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.

LILRB3 Supports Immunosuppressive Activity of Myeloid Cells and Tumor Development.

The existing T cell-centered immune checkpoint blockade therapies have been successful in treating some but not all patients with cancer. Immunosuppressive myeloid cells, including myeloid-derived suppressor cells (MDSC), that inhibit antitumor immunity and support multiple steps of tumor development are recognized as one of the major obstacles in cancer treatment. Leukocyte Ig-like receptor subfamily B3 (LILRB3), an immune inhibitory receptor containing tyrosine-based inhibitory motifs (ITIM), is expressed solely on myeloid cells. However, it is unknown whether LILRB3 is a critical checkpoint receptor in regulating the activity of immunosuppressive myeloid cells, and whether LILRB3 signaling can be blocked to activate the immune system to treat solid tumors. Here, we report that galectin-4 and galectin-7 induce activation of LILRB3 and that LILRB3 is functionally expressed on immunosuppressive myeloid cells. In some samples from patients with solid cancers, blockade of LILRB3 signaling by an antagonistic antibody inhibited the activity of immunosuppressive myeloid cells. Anti-LILRB3 also impeded tumor development in myeloid-specific LILRB3 transgenic mice through a T cell-dependent manner. LILRB3 blockade may prove to be a novel approach for immunotherapy of solid cancers.

Effects of Different Heterogeneous Nutrient Environments on the Growth and Activities of Enzymes in the Roots of Fokienia hodginsii Families.

Currently, research on the F. hodginsii asexual lineage primarily focuses on the screening of growth traits and the control of single fertilizer applications. The effects of the heterogeneity of soil nutrients on root growth and activity have not been studied in detail. Therefore, we propose forest management measures to improve the foraging ability of forest trees in conjunction with stand productivity. In this experiment, annual containerized seedlings of 10 free-pollinated F. hodginsii lines from a primary asexual seed orchard were used as test subjects, and three heterogeneous nutrient environments of nitrogen (N), phosphorus (P), and potassium (K) were constructed. In contrast, homogeneous nutrient environments were used as the control to carry out potting experiments, to study the growth of F. hodginsii lines and the differences in the activities of root enzymes under the three heterogeneous nutrient environments, and to carry out the comprehensive evaluation using the principal component and cluster analysis method. The results were as follows: (1) The seedling height of F. hodginsii family lines under a homogeneous nutrient environment was significantly higher than that of all heterogeneous nutrient environments; the diameter of the ground was the highest under N heterogeneous nutrient environment and significantly higher than that of all the other nutrient environments; the biomass of the root system was the highest under P heterogeneous nutrient environment, which was significantly higher than that of homogeneous nutrient environment and K heterogeneous nutrient environment. The catalase (CAT) activity of F. hodginsii roots was higher than that of homogeneous nutrients in all heterogeneous nutrient environments but not significant, and the superoxide dismutase (SOD) activity was slightly higher than that of K heterogeneous and homogeneous nutrient environments in N and P heterogeneous nutrient environments. SOD activity was slightly higher than that of K heterogeneous and homogeneous nutrient environments under N, and P. peroxidase (POD) activity in the F. hodginsii root system was the highest under the P heterogeneous nutrient environment, which was significantly higher than that of the other nutrient environments. Unlike the activities of the enzymes, the content of malondialdehyde (MDA) in the roots of F. hodginsii was higher in the heterogeneous environment than in all the other nutrient environments. (2) Under N and P heterogeneous nutrient environments, lines 552 and 590 had higher seedling height, ground diameter, and root enzyme activity, while root biomass was highest in line 544; and under K heterogeneous nutrient environments, line 591 had higher seedling height, ground diameter, and root enzyme activity while root biomass was highest in line 551. In contrast to the patterns of seedling height, accumulation of root biomass and activities of root enzymes, family No. 590 had the highest ground diameter of all the F. hodginsii families under the heterogeneous nutrient environments. Family No. 547 had the highest MDA content. In conclusion, it can be seen that N heterogeneous and homogeneous nutrient environments can significantly increase the seedling height and diameter of F. hodginsii compared with P and K heterogeneous nutrient environments, and N and P heterogeneous nutrient environments can also increase the root biomass, root enzyme activities and significantly reduce the MDA content of F. hodginsii. According to the principal component analysis and cluster analysis, it can be seen that among the 10 F. hodginsii family lines, family lines 590 and 552 have higher evaluation in growth, root biomass accumulation, and enzyme activity.

Mapping the protein-protein interactome in the tumor immune microenvironment.

