Binding of LAG-3 to stable peptide-MHC class II limits T cell function and suppresses autoimmunity and anti-cancer immunity.

Lymphocyte activation gene-3 (LAG-3) is a potent inhibitory co-receptor; yet, its functional ligand remains elusive, with distinct potential ligands identified. Here, we investigated the relative contribution of potential ligands, stable peptide-MHC class II complexes (pMHCII) and fibrinogen-like protein 1 (FGL1), to LAG-3 activity in vitro and in vivo. Binding of LAG-3 to stable pMHCII but not to FGL1 induced T cell suppression in vitro. Consistently, LAG-3 mutants lacking FGL1-binding capacity but not those lacking stable pMHCII-binding capacity retained suppressive activity in vitro. Accordingly, targeted disruption of stable pMHCII- but not FGL1-binding capacity of LAG-3 in NOD mice recapitulated diabetes exacerbation by LAG-3 deficiency. Additionally, the loss of stable pMHCII-binding capacity of LAG-3 augmented anti-cancer immunity comparably with LAG-3 deficiency in C57BL/6 mice. These results identify stable pMHCII as a functional ligand of LAG-3 both in autoimmunity and anti-cancer immunity. Thus, stable pMHCII-LAG-3 interaction is a potential therapeutic target in human diseases.

A SARS-CoV-2 antibody broadly neutralizes SARS-related coronaviruses and variants by coordinated recognition of a virus-vulnerable site.

Potent neutralizing SARS-CoV-2 antibodies often target the spike protein receptor-binding site (RBS), but the variability of RBS epitopes hampers broad neutralization of multiple sarbecoviruses and drifted viruses. Here, using humanized mice, we identified an RBS antibody with a germline VH gene that potently neutralized SARS-related coronaviruses, including SARS-CoV and SARS-CoV-2 variants. X-ray crystallography revealed coordinated recognition by the heavy chain of non-RBS conserved sites and the light chain of RBS with a binding angle mimicking the angiotensin-converting enzyme 2 (ACE2) receptor. The minimum footprints in the hypervariable region of RBS contributed to the breadth of neutralization, which was enhanced by immunoglobulin G3 (IgG3) class switching. The coordinated binding resulted in broad neutralization of SARS-CoV and emerging SARS-CoV-2 variants of concern. Low-dose therapeutic antibody treatment in hamsters reduced the virus titers and morbidity during SARS-CoV-2 challenge. The structural basis for broad neutralizing activity may inform the design of a broad spectrum of therapeutics and vaccines.

2-thiouridine is a broad-spectrum antiviral nucleoside analogue against positive-strand RNA viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections are causing significant morbidity and mortality worldwide. Furthermore, over 1 million cases of newly emerging or re-emerging viral infections, specifically dengue virus (DENV), are known to occur annually. Because no virus-specific and fully effective treatments against these or many other viruses have been approved, there is an urgent need for novel, effective therapeutic agents. Here, we identified 2-thiouridine (s2U) as a broad-spectrum antiviral ribonucleoside analogue that exhibited antiviral activity against several positive-sense single-stranded RNA (ssRNA+) viruses, such as DENV, SARS-CoV-2, and its variants of concern, including the currently circulating Omicron subvariants. s2U inhibits RNA synthesis catalyzed by viral RNA-dependent RNA polymerase, thereby reducing viral RNA replication, which improved the survival rate of mice infected with DENV2 or SARS-CoV-2 in our animal models. Our findings demonstrate that s2U is a potential broad-spectrum antiviral agent not only against DENV and SARS-CoV-2 but other ssRNA+ viruses.

Structure of the human galanin receptor 2 bound to galanin and Gq reveals the basis of ligand specificity and how binding affects the G-protein interface.

Galanin is a neuropeptide expressed in the central and peripheral nervous systems, where it regulates various processes including neuroendocrine release, cognition, and nerve regeneration. Three G-protein coupled receptors (GPCRs) for galanin have been discovered, which is the focus of efforts to treat diseases including Alzheimer's disease, anxiety, and addiction. To understand the basis of the ligand preferences of the receptors and to assist structure-based drug design, we used cryo-electron microscopy (cryo-EM) to solve the molecular structure of GALR2 bound to galanin and a cognate heterotrimeric G-protein, providing a molecular view of the neuropeptide binding site. Mutant proteins were assayed to help reveal the basis of ligand specificity, and structural comparison between the activated GALR2 and inactive hß2AR was used to relate galanin binding to the movements of transmembrane (TM) helices and the G-protein interface.

