immunotherapy. Although the conformational state of the CD47-SIRPα complex has been revealed through crystallographic studies, further characterization is needed to fully understand the binding mechanism and to identify the hot spot residues involved. In this study, molecular dynamics (MD) simulations were carried out for the complexes of CD47 with two SIRPα variants (SIRPαv1, SIRPαv2) and the commercially available anti-CD47 monoclonal antibody (B6H12.2). The calculated binding free energy of CD47-B6H12.2 is lower than that of CD47-SIRPαv1 and CD47-SIRPαv2 in all the three simulations, indicating that CD47-B6H12.2 has a higher binding affinity than the other two complexes. Moreover, the dynamical cross-correlation matrix reveals that the CD47 protein shows more correlated motions when it binds to B6H12.2. Significant effects were observed in the energy and structural analyses of the residues (Glu35, Tyr37, Leu101, Thr102, Arg103) in the C strand and FG region of CD47 when it binds to the SIRPα variants. The critical residues (Leu30, Val33, Gln52, Lys53, Thr67, Arg69, Arg95, and Lys96) were identified in SIRPαv1 and SIRPαv2, which surround the distinctive groove regions formed by the B2C, C'D, DE, and FG loops. Moreover, the crucial groove structures of the SIRPα variants shape into obvious druggable sites. The C'D loops on the binding interfaces undergo notable dynamical changes throughout the simulation. For B6H12.2, the residues Tyr32LC, His92LC, Arg96LC, Tyr32HC, Thr52HC, Ser53HC, Ala101HC, and Gly102HC in its initial half of the light and heavy chains exhibit obvious energetic and structural impacts upon binding with CD47. The elucidation of the binding mechanism of SIRPαv1, SIRPαv2, and B6H12.2 with CD47 could provide novel perspectives for the development of inhibitors targeting CD47-SIRPα.

All-Atom Simulations Reveal the Intricacies of Signal Transduction upon Binding of the HLA-E Ligand to the Transmembrane Inhibitory CD94/NKG2A Receptor.

Natural killer (NK) cells play an important role in the innate immune response against tumors and various pathogens such as viruses and bacteria. Their function is controlled by a wide array of activating and inhibitory receptors, which are expressed on their cell surface. Among them is a dimeric NKG2A/CD94 inhibitory transmembrane (TM) receptor which specifically binds to the non-classical MHC I molecule HLA-E, which is often overexpressed on the surface of senescent and tumor cells. Using the Alphafold 2 artificial intelligence system, we constructed the missing segments of the NKG2A/CD94 receptor and generated its complete 3D structure comprising extracellular (EC), TM, and intracellular regions, which served as a starting point for the multi-microsecond all-atom molecular dynamics simulations of the receptor with and without the bound HLA-E ligand and its nonameric peptide. The simulated models revealed that an intricate interplay of events is taking place between the EC and TM regions ultimately affecting the intracellular immunoreceptor tyrosine-based inhibition motif (ITIM) regions that host the point at which the signal is transmitted further down the inhibitory signaling cascade. Signal transduction through the lipid bilayer was also coupled with the changes in the relative orientation of the NKG2A/CD94 TM helices in response to linker reorganization, mediated by fine-tuned interactions in the EC region of the receptor, taking place after HLA-E binding. This research provides atomistic details of the cells' protection mechanism against NK cells and broadens the knowledge regarding the TM signaling of ITIM-bearing receptors.

Serum soluble triggering receptor expressed on myeloid cells-2 was not altered by rTMS in patients with treatment-resistant depression.

AIM: Repetitive transcranial magnetic stimulation (rTMS) is one of the most effective and minimally invasive treatments for treatment-resistant depression (TRD). However, the mechanism underlying the therapeutic effects of rTMS in patients with TRD remains unclear. In recent years, the pathogenesis of depression has been closely associated with chronic inflammation and microglia are believed to play an important role in chronic inflammation. Triggering receptor expressed on myeloid cells-2 (TREM2) plays an important role in microglial neuroinflammatory regulation. In this study, we investigated the changes in peripheral soluble TREM2 (sTREM2) before and after rTMS treatment in patients with TRD. METHODS: Twenty-six patients with TRD were enrolled in this frequency (10 Hz) rTMS study. Depressive symptoms, cognitive function, and serum sTREM2 concentrations were measured at baseline and the end of the 6-week rTMS treatment. RESULTS: This study showed that rTMS ameliorated depressive symptoms and partially improved cognitive dysfunction in TRD. However, rTMS treatment did not alter serum sTREM2 levels. CONCLUSIONS: This is the first sTREM2 study in patients with TRD who underwent rTMS treatment. These results suggest that serum sTREM2 may not be relevant for the mechanism underlying the therapeutic effect of rTMS in patients with TRD. Future studies should confirm the present findings using a larger patient sample and a sham rTMS procedure, as well as CSF sTREM2. Furthermore, a longitudinal study should be conducted to clarify the effects of rTMS on sTREM2 levels.

