Microglia LILRB4 upregulation reduces brain damage after acute ischemic stroke by limiting CD8+ T cell recruitment.

BACKGROUND: Leukocyte immunoglobulin-like receptor B4 (LILRB4) plays a significant role in regulating immune responses. LILRB4 in microglia might influence the infiltration of peripheral T cells. However, whether and how LILRB4 expression aggravates brain damage after acute ischemic stroke remains unclear. This study investigates the role of LILRB4 in modulating the immune response and its potential protective effects against ischemic brain injury in mice. METHODS AND RESULTS: Microglia-specific LILRB4 conditional knockout (LILRB4-KO) and overexpression transgenic (LILRB4-TG) mice were constructed by a Cre-loxP system. Then, they were used to investigate the role of LILRB4 after ischemic stroke using a transient middle cerebral artery occlusion (tMCAO) mouse model. Spatial transcriptomics analysis revealed increased LILRB4 expression in the ischemic hemisphere. Single-cell RNA sequencing (scRNA-seq) identified microglia-cluster3, an ischemia-associated microglia subcluster with elevated LILRB4 expression in the ischemic brain. Flow cytometry and immunofluorescence staining showed increased CD8+ T cell infiltration into the brain in LILRB4-KO-tMCAO mice. Behavioral tests, cortical perfusion maps, and infarct size measurements indicated that LILRB4-KO-tMCAO mice had more severe functional deficits and larger infarct sizes compared to Control-tMCAO and LILRB4-TG-tMCAO mice. T cell migration assays demonstrated that LILRB4-KD microglia promoted CD8+ T cell recruitment and activation in vitro, which was mitigated by CCL2 inhibition and recombinant arginase-1 addition. The scRNA-seq and spatial transcriptomics identified CCL2 was predominantly secreted from activated microglia/macrophage and increased CCL2 expression in LILRB4-KD microglia, suggesting a chemokine-mediated mechanism of LILRB4. CONCLUSION: LILRB4 in microglia plays a crucial role in modulating the post-stroke immune response by regulating CD8+ T cell infiltration and activation. Knockout of LILRB4 exacerbates ischemic brain injury by promoting CD8+ T cell recruitment. Overexpression of LILRB4, conversely, offers neuroprotection. These findings highlight the therapeutic potential of targeting LILRB4 and its downstream pathways to mitigate immune-mediated damage in ischemic stroke.

Molecular regulatory mechanism of LILRB4 in the immune response.

Immune diseases are caused by the imbalance of immune regulation. This imbalance is regulated by many factors, both negative and positive. Leukocyte immunoglobulin-like receptor B4 (LILRB4) is a member of leukocyte immunoglobulin-like receptors (LILRs). LILRs are expressed constitutively on the surface of multiple immune cells which associate with membrane adaptors to signal through multi- ple cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or immunoreceptor tyro-sine-based activation motifs (ITAMs). Through ITIM, LILRB4 could recruit the src homology domain type-2-containing tyrosine phosphatase 1 or 2 (SHP-1 or SHP-2) into the cell membrane. In addition, many factors can induce the expression of LILRB4, such as vitamin D, interferon and so on. Studies have demonstrated that LILRB4 had a negative regulatory role in various of immune diseases. The present review intends to expound the structure and function of LILRB4, as well as its regulators and receptors in the immune cells, so as to provide a theoretical basis for immune disease therapy.

Leukocyte immunoglobulin-like receptor B4 (LILRB4) negatively mediates the pathological cardiac hypertrophy by suppressing fibrosis, inflammation and apoptosis via the activation of NF-κB signaling.

