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Casitas B lymphoma-b (Cbl-b) is an E3 ubiquitin ligase that serves as a critical brake on immune cell activations. It negatively fine-tunes immune responses through ubiquitination of signaling proteins. Cbl-b deficiency in mice leads to spontaneous tumor rejection and induces only mild, non-lethal autoimmunity, highlighting its potential as a promising immunotherapy target. NX-1607 is the first oral small molecule inhibitor of Cbl-b and it is currently in phase I clinical trial. A recent publication employed high-throughput drug combination screening to systematically elucidate the molecular pathways by which NX-1607 enhances T cell activation. The authors administered 81 well-characterized inhibitors to Jurkat T cells under CD3-stimulated and unstimulated conditions in the presence of NX-1607. Inhibitors of the MAPK/ERK pathway significantly attenuated NX-1607-enhanced T cell activation as indicated by CD69 markers. Proto-oncogene tyrosine-protein kinase Src (SRC) family kinase inhibitors also reduced both CD69 activation markers and the phosphorylation of PLCγ1 and HCLS1 which are upstream of MAPK/ERK pathways. These findings were validated in an immunocompetent B-cell lymphoma mouse model, where NX-1607 consistently upregulated PLCγ1 and ERK phosphorylation. Together, this work delineates a PLCγ1-MAPK/ERK axis through which Cbl-b inhibition unleashes T cell activation, providing critical insight for the development of NX-1607 based immunotherapy.

Deriving hematopoietic stem cells (HSCs) from human pluripotent stem cells is one of major goals in stem cell and hematological research. To induce HSCs from human pluripotent stem cells, many attempts to mimic embryonic development through stepwise exposure to morphogens. HSCs arise from dorsal aorta of embryos then migrate and settle in the bone marrow. Development and maintenance of HSCs are controlled by the microenvironmental cues around the blood vessels (called vascular niche) through morphogens and cytokines. Vascular niche serves as a common mechanism from embryo development to life-long maintenance of HSCs. In this chapter, I discuss that how vascular niche regulates development and maintenance of HSCs and exemplify the role of vascular niche to exquisitely induce HSCs from human pluripotent stem cells.

The rapid emergence of spatial transcriptomics (ST) technologies is revolutionizing our understanding of tissue spatial architecture and biology. Although current ST methods, whether based on next-generation sequencing (seq-based approaches) or fluorescence in situ hybridization (image-based approaches), offer valuable insights, they face limitations either in cellular resolution or transcriptome-wide profiling. To address these limitations, we present SpatialScope, a unified approach integrating scRNA-seq reference data and ST data using deep generative models. With innovation in model and algorithm designs, SpatialScope not only enhances seq-based ST data to achieve single-cell resolution, but also accurately infers transcriptome-wide expression levels for image-based ST data. We demonstrate SpatialScope’s utility through simulation studies and real data analysis from both seq-based and image-based ST approaches. SpatialScope provides spatial characterization of tissue structures at transcriptome-wide single-cell resolution, facilitating downstream analysis, including detecting cellular communication through ligand-receptor interactions, localizing cellular subtypes, and identifying spatially differentially expressed genes. Spatial transcriptomics (ST) is transforming tissue analysis but has limitations. Here, authors introduce SpatialScope, an integrated approach combining scRNA-seq and ST data using deep generative models, enabling comprehensive spatial characterisation at transcriptome-wide single-cell resolution.

Macrophages absorbing cells infected with viable SARS-CoV-2 particles fail to transition into an anti-inflammatory state, potentially contributing to a damaging immune reaction linked to severe forms of COVID-19.

Microglia, the CNS-resident immune cells, are implicated in many neurological diseases. Nearly one in six of the world’s population suffers from neurological disorders, encompassing neurodegenerative and neuroautoimmune diseases, most with dysregulated neuroinflammation involved. Activated microglia become phagocytotic and secret various immune molecules, which are mediators of the brain immune microenvironment. Given their ability to penetrate through the blood–brain barrier in the neuroinflammatory context and their close interaction with neurons and other glial cells, microglia are potential therapeutic delivery vehicles and modulators of neuronal activity. Re-engineering microglia to treat neurological diseases is, thus, increasingly gaining attention. By altering gene expression, re-programmed microglia can be utilized to deliver therapeutics to targeted sites and control neuroinflammation in various neuroinflammatory diseases. This review addresses the current development in microglial engineering, including genetic targeting and therapeutic modulation. Furthermore, we discuss limitations to the genetic engineering techniques and models used to test the functionality of re-engineered microglia, including cell culture and animal models. Finally, we will discuss future directions for the application of engineered microglia in treating neurological diseases. Graphical Abstract

