Ligand binding initiates single-molecule integrin conformational activation.

Integrins link the extracellular environment to the actin cytoskeleton in cell migration and adhesiveness. Rapid coordination between events outside and inside the cell is essential. Single-molecule fluorescence dynamics show that ligand binding to the bent-closed integrin conformation, which predominates on cell surfaces, is followed within milliseconds by two concerted changes, leg extension and headpiece opening, to give the high-affinity integrin conformation. The extended-closed integrin conformation is not an intermediate but can be directly accessed from the extended-open conformation and provides a pathway for ligand dissociation. In contrast to ligand, talin, which links the integrin ß-subunit cytoplasmic domain to the actin cytoskeleton, modestly stabilizes but does not induce extension or opening. Integrin activation is thus initiated by outside-in signaling and followed by inside-out signaling. Our results further imply that talin binding is insufficient for inside-out integrin activation and that tensile force transmission through the ligand-integrin-talin-actin cytoskeleton complex is required.

A potent pan-sarbecovirus neutralizing antibody resilient to epitope diversification.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution has resulted in viral escape from clinically authorized monoclonal antibodies (mAbs), creating a need for mAbs that are resilient to epitope diversification. Broadly neutralizing coronavirus mAbs that are sufficiently potent for clinical development and retain activity despite viral evolution remain elusive. We identified a human mAb, designated VIR-7229, which targets the viral receptor-binding motif (RBM) with unprecedented cross-reactivity to all sarbecovirus clades, including non-ACE2-utilizing bat sarbecoviruses, while potently neutralizing SARS-CoV-2 variants since 2019, including the recent EG.5, BA.2.86, and JN.1. VIR-7229 tolerates extraordinary epitope variability, partly attributed to its high binding affinity, receptor molecular mimicry, and interactions with RBM backbone atoms. Consequently, VIR-7229 features a high barrier for selection of escape mutants, which are rare and associated with reduced viral fitness, underscoring its potential to be resilient to future viral evolution. VIR-7229 is a strong candidate to become a next-generation medicine.

A neural circuit for male sexual behavior and reward.

Male sexual behavior is innate and rewarding. Despite its centrality to reproduction, a molecularly specified neural circuit governing innate male sexual behavior and reward remains to be characterized. We have discovered a developmentally wired neural circuit necessary and sufficient for male mating. This circuit connects chemosensory input to BNSTprTac1 neurons, which innervate POATacr1 neurons that project to centers regulating motor output and reward. Epistasis studies demonstrate that BNSTprTac1 neurons are upstream of POATacr1 neurons, and BNSTprTac1-released substance P following mate recognition potentiates activation of POATacr1 neurons through Tacr1 to initiate mating. Experimental activation of POATacr1 neurons triggers mating, even in sexually satiated males, and it is rewarding, eliciting dopamine release and self-stimulation of these cells. Together, we have uncovered a neural circuit that governs the key aspects of innate male sexual behavior: motor displays, drive, and reward.

A tissue injury sensing and repair pathway distinct from host pathogen defense.

Pathogen infection and tissue injury are universal insults that disrupt homeostasis. Innate immunity senses microbial infections and induces cytokines/chemokines to activate resistance mechanisms. Here, we show that, in contrast to most pathogen-induced cytokines, interleukin-24 (IL-24) is predominately induced by barrier epithelial progenitors after tissue injury and is independent of microbiome or adaptive immunity. Moreover, Il24 ablation in mice impedes not only epidermal proliferation and re-epithelialization but also capillary and fibroblast regeneration within the dermal wound bed. Conversely, ectopic IL-24 induction in the homeostatic epidermis triggers global epithelial-mesenchymal tissue repair responses. Mechanistically, Il24 expression depends upon both epithelial IL24-receptor/STAT3 signaling and hypoxia-stabilized HIF1α, which converge following injury to trigger autocrine and paracrine signaling involving IL-24-mediated receptor signaling and metabolic regulation. Thus, parallel to innate immune sensing of pathogens to resolve infections, epithelial stem cells sense injury signals to orchestrate IL-24-mediated tissue repair.