Pathological cardiac hypertrophy is a leading cause of morbidity and mortality in the world. However, it is still unclear the molecular mechanism revealing the progression of the disease. In the study, we illustrated that the expression of leukocyte immunoglobulin-like receptor B4 (LILRB4), associated with the pathological development of various inflammatory diseases, was down-regulated in pressure overload-induced hearts of patients and mice. LILRB4-knockout mice developed cardiac hypertrophy and heart failure by promoting cardiac dysfunction, fibrosis, inflammation and apoptosis. Mechanistically, transforming growth factor ß1 (TGF-ß1) expression was significantly promoted by LILRB4 deficiency in hearts of mice after aortic banding (AB) surgery. AB-induced inflammation in cardiac tissues was accelerated by LILRB4 deletion through elevating nuclear factor κB (NF-κB) signaling pathway. Furthermore, apoptosis triggered by AB operation in heart tissues was markedly enhanced in LILRB4-KO mice through promoting Caspase-3 activation. Importantly, the in vitro study indicated that LILRB4 knockdown-promoted fibrosis; inflammation and apoptosis were largely via the NF-κB signaling. Therefore, the findings above identified LILRB4 might be a negative regulator of cardiac remodeling, illustrating that LILRB4 represented as a therapeutic target for the prevention of cardiac hypertrophy and heart failure.

Immune Checkpoint Inhibitors in Acute Myeloid Leukemia: Novel Combinations and Therapeutic Targets.

PURPOSE OF REVIEW: Immune checkpoint therapy has dramatically changed the therapeutic landscape of solid malignancies. Here, we review the scientific rationale and current data evaluating immune checkpoint inhibitors in acute myeloid leukemia (AML). RECENT FINDINGS: Immune checkpoint inhibitor monotherapy has shown limited clinical activity in AML. Initial results from early-phase clinical trials suggest that rational combinations of immune checkpoint inhibition with hypomethylating agents (HMAs) are safe and potentially more promising. There are currently no data directly comparing immune checkpoint inhibition to standard therapies. Emerging immune targets more specific for leukemia cells including LILRB4 may improve future therapeutic efficacy. The success of immune checkpoint inhibition in AML has been modest to date. However, an improved understanding of the biology and the use of rational combinations has potential to improve rates of durable responses. Multiple clinical trials in AML are currently evaluating the use of immune checkpoints alone and in combination.

LILRB4 deficiency aggravates the development of atherosclerosis and plaque instability by increasing the macrophage inflammatory response via NF-κB signaling.

Atherosclerosis is a chronic inflammatory disease. LILRB4 is associated with the pathological processes of various inflammatory diseases. However, the potential function and underlying mechanisms of LILRB4 in atherogenesis remain to be investigated. In this study, LILRB4 expression was examined in both human and mouse atherosclerotic plaques. The effects and possible mechanisms of LILRB4 in atherogenesis and plaque instability were evaluated in LILRB4-/-ApoE-/- and ApoE-/- mice fed a high-fat diet. We found that LILRB4 was located primarily in macrophages, and its expression was up-regulated in atherosclerotic lesions from human coronary arteries and mouse aortic roots. LILRB4 deficiency significantly accelerated the development of atherosclerotic lesions and increased the instability of plaques, as evidenced by the increased infiltration of lipids, decreased amount of collagen components and smooth muscle cells. Moreover, LILRB4 deficiency in bone marrow-derived cells promoted the development of atherosclerosis. In vivo and in vitro analyses revealed that the pro-inflammatory effects of LILRB4 deficiency were mediated by the increased activation of NF-κB signaling due to decreased Shp1 phosphorylation. In conclusion, the present study indicates that LILRB4 deficiency promotes atherogenesis, at least partly, through reduced Shp1 phosphorylation, which subsequently enhances the NF-κB-mediated inflammatory response. Thus, targeting the "LILRB4-Shp1" axis may be a novel therapeutic approach for atherosclerosis.

Human CD43+ B cells are closely related not only to memory B cells phenotypically but also to plasmablasts developmentally in healthy individuals.