Background Chimeric antigen receptor (CAR)-NK cell therapy has shown remarkable clinical efficacy and safety in the treatment of hematological malignancies. However, this efficacy was limited in solid tumors owing to hostile tumor microenvironment (TME). Radiotherapy is commonly used for solid tumors and proved to improve the TME. Therefore, the combination with radiotherapy would be a potential strategy to improve therapeutic efficacy of CAR-NK cells for solid tumors. Methods Glypican-3 (GPC3) was used as a target antigen of CAR-NK cell for hepatocellular carcinoma (HCC). To promote migration towards HCC, CXCR2-armed CAR-NK92 cells targeting GPC3 were first developed, and their cytotoxic and migration activities towards HCC cells were evaluated. Next, the effects of irradiation on the anti-tumor activity of CAR-NK92 cells were assessed in vitro and in HCC-bearing NCG mice. Lastly, to demonstrate the potential mechanism mediating the sensitized effect of irradiation on CAR-NK cells, the differential gene expression profiles induced by irradiation were analyzed and the expression of some important ligands for the NK-cell activating receptors were further determined by qRT-PCR and flow cytometry. Results In this study, we developed CXCR2-armed GPC3-targeting CAR-NK92 cells that exhibited specific and potent killing activity against HCC cells and the enhanced migration towards HCC cells. Pretreating HCC cells with irradiation enhanced in vitro anti-HCC effect and migration activity of CXCR2-armed CAR-NK92 cells. We further found that only high-dose (8 Gy) but not low-dose (2 Gy) irradiation in one fraction could significantly enhanced in vivo anti-HCC activity of CXCR2-armed CAR-NK92 cells. Irradiation with 8 Gy significantly up-regulated the expression of NK cell-activating ligands on HCC cells. Conclusions Our results indicate the evidence that irradiation could efficiently enhance the anti-tumor effect of CAR-NK cells in solid tumor model. The combination with radiotherapy would be an attractive strategy to improve therapeutic efficacy of CAR-NK cells for solid tumors.

Haematological malignancies comprise a diverse set of lymphoid and myeloid neoplasms which can arise during any stage of haematopoiesis in the bone marrow. Accumulating evidence suggests that chronic inflammation generated by inflammatory cytokines secreted by tumour and the tumour-associated cells within the bone marrow microenvironment initiates signalling pathways in malignant cells, resulting in activation of master transcription factors including Smads, STAT3, and NF-κB which confer cancer stem cell phenotypes and drive disease progression. Deciphering the molecular mechanisms for how immune cells interact with malignant cells to induce such epigenetic modifications, specifically DNA methylation, histone modification, expression of miRNAs and lnRNAs to perturbate haematopoiesis could provide new avenues for developing novel targeted therapies for haematological malignancies. Here, the complex positive and negative feedback loops involved in inflammatory cytokine-induced cancer stem cell generation and drug resistance are reviewed to highlight the clinical importance of immune-epigenetic crosstalk in haematological malignancies.

The tumor microenvironment encompasses various innate immune cells which regulate tumor progression. Exploiting innate immune cells is a new frontier of cancer immunotherapy. However, the classical surface markers for cell-type classification cannot always well-conclude the phenotype, which will further hinge our understanding. The innate immune cells include dendritic cells, monocytes/macrophages, natural killer cells, and innate lymphoid cells. They play important roles in tumor growth and survival, in some cases promoting cancer, in other cases negating cancer. The precise characterization of innate immune cells at the single-cell level will boost the potential of cancer immunotherapy. With the development of single-cell RNA sequencing technology, the transcriptome of each cell in the tumor microenvironment can be dissected at a single-cell level, which paves a way for a better understanding of the cell type and its functions. Here, we summarize the subtypes and functions of innate immune cells in the tumor microenvironment based on recent literature on single-cell technology. We provide updates on recent achievements and prospects for how to exploit novel functions of tumor-associated innate immune cells and target them for cancer immunotherapy.

