Journal of Experimental Medicine最新文献

Mapping the causal circuits that shape the phenotypic and functional landscape of immune cells remains a formidable challenge. Recent advances in pooled CRISPR-based screens, coupled with multiplexed single-cell profiling and imaging-based spatial readouts, make this goal increasingly attainable. In this Perspective, we discuss how CRISPR-based genetic screens will fundamentally transform our understanding of immunobiology. We highlight the applications of state-of-the-art, high-throughput pooled perturbation approaches, including emerging methodologies for bulk, single-cell, and spatial CRISPR screens, to advance our understanding of immunity and in vivo biology. Additionally, we summarize new strategies to address the complexity of combinatorial perturbations to uncover genetic interactions and mechanistic drivers of immunity at unprecedented scale and resolution. By integrating CRISPR screening data with experimental insights, we advocate a new framework in immunology research that leverages perturbation-driven regulatory effects and networks to discover new therapeutic targets and establish causal systems biology and immunology for advancing immunological knowledge and therapeutic application.

Gut-associated lymphoid tissues (GALT) represent major sites of adaptive immune priming in the intestine, yet our understanding of human GALT diversity and function remains limited. Here, we used single-cell RNA sequencing, flow cytometry, and confocal laser microscopy to map the fibroblast (FB) landscape of human GALT, including that of Peyer's patches (PP), mucosal isolated lymphoid follicles (M-ILF), and submucosal ILF (SM-ILF). We identify CD24 as a marker that distinguishes GALT from other intestinal FB and demonstrate that CD24+ FB consist of distinct subsets that locate within discrete niches. We show that the composition and transcriptional profile of M-ILF and SM-ILF FB differs with SM-ILF FB appearing more focused at providing T cell support. Finally, we find the transcription profile of PP T zone reticular cells to be altered in Crohn's disease and that cells with a GALT FB-like profile can be detected in other chronic inflammatory diseases. Collectively, our findings provide an important framework for understanding GALT diversity and function.

Tuberculosis (TB) typically causes lung destruction and fibrosis, leading to ∼1.3 million deaths annually. The cellular drivers of human TB immunopathology remain poorly defined. We performed single-cell RNA sequencing and spatial transcriptomics on lung tissues from TB-infected and TB-negative individuals, identifying 30 distinct immune, parenchymal, and stromal cell subsets. Several were linked to TB pathology and corroborated through immunohistochemistry, flow cytometry, and independent human datasets. Fibroblasts were identified as major drivers in both active TB granuloma and TB-diseased lung tissue. In particular, the MMP1+CXCL5+ fibroblast subset, expressing a myofibroblast-like gene signature, was associated with severe disease and higher bacterial burden in nonhuman primate granulomas. Network analyses revealed cross talk between MMP1+CXCL5+ fibroblasts and SPP1+ macrophages within the granuloma cuff, which has been reported in other disease contexts, and may play an important role in TB immunopathology. Our findings highlight previously unappreciated cell populations and potential targets for novel TB therapies.

Mechanical force generated by blood flow stimulates emergence of the first hematopoietic stem cells (HSCs) that populate the blood system. Force drives the transition of HSC precursors from an endothelial to hematopoietic identity, yet the molecular regulation of this fate switch remains poorly understood. We report that shear stress triggers adaptation in mitochondrial composition, ultrastructure, and function, which are essential for hematopoietic fate and engraftment potential. Shear stress remodels mitochondria in hemogenic endothelium by promoting mitochondrial gene transcription and protein synthesis. Laminar flow selectively initiates translation of 5' terminal polypyrimidine (5'TOP) motif-containing transcripts, which commonly encode ribosome and translation machinery. Flow-responsive metabolic reprogramming depends upon mechanistic target of rapamycin (mTOR) activation and is stymied when ribosome activity or mTOR is blocked. Conversely, chemical induction of mTOR mimics the effects of force on mitochondria and blood reconstituting potential and also partially rescues hematopoiesis in heartbeat mutants in utero. These findings identify mechanometabolism as a determinant of hematopoietic fate that could inform engineering of HSCs for disease modeling and treatment.

Little is known about the role of autophagy in the human humoral immune system. Here, we found that in B cells, genetic ablation of FIP200, a mammalian metabolic sensor that regulates autophagy in response to a range of stimuli, led to diminished humoral immune responses in mice. FIP200-deficient B cells displayed decreased differentiation into plasma cells, as well as mitochondrial dysfunction, alterations in heme biosynthesis, and significant cell death. Notably, the addition of heme was sufficient to rescue plasma cell differentiation of FIP200-deficient B cells. Thus, FIP200 determines B cell fates by controlling mitophagy and metabolic reprogramming.

Central tolerance, which relies on the presentation of self-antigens by mTECs and DCs, prevents autoimmunity by eliminating self-reactive T cells. While mTECs produce self-antigens autonomously, DCs acquire them from mTECs via cooperative antigen transfer (CAT). We previously showed that mTEC and DC subsets exhibit preferential pairing in CAT, providing a rationale for the existence of molecular determinants underpinning this pairing and its outcome. Here, we compared the transcriptomes of CAT-experienced and CAT-inexperienced DCs and identified Claudin 1 as a molecule involved in CAT and type 1 DC (DC1) maturation. DC1-specific ablation of Claudin 1 resulted in decreased CAT to late mature DC1s and dramatically diminished DC1 maturation. These phenotypes correlated with the displacement of DC1s from mTECs and their decreased expression of MHCII pathway genes. This translated into impaired Treg selection and clonal deletion, ultimately manifesting in symptoms of multiorgan autoimmunity and shortened lifespan. Collectively, our results identify thymic DC1-derived Claudin 1 as a regulator of immune tolerance.

Runt-related transcription (RUNX) factors play a key role in T cell development. At the T-lineage commitment checkpoint, RUNX1 undergoes dynamic partner switching, resulting in its redeployment. Here, we investigated the functional differences in RUNX factors between the lymphoid progenitor (LP)- and Notch-stimulated earliest T progenitor stages (Phase 1). We identified CCCTC-binding factor (CTCF) as an LP-specific RUNX1-interacting partner, with LP-specific RUNX1-binding genomic sites significantly enriched for CTCF consensus motifs and co-occupied by CTCF. On Notch stimulation, Notch1 intracellular domain directly interacts with RUNX1 and recruits the RUNX1/Mediator/p300 transcriptional activation complex to Notch-regulated T-signature gene loci. CRISPR/Cas9-mediated stage-specific deletion of RUNX factors and their binding partners revealed that the RUNX1/CTCF complex in LP negatively regulates T-signature gene expression, whereas the RUNX1/Mediator/p300 complex in Phase 1 promotes it. Our findings highlight the crucial role of Notch-mediated functional conversion of RUNX factors, including protein complex reorganization and genomic redeployment in initiating T-lineage program.