CD20(+)CD27(+)CD43(+) B (CD43(+) B) cells have been newly defined among PBMCs and proposed to be human B1 cells. However, it is controversial as to whether they are orthologs of murine B1 cells and how they are related to other B-cell populations, particularly CD20(+)CD27(+)CD43(-) memory B cells and CD20(low)CD27(high)CD43(high) plasmablasts. Our objective is to identify phenotypically the position of CD43(+) B cells among peripheral B-lineage cell compartments in healthy donors, with reference to B-cell subsets from patients with systemic lupus erythematosus (SLE). We found that CD43(+) B cells among PBMCs from healthy subjects were indistinguishable phenotypically from memory B cells in terms of surface markers, and spontaneous in vitro Ig and IL-10 secretion capability, but quite different from plasmablasts. However, a moderate correlation was found in the frequency of CD43(+) B cells with that of plasmablasts in healthy donors but not in SLE patients. An in vitro differentiation experiment indicated that CD43(+) B cells give rise to plasmablasts more efficiently than do memory B cells, suggesting that they are more closely related to plasmablasts developmentally than are memory B cells, which is also supported by quantitative PCR analysis of mRNA expression of B-cell and plasma cell signature genes. Thus, we conclude that, in healthy individuals, CD43(+) B cells are closely related not only to memory B cells phenotypically but also to plasmablasts developmentally, although the developmental origin of CD43(+) B cells is not necessarily the same as that of plasmablasts.

Lilrb4 ameliorates ileal injury in rats with hemorrhagic shock and suppresses the activation of NF-κB signaling pathway.

Hemorrhagic shock (HS) leads to intestinal damage and subsequent multiple organ dysfunction syndrome. Intestinal barrier dysfunction is the main cause of multiple organ failure associated with HS. Leukocyte immunoglobulin-like receptor B4 (Lilrb4) belongs to the Ig superfamily and is a vital natural immunomodulatory receptor. The purpose of this study was to identify the role and molecular mechanism of Lilrb4 in HS-induced ileal injury. In this work, HS was established by femoral artery cannula and 90 min of HS (blood pressure, 35-40 mmHg), followed by resuscitation. RNA sequencing analysis showed that Lilrb4 was highly expressed in the ileum of HS rats. As observed, HS rats exhibited severe ileal injury, characterized by enlarged subepithelial space, edema, exfoliation and extensive loss of villi. Whereas, lentivirus system-mediated Lilrb4 overexpression considerably mitigated these alterations. HS led to increased release of markers associated with intestinal injury, which was effectively reversed by Lilrb4 overexpression. In addition, after resuscitation, Lilrb4 overexpression inhibited HS-triggered inflammatory response, as evidenced by decreased levels of proinflammatory cytokines. Lilrb4 also inhibited the activation of NF-κB signal induced by HS. Notably, Lilrb4 modulated the balance of regulatory T (Treg)-T helper 17 (Th17) cells in the mesenteric lymph node (MLN), which may also contribute to its protective role in HS progression. In aggregate, these findings confirmed that Lilrb4 overexpression protected against ileal injury caused by HS, indicating that Lilrb4 may be a potential candidate for the treatment of HS.

LILRB4 on multiple myeloma cells promotes bone lesion by p-SHP2/NF-κB/RELT signal pathway.

BACKGROUND: Leukocyte Ig-like receptor B family 4 (LILRB4) as an immune checkpoint on myeloid cells is a potential target for tumor therapy. Extensive osteolytic bone lesion is the most characteristic feature of multiple myeloma. It is unclear whether ectopic LILRB4 on multiple myeloma regulates bone lesion. METHODS: The conditioned medium (CM) from LILRB4-WT and -KO cells was used to analyze the effects of LILRB4 on osteoclasts and osteoblasts. Xenograft, syngeneic and patient derived xenograft models were constructed, and micro-CT, H&E staining were used to observe the bone lesion. RNA-seq, cytokine array, qPCR, the activity of luciferase, Co-IP and western blotting were used to clarify the mechanism by which LILRB4 mediated bone damage in multiple myeloma. RESULTS: We comprehensively analyzed the expression of LILRB4 in various tumor tissue arrays, and found that LILRB4 was highly expressed in multiple myeloma samples. The patient's imaging data showed that the higher the expression level of LILRB4, the more serious the bone lesion in patients with multiple myeloma. The conditioned medium from LILRB4-WT not -KO cells could significantly promote the differentiation and maturation of osteoclasts. Xenograft, syngeneic and patient derived xenograft models furtherly confirmed that LILRB4 could mediate bone lesion of multiple myeloma. Next, cytokine array was performed to identify the differentially expressed cytokines, and RELT was identified and regulated by LILRB4. The overexpression or exogenous RELT could regenerate the bone damage in LILRB4-KO cells in vitro and in vivo. The deletion of LILRB4, anti-LILRB4 alone or in combination with bortezomib could significantly delay the progression of bone lesion of multiple myeloma. CONCLUSIONS: Our findings indicated that LILRB4 promoted the bone lesion by promoting the differentiation and mature of osteoclasts through secreting RELT, and blocking LILRB4 singling pathway could inhibit the bone lesion.