The cell-to-cell communication primarily occurs through cell-surface and secreted proteins, which form a sophisticated network that coordinates systemic immune function. Uncovering these protein-protein interactions (PPIs) is indispensable for understanding the molecular mechanism and elucidating immune system aberrances under diseases. Traditional biological studies typically focus on a limited number of PPI pairs due to the relative low throughput of commonly used techniques. Encouragingly, classical methods have advanced, and many new systems tailored for large-scale protein-protein screening have been developed and successfully utilized. These high-throughput PPI investigation techniques have already made considerable achievements in mapping the immune cell interactome, enriching PPI databases and analysis tools, and discovering therapeutic targets for cancer and other diseases, which will definitely bring unprecedented insight into this field.

A new mechanistic insight into the association between environmental perfluorooctane sulfonic acid (PFOS) exposure and attention deficit and hyperactivity disorder (ADHD)-like behavior.

Perfluorooctane sulfonic acid (PFOS) is one of the main residual environmental pollutants that threaten human health. PFOS exposure is positively correlated with the prevalence of attention deficit hyperactivity disorder (ADHD); however, the underlying mechanism is unknown. Given that dopamine (DA) is a crucial target for PFOS and that its dysfunction is a key role in ADHD development, it is speculated that PFOS exposure contributes to the occurrence of ADHD to some extent by disrupting DA homeostasis. To establish the relationship between PFOS exposure, DA dysfunction, and ADHD-like behavior, adult zebrafish were exposed to PFOS for 21 days using PFOS concentrations in the serum of patients with ADHD as the reference exposure dose. Results showed that PFOS caused ADHD-like behaviors, with the presence of the slightly elevated percentage of time spent in movement and prolonged time spent in reaching the target zone in the T-maze. Hyperactivity and cognitive ability impairment were more severe with increasing PFOS concentrations. Further investigation showed that PFOS exposure resulted in a decrease in the DA content, accompanied by a decrease in the number of dopaminergic neurons and a disturbance in the transcription profiles of genes associated with the dopaminergic system. Treatment with Ritalin effectively alleviated PFOS-induced ADHD-like behavior and restored DA levels, number of dopaminergic neurons, and expression of DA metabolism-related genes, suggesting that PFOS exposure induced ADHD-like behavior by triggering DA secretion disorder. This study enriches our understanding of the pathogenic mechanisms underlying ADHD development and emphasizes the importance of focusing on the health risks pertaining to environmental exposure.

The Metabolism and Immune Environment in Diffuse Large B-Cell Lymphoma.

Cells utilize different metabolic processes to maintain their growth and differentiation. Tumor cells have made some metabolic changes to protect themselves from malnutrition. These metabolic alterations affect the tumor microenvironment and macroenvironment. Developing drugs targeting these metabolic alterations could be a good direction. In this review, we briefly introduce metabolic changes/regulations of the tumor macroenvironment and microenvironment and summarize potential drugs targeting the metabolism in diffuse large B-cell lymphoma.

Neutrophils as key regulators of tumor immunity that restrict immune checkpoint blockade in liver cancer.

OBJECTIVE: Liver cancer is a deadly malignancy associated with high mortality and morbidity. Less than 20% of patients with advanced liver cancer respond to a single anti-PD-1 treatment. The high heterogeneity of neutrophils in the tumor immune microenvironment in liver cancer may contribute to resistance to immune checkpoint blockade (ICB). However, the underlying mechanism remains largely unknown. METHODS: We established an orthotopic liver cancer model by using transposable elements to integrate the oncogenes Myc and KrasG12D into the genome in liver cells from conditional Trp53 null/null mice (pTMK/Trp53-/-). Flow cytometry and immunohistochemistry were used to assess the changes in immune cells in the tumor microenvironment. An ex vivo coculture assay was performed to test the inhibitory effects of tumor-associated neutrophils (TANs) on CD8+ T cells. The roles of neutrophils, T cells, and NK cells were validated through antibody-mediated depletion. The efficacy of the combination of neutrophil depletion and ICB was evaluated. RESULTS: Orthotropic pTMK/Trp53-/- mouse liver tumors displayed a moderate response to anti-Ly6G treatment but not PD-1 blockade. Depletion of neutrophils increased the infiltration of CD8+ T cells and decreased the number of exhausted T cells in the tumor microenvironment. Furthermore, depletion of either CD8+ T or NK cells abrogated the antitumor efficacy of anti-Ly6G treatment. Moreover, the combination of anti-Ly6G with anti-PD-L1 enhanced the infiltration of cytotoxic CD8+ T cells and thereafter resulted in a significantly greater decrease in tumor burden. CONCLUSIONS: Our data suggest that TANs may contribute to the resistance of liver cancer to ICB, and combining TAN depletion with T cell immunotherapy synergistically increases antitumor efficacy.