Chlamydia trachomatis L2 434/Bu readily activates glycolysis under hypoxia for efficient metabolism.

To understand why Chlamydia trachomatis (Ct) (L2/434/Bu) favors hypoxia, we examined the dynamics of infected cells using a glycolysis-related PCR array and metabolomic analysis, along with the perturbation of nucleotide synthesis. Our findings revealed that, compared to normoxia, hypoxia with infection significantly and selectively upregulates the expression of genes related to glycolysis, glycogen degradation, and the pentose phosphate pathway. Furthermore, hypoxia induced a significant decrease in metabolite levels, particularly methionine-related metabolites, independent of infection, indicating efficient metabolism under hypoxia. Additionally, the perturbation of nucleotide synthesis with adenosine derivatives impaired Ct growth. Collectively, our results suggest that Ct favors a hypoxic environment with efficient metabolism, in which Ct readily activates glycolysis responsible for stable nucleotide synthesis as well as ATP supply.

Genome-wide CRISPR screens identify CD48 defining susceptibility to NK cytotoxicity in peripheral T-cell lymphomas.

Adult T-cell leukemia/lymphoma (ATLL) is one of the aggressive peripheral T-cell neoplasms with a poor prognosis. Accumulating evidence demonstrates that escape from adaptive immunity is a hallmark of ATLL pathogenesis. However, the mechanisms by which ATLL cells evade natural killer (NK)-cell-mediated immunity have been poorly understood. Here we show that CD48 expression in ATLL cells determines the sensitivity for NK-cell-mediated cytotoxicity against ATLL cells. We performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening using 2 ATLL-derived cell lines and discovered CD48 as one of the best-enriched genes whose knockout conferred resistance to YT1-NK cell line-mediated cytotoxicity. The ability of CD48-knockout ATLL cells to evade NK-cell effector function was confirmed using human primary NK cells with reduced interferon-γ (IFNγ) induction and degranulation. We found that primary ATLL cells had reduced CD48 expression along with disease progression. Furthermore, other subgroups among aggressive peripheral T-cell lymphomas (PTCLs) also expressed lower concentrations of CD48 than normal T cells, suggesting that CD48 is a key molecule in malignant T-cell evasion of NK-cell surveillance. Thus, this study demonstrates that CD48 expression is likely critical for malignant T-cell lymphoma cell regulation of NK-cell-mediated immunity and provides a rationale for future evaluation of CD48 as a molecular biomarker in NK-cell-associated immunotherapies.

Virological characteristics of the SARS-CoV-2 Omicron EG.5.1 variant.

In middle to late 2023, a sublineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron XBB, EG.5.1 (a progeny of XBB.1.9.2), is spreading rapidly around the world. We performed multiscale investigations, including phylogenetic analysis, epidemic dynamics modeling, infection experiments using pseudoviruses, clinical isolates, and recombinant viruses in cell cultures and experimental animals, and the use of human sera and antiviral compounds, to reveal the virological features of the newly emerging EG.5.1 variant. Our phylogenetic analysis and epidemic dynamics modeling suggested that two hallmark substitutions of EG.5.1, S:F456L and ORF9b:I5T are critical to its increased viral fitness. Experimental investigations on the growth kinetics, sensitivity to clinically available antivirals, fusogenicity, and pathogenicity of EG.5.1 suggested that the virological features of EG.5.1 are comparable to those of XBB.1.5. However, cryo-electron microscopy revealed structural differences between the spike proteins of EG.5.1 and XBB.1.5. We further assessed the impact of ORF9b:I5T on viral features, but it was almost negligible in our experimental setup. Our multiscale investigations provide knowledge for understanding the evolutionary traits of newly emerging pathogenic viruses, including EG.5.1, in the human population.

Identification of RPL15 60S Ribosomal Protein as a Novel Topotecan Target Protein That Correlates with DAMP Secretion and Antitumor Immune Activation.