LILRB4 Checkpoint for Immunotherapy: Structure, Mechanism and Disease Targets.

LILRB4, a myeloid inhibitory receptor belonging to the family of leukocyte immunoglobulin-like receptors (LILRs/LIRs), plays a pivotal role in the regulation of immune tolerance. LILRB4 primarily mediates suppressive immune responses by transmitting inhibitory signals through immunoreceptor tyrosine-based inhibitory motifs (ITIMs). This immune checkpoint molecule has gained considerable attention due to its potent regulatory functions. Its ability to induce effector T cell dysfunction and promote T suppressor cell differentiation has been demonstrated, indicating the therapeutic potential of LILRB4 for modulating excessive immune responses, particularly in autoimmune diseases or the induction of transplant tolerance. Additionally, through intervening with LILRB4 molecules, immune system responsiveness can be adjusted, representing significant value in areas such as cancer treatment. Thus, LILRB4 has emerged as a key player in addressing autoimmune diseases, transplant tolerance induction, and other medical issues. In this review, we provide a comprehensive overview of LILRB4, encompassing its structure, expression, and ligand molecules as well as its role as a tolerance receptor. By exploring the involvement of LILRB4 in various diseases, its significance in disease progression is emphasized. Furthermore, we propose that the manipulation of LILRB4 represents a promising immunotherapeutic strategy and highlight its potential in disease prevention, treatment and diagnosis.

Downregulation of LILRB4 Promotes Human Aortic Smooth Muscle Cell Contractile Phenotypic Switch and Apoptosis in Aortic Dissection.

Aortic dissection (AD) is a severe vascular disease with high rates of mortality and morbidity. However, the underlying molecular mechanisms of AD remain unclear. Differentially expressed genes (DEGs) were screened by bioinformatics methods. Alterations of histopathology and inflammatory factor levels in ß-aminopropionitrile (BAPN)-induced AD mouse model were evaluated through Hematoxylin-Eosin (HE) staining and Enzyme-linked immunosorbent assay (ELISA), respectively. Reverse transcription quantitative real-time polymerase chain reaction was performed to detect DEGs expression. Furthermore, the role of LILRB4 in AD was investigated through Cell Counting Kit-8 (CCK-8), wound healing, and flow cytometry. Western blotting was employed to assess the phenotypic switch and extracellular matrix (ECM)-associated protein expressions in platelet-derived growth factor-BB (PDGF-BB)-stimulated in vitro model of AD. In the AD mouse model, distinct dissection formation was observed. TNF-α, IL-1ß, IL-8, and IL-6 levels were higher in the AD mouse model than in the controls. Six hub genes were identified, including LILRB4, TIMP1, CCR5, CCL7, MSR1, and CLEC4D, all of which were highly expressed. Further exploration revealed that LILRB4 knockdown inhibited the cell vitality and migration of PDGF-BB-induced HASMCs while promoting apoptosis and G0/G1 phase ratio. More importantly, LILRB4 knockdown promoted the protein expression of α-SMA and SM22α, while decreasing the expression of Co1, MMP2, and CTGF, which suggested that LILRB4 silencing promoted contractile phenotypic transition and ECM stability. LILRB4 knockdown inhibits the progression of AD. Our study provides a new potential target for the clinical treatment of AD.

Discovery of galectin-8 as an LILRB4 ligand driving M-MDSCs defines a class of antibodies to fight solid tumors.