The BET PROTAC inhibitor dBET6 protects against retinal degeneration and inhibits the cGAS-STING in response to light damage.

BACKGROUND: Chronic inflammation significantly contributes to photoreceptor death in blinding retinal diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Bromodomain and extraterminal domain (BET) proteins are epigenetic readers that act as key proinflammatory factors. We recently found the first-generation BET inhibitor JQ1 alleviated sodium iodate-induced retinal degeneration by suppressing cGAS-STING innate immunity. Here, we investigated the effects and mechanism of dBET6, a proteolysis­targeting chimera (PROTAC) small molecule that selectively degrades BET by the ubiquitin‒proteasome system, in light-induced retinal degeneration. METHODS: Mice were exposed to bright light to induce retinal degeneration, and the activation of cGAS-STING was determined by RNA-sequencing and molecular biology. Retinal function, morphology, photoreceptor viability and retinal inflammation were examined in the presence and absence of dBET6 treatment. RESULTS: Intraperitoneal injection of dBET6 led to the rapid degradation of BET protein in the retina without detectable toxicity. dBET6 improved retinal responsiveness and visual acuity after light damage (LD). dBET6 also repressed LD-induced retinal macrophages/microglia activation, Müller cell gliosis, photoreceptor death and retinal degeneration. Analysis of single-cell RNA-sequencing results revealed cGAS-STING components were expressed in retinal microglia. LD led to dramatic activation of the cGAS-STING pathway, whereas dBET6 suppressed LD-induced STING expression in reactive macrophages/microglia and the related inflammatory response. CONCLUSIONS: This study indicates targeted degradation of BET by dBET6 exerts neuroprotective effects by inhibiting cGAS-STING in reactive retinal macrophages/microglia, and is expected to become a new strategy for treatment of retinal degeneration.

IgG4-related ophthalmic disease masquerading as ciliary body tumors and scleritis in both eyes: a case report.

BACKGROUND: To report a rare case of IgG4-related ophthalmic disease (IgG4-ROD) manifesting as intraocular masses and scleritis in both eyes in a 61-year-old male and to investigate the changes in multimodal imaging features of the lesion sites and helper T-cell type 1 (Th 1)/Th 2/Th 17 cytokine levels in the aqueous humor. CASE PRESENTATION: A patient with IgG4-ROD seemingly manifested with an intraocular tumor in the left eye and sequentially, with an inflammatory mass in the ciliary body and scleritis in the right eye. The patient complained of vision loss of 6 months duration in the left eye at his first visit. With a preliminary diagnosis of an intraocular tumor, enucleation of the left eyeball and histopathological examination were performed. Approximately 3 months later, the patient started to experience headache, eye pain, and declining vision in the right eye. Ophthalmic imaging revealed a ciliary mass and scleritis. Th 1/Th 2/Th 17 cytokine levels and multimodal imaging findings were analyzed before and after corticosteroid treatment. Histopathological examination and immunohistochemistry (IHC) of the enucleated left eye demonstrated lymphoplasmacytic infiltration with an IgG4+/IgG+ cell ratio of approximately 40%, pointing to the diagnosis of probable IgG4-ROD. Long-term treatment with corticosteroids led to significant improvement in the signs and symptoms of the left eye. Th 1/Th 2/Th 17 cytokine profile monitoring of the aqueous humor and multimodal imaging of the right eye showed gradual regression of the mass and attenuation of ocular inflammation during treatment. CONCLUSIONS: Patients with an atypical presentation of IgG4-ROD, such as intraocular masses and scleritis, are likely to experience a significant delay in diagnosis. This case demonstrates the significance of IgG4-ROD in the differential diagnosis of intraocular tumors and ocular inflammation. IgG4-RD is a newly diagnosed disease with multi-organ involvement and little is known about its pathogenesis, particularly in the eye. The present case will open new challenges in the clinico-pathological diagnosis and research of this disease. Combined investigations of multimodal imaging and cytokine level detection of intraocular fluid provide a new and effective way to monitor disease progression.

Effects of conversion of natural forest to plantations on the abundance of nitrite reducing genes in soil aggregates in subtropical forest region.

