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Cellular functions are strongly dependent on surrounding cells and environmental factors. Current technologies are limited in their ability to characterize the spatial location and gene programs of cells in poorly structured and dynamic niches. We developed a method, NICHE-seq, that combines photoactivatable fluorescent reporters, two-photon microscopy, and single-cell RNA sequencing (scRNA-seq) to infer the cellular and molecular composition of niches. We applied NICHE-seq to examine the high-order assembly of immune cell networks. NICHE-seq is highly reproducible in spatial tissue reconstruction, enabling identification of rare niche-specific immune subpopulations and gene programs, including natural killer cells within infected B cell follicles and distinct myeloid states in the spleen and tumor. This study establishes NICHE-seq as a broadly applicable method for elucidating high-order spatial organization of cell types and their molecular pathways.

Understanding the mechanism that leads to immune dysfunction in severe coronavirus disease 2019 (COVID-19) is crucial for the development of effective treatment. Here, using single-cell RNA sequencing, we characterized the peripheral blood mononuclear cells (PBMCs) from uninfected controls and COVID-19 patients and cells in paired broncho-alveolar lavage fluid (BALF). We found a close association of decreased dendritic cells (DCs) and increased monocytes resembling myeloid-derived suppressor cells (MDSCs), which correlated with lymphopenia and inflammation in the blood of severe COVID-19 patients. Those MDSC-like monocytes were immune-paralyzed. In contrast, monocyte-macrophages in BALFs of COVID-19 patients produced massive amounts of cytokines and chemokines, but secreted little interferons. The frequencies of peripheral T cells and NK cells were significantly decreased in severe COVID-19 patients, especially for innate-like T and various CD8 + T cell subsets, compared to healthy controls. In contrast, the proportions of various activated CD4 + T cell subsets among the T cell compartment, including Th1, Th2, and Th17-like cells were increased and more clonally expanded in severe COVID-19 patients. Patients’ peripheral T cells showed no sign of exhaustion or augmented cell death, whereas T cells in BALFs produced higher levels of IFNG , TNF , CCL4 , CCL5 , etc. Paired TCR tracking indicated abundant recruitment of peripheral T cells to the severe patients’ lung. Together, this study comprehensively depicts how the immune cell landscape is perturbed in severe COVID-19.

Multiple sclerosis (MS) is characterized by pathological inflammation that results from the recruitment of lymphoid and myeloid immune cells from the blood into the brain. Due to subset heterogeneity, defining the functional roles of the various cell subsets in acute and chronic stages of MS has been challenging. Here, we used index and transcriptional single-cell sorting to characterize the mononuclear phagocytes that infiltrate the central nervous system from the periphery in mice with experimentally induced autoimmune encephalomyelitis, a model of MS. We identified eight monocyte and three dendritic cell subsets at acute and chronic disease stages in which the defined transcriptional programs pointed toward distinct functions. Monocyte-specific cell ablation identified Cxcl10 + and Saa3 + monocytic subsets with a pathogenic potential. Transfer experiments with different monocyte and precursor subsets indicated that these Cxcl10 + and Saa3 + pathogenic cells were not derived from Ly6C + monocytes but from early myeloid cell progenitors. These results suggest that blocking specific pathogenic monocytic subsets, including Cxcl10 + and Saa3 + monocytes, could be used for targeted therapeutic interventions. Mildner and colleagues characterize two subsets ( Cxcl10 + and Saa3 + ) of monocytes with pathogenic potential in the central nervous system of mice with experimentally induced autoimmune encephalomyelitis and show these pathogenic cells are not derived from Ly6C + monocytes, but from early myeloid cell progenitors.

Historically, sharing T cell receptors （TCRs） between individuals has been speculated to be impossible, consider- ing the dramatic discrepancy between the potential enormity of the TCR repertoire and the limited number of T cells generated in each individual. However, public T cell response, in which multiple individuals share identical TCRs in responding to a same antigenic epitope, has been extensively observed in a variety of immune responses across many species. Public T cell responses enable individuals within a population to generate similar antigen-specific TCRs against certain ubiquitous pathogens, leading to favorable biological outcomes. However, the relatively concentrated feature of TCR repertoire may limit T cell response in a population to some other pathogens. It could be a great ben- efit for human health if public T cell responses can be manipulated. Therefore, the mechanistic insight of public TCR generation is important to know. Recently, high-throughput DNA sequencing has revolutionized the study of immune receptor repertoires, which allows a much better understanding of the factors that determine the overlap of TCR repertoire among individuals. Here, we summarize the current knowledge on public T-cell response and discuss fu- ture challenges in this field.