LILRB4 is an immunosuppressive receptor, and its targeting drugs are undergoing multiple preclinical and clinical trials. Currently, the absence of a functional LILRB4 ligand in solid tumors not only limits the strategy of early antibody screening but also leads to the lack of companion diagnostic (CDx) criteria, which is critical to the objective response rate in early-stage clinical trials. Here, we show that galectin-8 (Gal-8) is a high-affinity functional ligand of LILRB4, and its ligation induces M-MDSC by activating STAT3 and inhibiting NF-κB. Significantly, Gal-8, but not APOE, can induce MDSC, and both ligands bind LILRB4 noncompetitively. Gal-8 expression promotes in vivo tumor growth in mice, and the knockout of LILRB4 attenuates tumor growth in this context. Antibodies capable of functionally blocking Gal-8 are able to suppress tumor growth in vivo. These results identify Gal-8 as an MDSC-driving ligand of LILRB4, and they redefine a class of antibodies for solid tumors.

LILRB4 knockdown inhibits aortic dissection development by regulating pyroptosis and the JAK2/STAT3 signaling pathway.

Aortic dissection (AD) is a life-threatening condition with a high mortality rate and without effective pharmacological therapies. Our previous study illustrated that leukocyte immunoglobulin-like receptor B4 (LILRB4) knockdown promoted the contractile phenotypic switch and apoptosis of AD cells. This study aimed to further investigate the role of LILRB4 in animal models of AD and elucidate its underlying molecular mechanisms. Animal models of AD were established using 0.1% beta-aminopropionitrile and angiotensin II and an in vitro model was developed using platelet-derived growth factor BB (PDGF-BB). The effects of LILRB4 knockdown on histopathological changes, pyroptosis, phenotype transition, extracellular matrix (ECM), and Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) pathways were assessed using a series of in vivo and in vitro assays. The effects of the JAK2 inhibitor AG490 on AD cell function, phenotypic transition, and ECM were explored. LILRB4 was highly expressed in AD and its knockdown increased survival rate, reduced AD incidence, and alleviated histopathological changes in the AD mouse model. Furthermore, LILRB4 knockdown promoted contractile phenotype switch, stabilized the ECM, and inhibited pyroptosis. Mechanistically, LILRB4 knockdown inhibited the JAK2/STAT3 signaling pathway. JAK2 inhibitor AG490 inhibited cell viability and migration, enhanced apoptosis, induced G0/G1 cell cycle arrest, and suppressed S-phase progression in PDGF-BB-stimulated human aortic smooth muscle cells. LILRB4 knockdown suppresses AD development by inhibiting pyroptosis and the JAK2/STAT3 signaling pathway.

Fc gamma receptors promote antibody-induced LILRB4 internalization and immune regulation of monocytic AML.

The immune checkpoint leukocyte immunoglobulin-like receptor B4 (LILRB4) is found specifically on the cell surface of acute monocytic leukemia (monocytic AML), an aggressive and common subtype of AML. We have developed a humanized monoclonal IgG1 LILRB4-blocking antibody (h128-3), which improved immune regulation but reduced cell surface expression of LILRB4 in monocytic AML models by 40-60%. Interestingly, most of this effect was neutralized by mutation of the Fc region of the antibody (h128-3/N297A), which prevents interaction with Fc gamma receptors (FcγRs). This suggested that there is FcγR-dependent antigenic modulation underlying h128-3's effects, a mechanism known to alter the function of antibodies targeting B-cell malignancies. We disrupted the Fc-FcγR interaction pharmacologically and with stable CRISPR-Cas9-mediated genetic knockout of FcγRs in monocytic AML cell lines to investigate the role of FcγR-dependent antigenic modulation in the regulation of LILRB4 by h128-3. When FcγRI is inhibited or removed from the surface of monocytic AML cells, h128-3 cannot optimally perform its blocking function, resulting in activation of the LILRB4 inhibitory receptor and leading to a 15-25% decrease in T-cell-mediated cytotoxicity in vitro. In the absence of FcγRI, scaffolding by FcγRIIa allows h128-3 to maintain LILRB4-blocking function. Here we define a FcγR-dependent antigenic modulation mechanism underlying the function of an immunoreceptor blocking antibody for the first time in myeloid malignancy. This research will facilitate the development of safe, precision-targeted antibody therapeutics in myeloid malignancies with greater potency and efficacy.