BTLA inhibition has a dominant role in the cis-complex of BTLA and HVEM.

The engagement of the herpesvirus entry mediator (HVEM, TNFRSF14) by the B and T lymphocyte attenuator (BTLA) represents a unique interaction between an activating receptor of the TNFR-superfamily and an inhibitory receptor of the Ig-superfamily. BTLA and HVEM have both been implicated in the regulation of human T cell responses, but their role is complex and incompletely understood. Here, we have used T cell reporter systems to dissect the complex interplay of HVEM with BTLA and its additional ligands LIGHT and CD160. Co-expression with LIGHT or CD160, but not with BTLA, induced strong constitutive signaling via HVEM. In line with earlier reports, we observed that in cis interaction of BTLA and HVEM prevented HVEM co-stimulation by ligands on surrounding cells. Intriguingly, our data indicate that BTLA mediated inhibition is not impaired in this heterodimeric complex, suggesting a dominant role of BTLA co-inhibition. Stimulation of primary human T cells in presence of HVEM ligands indicated a weak costimulatory capacity of HVEM potentially owed to its in cis engagement by BTLA. Furthermore, experiments with T cell reporter cells and primary T cells demonstrate that HVEM antibodies can augment T cell responses by concomitantly acting as checkpoint inhibitors and co-stimulation agonists.

Crystal Structure of Human CD47 in Complex with Engineered SIRPα.D1(N80A).

Background: Targeting the CD47/SIRPα signaling pathway represents a novel approach to enhance anti-tumor immunity. However, the crystal structure of the CD47/SIRPα has not been fully studied. This study aims to analyze the structure interface of the complex of CD47 and IMM01, a novel recombinant SIRPα-Fc fusion protein. Methods: IMM01-Fab/CD47 complex was crystalized, and diffraction images were collected. The complex structure was determined by molecular replacement using the program PHASER with the CD47-SIRPαv2 structure (PDB code 2JJT) as a search model. The model was manually built using the COOT program and refined using TLS parameters in REFMAC from the CCP4 program suite. Results: Crystallization and structure determination analysis of the interface of IMM01/CD47 structure demonstrated CD47 surface buried by IMM01. Comparison with the literature structure (PDB ID 2JJT) showed that the interactions of IMM01/CD47 structure are the same. All the hydrogen bonds that appear in the literature structure are also present in the IMM01/CD47 structure. These common hydrogen bonds are stable under different crystal packing styles, suggesting that these hydrogen bonds are important for protein binding. In the structure of human CD47 in complex with human SIRPα, except SER66, the amino acids that form hydrogen bonds are all conserved. Furthermore, comparing with the structure of PDB ID 2JJT, the salt bridge interaction from IMM01/CD47 structure are very similar, except the salt bridge bond between LYS53 in IMM01 and GLU106 in CD47, which only occurs between the B and D chains. However, as the side chain conformation of LYS53 in chain A is slightly different, the salt bridge bond is absent between the A and C chains. At this site between chain A and chain C, there are a salt bridge bond between LYS53 (A) and GLU104 (C) and a salt bridge bond between HIS56 (A) and GLU106 (C) instead. According to the sequence alignment results of SIRPα, SIRPß and SIRPγ in the literature of PDB ID 2JJT, except ASP100, the amino acids that form common salt bridge bonds are all conserved. Conclusion: Our data demonstrated crystal structure of the IMM01/CD47 complex and provides a structural basis for the structural binding interface and future clinical applications.

NMR analysis suggests the terminal domains of Robo1 remain extended but are rigidified in the presence of heparan sulfate.