Over the last three decades, microglia have been recognized as essential components of central nervous system (CNS) development, homeostasis, immune surveillance, and neurodegenerative pathogenesis. Historically, microglia were regarded as exclusive to the CNS, based on the absence of cells bearing microglial morphology, transcriptional identity, and ontogeny in tissues outside the CNS in standard mouse and rat models. However, recent studies, including those from our group, have identified cells in peripheral tissues of humans and other vertebrates that share the transcriptomic signature and yolk sac-derived ontogeny characteristic of CNS microglia. These findings suggest that the microglial lineage represents a broader spectrum of tissue-resident macrophages with specialized roles across organs. Microglia constitute one of the most evolutionarily ancient macrophage subtypes, with IBA1-positive cells already identifiable in the brain of the annelid Hirudo medicinalis. Wu et al's recent identification of microglial cells in the peripheral nervous system (PNS) provides compelling evidence supporting the notion that homologous cell types may evolve independently in distinct anatomical niches. In evolutionary biology, homology denotes descent from a common ancestor, irrespective of phenotypic similarity. Conventionally, cell type homology within and between species has primarily been inferred from shared anatomical location, phenotype, or function.

The eukaryotic asparagine (N)-linked glycan is pre-assembled as a fourteen-sugar oligosaccharide on a lipid carrier in the endoplasmic reticulum (ER). Seven sugars are first added to dolichol pyrophosphate (PP-Dol) on the cytoplasmic face of the ER, generating Man5GlcNAc2-PP-Dol (M5GN2-PP-Dol). M5GN2-PP-Dol is then flipped across the bilayer into the lumen by an ER translocator. Genetic studies identified Rft1 as the M5GN2-PP-Dol flippase in vivo but are at odds with biochemical data suggesting Rft1 is dispensable for flipping in vitro. Thus, the question of whether Rft1 plays a direct or an indirect role during M5GN2-PP-Dol translocation has been controversial for over two decades. We describe a completely reconstituted in vitro assay for M5GN2-PP-Dol translocation and demonstrate that purified Rft1 catalyzes the translocation of M5GN2-PP-Dol across the lipid bilayer. These data, combined with in vitro results demonstrating substrate selectivity and rft1∆ phenotypes, confirm the molecular identity of Rft1 as the M5GN2-PP-Dol ER flippase. Whether Rft1 plays a role during M5GN2-PP-Dol translocation has been controversial for over two decades. In this work, a reconstituted in vitro assay demonstrates that purified Rft1 is sufficient to flip M5GN2-PP-Dol across the lipid bilayer.

Profiling heterologous cell types within tumors is essential to decipher tumor microenvironment that shapes tumor progress and determines the outcome of therapeutic response. Here, we comprehensively characterized transcriptomes of 34,037 single cells obtained from 12 treatment‐naïve patients with colorectal cancer. Our comprehensive evaluation revealed attenuated B‐cell antigen presentation, distinct regulatory T‐cell clusters with different origin and novel polyfunctional tumor associated macrophages associated with CRC. Moreover, we identified expanded XCL1+ T‐cell clusters associated with tumor mutational burden high status. We further explored the underlying molecular mechanisms by profiling epigenetic landscape and inferring transcription factor motifs using single‐cell ATAC‐seq. Our dataset and analysis approaches herein provide a rich resource for further study of the impact of immune cells and translational research for human colorectal cancer. 1) Single‐cell profiling of clinical samples of 12 human colorectal cancer patients. 2) Tumor mutational burden states of human CRC contribute to distinct immune profile patterns. 3) Single‐cell analysis in human CRC identified phenotypic and functional diversity of tumor‐associated macrophages and T cells.