Damage-associated molecular patterns (DAMPs) contribute to antitumor immunity during cancer chemotherapy. We previously demonstrated that topotecan (TPT), a topoisomerase I inhibitor, induces DAMP secretion from cancer cells, which activates STING-mediated antitumor immune responses. However, how TPT induces DAMP secretion in cancer cells is yet to be elucidated. Here, we identified RPL15, a 60S ribosomal protein, as a novel TPT target and showed that TPT inhibited preribosomal subunit formation via its binding to RPL15, resulting in the induction of DAMP-mediated antitumor immune activation independent of TOP1. TPT inhibits RPL15-RPL4 interactions and decreases RPL4 stability, which is recovered by CDK12 activity. RPL15 knockdown induced DAMP secretion and increased the CTL population but decreased the regulatory T cell population in a B16-F10 murine melanoma model, which sensitized B16-F10 tumors against PD-1 blockade. Our study identified a novel TPT target protein and showed that ribosomal stress is a trigger of DAMP secretion, which contributes to antitumor immunotherapy.

Impact of Micropolymorphism Outside the Peptide Binding Groove in the Clinically Relevant Allele HLA-C*14 on T Cell Responses in HIV-1 Infection.

There is increasing evidence for the importance of human leukocyte antigen C (HLA-C)-restricted CD8+ T cells in HIV-1 control, but these responses are relatively poorly investigated. The number of HLA-C-restricted HIV-1 epitopes identified is much smaller than those of HLA-A-restricted or HLA-B-restricted ones. Here, we utilized a mass spectrometry-based approach to identify HIV-1 peptides presented by HLA-C*14:03 protective and HLA-C*14:02 nonprotective alleles. We identified 25 8- to 11-mer HLA-I-bound HIV-1 peptides from HIV-1-infected HLA-C*14:02+/14:03+ cells. Analysis of T cell responses to these peptides identified novel 6 T cell epitopes targeted in HIV-1-infected HLA-C*14:02+/14:03+ subjects. Analyses using HLA stabilization assays demonstrated that all 6 epitope peptides exhibited higher binding to and greater cell surface stabilization of HLA-C*14:02 than HLA-C*14:03. T cell response magnitudes were typically higher in HLA-C*14:02+ than HLA-C*14:03+ individuals, with responses to the Pol KM9 and Nef epitopes being significantly higher. The results show that HLA-C*14:02 can elicit stronger T cell responses to HIV-1 than HLA-C*14:03 and suggest that the single amino acid difference between these HLA-C14 subtypes at position 21, outside the peptide-binding groove, indirectly influences the stability of peptide-HLA-C*14 complexes and induction/expansion of HIV-specific T cells. Taken together with a previous finding that KIR2DL2+ NK cells recognized HLA-C*14:03+ HIV-1-infected cells more than HLA-C*14:02+ ones, the present study indicates that these HLA-C*14 subtypes differentially impact HIV-1 control by T cells and NK cells. IMPORTANCE Some human leukocyte antigen (HLA) class I alleles are associated with good clinical outcomes in HIV-1 infection and are called protective HLA alleles. Identification of T cell epitopes restricted by protective HLA alleles can give important insight into virus-immune system interactions and inform design of immune-based prophylactic/therapeutic strategies. Although epitopes restricted by many protective HLA-A/B alleles have been identified, protective HLA-C alleles are relatively understudied. Here, we identified 6 novel T cell epitopes presented by both HLA-C*14:02 (no association with protection) and HLA-C*14:03 (protective) using a mass spectrometry-based immunopeptidome profiling approach. We found that these peptides bound to and stabilized HLA-C*14:02 better than HLA-C*14:03 and observed differences in induction/expansion of epitope-specific T cell responses in HIV-infected HLA-C*14:02+ versus HLA-C*14:03+ individuals. These results enhance understanding of how the microstructural difference at position 21 between these HLA-C*14 subtypes may influence cellular immune responses involved in viral control in HIV-1 infection.

SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells.

The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.

Protein kinase Cß is involved in cigarette smoke gas phase-induced ferroptosis in J774 macrophages.