Human roundabout 1 (hRobo1) is an extracellular receptor glycoprotein that plays important roles in angiogenesis, organ development, and tumor progression. Interaction between hRobo1 and heparan sulfate (HS) has been shown to be essential for its biological activity. To better understand the effect of HS binding we engineered a lanthanide-binding peptide sequence (Loop) into the Ig2 domain of hRobo1. Native mass spectrometry was used to verify that loop introduction did not inhibit HS binding or conformational changes previously suggested by gas phase ion mobility measurements. NMR experiments measuring long-range pseudocontact shifts were then performed on 13C-methyl labeled hRobo1-Ig1-2-Loop in HS-bound and unbound forms. The magnitude of most PCSs for methyl groups in the Ig1 domain increase in the bound state confirming a change in the distribution of interdomain geometries. A grid search over Ig1 orientations to optimize the fit of data to a single conformer for both forms produced two similar structures, both of which differ from existing X-ray crystal structures and structures inferred from gas-phase ion mobility measurements. The structures and degree of fit suggest that the hRobo1-Ig1-2 structure changes slightly and becomes more rigid on HS binding. This may have implications for Robo-Slit signaling.

Targeting the HVEM protein using a fragment of glycoprotein D to inhibit formation of the BTLA/HVEM complex.

Cancer immunotherapy using blockade of immune checkpoints is mainly based on monoclonal antibodies. Despite the tremendous success achieved by using those molecules to block immune checkpoint proteins, antibodies possess some weaknesses, which means that there is still a need to search for new compounds as alternatives to antibodies. Many current approaches are focused on use of peptides/peptidomimetics to destroy receptor/ligand interactions. Our studies concern blockade of the BTLA/HVEM complex, which generates an inhibitory effect on the immune response resulting in tolerance to cancer cells. To design inhibitors of such proteins binding we based our work on the amino acid sequence and structure of a ligand of HVEM protein, namely glycoprotein D, which possesses the same binding site on HVEM as BTLA protein. To disrupt the BTLA and HVEM interaction we designed several peptides, all fragments of glycoprotein D, and tested their binding to HVEM using SPR and their ability to inhibit the BTLA/HVEM complex formation using ELISA tests and cellular reporter platforms. That led to identification of two peptides, namely gD(1-36)(K10C-D30C) and gD(1-36)(A12C-L25C), which interact with HVEM and possess blocking capacities. Both peptides are not cytotoxic to human PBMCs, and show stability in human plasma. We also studied the 3D structure of the gD(1-36)(K10C-D30C) peptide using NMR and molecular modeling methods. The obtained data reveal that it possesses an unstructured conformation and binds to HVEM in the same location as gD and BTLA. All these results suggest that peptides based on the binding fragment of gD protein represent promising immunomodulation agents for future cancer immunotherapy.

SIRPα-specific monoclonal antibody enables antibody-dependent phagocytosis of neuroblastoma cells.

Immunotherapy with anti-GD2 monoclonal antibodies (mAbs) provides some benefits for patients with neuroblastoma (NB). However, the therapeutic efficacy remains limited, and treatment is associated with significant neuropathic pain. Targeting O-acetylated GD2 (OAcGD2) by 8B6 mAb has been proposed to avoid pain by more selective tumor cell targeting. Thorough understanding of its mode of action is necessary to optimize this treatment strategy. Here, we found that 8B6-mediated antibody-dependent cellular phagocytosis (ADCP) performed by macrophages is a key effector mechanism. But efficacy is limited by upregulation of CD47 expression on neuroblastoma cells in response to OAcGD2 mAb targeting, inhibiting 8B6-mediated ADCP. Antibody specific for the CD47 receptor SIRPα on macrophages restored 8B6-induced ADCP of CD47-expressing NB cells and improved the antitumor activity of 8B6 mAb therapy. These results identify ADCP as a critical mechanism for tumor cytolysis by anti-disialoganglioside mAb and support a combination with SIRPα blocking agents for effective neuroblastoma therapy.

The Glycoprotein 340's Scavenger Receptor Cysteine-Rich Domain Promotes Adhesion of Staphylococcus aureus and Pseudomonas aeruginosa to Contact Lens Polymers.

