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A cell’s phenotype and function are influenced by dynamic interactions with its microenvironment. To examine cellular spatiotemporal activity, we developed SPACECAT—Spatially PhotoActivatable Color Encoded Cell Address Tags—to annotate, track, and isolate cells while preserving viability. In SPACECAT, samples are stained with photocaged fluorescent molecules, and cells are labeled by uncaging those molecules with user-patterned near-UV light. SPACECAT offers single-cell precision and temporal stability across diverse cell and tissue types. Illustratively, we target crypt-like regions in patient-derived intestinal organoids to enrich for stem-like and actively mitotic cells, matching literature expectations. Moreover, we apply SPACECAT to ex vivo tissue sections from four healthy organs and an autochthonous lung tumor model. Lastly, we provide a computational framework to identify spatially-biased transcriptome patterns and enriched phenotypes. This minimally perturbative and broadly applicable method links cellular spatiotemporal and/or behavioral phenotypes with diverse downstream assays, enabling insights into the connections between tissue microenvironments and (dys)function. Spatial transcriptomics aims to pair omic data with tissue structure. Here the authors report Spatially PhotoActivatable Colour Encoded Cell Address Tags (SPACECAT) to track and isolate live cells by location; this enables spatially informed downstream assays like scRNA-seq and flow cytometry.

We present a scalable, integrated strategy for coupled protein and RNA detection from single cells. Our approach leverages the DNA polymerase activity of reverse transcriptase to simultaneously perform proximity extension assays and complementary DNA synthesis in the same reaction. Using the Fluidigm C1™ system, we profile the transcriptomic and proteomic response of a human breast adenocarcinoma cell line to a chemical perturbation, benchmarking against in situ hybridizations and immunofluorescence staining, as well as recombinant proteins, ERCC Spike-Ins, and population lysate dilutions. Through supervised and unsupervised analyses, we demonstrate synergies enabled by simultaneous measurement of single-cell protein and RNA abundances. Collectively, our generalizable approach highlights the potential for molecular metadata to inform highly-multiplexed single-cell analyses.

Cellular immunity is critical for controlling intracellular pathogens, but individual cellular dynamics and cell–cell cooperativity in evolving human immune responses remain poorly understood. Single-cell RNA-sequencing (scRNA-seq) represents a powerful tool for dissecting complex multicellular behaviors in health and disease 1 , 2 and nominating testable therapeutic targets 3 . Its application to longitudinal samples could afford an opportunity to uncover cellular factors associated with the evolution of disease progression without potentially confounding inter-individual variability 4 . Here, we present an experimental and computational methodology that uses scRNA-seq to characterize dynamic cellular programs and their molecular drivers, and apply it to HIV infection. By performing scRNA-seq on peripheral blood mononuclear cells from four untreated individuals before and longitudinally during acute infection 5 , we were powered within each to discover gene response modules that vary by time and cell subset. Beyond previously unappreciated individual- and cell-type-specific interferon-stimulated gene upregulation, we describe temporally aligned gene expression responses obscured in bulk analyses, including those involved in proinflammatory T cell differentiation, prolonged monocyte major histocompatibility complex II upregulation and persistent natural killer (NK) cell cytolytic killing. We further identify response features arising in the first weeks of infection, for example proliferating natural killer cells, which potentially may associate with future viral control. Overall, our approach provides a unified framework for characterizing multiple dynamic cellular responses and their coordination. Single-cell RNA-sequencing of blood from HIV-1-infected individuals obtained before initiation of antiretroviral therapy provides insights into the initial immune responses during early infection that might shape future outcomes.