Cigarette smoking is a risk factor for respiratory infection caused by immune cell dysfunction. Cigarette smoke is divided into tar and gas phases. Although the gas phase induces cell death in various cell types, the mechanism for gas phase-induced cell death remains to be clarified. In this study, we have examined the effects of cigarette smoke gas phase on J774 macrophages. Cigarette smoke gas phase and cytotoxic factors in the gas phase induced protein kinase C (PKC)-dependent ferroptosis. Pharmacological studies using isoform-specific PKC inhibitors have revealed that PKCß is involved in cigarette smoke gas phase-induced ferroptosis in J774 macrophages.

An Electron-Deficient CpE Iridium(III) Catalyst: Synthesis, Characterization, and Application to Ether-Directed C-H Amidation.

The synthesis, characterization, and catalytic performance of an iridium(III) catalyst with an electron-deficient cyclopentadienyl ligand ([CpE IrI2 ]2 ) are reported. The [CpE IrI2 ]2 catalyst was synthesized by complexation of a precursor of the CpE ligand with [Ir(cod)OAc]2 , followed by oxidation, desilylation, and removal of the COD ligand. The electron-deficient [CpE IrI2 ]2 catalyst enabled C-H amidation reactions assisted by a weakly coordinating ether directing group. Experimental mechanistic studies and DFT calculations suggested that the high catalytic performance of [CpE IrI2 ]2 is due to its electron-deficient nature, which accelerates both C-H activation and IrV -nitrenoid formation.

Dihydromaniwamycin E, a Heat-Shock Metabolite from Thermotolerant Streptomyces sp. JA74, Exhibiting Antiviral Activity against Influenza and SARS-CoV-2 Viruses.

Dihydromaniwamycin E (1), a new maniwamycin derivative featuring an azoxy moiety, has been isolated from the culture extract of thermotolerant Streptomyces sp. JA74 along with the known analogue maniwamycin E (2). Compound 1 is produced only by cultivation of strain JA74 at 45 °C, and this type of compound has been previously designated a "heat shock metabolite (HSM)" by our research group. Compound 2 is detected as a production-enhanced metabolite at high temperature. Structures of 1 and 2 are elucidated by NMR and MS spectroscopic analyses. The absolute structure of 1 is determined after the total synthesis of four stereoisomers. Though the absolute structure of 2 has been proposed to be the same as the structure of maniwamycin D, the NMR and the optical rotation value of 2 are in agreement with those of maniwamycin E. Therefore, this study proposes a structural revision of maniwamycins D and E. Compounds 1 and 2 show inhibitory activity against the influenza (H1N1) virus infection of MDCK cells, demonstrating IC50 values of 25.7 and 63.2 µM, respectively. Notably, 1 and 2 display antiviral activity against SARS-CoV-2, the causative agent of COVID-19, when used to infect 293TA and VeroE6T cells, with 1 and 2 showing IC50 values (for infection of 293TA cells) of 19.7 and 9.7 µM, respectively. The two compounds do not exhibit cytotoxicity in these cell lines at those IC50 concentrations.

Synthesis and Anti-dengue Virus Activity of 5-Ethynylimidazole-4-carboxamide (EICA) Nucleotide Prodrugs.

We previously showed that 5-ethynyl-(1-ß-D-ribofuranosyl)imidazole-4-carboxamide (1; EICAR) is a potent anti-dengue virus (DENV) compound but is cytotoxic to some cell lines, while its 4-thio derivative, 5-ethynyl-(4-thio-1-ß-D-ribofuranosyl)imidazole-4-carboxamide (2; 4'-thioEICAR), has less cytotoxicity but also less anti-DENV activity. Based on the hypothesis that the lower anti-DENV activity of 2 is due to reduced susceptibility to phosphorylation by cellular kinase(s), we investigated whether a monophosphate prodrug of 2 can improve its activity. Here, we first prepared two types of prodrug of 1, which revealed that the S-acyl-2-thioethyl (SATE) prodrug had stronger anti-DENV activity than the aryloxyphosphoramidate (so-called ProTide) prodrug. Based on these findings, we next prepared the SATE prodrug of 4'-thioEICAR 18. As expected, the resulting 18 showed potent anti-DENV activity, which was comparable to that of 1; however, its cytotoxicity was also increased relative to 2. Our findings suggest that prodrugs of 4'-thioribonucleoside derivatives such as EICAR (1) represent an effective approach to developing potent biologically active compounds; however, the balance between antiviral activity and cytotoxicity remains to be addressed.