Large proportion of natural forest has been transformed into plantations in subtropical regions, with consequences on forest ecosystem structure and function. In order to understand the responses of two nitrite reducing genes (nirK and nirS) in N2O production to forest conversion, we collected soil samples from Castanopsis carlesii natural forest, Cunninghamia lanceolata plantation and Pinus massoniana plantation and examined the abundance of nirK and nirS genes in soils and aggregates. Results showed that forest conversion increased soil pH, while decreased soil ammonium content. Forest conversion did not influence the mass proportion of soil aggregates. The abundance of nirK and nirS genes varied in aggregates with different particle sizes. The abundance of nirK and nirS genes was the highest in small macraoaggregates and the lowest in the silt-clay particles. Moreover, the abundance of nirK was significantly higher than that of nirS in soils of all forest types, indicating that nirK dominated in the acidic forest soils. Conversion of natural forest to plantations significantly increased the abundance of nirK and nirS genes in the bulk soil and aggregates, indicating that forest conversion would be beneficial for the growth of microorganisms bearing nirK and nirS genes, which might be associated with the increases of soil pH. Taken together, conversion of natural forest to C. lanceolata plantation or P. massoniana plantation significantly increased the abundance of nirK and nirS in soils and aggregates, but did not affect the mass proportions of aggregates.

Effects of single and combined exposure to zinc and two tetracycline antibiotics on zebrafish at the early stage.

Tetracycline antibiotics (TCs) and heavy metals are commonly used in livestock and poultry farming, leading to their coexistence in the aquatic environment. This coexistence causes combined toxicity to aquatic organisms. Here, zebrafish embryos were exposed to chlortetracycline (CTC), oxytetracycline (OTC), zinc chloride (ZnCl2), and their combinations for 120 h to evaluate their adverse effects on the growth, antioxidant system, immune system, and endocrine system during the early stage of life. OTC/ZnCl2 combined exposure significantly reduced the body weight, whereas the TCs/ZnCl2 combination significantly increased the heart rate of zebrafish larvae, suggesting growth impairment induced by TCs and ZnCl2. Further, combined groups showed more prominent toxicity to the antioxidant system than single groups, as revealed by related levels of enzyme activity and gene expression. In addition, the levels of most pro-inflammatory genes were downregulated, and those of NF-κB-related genes were upregulated in all treatment groups, indicating an immunosuppressive response and the potential role of NF-κB signaling, while the combined treatment was not more toxic than TCs or ZnCl2 alone. Similarly, hormone and endocrine related gene levels were determined. Although both single and combined exposures caused certain endocrine-disrupting effects, the combined exposure did not result in higher toxicity than a single exposure. Our findings showed that a mixture of TCs and ZnCl2 might exert greater toxic effects as compared to a single compound on some systems, providing fundamental data on the toxic effects of single and combined TC and ZnCl2 exposure on aquatic organisms, although studies are needed to explore the underlying mechanisms.

Response of the root morphological structure of Fokienia hodginsii seedlings to competition from neighboring plants in a heterogeneous nutrient environment.

Introduction: Critical changes often occur in Fokienia hodginsii seedlings during the process of growth owing to differences in the surrounding environment. The most common differences are heterogeneous nutrient environments and competition from neighboring plants. Methods: In this study, we selected one-year-old, high-quality Fokienia hodginsii seedlings as experimental materials. Three planting patterns were established to simulate different competitive treatments, and seedlings were also exposed to three heterogeneous nutrient environments and a homogeneous nutrient environment (control) to determine their effect on the root morphology and structure of F. hodginsii seedlings. Results: Heterogeneous nutrient environments, compared with a homogeneous environment, significantly increased the dry matter accumulation and root morphology indexes of the root system of F. hodginsii, which proliferated in nutrient-rich patches, and the P heterogeneous environment had the most significant enhancement effect, with dry matter accumulation 70.2%, 7.0%, and 27.0% higher than that in homogeneous and N and K heterogeneous environments, respectively. Homogeneous environments significantly increased the specific root length and root area of the root system; the dry matter mass and morphological structure of the root system of F. hodginsii with a heterospecific neighbor were higher than those under conspecific neighbor and single-plant treatments, and the root area of the root system under the conspecific neighbor treatment was higher than that under the heterospecific neighbor treatment, by 20% and 23%, respectively. Moreover, the root system under heterospecific neighbor treatment had high sensitivity; the heterogeneous nutrient environment increased the mean diameter of the fine roots of the seedlings of F. hodginsii and the diameter of the vascular bundle, and the effect was most significant in the P heterogeneous environment, exceeding that in the N and K heterogeneous environments. The effect was most significant in the P heterogeneous environment, which increased fine root diameter by 20.5% and 10.3%, respectively, compared with the homogeneous environment; in contrast, the fine root vascular ratio was highest in the homogeneous environment, and most of the indicators of the fine root anatomical structure in the nutrient-rich patches were of greater values than those in the nutrient-poor patches in the different heterogeneous environments; competition promoted most of the indicators of the fine root anatomical structure of F. hodginsii seedlings. According a principal component analysis (PCA), the N, Pm and K heterogeneous environments with heterospecific neighbors and the P heterogeneous environment with a conspecific neighbor had higher evaluation in the calculation of eigenvalues of the PCA. Discussion: The root dry matter accumulation, root morphology, and anatomical structure of F. hodginsii seedlings in the heterogeneous nutrient environment were more developed than those in the homogeneous nutrient environment. The effect of the P heterogeneous environment was the most significant. The heterospecific neighbor treatment was more conducive to the expansion and development of root morphology of F. hodginsii seedlings than were the conspecific neighbor and single-plant treatments.