Leukocyte Ig-like receptor B4 (Lilrb4a) alleviates cardiac dysfunction and isoproterenol-induced arrhythmogenic remodeling associated with cardiac fibrosis and inflammation.

BACKGROUND: Heart failure is usually accompanied by activation of the sympathetic nerve, and excessive activation of the sympathetic nerve promotes cardiac remodeling and cardiac dysfunction. In the isoproterenol (ISO)-induced animal model, it is often accompanied by myocardial hypertrophy, fibrosis, and inflammation. Leukocyte immunoglobulin-like receptor B4a (Lilrb4a), an immunosuppressive regulatory receptor, plays a vital role in cardiovascular disease. However, the effect of Lilrb4a on ventricular arrhythmia in an ISO-induced mouse model remains unclear. OBJECTIVE: The purpose of this study was to explore the role and molecular mechanism of Lilrb4a in ISO-induced arrhythmogenic remodeling. METHODS: Lilrb4a knockout mice and Lilrb4a overexpression mice were infused with ISO (15 mg/kg per 24 hours, 4 weeks). Echocardiography and histology evaluations of myocardial hypertrophy and cardiac structural remodeling were conducted. Surface electrocardiography and electrophysiologic examination were used to evaluate cardiac electrical remodeling and susceptibility to ventricular arrhythmias. Quantitative reverse transcriptase-polymerase chain reaction analysis and Western blotting were used to detect the expression levels of ion channel proteins and signal pathway proteins. RESULTS: The results discovered that ISO induced cardiac hypertrophy, fibrosis, and inflammation and led to electrical remodeling and the occurrence of ventricular arrhythmias. Lilrb4a alleviated cardiac structural and electrical remodeling and protected against the occurrence of ventricular arrhythmias in ISO-induced mice by gain-of-function or loss-of-function approaches. The mechanism is that Lilrb4a inhibited NF-κB signaling and MAPK signaling activation mediated by transforming growth factor kinase 1. CONCLUSION: Lilrb4a alleviates cardiac dysfunction and ISO-induced arrhythmogenic remodeling associated with cardiac fibrosis and inflammation through the regulation of NF-κB signaling and MAPK signaling activation.

LILRB4 regulates the function of decidual MDSCs via the SHP-2/STAT6 pathway during Toxoplasma gondii infection.

BACKGROUND: Toxoplasma gondii infection can cause adverse pregnancy outcomes, such as recurrent abortion, fetal growth restriction and infants with malformations, among others. Decidual myeloid-derived suppressor cells (dMDSCs) are a novel immunosuppressive cell type at the fetal-maternal interface which play an important role in sustaining normal pregnancy that is related to their high expression of the inhibitory molecule leukocyte immunoglobulin-like receptor B4 (LILRB4). It has been reported that the expression of LILRB4 is downregulated on decidual macrophages after T. gondii infection, but it remains unknown whether T. gondii infection can induce dMDSC dysfunction resulting from the change in LILRB4 expression. METHODS: LILRB4-deficient (LILRB4-/-) pregnant mice infected with T. gondii with associated adverse pregnancy outcomes, and anti-LILRB4 neutralized antibodies-treated infected human dMDSCs were used in vivo and in vitro experiments, respectively. The aim was to investigate the effect of LILRB4 expression on dMDSC dysfunction induced by T. gondii infection. RESULTS: Toxoplasma gondii infection was observed to reduce STAT3 phosphorylation, resulting in decreased LILRB4 expression on dMDSCs. The levels of the main functional molecules (arginase-1 [Arg-1], interleukin-10 [IL-10]) and main signaling molecules (phosphorylated Src-homology 2 domain-containing protein tyrosine phosphatase [p-SHP2], phosphorylated signal transducer and activator of transcription 6 [p-STAT6]) in dMDSCs were all significantly reduced in human and mouse dMDSCs due to the decrease of LILRB4 expression induced by T. gondii infection. SHP-2 was found to directly bind to STAT6 and STAT6 to bind to the promoter of the Arg-1 and IL-10 genes during T. gondii infection. CONCLUSIONS: The downregulation of LILRB4 expression on dMDSCs induced by T. gondii infection could regulate the expression of Arg-1 and IL-10 via the SHP-2/STAT6 pathway, resulting in the dysfunction of dMDSCs, which might contribute to adverse outcomes during pregnancy by T. gondii infection.