Contact lenses are biomaterials worn on the eye to correct refractive errors. Bacterial adhesion and colonization of these lenses results in adverse events, such as microbial keratitis. The adsorption of tear proteins to contact lens materials enhances bacterial adhesion. Glycoprotein 340 (Gp340), a tear component, is known to promote microbial colonization in the oral cavity; however, it has not been investigated in any contact lens-related adverse event. Therefore, this study examined the adsorption of Gp340 and its recombinantly expressed scavenger receptor cysteine-rich (iSRCR1Gp340) domain on two common contact lens materials, etafilcon A and lotrafilcon B, and the concomitant effects on the adherence of clinical isolates of microbial keratitis causative agents, Pseudomonas aeruginosa (PA6206; PA6294), and Staphylococcus aureus (SA38; USA300). Across all strains and materials, iSRCR1Gp340 enhanced adherence of bacteria in a dose-dependent manner. However, iSRCR1Gp340 did not modulate the lysozyme's or lactoferrin's effects on bacterial adhesion to the contact lens. The Gp340 binding serine-rich surface protein (SraP) significantly enhanced the binding of USA300 to iSRCR1Gp340-coated lenses. In addition, iSRCR1Gp340-coated surfaces had significantly diminished biofilms with the SraP mutant (ΔSraP), and there was a further reduction in biofilms with the sortase A mutant (ΔSrtA), indicating the likely involvement of additional surface proteins. Finally, the binding affinities between iSRCR1Gp340 and SraP were determined using surface plasmon resonance (SPR), where the complete SraP binding region displayed nanomolar affinity, whereas its smaller fragments adhered with micromolar affinities. This study concludes that Gp340 and its SRCR domains play an important role in bacterial adhesion to the contact lens.

The US3 Kinase of Herpes Simplex Virus Phosphorylates the RNA Sensor RIG-I To Suppress Innate Immunity.

Recent studies have demonstrated that the signaling activity of the cytosolic pathogen sensor retinoic acid-inducible gene-I (RIG-I) is modulated by a variety of posttranslational modifications (PTMs) to fine-tune the antiviral type I interferon (IFN) response. Whereas K63-linked ubiquitination of the RIG-I caspase activation and recruitment domains (CARDs) catalyzed by TRIM25 or other E3 ligases activates RIG-I, phosphorylation of RIG-I at S8 and T170 represses RIG-I signal transduction by preventing the TRIM25-RIG-I interaction and subsequent RIG-I ubiquitination. While strategies to suppress RIG-I signaling by interfering with its K63-polyubiquitin-dependent activation have been identified for several viruses, evasion mechanisms that directly promote RIG-I phosphorylation to escape antiviral immunity are unknown. Here, we show that the serine/threonine (Ser/Thr) kinase US3 of herpes simplex virus 1 (HSV-1) binds to RIG-I and phosphorylates RIG-I specifically at S8. US3-mediated phosphorylation suppressed TRIM25-mediated RIG-I ubiquitination, RIG-I-MAVS binding, and type I IFN induction. We constructed a mutant HSV-1 encoding a catalytically-inactive US3 protein (K220A) and found that, in contrast to the parental virus, the US3 mutant HSV-1 was unable to phosphorylate RIG-I at S8 and elicited higher levels of type I IFNs, IFN-stimulated genes (ISGs), and proinflammatory cytokines in a RIG-I-dependent manner. Finally, we show that this RIG-I evasion mechanism is conserved among the alphaherpesvirus US3 kinase family. Collectively, our study reveals a novel immune evasion mechanism of herpesviruses in which their US3 kinases phosphorylate the sensor RIG-I to keep it in the signaling-repressed state. IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes lifelong latency in the majority of the human population worldwide. HSV-1 occasionally reactivates to produce infectious virus and to facilitate dissemination. While often remaining subclinical, both primary infection and reactivation occasionally cause debilitating eye diseases, which can lead to blindness, as well as life-threatening encephalitis and newborn infections. To identify new therapeutic targets for HSV-1-induced diseases, it is important to understand the HSV-1-host interactions that may influence infection outcome and disease. Our work uncovered direct phosphorylation of the pathogen sensor RIG-I by alphaherpesvirus-encoded kinases as a novel viral immune escape strategy and also underscores the importance of RNA sensors in surveilling DNA virus infection.

Biomimetic design of inhibitors of immune checkpoint LILRB4.

Immune checkpoint inhibitors have become a hot spot in the treatment of acute myeloid leukemia (AML), the most common acute leukemia (blood cancer) in adults. In the present study, molecular insights into the molecular interactions between an immune checkpoint leukocyte immunoglobulin-like receptor b4 (LILRB4) and its mAb h128-3 was explored using molecular dynamics (MD) simulation for the biomimetic design of peptide inhibitor of LILRB4. Both hydrophobic interaction and electrostatic interaction were found favorable for the binding of the mAb h128-3 on LILRB4, and hydrophobic interaction was identified as the main driving force. The key amino acid residues for the binding of mAb h128-3 on LILRB4 were identified as Y93, D94, D106, Y34, S103, W107, Y61, N30, E27, Y33, Y59, W95, S92 through MM-PBSA (molecular mechanics-Poisson-Boltzmann surface area) method. Based on this, an inhibitor library with the sequence of SXDXYXSY (Where X is an arbitrary amino acid residue) were designed. Two peptide inhibitors, SADHYHSY and SVDWYHSY were obtained through screening using molecular docking and MD simulations, and then validated by successful blocking of LILRB4 through the covering of LILRB4 surface by these inhibitors. These results would be helpful for the research and development of therapies for AML.