Cell atlas of the immune microenvironment in gastrointestinal cancers: Dendritic cells and beyond.

Gastrointestinal (GI) cancers occur in the alimentary tract and accessory organs. They exert a global burden with high morbidity and mortality. Inside the tumor microenvironment, dendritic cells (DCs) are the most efficient antigen-presenting cells and are necessary for adaptive immune responses such as T and B-cell maturation. However, the subsets of DCs revealed before were mostly based on flow cytometry and bulk sequencing. With the development of single-cell RNA sequencing (scRNA-seq), the tumor and microenvironment heterogeneity of GI cancer has been illustrated. In this review, we summarize the classification and development trajectory of dendritic cells at the single-cell level in GI cancer. Additionally, we focused on the interaction of DCs with T cells and their effect on the response to immunotherapy. Specifically, we focused on the newly identified tumor-infiltrating dendritic cells and discuss their potential function in antitumor immunity.

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The reactive nitrogen deposition in subtropical region of China has been increasing annually, which affects biogeochemical processes in forest soils. In this study, three treatments were established, including control (no N addition, CK), low nitrogen deposition (40 kg·hm-2·a-1, LN), and high nitrogen deposition (80 kg·hm-2·a-1, HN) to study the response of denitrifying functional genes and potential N2O emissions to simulated nitrogen deposition in the soils of a natural Castanopsis carlesii forest. Results showed that HN significantly decreased soil potential N2O emission, while 8-year nitrogen deposition did not affect the abundances of nirS, nirK, nosZ Ⅰ and nosZ Ⅱ. However, the abundance of nosZ Ⅰwas significantly higher than nosZ Ⅱ in all the treatments, indicating that nosZ Ⅰ dominated over nosZ Ⅱ in the acidic soils. HN significantly decreased the ratio of (nirK+nirS)/(nosZ Ⅰ+nosZ Ⅱ), which was positively correlated with soil pH. The results suggested that long-term high nitrogen deposition reduced soil pH and the abundance ratio of (nirK+nirS)/(nosZ Ⅰ+nosZ Ⅱ), which subsequently reduced the potential N2O emission.

Myeloid checkpoints for cancer immunotherapy.

Myeloid checkpoints are receptors on the myeloid cell surface which can mediate inhibitory signals to modulate anti-tumor immune activities. They can either inhibit cellular phagocytosis or suppress T cells and are thus involved in the pathogenesis of various diseases. In the tumor microenvironment, besides killing tumor cells by phagocytosis or activating anti-tumor immunity by tumor antigen presentation, myeloid cells could execute pro-tumor efficacies through myeloid checkpoints by interacting with counter-receptors on other immune cells or cancer cells. In summary, myeloid checkpoints may be promising therapeutic targets for cancer immunotherapy.

Liver tumour immune microenvironment subtypes and neutrophil heterogeneity.