Maternal genomic imprinting is crucial for the maintenance of adult stem cells, which is accomplished by maintaining long-term haematopoietic stem cell quiescence. Epigenetic control of adult stem cells A new study by Linheng Li and colleagues investigates the effect of deletion of the H19 differentially methylated region (H19-DMR) in haematopoietic stem cells. The DMR is known to control expression of the imprinted H19 and Igf2 genes from the H19 – Igf2 locus, restricting H19 expression to the maternal allele and Igf2 to the paternal allele. The authors report the predominant expression of a list of maternally expressed growth-restricting imprinted genes in long-term haematopoietic stem cells (LT-HSCs), but not in proliferating short-term HSCs, suggesting a crucial role for genomic imprinting in maintaining quiescent LT-HSCs. The epigenetic regulation of imprinted genes by monoallelic DNA methylation of either maternal or paternal alleles is critical for embryonic growth and development 1 . Imprinted genes were recently shown to be expressed in mammalian adult stem cells to support self-renewal of neural and lung stem cells 2 , 3 , 4 ; however, a role for imprinting per se in adult stem cells remains elusive. Here we show upregulation of growth-restricting imprinted genes, including in the H19–Igf2 locus 5 , in long-term haematopoietic stem cells and their downregulation upon haematopoietic stem cell activation and proliferation. A differentially methylated region upstream of H19 (H19-DMR), serving as the imprinting control region, determines the reciprocal expression of H19 from the maternal allele and Igf2 from the paternal allele 1 . In addition, H19 serves as a source of miR-675, which restricts Igf1r expression 6 . We demonstrate that conditional deletion of the maternal but not the paternal H19-DMR reduces adult haematopoietic stem cell quiescence, a state required for long-term maintenance of haematopoietic stem cells, and compromises haematopoietic stem cell function. Maternal-specific H19-DMR deletion results in activation of the Igf2–Igfr1 pathway, as shown by the translocation of phosphorylated FoxO3 (an inactive form) from nucleus to cytoplasm and the release of FoxO3-mediated cell cycle arrest, thus leading to increased activation, proliferation and eventual exhaustion of haematopoietic stem cells. Mechanistically, maternal-specific H19-DMR deletion leads to Igf2 upregulation and increased translation of Igf1r, which is normally suppressed by H19 -derived miR-675. Similarly, genetic inactivation of Igf1r partly rescues the H19-DMR deletion phenotype. Our work establishes a new role for this unique form of epigenetic control at the H19–Igf2 locus in maintaining adult stem cells.

Background Oral squamous cell carcinoma (OSCC) overexpresses CD47, enabling immune evasion via a “don’t eat me” signal to macrophages. Although CD47 blockade shows promise, its efficacy is limited due to a lack of “eat me” signal and contact between macrophages and tumor cells. Objectives This study aimed to evaluate the synergistic anti-tumor effect of combining photothermal therapy (PTT) with CD47 blockade in OSCC and elucidate the underlying mechanisms. Methods In vitro phagocytosis was assessed by flow cytometry. In vivo anti-tumor efficacy was measured by tumor growth inhibition. Mechanistic studies included detection of immunogenic cell death (ICD) markers (ATP, HMGB1, calreticulin (CRT)), confocal microscopy for CRT-macrophage co-localization, analysis of ECM component expression, and immunofluorescence for macrophage infiltration. Results The combination of PTT and CD47 blockade significantly enhanced macrophage phagocytosis in vitro and strongly inhibited tumor growth in vivo. PTT induced ICD, as evidenced by the release of ATP and HMGB1, and the exposure of CRT on the cell membrane. Confocal microscopy confirmed co-localization of CRT-expressing tumor cells with macrophages. Furthermore, PTT down-regulated ECM components at transcriptional and protein levels, which correlated with increased macrophage infiltration into tumors. Conclusions PTT synergizes with CD47 blockade primarily by inducing CRT-dependent pro-phagocytic signaling to provide the “eat me” signal. In parallel, PTT downregulates ECM components, enabling the essential “come near me” process that facilitates macrophage infiltration into tumors. This dual approach significantly improves the macrophage based anti-tumor efficacy of CD47 blockade.