Xist ribonucleoproteins promote female sex-biased autoimmunity.

Autoimmune diseases disproportionately affect females more than males. The XX sex chromosome complement is strongly associated with susceptibility to autoimmunity. Xist long non-coding RNA (lncRNA) is expressed only in females to randomly inactivate one of the two X chromosomes to achieve gene dosage compensation. Here, we show that the Xist ribonucleoprotein (RNP) complex comprising numerous autoantigenic components is an important driver of sex-biased autoimmunity. Inducible transgenic expression of a non-silencing form of Xist in male mice introduced Xist RNP complexes and sufficed to produce autoantibodies. Male SJL/J mice expressing transgenic Xist developed more severe multi-organ pathology in a pristane-induced lupus model than wild-type males. Xist expression in males reprogrammed T and B cell populations and chromatin states to more resemble wild-type females. Human patients with autoimmune diseases displayed significant autoantibodies to multiple components of XIST RNP. Thus, a sex-specific lncRNA scaffolds ubiquitous RNP components to drive sex-biased immunity.

Engineered cell entry links receptor biology with single-cell genomics.

Cells communicate with each other via receptor-ligand interactions. Here, we describe lentiviral-mediated cell entry by engineered receptor-ligand interaction (ENTER) to display ligand proteins, deliver payloads, and record receptor specificity. We optimize ENTER to decode interactions between T cell receptor (TCR)-MHC peptides, antibody-antigen, and other receptor-ligand pairs. A viral presentation strategy allows ENTER to capture interactions between B cell receptor and any antigen. We engineer ENTER to deliver genetic payloads to antigen-specific T or B cells to selectively modulate cellular behavior in mixed populations. Single-cell readout of ENTER by RNA sequencing (ENTER-seq) enables multiplexed enumeration of antigen specificities, TCR clonality, cell type, and states of individual T cells. ENTER-seq of CMV-seropositive patient blood samples reveals the viral epitopes that drive effector memory T cell differentiation and inter-clonal vs. intra-clonal phenotypic diversity targeting the same epitope. ENTER technology enables systematic discovery of receptor specificity, linkage to cell fates, and antigen-specific cargo delivery.

Lymph node colonization induces tumor-immune tolerance to promote distant metastasis.

For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and, therefore, an important prognostic factor. Beyond its utility as a biomarker, whether and how LN metastasis plays an active role in shaping distant metastasis remains an open question. Here, we develop a syngeneic melanoma mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that an epigenetically instilled tumor-intrinsic interferon response program confers enhanced LN metastatic potential by enabling the evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce antigen-specific regulatory T cells, and generate tumor-specific immune tolerance that subsequently facilitates distant tumor colonization. These effects extend to human cancers and other murine cancer models, implicating a conserved systemic mechanism by which malignancies spread to distant organs.

Infection trains the host for microbiota-enhanced resistance to pathogens.

The microbiota shields the host against infections in a process known as colonization resistance. How infections themselves shape this fundamental process remains largely unknown. Here, we show that gut microbiota from previously infected hosts display enhanced resistance to infection. This long-term functional remodeling is associated with altered bile acid metabolism leading to the expansion of taxa that utilize the sulfonic acid taurine. Notably, supplying exogenous taurine alone is sufficient to induce this alteration in microbiota function and enhance resistance. Mechanistically, taurine potentiates the microbiota's production of sulfide, an inhibitor of cellular respiration, which is key to host invasion by numerous pathogens. As such, pharmaceutical sequestration of sulfide perturbs the microbiota's composition and promotes pathogen invasion. Together, this work reveals a process by which the host, triggered by infection, can deploy taurine as a nutrient to nourish and train the microbiota, promoting its resistance to subsequent infection.

Discovery and functional interrogation of SARS-CoV-2 RNA-host protein interactions.

SARS-CoV-2 is the cause of a pandemic with growing global mortality. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with ChIRP-MS data from three other RNA viruses defined viral specificity of RNA-host protein interactions. Targeted CRISPR screens revealed that the majority of functional RNA-binding proteins protect the host from virus-induced cell death, and comparative CRISPR screens across seven RNA viruses revealed shared and SARS-specific antiviral factors. Finally, by combining the RNA-centric approach and functional CRISPR screens, we demonstrated a physical and functional connection between SARS-CoV-2 and mitochondria, highlighting this organelle as a general platform for antiviral activity. Altogether, these data provide a comprehensive catalog of functional SARS-CoV-2 RNA-host protein interactions, which may inform studies to understand the host-virus interface and nominate host pathways that could be targeted for therapeutic benefit.