The heterogeneity of the tumour immune microenvironment (TIME), organized by various immune and stromal cells, is a major contributing factor of tumour metastasis, relapse and drug resistance1-3, but how different TIME subtypes are connected to the clinical relevance in liver cancer remains unclear. Here we performed single-cell RNA-sequencing (scRNA-seq) analysis of 189 samples collected from 124 patients and 8 mice with liver cancer. With more than 1 million cells analysed, we stratified patients into five TIME subtypes, including immune activation, immune suppression mediated by myeloid or stromal cells, immune exclusion and immune residence phenotypes. Different TIME subtypes were spatially organized and associated with chemokine networks and genomic features. Notably, tumour-associated neutrophil (TAN) populations enriched in the myeloid-cell-enriched subtype were associated with an unfavourable prognosis. Through in vitro induction of TANs and ex vivo analyses of patient TANs, we showed that CCL4+ TANs can recruit macrophages and that PD-L1+ TANs can suppress T cell cytotoxicity. Furthermore, scRNA-seq analysis of mouse neutrophil subsets revealed that they are largely conserved with those of humans. In vivo neutrophil depletion in mouse models attenuated tumour progression, confirming the pro-tumour phenotypes of TANs. With this detailed cellular heterogeneity landscape of liver cancer, our study illustrates diverse TIME subtypes, highlights immunosuppressive functions of TANs and sheds light on potential immunotherapies targeting TANs.

Blocking LAIR1 signaling in immune cells inhibits tumor development.

The current immune checkpoint blockade therapy has been successful in treating some cancers but not others. New molecular targets and therapeutic approaches of cancer immunology need to be identified. Leukocyte associated immunoglobulin like receptor 1 (LAIR1) is an immune inhibitory receptor expressing on most immune cell types. However, it remains a question whether we can specifically and actively block LAIR1 signaling to activate immune responses for cancer treatment. Here we report the development of specific antagonistic anti-LAIR1 monoclonal antibodies and studied the effects of LAIR1 blockade on the anti-tumor immune functions. The anti-LAIR1 antagonistic antibody stimulated the activities of T cells, natural killer cells, macrophages, and dendritic cells in vitro. The single-cell RNA sequencing analysis of intratumoral immune cells in syngeneic human LAIR1 transgenic mice treated with control or anti-LAIR1 antagonist antibodies indicates that LAIR1 signaling blockade increased the numbers of CD4 memory T cells and inflammatory macrophages, but decreased those of pro-tumor macrophages, regulatory T cells, and plasmacytoid dendritic cells. Importantly, the LAIR1 blockade by the antagonistic antibody inhibited the activity of immunosuppressive myeloid cells and reactivated T cells from cancer patients in vitro and impeded tumor metastasis in a humanized mouse model. Blocking LAIR1 signaling in immune cells represents a promising strategy for development of anti-cancer immunotherapy.

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.

LILRB2-mediated TREM2 signaling inhibition suppresses microglia functions.

BACKGROUND: Microglia plays crucial roles in Alzheimer's disease (AD) development. Triggering receptor expressed on myeloid cells 2 (TREM2) in association with DAP12 mediates signaling affecting microglia function. Here we study the negative regulation of TREM2 functions by leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), an inhibitory receptor bearing ITIM motifs. METHODS: To specifically interrogate LILRB2-ligand (oAß and PS) interactions and microglia functions, we generated potent antagonistic LILRB2 antibodies with sub-nanomolar level activities. The biological effects of LILRB2 antagonist antibody (Ab29) were studied in human induced pluripotent stem cell (iPSC)-derived microglia (hMGLs) for migration, oAß phagocytosis, and upregulation of inflammatory cytokines. Effects of the LILRB2 antagonist antibody on microglial responses to amyloid plaques were further studied in vivo using stereotaxic grafted microglia in 5XFAD mice. RESULTS: We confirmed the expression of both LILRB2 and TREM2 in human brain microglia using immunofluorescence. Upon co-ligation of the LILRB2 and TREM2 by shared ligands oAß or PS, TREM2 signaling was significantly inhibited. We identified a monoclonal antibody (Ab29) that blocks LILRB2/ligand interactions and prevents TREM2 signaling inhibition mediated by LILRB2. Further, Ab29 enhanced microglia phagocytosis, TREM2 signaling, migration, and cytokine responses to the oAß-lipoprotein complex in hMGL and microglia cell line HMC3. In vivo studies showed significantly enhanced clustering of microglia around plaques with a prominent increase in microglial amyloid plaque phagocytosis when 5XFAD mice were treated with Ab29. CONCLUSIONS: This study revealed for the first time the molecular mechanisms of LILRB2-mediated inhibition of TREM2 signaling in microglia and demonstrated a novel approach of enhancing TREM2-mediated microglia functions by blocking LILRB2-ligand interactions. Translationally, a LILRB2 antagonist antibody completely rescued the inhibition of TREM2 signaling by LILRB2, suggesting a novel therapeutic strategy for improving microglial functions.