Prognostic impact of LILRB4 expression on tumor-infiltrating cells in resected non-small cell lung cancer.

BACKGROUND: Leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4/ILT3) is an up-and-coming molecule that promotes immune evasion. We have previously reported that LILRB4 facilitates myeloid-derived suppressor cells (MDSCs)-mediated tumor metastasis in mice. This study aimed to investigate the impact of the LILRB4 expression levels on tumor-infiltrating cells on the prognosis of non-small cell lung cancer (NSCLC) patients. METHODS: We immunohistochemically evaluated the LILRB4 expression levels of completely resected 239 NSCLC specimens. Whether the blocking of LILRB4 on human PBMC-derived CD33+ MDSCs inhibited the migration ability of lung cancer cells was also examined using transwell migration assay. RESULTS: The LILRB4 high group, in which patients with a high LILRB4 expression level on tumor-infiltrating cells, showed a shorter overall survival (OS) (p = 0.013) and relapse-free survival (RFS) (p = 0.0017) compared to the LILRB4 low group. Multivariate analyses revealed that a high LILRB4 expression was an independent factor for postoperative recurrence, poor OS and RFS. Even in the cohort background aligned by propensity score matching, OS (p = 0.023) and RFS (p = 0.0046) in the LILRB4 high group were shorter than in the LILRB4 low group. Some of the LILRB4 positive cells were positive for MDSC markers, CD33 and CD14. Transwell migration assay demonstrated that blocking LILRB4 significantly inhibited the migration of human lung cancer cells cocultured with CD33+ MDSCs. CONCLUSION: Together, signals through LILRB4 on tumor-infiltrating cells, including MDSCs, play an essential role in promoting tumor evasion and cancer progression, impacting the recurrence and poor prognosis of patients with resected NSCLC.

Antibody therapies for the treatment of acute myeloid leukemia: exploring current and emerging therapeutic targets.

INTRODUCTION: Acute myeloid leukemia (AML) is the most common and deadly type of leukemia affecting adults. It is typically managed with rounds of non-targeted chemotherapy followed by hematopoietic stem cell transplants, but this is only possible in patients who can tolerate these harsh treatments and many are elderly and frail. With the identification of novel tumor-specific cell surface receptors, there is great conviction that targeted antibody therapies will soon become available for these patients. AREAS COVERED: In this review, we describe the current landscape of known target receptors for monospecific and bispecific antibody-based therapeutics for AML. Here, we characterize each of the receptors and targeted antibody-based therapeutics in development, illustrating the rational design behind each therapeutic compound. We then discuss the bispecific antibodies in development and how they improve immune surveillance of AML. For each therapeutic, we also summarize the available pre-clinical and clinical data, including data from discontinued trials. EXPERT OPINION: One antibody-based therapeutic has already been approved for AML treatment, the CD33-targeting antibody-drug conjugate, gemtuzumab ozogamicin. Many more are currently in pre-clinical and clinical studies. These antibody-based therapeutics can perform tumor-specific, elaborate cytotoxic functions and there is growing confidence they will soon lead to personalized, safe AML treatment options that induce durable remissions.

LILRB4 promotes tumor metastasis by regulating MDSCs and inhibiting miR-1 family miRNAs.