This work identifies a role for intestinal epithelial cell (IEC)-intrinsic expression of histone deacetylase 3 in regulating commensal-bacteria-dependent gene expression and intestinal homeostasis; IEC-specific HDAC3 deficiency gives rise to Paneth cell abnormalities, impaired intestinal barrier function, and increased DSS-induced intestinal inflammation in commensal-bacteria-containing, but not germ-free, mice. HDAC3 role in intestinal homeostasis Epigenetic mechanisms alter the transcriptional response to environmental cues and thus represent a mechanism that can link host genetic predisposition and environmental triggers in the pathogenesis of inflammatory bowel disease (IBD). This study identifies a previously unrecognized role for intestinal epithelial cell (IEC)-intrinsic expression of the epigenome-modifying enzyme histone deacetylase 3 (HDAC3) in regulating intestinal barrier function and susceptibility to commensal-driven inflammation. IEC-specific HDAC3 deficiency in mice is shown to give rise to Paneth-cell abnormalities, rectal prolapse, increased susceptibility to dextran sodium sulphate-induced colitis and increased barrier permeability in commensal-containing but not germ-free animals. The development and severity of inflammatory bowel diseases and other chronic inflammatory conditions can be influenced by host genetic and environmental factors, including signals derived from commensal bacteria 1 , 2 , 3 , 4 , 5 , 6 . However, the mechanisms that integrate these diverse cues remain undefined. Here we demonstrate that mice with an intestinal epithelial cell (IEC)-specific deletion of the epigenome-modifying enzyme histone deacetylase 3 (HDAC3 ΔIEC mice) exhibited extensive dysregulation of IEC-intrinsic gene expression, including decreased basal expression of genes associated with antimicrobial defence. Critically, conventionally housed HDAC3 ΔIEC mice demonstrated loss of Paneth cells, impaired IEC function and alterations in the composition of intestinal commensal bacteria. In addition, HDAC3 ΔIEC mice showed significantly increased susceptibility to intestinal damage and inflammation, indicating that epithelial expression of HDAC3 has a central role in maintaining intestinal homeostasis. Re-derivation of HDAC3 ΔIEC mice into germ-free conditions revealed that dysregulated IEC gene expression, Paneth cell homeostasis and intestinal barrier function were largely restored in the absence of commensal bacteria. Although the specific mechanisms through which IEC-intrinsic HDAC3 expression regulates these complex phenotypes remain to be determined, these data indicate that HDAC3 is a critical factor that integrates commensal-bacteria-derived signals to calibrate epithelial cell responses required to establish normal host–commensal relationships and maintain intestinal homeostasis.

Mice engrafted with components of a human immune system have become widely-used models for studying aspects of human immunity and disease. However, a defined methodology to objectively measure and compare the quality of the human immune response in different models is lacking. Here, by taking advantage of the highly immunogenic live-attenuated yellow fever virus vaccine YFV-17D, we provide an in-depth comparison of immune responses in human vaccinees, conventional humanized mice, and second generation humanized mice. We demonstrate that selective expansion of human myeloid and natural killer cells promotes transcriptomic responses akin to those of human vaccinees. These enhanced transcriptomic profiles correlate with the development of an antigen-specific cellular and humoral response to YFV-17D. Altogether, our approach provides a robust scoring of the quality of the human immune response in humanized mice and highlights a rational path towards developing better pre-clinical models for studying the human immune response and disease. Humanized mice are an enabling technology to explore human immunity and disease. Here, Douam et al. provide an in-depth comparison of immune responses to yellow fever vaccine in human vaccinees, conventional and second-generation humanized mice and define a workflow to evaluate and refine these models.

Many patients infected with coronaviruses, such as SARS-CoV-2 and NL63 that use ACE2 receptors to infect cells, exhibit gastrointestinal symptoms and viral proteins are found in the human gastrointestinal tract, yet little is known about the inflammatory and pathological effects of coronavirus infection on the human intestine. Here, we used a human intestine-on-a-chip (Intestine Chip) microfluidic culture device lined by patient organoid-derived intestinal epithelium interfaced with human vascular endothelium to study host cellular and inflammatory responses to infection with NL63 coronavirus. These organoid-derived intestinal epithelial cells dramatically increased their ACE2 protein levels when cultured under flow in the presence of peristalsis-like mechanical deformations in the Intestine Chips compared to when cultured statically as organoids or in Transwell inserts. Infection of the intestinal epithelium with NL63 on-chip led to inflammation of the endothelium as demonstrated by loss of barrier function, increased cytokine production, and recruitment of circulating peripheral blood mononuclear cells (PBMCs). Treatment of NL63 infected chips with the approved protease inhibitor drug, nafamostat, inhibited viral entry and resulted in a reduction in both viral load and cytokine secretion, whereas remdesivir, one of the few drugs approved for COVID19 patients, was not found to be effective and it also was toxic to the endothelium. This model of intestinal infection was also used to test the effects of other drugs that have been proposed for potential repurposing against SARS-CoV-2. Taken together, these data suggest that the human Intestine Chip might be useful as a human preclinical model for studying coronavirus related pathology as well as for testing of potential anti-viral or anti-inflammatory therapeutics.