HVEM structures and mutants reveal distinct functions of binding to LIGHT and BTLA/CD160.

HVEM is a TNF (tumor necrosis factor) receptor contributing to a broad range of immune functions involving diverse cell types. It interacts with a TNF ligand, LIGHT, and immunoglobulin (Ig) superfamily members BTLA and CD160. Assessing the functional impact of HVEM binding to specific ligands in different settings has been complicated by the multiple interactions of HVEM and HVEM binding partners. To dissect the molecular basis for multiple functions, we determined crystal structures that reveal the distinct HVEM surfaces that engage LIGHT or BTLA/CD160, including the human HVEM-LIGHT-CD160 ternary complex, with HVEM interacting simultaneously with both binding partners. Based on these structures, we generated mouse HVEM mutants that selectively recognized either the TNF or Ig ligands in vitro. Knockin mice expressing these muteins maintain expression of all the proteins in the HVEM network, yet they demonstrate selective functions for LIGHT in the clearance of bacteria in the intestine and for the Ig ligands in the amelioration of liver inflammation.

Evolving A RIG-I Antagonist: A Modified DNA Aptamer Mimics Viral RNA.

Vertebrate organisms express a diversity of protein receptors that recognize and respond to the presence of pathogenic molecules, functioning as an early warning system for infection. As a result of mutation or dysregulated metabolism, these same innate immune receptors can be inappropriately activated, leading to inflammation and disease. One of the most important receptors for detection and response to RNA viruses is called RIG-I, and dysregulation of this protein is linked with a variety of disease states. Despite its central role in inflammatory responses, antagonists for RIG-I are underdeveloped. In this study, we use invitro selection from a pool of modified DNA aptamers to create a high affinity RIG-I antagonist. A high resolution crystal structure of the complex reveals molecular mimicry between the aptamer and the 5'-triphosphate terminus of viral ligands, which bind to the same amino acids within the CTD recognition platform of the RIG-I receptor. Our study suggests a powerful, generalizable strategy for generating immunomodulatory drugs and mechanistic tool compounds.

Nanocages displaying SIRP gamma clusters combined with prophagocytic stimulus of phagocytes potentiate anti-tumor immunity.

Antigen-presenting cells (APCs), including macrophages and dendritic cells (DCs), play a crucial role in bridging innate and adaptive immunity; thereby, innate immune checkpoint blockade-based therapy is an attractive approach for the induction of sustainable tumor-specific immunity. The interaction between the cluster of differentiation 47 (CD47) on tumor and signal-regulatory protein alpha (SIRPα) on phagocytic cells inhibits the phagocytic function of APCs, acting as a "don't eat me" signal. Accordingly, CD47 blockade is known to increase tumor cell phagocytosis, eliciting tumor-specific CD8+ T-cell immunity. Here, we introduced a nature-derived nanocage to deliver SIRPγ for blocking of antiphagocytic signaling through binding to CD47 and combined it with prophagocytic stimuli using a metabolic reprogramming reagent for APCs (CpG-oligodeoxynucleotides). Upon delivering the clustered SIRPγ variant, the nanocage showed enhanced CD47 binding profiles on tumor cells, thereby promoting active engulfment by phagocytes. Moreover, combination with CpG potentiated the prophagocytic ability, leading to the establishment of antitumorigenic surroundings. This combination treatment could competently inhibit tumor growth by invigorating APCs and CD8+ T-cells in TMEs in B16F10 orthotopic tumor models, known to be resistant to CD47-targeting therapeutics. Collectively, enhanced delivery of an innate immune checkpoint antagonist with metabolic modulation stimuli of immune cells could be a promising strategy for arousing immune responses against cancer.

TLR2 Potentiates SR-Marco-Mediated Neuroinflammation by Interacting with the SRCR Domain.