Affirming NIH's commitment to addressing structural racism in the biomedical research enterprise.

NIH has acknowledged and committed to ending structural racism. The framework for NIH's approach, summarized here, includes understanding barriers; developing robust health disparities/equity research; improving its internal culture; being transparent and accountable; and changing the extramural ecosystem so that diversity, equity, and inclusion are reflected in funded research and the biomedical workforce.

Epigenetic regulation of T cell exhaustion.

Chronic antigen stimulation during viral infections and cancer can lead to T cell exhaustion, which is characterized by reduced effector function and proliferation, and the expression of inhibitory immune checkpoint receptors. Recent studies have demonstrated that T cell exhaustion results in wholescale epigenetic remodeling that confers phenotypic stability to these cells and prevents T cell reinvigoration by checkpoint blockade. Here, we review foundational technologies to profile the epigenome at multiple scales, including mapping the locations of transcription factors and histone modifications, DNA methylation and three-dimensional genome conformation. We discuss how these technologies have elucidated the development and epigenetic regulation of exhausted T cells and functional implications across viral infection, cancer, autoimmunity and engineered T cell therapies. Finally, we cover emerging multi-omic and genome engineering technologies, current and upcoming opportunities to apply these to T cell exhaustion, and therapeutic opportunities for T cell engineering in the clinic.

Divergent clonal differentiation trajectories of T cell exhaustion.

Chronic antigen exposure during viral infection or cancer promotes an exhausted T cell (Tex) state with reduced effector function. However, whether all antigen-specific T cell clones follow the same Tex differentiation trajectory remains unclear. Here, we generate a single-cell multiomic atlas of T cell exhaustion in murine chronic viral infection that redefines Tex phenotypic diversity, including two late-stage Tex subsets with either a terminal exhaustion (Texterm) or a killer cell lectin-like receptor-expressing cytotoxic (TexKLR) phenotype. We use paired single-cell RNA and T cell receptor sequencing to uncover clonal differentiation trajectories of Texterm-biased, TexKLR-biased or divergent clones that acquire both phenotypes. We show that high T cell receptor signaling avidity correlates with Texterm, whereas low avidity correlates with effector-like TexKLR fate. Finally, we identify similar clonal differentiation trajectories in human tumor-infiltrating lymphocytes. These findings reveal clonal heterogeneity in the T cell response to chronic antigen that influences Tex fates and persistence.

Escherichia coli in Auto(trophic) Mode.

It is challenging to convert a heterotrophic organism that loves sugars and other multicarbon compounds as energy and carbon sources into an autotroph that builds all biomass from carbon dioxide. In this issue, Gleizer et al. demonstrate how this can be achieved.

Single-Cell RNA-Seq Reveals AML Hierarchies Relevant to Disease Progression and Immunity.

Acute myeloid leukemia (AML) is a heterogeneous disease that resides within a complex microenvironment, complicating efforts to understand how different cell types contribute to disease progression. We combined single-cell RNA sequencing and genotyping to profile 38,410 cells from 40 bone marrow aspirates, including 16 AML patients and five healthy donors. We then applied a machine learning classifier to distinguish a spectrum of malignant cell types whose abundances varied between patients and between subclones in the same tumor. Cell type compositions correlated with prototypic genetic lesions, including an association of FLT3-ITD with abundant progenitor-like cells. Primitive AML cells exhibited dysregulated transcriptional programs with co-expression of stemness and myeloid priming genes and had prognostic significance. Differentiated monocyte-like AML cells expressed diverse immunomodulatory genes and suppressed T cell activity in vitro. In conclusion, we provide single-cell technologies and an atlas of AML cell states, regulators, and markers with implications for precision medicine and immune therapies. VIDEO ABSTRACT.

Distinct epigenomic landscapes underlie tissue-specific memory T cell differentiation.