Chronic Lymphocytic Leukemia (CLL) is defined by a perturbed B-cell receptor-mediated signaling machinery. We aimed to model differential signaling behavior between B cells from CLL and healthy individuals to pinpoint modes of dysregulation. We developed an experimental methodology combining immunophenotyping, multiplexed phosphospecific flow cytometry, and multifactorial statistical modeling. Utilizing patterns of signaling network covariance, we modeled BCR signaling in 67 CLL patients using Partial Least Squares Regression (PLSR). Results from multidimensional modeling were validated using an independent test cohort of 38 patients. We identified a dynamic and variable imbalance between proximal (pSYK, pBTK) and distal (pPLCγ2, pBLNK, ppERK) phosphoresponses. PLSR identified the relationship between upstream tyrosine kinase SYK and its target, PLCγ2, as maximally predictive and sufficient to distinguish CLL from healthy samples, pointing to this juncture in the signaling pathway as a hallmark of CLL B cells. Specific BCR pathway signaling signatures that correlate with the disease and its degree of aggressiveness were identified. Heterogeneity in the PLSR response variable within the B cell population is both a characteristic mark of healthy samples and predictive of disease aggressiveness. Single-cell multidimensional analysis of BCR signaling permitted focused analysis of the variability and heterogeneity of signaling behavior from patient-to-patient, and from cell-to-cell. Disruption of the pSYK/pPLCγ2 relationship is uncovered as a robust hallmark of CLL B cell signaling behavior. Together, these observations implicate novel elements of the BCR signal transduction as potential therapeutic targets.

Cytokine-producing innate lymphoid cells are found at mucosal surfaces. Artis and Wherry and their colleagues show that innate 'nuocyte-like' cells accumulate in virus-infected lungs and contribute to the repair of tissues. Innate lymphoid cells (ILCs), a heterogeneous cell population, are critical in orchestrating immunity and inflammation in the intestine, but whether ILCs influence immune responses or tissue homeostasis at other mucosal sites remains poorly characterized. Here we identify a population of lung-resident ILCs in mice and humans that expressed the alloantigen Thy-1 (CD90), interleukin 2 (IL-2) receptor α-chain (CD25), IL-7 receptor α-chain (CD127) and the IL-33 receptor subunit T1-ST2. Notably, mouse ILCs accumulated in the lung after infection with influenza virus, and depletion of ILCs resulted in loss of airway epithelial integrity, diminished lung function and impaired airway remodeling. These defects were restored by administration of the lung ILC product amphiregulin. Collectively, our results demonstrate a critical role for lung ILCs in restoring airway epithelial integrity and tissue homeostasis after infection with influenza virus.

Group 3 innate lymphoid cells are shown to process and present antigen and to control CD4 + T-cell responses to intestinal commensal bacteria through an MHC-class-II-dependent mechanism. Immune response to intestinal bacteria The recently characterized innate lymphoid cells (ILCs) can be classified functionally into three groups. Group 1 ILCs produce interferon-γ, group 2 cells express interleukin (IL)-5, IL-13 and amphiregulin, and group 3 ILCs produce IL-17A and IL-22. The function of ILCs in the presence of adaptive immunity and their potential to influence adaptive immune cell responses are largely unknown. A study in mice now shows that group 3 ILCs process and present antigen and control CD4 + T-cell responses to intestinal commensal bacteria through an MHC-class-II-dependent mechanism. This finding may be relevant to the pathogenesis of chronic human diseases associated with inflammatory host immune responses to commensal bacteria. Innate lymphoid cells (ILCs) are a recently characterized family of immune cells that have critical roles in cytokine-mediated regulation of intestinal epithelial cell barrier integrity 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 . Alterations in ILC responses are associated with multiple chronic human diseases, including inflammatory bowel disease, implicating a role for ILCs in disease pathogenesis 3 , 8 , 11 , 12 , 13 . Owing to an inability to target ILCs selectively, experimental studies assessing ILC function have predominantly used mice lacking adaptive immune cells 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 . However, in lymphocyte-sufficient hosts ILCs are vastly outnumbered by CD4 + T cells, which express similar profiles of effector cytokines. Therefore, the function of ILCs in the presence of adaptive immunity and their potential to influence adaptive immune cell responses remain unknown. To test this, we used genetic or antibody-mediated depletion strategies to target murine ILCs in the presence of an adaptive immune system. We show that loss of retinoic-acid-receptor-related orphan receptor-γt-positive (RORγt + ) ILCs was associated with dysregulated adaptive immune cell responses against commensal bacteria and low-grade systemic inflammation. Remarkably, ILC-mediated regulation of adaptive immune cells occurred independently of interleukin (IL)-17A, IL-22 or IL-23. Genome-wide transcriptional profiling and functional analyses revealed that RORγt + ILCs express major histocompatibility complex class II (MHCII) and can process and present antigen. However, rather than inducing T-cell proliferation, ILCs acted to limit commensal bacteria-specific CD4 + T-cell responses. Consistent with this, selective deletion of MHCII in murine RORγt + ILCs resulted in dysregulated commensal bacteria-dependent CD4 + T-cell responses that promoted spontaneous intestinal inflammation. These data identify that ILCs maintain intestinal homeostasis through MHCII-dependent interactions with CD4 + T cells that limit pathological adaptive immune cell responses to commensal bacteria.