Microglial activation-induced neuroinflammation is critical in the pathogenesis of neurodegenerative diseases. Activated microglia are regulated mainly by innate pattern recognition receptors (PRRs) on their surface, of which macrophage receptor with collagenous structure (Marco) is a well-characterized scavenger receptor constitutively expressed on specific subsets of macrophages, including microglia. Increasing evidence has shown that Marco is involved in the pathogenesis of a range of inflammatory processes. However, research on the role of Marco in regulating neuroinflammation has reported conflicting results. In the present study, we examined the role Marco played in triggering neuroinflammation and its underlying mechanisms. The results demonstrated that silencing the Marco gene resulted in a significantly reduced neuroinflammatory response and vice versa. α-Syn stimulation in Marco overexpressing cells induced a pronounced inflammatory response, suggesting that Marco alone could trigger an inflammatory response. We also found that TLR2 significantly promoted Marco-mediated neuroinflammation, indicating TLR2 was an important co-receptor of Marco. Knocking down the TLR2 gene in microglia and mouse substantia nigra resulted in decreased expression of Marco. Subsequent mechanistic studies showed that deleting the SRCR domain of Marco resulted in disruption of the inflammatory response and the interaction between TLR2 and Marco. This suggested that TLR2 binds directly to the SRCR domain of Marco and regulates Marco-mediated neuroinflammation. In summary, this investigation revealed that TLR2 could potentiate Marco-mediated neuroinflammation by interacting with the SRCR domain of Marco, providing a new target for inhibiting neuroinflammation in neurodegenerative diseases.

Selection and Characterization of FD164, a High-Affinity Signal Regulatory Protein α Variant with Balanced Safety and Effectiveness, from a Targeted Epitope Mammalian Cell-Displayed Antibody Library.

Phagocytic resistance plays a key role in tumor-mediated immune escape, so phagocytosis immune checkpoints are a potential target for cancer immunotherapy. CD47 is one of the important phagocytosis immune checkpoints; thus, blocking the interaction between CD47 and signal regulatory protein α (SIRPα) may provide new options for cancer treatment. Using computer-aided targeted epitope mammalian cell-displayed antibody library, we screened and obtained an engineered SIRPα variant fragment crystallizable fusion protein, FD164, with higher CD47-binding activity than wild-type SIRPα Compared with wild-type SIRPα, FD164 has approximately 3-fold higher affinity for binding to CD47, which further enhanced its phagocytic effect in vitro and tumor suppressor activity in vivo. FD164 maintains the similar antitumor activity of the clinical research drug Hu5F9 in the mouse xenograft model. Furthermore, FD164 combined with rituximab can significantly improve the effect of single-agent therapy. On the other hand, compared with Hu5F9, FD164 does not cause hemagglutination, and its ability to bind to red blood cells or white blood cells is weaker at the same concentration. Finally, it was confirmed by computer structure prediction and alanine scanning experiments that the N45, E47, 52TEVYVK58, K60, 115EVTELTRE122, and E124 residues of CD47 are important for SIRPα or FD164 recognition. Briefly, we obtained a high-affinity SIRPα variant FD164 with balanced safety and effectiveness. SIGNIFICANCE STATEMENT: Up to now, few clinically marketed drugs targeting CD47 have been determined to be effective and safe. FD164, a potential signal regulatory protein α variant fragment crystallizable protein with balanced safety and effectiveness, could provide a reference for the development of antitumor drugs.

Pathogen effector recognition-dependent association of NRG1 with EDS1 and SAG101 in TNL receptor immunity.

Plants utilise intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors to detect pathogen effectors and activate local and systemic defence. NRG1 and ADR1 "helper" NLRs (RNLs) cooperate with enhanced disease susceptibility 1 (EDS1), senescence-associated gene 101 (SAG101) and phytoalexin-deficient 4 (PAD4) lipase-like proteins to mediate signalling from TIR domain NLR receptors (TNLs). The mechanism of RNL/EDS1 family protein cooperation is not understood. Here, we present genetic and molecular evidence for exclusive EDS1/SAG101/NRG1 and EDS1/PAD4/ADR1 co-functions in TNL immunity. Using immunoprecipitation and mass spectrometry, we show effector recognition-dependent interaction of NRG1 with EDS1 and SAG101, but not PAD4. An EDS1-SAG101 complex interacts with NRG1, and EDS1-PAD4 with ADR1, in an immune-activated state. NRG1 requires an intact nucleotide-binding P-loop motif, and EDS1 a functional EP domain and its partner SAG101, for induced association and immunity. Thus, two distinct modules (NRG1/EDS1/SAG101 and ADR1/EDS1/PAD4) mediate TNL receptor defence signalling.