The plant membrane-trafficking system plays a crucial role in maintaining proper cellular functions and responding to various developmental and environmental cues. Thus far, our knowledge of the maize membrane-trafficking system is still limited. In this study, we systematically identified 479 membrane-trafficking genes from the maize genome using orthology search and studied their functions by integrating transcriptome and evolution analyses. These genes encode the components of coated vesicles, AP complexes, autophagy, ESCRTs, retromers, Rab GTPases, tethering factors, and SNAREs. The maize genes exhibited diverse but coordinated expression patterns, with 249 genes showing elevated expression in reproductive tissues. Further WGCNA analysis revealed that five COPII components and four Rab GTPases had high connectivity with protein biosynthesis during endosperm development and that eight components of autophagy, ESCRT, Rab, and SNARE were strongly co-upregulated with defense-related genes and/or with secondary metabolic processes to confer basal resistance to Fusarium graminearum . In addition, we identified 39 membrane-trafficking genes with strong selection signals during maize domestication and/or improvement. Among them, ZmSec23a and ZmVPS37A were selected for kernel oil production during improvement and pathogen resistance during domestication, respectively. In summary, these findings will provide important hints for future appreciation of the functions of membrane-trafficking genes in maize.

The functional activity and differentiation potential of cells are determined by their interactions with surrounding cells. Approaches that allow unbiased characterization of cell states while at the same time providing spatial information are of major value to assess this environmental influence. However, most current techniques are hampered by a tradeoff between spatial resolution and cell profiling depth. Here, we develop a photocage-based technology that allows isolation and in-depth analysis of live cells from regions of interest in complex ex vivo systems, including primary human tissues. The use of a highly sensitive 4-nitrophenyl(benzofuran) cage coupled to a set of nanobodies allows high-resolution photo-uncaging of different cell types in areas of interest. Single-cell RNA-sequencing of spatially defined CD8 + T cells is used to exemplify the feasibility of identifying location-dependent cell states. The technology described here provides a valuable tool for the analysis of spatially defined cells in diverse biological systems, including clinical samples. The development of a photocage-nanobody based technology enabled in-depth analysis of live cells from tissues while retaining their spatial information.

Background Angiogenesis is indispensable for the sustained survival and progression of both embryonic development and tumorigenesis. This intricate process is tightly regulated by a multitude of pro‐angiogenic genes. The presence of gene modules facilitating angiogenesis has been substantiated in both embryonic development and the context of tumor proliferation. However, it remains unresolved whether the pro‐angiogenic gene modules expressed during embryonic development also exist in tumors. Methods This study performed a pan‐cancer single‐cell RNA sequencing (scRNA‐seq) analysis on samples from 332 patients across seven cancer types: thyroid carcinoma, lung cancer, breast cancer, hepatocellular carcinoma, colorectal cancer, ovarian carcinoma, and prostate adenocarcinoma. Data processing was carried out using the Seurat R package, with rigorous quality control to filter high‐quality cells and mitigate batch effects across datasets. We used principal component analysis (PCA), shared nearest neighbor graph‐based clustering, and Uniform Manifold Approximation and Projection (UMAP) to visualize cell types and identify distinct cell clusters. Myeloid cell subpopulations were further analyzed for the expression of embryonic pro‐angiogenic gene modules (EPGM) and tumor pro‐angiogenic gene modules (TPGM). Results The analysis identified nine major cell types within the tumor microenvironment, with myeloid cells consistently exhibiting elevated expression of both tumor pro‐angiogenic gene modules (TPGM) and EPGM across all tumor types. In particular, myeloid cells, including macrophages and monocytes, showed high EPGM expression, indicating an active role of embryonic pro‐angiogenesis pathways in tumors. A subset analysis revealed 20 distinct myeloid subtypes with varying EPGM and TPGM expression across different cancers. Treatment and disease stage influenced these gene expressions, with certain subtypes, such as HSPAhi/STAT1+ macrophages in breast cancer, displaying reduced pro‐angiogenic gene activity post‐treatment. Conclusion This study provides evidence that tumors may exploit EPGM to enhance vascularization and support sustained growth, as evidenced by the elevated EPGM expression in tumor‐associated myeloid cells. The consistent presence of EPGM in TAMs across multiple cancer types suggests a conserved mechanism wherein tumors harness embryonic angiogenic pathways to facilitate their progression. Distinct EPGM expression patterns in specific myeloid cell subsets indicate potential therapeutic targets, particularly in cases where EPGM activation contributes to resistance against anti‐angiogenic therapies. These findings shed new light on the molecular mechanisms underlying tumor angiogenesis and highlight the prognostic relevance of EPGM expression in cancer, underscoring its potential as a biomarker for clinical applications.