The memory CD8+ T cell pool contains phenotypically and transcriptionally heterogeneous subsets with specialized functions and recirculation patterns. Here, we examined the epigenetic landscape of CD8+ T cells isolated from seven non-lymphoid organs across four distinct infection models, alongside their circulating T cell counterparts. Using single-cell transposase-accessible chromatin sequencing (scATAC-seq), we found that tissue-resident memory T (TRM) cells and circulating memory T (TCIRC) cells develop along distinct epigenetic trajectories. We identified organ-specific transcriptional regulators of TRM cell development, including FOSB, FOS, FOSL1, and BACH2, and defined an epigenetic signature common to TRM cells across organs. Finally, we found that although terminal TEX cells share accessible regulatory elements with TRM cells, they are defined by TEX-specific epigenetic features absent from TRM cells. Together, this comprehensive data resource shows that TRM cell development is accompanied by dynamic transcriptome alterations and chromatin accessibility changes that direct tissue-adapted and functionally distinct T cell states.

Mechanotransduction via endothelial adhesion molecule CD31 initiates transmigration and reveals a role for VEGFR2 in diapedesis.

Engagement of platelet endothelial cell adhesion molecule 1 (PECAM, PECAM-1, CD31) on the leukocyte pseudopod with PECAM at the endothelial cell border initiates transendothelial migration (TEM, diapedesis). We show, using fluorescence lifetime imaging microscopy (FLIM), that physical traction on endothelial PECAM during TEM initiated the endothelial signaling pathway. In this role, endothelial PECAM acted as part of a mechanotransduction complex with VE-cadherin and vascular endothelial growth factor receptor 2 (VEGFR2), and this predicted that VEGFR2 was required for efficient TEM. We show that TEM required both VEGFR2 and the ability of its Y1175 to be phosphorylated, but not VEGF or VEGFR2 endogenous kinase activity. Using inducible endothelial-specific VEGFR2-deficient mice, we show in three mouse models of inflammation that the absence of endothelial VEGFR2 significantly (by ≥75%) reduced neutrophil extravasation by selectively blocking diapedesis. These findings provide a more complete understanding of the process of transmigration and identify several potential anti-inflammatory targets.

A Cre-deleter specific for embryo-derived brain macrophages reveals distinct features of microglia and border macrophages.

Genetic tools to target microglia specifically and efficiently from the early stages of embryonic development are lacking. We generated a constitutive Cre line controlled by the microglia signature gene Crybb1 that produced nearly complete recombination in embryonic brain macrophages (microglia and border-associated macrophages [BAMs]) by the perinatal period, with limited recombination in peripheral myeloid cells. Using this tool in combination with Flt3-Cre lineage tracer, single-cell RNA-sequencing analysis, and confocal imaging, we resolved embryonic-derived versus monocyte-derived BAMs in the mouse cortex. Deletion of the transcription factor SMAD4 in microglia and embryonic-derived BAMs using Crybb1-Cre caused a developmental arrest of microglia, which instead acquired a BAM specification signature. By contrast, the development of genuine BAMs remained unaffected. Our results reveal that SMAD4 drives a transcriptional and epigenetic program that is indispensable for the commitment of brain macrophages to the microglia fate and highlight Crybb1-Cre as a tool for targeting embryonic brain macrophages.

Building a Translational Microbiome Toolbox.

Designing successful microbiota-based therapies requires in-depth understanding of the ecological foundations of this community. In this issue, two studies by Whitaker et al. and Lim et al. provide refined genetic tools for dissecting the spatial organization and temporal dynamics of bacterial communities at the single-cell and -gene levels.

Comprehensive Analysis of Hypermutation in Human Cancer.

We present an extensive assessment of mutation burden through sequencing analysis of >81,000 tumors from pediatric and adult patients, including tumors with hypermutation caused by chemotherapy, carcinogens, or germline alterations. Hypermutation was detected in tumor types not previously associated with high mutation burden. Replication repair deficiency was a major contributing factor. We uncovered new driver mutations in the replication-repair-associated DNA polymerases and a distinct impact of microsatellite instability and replication repair deficiency on the scale of mutation load. Unbiased clustering, based on mutational context, revealed clinically relevant subgroups regardless of the tumors' tissue of origin, highlighting similarities in evolutionary dynamics leading to hypermutation. Mutagens, such as UV light, were implicated in unexpected cancers, including sarcomas and lung tumors. The order of mutational signatures identified previous treatment and germline replication repair deficiency, which improved management of patients and families. These data will inform tumor classification, genetic testing, and clinical trial design.