Application of Acoustic Ejection MS System to High-Throughput Screening for SARS-CoV-2 3CL Protease Inhibitors.

MS is a powerful methodology for chemical screening to directly quantify substrates and products of enzymes, but its low throughput has been an issue. Recently, an acoustic liquid-handling apparatus (Echo®) used for rapid nano-dispensing has been coupled to a high-sensitivity mass spectrometer to create the Echo® MS system, and we applied this system to screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CL protease inhibitors. Primary screening of 32033 chemical samples was completed in 12 h. Among the hits showing selective, dose-dependent 3CL-inhibitory activity, 8 compounds showed antiviral activity in cell-based assay.

Molecular mechanism of the recognition of bacterially cleaved immunoglobulin by the immune regulatory receptor LILRA2.

Human leukocyte immunoglobulin-like receptors (LILRs) typically regulate immune activation by binding to the human leukocyte antigen class I molecules. LILRA2, a member of the LILR family, was recently reported to bind to other unique ligands, the bacterially degraded Igs (N-truncated Igs), for the activation of immune cells. Therefore, LILRA2 is currently attracting significant attention as a novel innate immune receptor. However, the detailed recognition mechanisms required for this interaction remain unclear. In this study, using several biophysical techniques, we uncovered the molecular mechanism of N-truncated Ig recognition by LILRA2. Surface plasmon resonance analysis disclosed that LILRA2 specifically binds to N-truncated Ig with weak affinity (Kd = 4.8 µm) and fast kinetics. However, immobilized LILRA2 exhibited a significantly enhanced interaction with N-truncated Ig due to avidity effects. This suggests that cell surface-bound LILRA2 rapidly monitors and identifies bi- or multivalent abnormal N-truncated Igs through specific cross-linking to induce immune activation. Van't Hoff analysis revealed that this interaction is enthalpy-driven, with a small entropy loss, and results from differential scanning calorimetry indicated the instability of the putative LILRA2-binding site, the Fab region of the N-truncated Ig. Atomic force microscopy revealed that N truncation does not cause significant structural changes in Ig. Furthermore, mutagenesis analysis identified the hydrophobic region of LILRA2 domain 2 as the N-truncated Ig-binding site, representing a novel ligand-binding site for the LILR family. These results provide detailed insights into the molecular regulation of LILR-mediated immune responses targeting ligands that have been modified by bacteria.

The Measles Virus V Protein Binding Site to STAT2 Overlaps That of IRF9.

Measles virus (MeV) is a highly immunotropic and contagious pathogen that can even diminish preexisting antibodies and remains a major cause of childhood morbidity and mortality worldwide despite the availability of effective vaccines. MeV is one of the most extensively studied viruses with respect to the mechanisms of JAK-STAT antagonism. Of the three proteins translated from the MeV P gene, P and V are essential for inactivation of this pathway. However, the lack of data from direct analyses of the underlying interactions means that the detailed molecular mechanism of antagonism remains unresolved. Here, we prepared recombinant MeV V protein, which is responsible for human JAK-STAT antagonism, and a panel of variants, enabling the biophysical characterization of V protein, including direct V/STAT1 and V/STAT2 interaction assays. Unambiguous direct interactions between the host and viral factors, in the absence of other factors such as Jak1 or Tyk2, were observed, and the dissociation constants were quantified for the first time. Our data indicate that interactions between the C-terminal region of V and STAT2 is 1 order of magnitude stronger than that of the N-terminal region of V and STAT1. We also clarified that these interactions are completely independent of each other. Moreover, results of size exclusion chromatography demonstrated that addition of MeV-V displaces STAT2-core, a rigid region of STAT2 lacking the N- and C-terminal domains, from preformed complexes of STAT2-core/IRF-associated domain (IRF9). These results provide a novel model whereby MeV-V can not only inhibit the STAT2/IRF9 interaction but also disrupt preassembled interferon-stimulated gene factor 3.IMPORTANCE To evade host immunity, many pathogenic viruses inactivate host Janus kinase signal transducer and activator of transcription (STAT) signaling pathways using diverse strategies. Measles virus utilizes P and V proteins to counteract this signaling pathway. Data derived largely from cell-based assays have indicated several amino acid residues of P and V proteins as important. However, biophysical properties of V protein or its direct interaction with STAT molecules using purified proteins have not been studied. We have developed novel molecular tools enabling us to identify a novel molecular mechanism for immune evasion whereby V protein disrupts critical immune complexes, providing a clear strategy by which measles virus can suppress interferon-mediated antiviral gene expression.