Myeloid-derived suppressor cells (MDSCs) are a population of immune suppressive cells that are involved in tumor-associated immunosuppression, and dominate tumor progression and metastasis. In this study, we report that the leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4, murine ortholog gp49B) orchestrates the polarization of MDSCs to exhibit pro-tumor phenotypes. We found that gp49B deficiency inhibited tumor metastases of cancer cells, and reduced tumor-infiltration of monocytic MDSCs (M-MDSCs) in tumor-bearing mice. Gp49B-/- MDSCs inhibited pro-tumor immune responses, such as activation of Treg cells, promotion of cancer cell migration, and stimulation of tumor angiogenesis. Treatment of wild-type tumor-bearing mice with gp49B-/- M-MDSCs reduced cancer metastasis. Furthermore, gp49B knockout affected plasma exosome composition in terms of increased miR-1 family microRNAs (miRNAs) expression, which correlates with the upregulation of gp49B-/- MDSC-derived anti-tumor miRNAs. Collectively, our findings reveal that LILRB4/gp49B promotes MDSC-mediated tumor metastasis by regulating the M2-polarization of MDSCs and suppressing the secretion of miR-1 family miRNAs, which facilitate tumor migration and invasion. Abbreviations CTLA-4: cytotoxic T-lymphocyte-associated protein-4; FBS: fetal bovine serum; G-MDSCs: granulocytic-MDSCs; GP49B: glycoprotein 49B; HE: hematoxylin-eosin; ICI: immune checkpoint inhibitor; ITIM: immunoreceptor tyrosine-based inhibition motif; LILRB4: leukocyte immunoglobulin-like receptor B4; M-CSF: macrophage colony stimulating factor; MDSC: myeloid-derived suppressor cell; M-MDSC: monocytic MDSC; MMP-9: metallopeptidase-9; mAb: monoclonal antibody; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PD-1: programmed death-1; PD-L1: programmed death ligand-1; PMN-MDSC: polymorphonuclear-MDSC; qRT-PCR: quantitative reverse transcription PCR; TAM: tumor associated macrophage; TME: tumor microenvironment; TMM: trimmed mean of M value; VEGFA: vascular endothelial growth factor A.

LILRB4, an immune checkpoint on myeloid cells.

Leukocyte immunoglobulin-like receptor B4 (LILRB4) is an inhibitory receptor in the LILR family mainly expressed on normal and malignant human cells of myeloid origin. By binding to ligands, LILRB4 is activated and subsequently recruits adaptors to cytoplasmic immunoreceptor tyrosine inhibitory motifs to initiate different signaling cascades, thus playing an important role in physiological and pathological conditions, including autoimmune diseases, microbial infections, and cancers. In normal myeloid cells, LILRB4 regulates intrinsic cell activation and differentiation. In disease-associated or malignant myeloid cells, LILRB4 is significantly correlated with disease severity or patient survival and suppresses T cells, thereby participating in the pathogenesis of various diseases. In summary, LILRB4 functions as an immune checkpoint on myeloid cells and may be a promising therapeutic target for various human immune diseases, especially for cancer immunotherapy.

RelB contributes to the survival, migration and lymphomagenesis of B cells with constitutively active CD40 signaling.

Activation of CD40-signaling contributes to the initiation, progression and drug resistance of B cell lymphomas. We contributed to this knowledge by showing that constitutive CD40-signaling in B cells induces B cell hyperplasia and finally B cell lymphoma development in transgenic mice. CD40 activates, among others, the non-canonical NF-ĸB signaling, which is constitutively activated in several human B cell lymphomas and is therefore presumed to contribute to lymphopathogenesis. This prompted us to study the regulatory role of the non-canonical NF-ĸB transcription factor RelB in lymphomagenesis. To this end, we crossed mice expressing a constitutively active CD40 receptor in B cells with conditional RelB-KO mice. Ablation of RelB attenuated pre-malignant B cell expansion, and resulted in an impaired survival and activation of long-term CD40-stimulated B cells. Furthermore, we found that hyperactivation of non-canonical NF-кB signaling enhances the retention of B cells in the follicles of secondary lymphoid organs. RNA-Seq-analysis revealed that several genes involved in B-cell migration, survival, proliferation and cytokine signaling govern the transcriptional differences modulated by the ablation of RelB in long-term CD40-stimulated B cells. Inactivation of RelB did not abrogate lymphoma development. However, lymphomas occurred with a lower incidence and had a longer latency period. In summary, our data suggest that RelB, although it is not strictly required for malignant transformation, accelerates the lymphomagenesis of long-term CD40-stimulated B cells by regulating genes involved in migration, survival and cytokine signaling.