Signaling in Host-Associated Microbial Communities.

Human-associated microbiota form and stabilize communities based on interspecies interactions. We review how these microbe-microbe and microbe-host interactions are communicated to shape communities over a human's lifespan, including periods of health and disease. Modeling and dissecting signaling in host-associated communities is crucial to understand their function and will open the door to therapies that prevent or correct microbial community dysfunction to promote health and treat disease.

Temporal Stability of the Human Skin Microbiome.

Biogeography and individuality shape the structural and functional composition of the human skin microbiome. To explore these factors' contribution to skin microbial community stability, we generated metagenomic sequence data from longitudinal samples collected over months and years. Analyzing these samples using a multi-kingdom, reference-based approach, we found that despite the skin's exposure to the external environment, its bacterial, fungal, and viral communities were largely stable over time. Site, individuality, and phylogeny were all determinants of stability. Foot sites exhibited the most variability; individuals differed in stability; and transience was a particular characteristic of eukaryotic viruses, which showed little site-specificity in colonization. Strain and single-nucleotide variant-level analysis showed that individuals maintain, rather than reacquire, prevalent microbes from the environment. Longitudinal stability of skin microbial communities generates hypotheses about colonization resistance and empowers clinical studies exploring alterations observed in disease states.

Tunable Single-Cell Extraction for Molecular Analyses.

Because of cellular heterogeneity, the analysis of endogenous molecules from single cells is of significant interest and has major implications. While micromanipulation or cell sorting followed by cell lysis is already used for subsequent molecular examinations, approaches to directly extract the content of living cells remain a challenging but promising alternative to achieving non-destructive sampling and cell-context preservation. Here, we demonstrate the quantitative extraction from single cells with spatiotemporal control using fluidic force microscopy. We further present a comprehensive analysis of the soluble molecules withdrawn from the cytoplasm or the nucleus, including the detection of enzyme activities and transcript abundances. This approach has uncovered the ability of cells to withstand extraction of up to several picoliters and opens opportunities to study cellular dynamics and cell-cell communication under physiological conditions at the single-cell level.

Detection of alternative DNA structures and its implications for human disease.

Around 3% of the genome consists of simple DNA repeats that are prone to forming alternative (non-B) DNA structures, such as hairpins, cruciforms, triplexes (H-DNA), four-stranded guanine quadruplexes (G4-DNA), and others, as well as composite RNA:DNA structures (e.g., R-loops, G-loops, and H-loops). These DNA structures are dynamic and favored by the unwinding of duplex DNA. For many years, the association of alternative DNA structures with genome function was limited by the lack of methods to detect them in vivo. Here, we review the recent advancements in the field and present state-of-the-art technologies and methods to study alternative DNA structures. We discuss the limitations of these methods as well as how they are beginning to provide insights into causal relationships between alternative DNA structures, genome function and stability, and human disease.

Macrophage inflammatory and regenerative response periodicity is programmed by cell cycle and chromatin state.

Cell cycle (CC) facilitates cell division via robust, cyclical gene expression. Protective immunity requires the expansion of pathogen-responsive cell types, but whether CC confers unique gene expression programs that direct the subsequent immunological response remains unclear. Here, we demonstrate that single macrophages (MFs) adopt different plasticity states in CC, which leads to heterogeneous cytokine-induced polarization, priming, and repolarization programs. Specifically, MF plasticity to interferon gamma (IFNG) is substantially reduced during S-G2/M, whereas interleukin 4 (IL-4) induces S-G2/M-biased gene expression, mediated by CC-biased enhancers. Additionally, IL-4 polarization shifts the CC-phase distribution of MFs toward the G2/M phase, providing a subpopulation-specific mechanism for IL-4-induced, dampened IFNG responsiveness. Finally, we demonstrate CC-dependent MF responses in murine and human disease settings in vivo, including Th2-driven airway inflammation and pulmonary fibrosis, where MFs express an S-G2/M-biased tissue remodeling gene program. Therefore, MF inflammatory and regenerative responses are gated by CC in a cyclical, phase-dependent manner.