Structure, solubility, and permeability relationships in a diverse middle molecule library.

To develop methodology to predict the potential druggability of middle molecules, we examined the structure, solubility, and permeability relationships of a diverse library (HKDL ver.1) consisting of 510 molecules (359 natural product derivatives, 76 non-natural products, 46 natural products, and 29 non-natural product derivatives). The library included peptides, depsipeptides, macrolides, and lignans, and 476 of the 510 compounds had a molecular weight in the range of 500-2000 Da. The solubility and passive diffusion velocity of the middle molecules were assessed using the parallel artificial membrane permeability assay (PAMPA). Quantitative values of solubility of 471 molecules and passive diffusion velocity of 287 molecules were obtained, and their correlations with the structural features of the molecules were examined. Based on the results, we propose a method to predict the passive diffusion characteristics of middle molecules from their three-dimensional structural features.

Enzootic nasal tumor virus type 2 envelope of goats acts as a retroviral oncogene in cell transformation.

Enzootic nasal tumor virus type 1 (ENTV-1) (ovine nasal tumor virus) and ENTV-2 (caprine nasal tumor virus) are known to be causative agents of enzootic nasal adenocarcinoma (ENA) in sheep and goats, respectively. Although the nucleotide and amino acid sequences of ENTV-1 and ENTV-2 are quite similar, they are recognized as phylogenetically distinct viruses. The envelope protein of ENTV-1 functions as an oncoprotein in the in vitro transformation of epithelial cells and fibroblasts. Thus, it is the primary determinant of in vivo tumorigenesis in ENA. As per our knowledge, no previous studies have reported in detail the role of ENTV-2 in ENA tumorigenesis. Here, in order to investigate the molecular mechanism of caprine ENA oncogenesis by ENTV-2, we have attempted to identify the transforming potential of ENTV-2 envelope, and investigated the activation of cell signaling pathways in oncogenic transformation. Our findings confirmed that ENTV-2 envelope was capable of inducing oncogenic transformation of rat cell lines in vitro. Further, we found that MAPK, Akt, and p38 were constitutively activated in ENTV-2 envelope-transformed clone cells. In addition, inhibitor experiments revealed that MEK-MAPK and PI3K-Akt signaling pathways are involved in the ENTV-2 envelope-induced cell transformation. These data indicate that ENTV-2 envelope could induce oncogenic transformation by signaling pathways that are also utilized by ENTV-1 envelope.

Structural and Functional Basis for LILRB Immune Checkpoint Receptor Recognition of HLA-G Isoforms.

Human leukocyte Ig-like receptors (LILR) LILRB1 and LILRB2 are immune checkpoint receptors that regulate a wide range of physiological responses by binding to diverse ligands, including HLA-G. HLA-G is exclusively expressed in the placenta, some immunoregulatory cells, and tumors and has several unique isoforms. However, the recognition of HLA-G isoforms by LILRs is poorly understood. In this study, we characterized LILR binding to the ß2-microglobulin (ß2m)-free HLA-G1 isoform, which is synthesized by placental trophoblast cells and tends to dimerize and multimerize. The multimerized ß2m-free HLA-G1 dimer lacked detectable affinity for LILRB1, but bound strongly to LILRB2. We also determined the crystal structure of the LILRB1 and HLA-G1 complex, which adopted the typical structure of a classical HLA class I complex. LILRB1 exhibits flexible binding modes with the α3 domain, but maintains tight contacts with ß2m, thus accounting for ß2m-dependent binding. Notably, both LILRB1 and B2 are oriented at suitable angles to permit efficient signaling upon complex formation with HLA-G1 dimers. These structural and functional features of ligand recognition by LILRs provide novel insights into their important roles in the biological regulations.