Dynamic CD8+ T cell responses to cancer immunotherapy in human regional lymph nodes are disrupted in metastatic lymph nodes.

CD8+ T cell responses are critical for anti-tumor immunity. While extensively profiled in the tumor microenvironment, recent studies in mice identified responses in lymph nodes (LNs) as essential; however, the role of LNs in human cancer patients remains unknown. We examined CD8+ T cells in human head and neck squamous cell carcinomas, regional LNs, and blood using mass cytometry, single-cell genomics, and multiplexed ion beam imaging. We identified progenitor exhausted CD8+ T cells (Tpex) that were abundant in uninvolved LN and clonally related to terminally exhausted cells in the tumor. After anti-PD-L1 immunotherapy, Tpex in uninvolved LNs reduced in frequency but localized near dendritic cells and proliferating intermediate-exhausted CD8+ T cells (Tex-int), consistent with activation and differentiation. LN responses coincided with increased circulating Tex-int. In metastatic LNs, these response hallmarks were impaired, with immunosuppressive cellular niches. Our results identify important roles for LNs in anti-tumor immune responses in humans.

A functional cellular framework for sex and estrous cycle-dependent gene expression and behavior.

Sex hormones exert a profound influence on gendered behaviors. How individual sex hormone-responsive neuronal populations regulate diverse sex-typical behaviors is unclear. We performed orthogonal, genetically targeted sequencing of four estrogen receptor 1-expressing (Esr1+) populations and identified 1,415 genes expressed differentially between sexes or estrous states. Unique subsets of these genes were distributed across all 137 transcriptomically defined Esr1+ cell types, including estrous stage-specific ones, that comprise the four populations. We used differentially expressed genes labeling single Esr1+ cell types as entry points to functionally characterize two such cell types, BNSTprTac1/Esr1 and VMHvlCckar/Esr1. We observed that these two cell types, but not the other Esr1+ cell types in these populations, are essential for sex recognition in males and mating in females, respectively. Furthermore, VMHvlCckar/Esr1 cell type projections are distinct from those of other VMHvlEsr1 cell types. Together, projection and functional specialization of dimorphic cell types enables sex hormone-responsive populations to regulate diverse social behaviors.

In situ identification of cellular drug targets in mammalian tissue.

The lack of tools to observe drug-target interactions at cellular resolution in intact tissue has been a major barrier to understanding in vivo drug actions. Here, we develop clearing-assisted tissue click chemistry (CATCH) to optically image covalent drug targets in intact mammalian tissues. CATCH permits specific and robust in situ fluorescence imaging of target-bound drug molecules at subcellular resolution and enables the identification of target cell types. Using well-established inhibitors of endocannabinoid hydrolases and monoamine oxidases, direct or competitive CATCH not only reveals distinct anatomical distributions and predominant cell targets of different drug compounds in the mouse brain but also uncovers unexpected differences in drug engagement across and within brain regions, reflecting rare cell types, as well as dose-dependent target shifts across tissue, cellular, and subcellular compartments that are not accessible by conventional methods. CATCH represents a valuable platform for visualizing in vivo interactions of small molecules in tissue.

Discovering dominant tumor immune archetypes in a pan-cancer census.

Cancers display significant heterogeneity with respect to tissue of origin, driver mutations, and other features of the surrounding tissue. It is likely that individual tumors engage common patterns of the immune system-here "archetypes"-creating prototypical non-destructive tumor immune microenvironments (TMEs) and modulating tumor-targeting. To discover the dominant immune system archetypes, the University of California, San Francisco (UCSF) Immunoprofiler Initiative (IPI) processed 364 individual tumors across 12 cancer types using standardized protocols. Computational clustering of flow cytometry and transcriptomic data obtained from cell sub-compartments uncovered dominant patterns of immune composition across cancers. These archetypes were profound insofar as they also differentiated tumors based upon unique immune and tumor gene-expression patterns. They also partitioned well-established classifications of tumor biology. The IPI resource provides a template for understanding cancer immunity as a collection of dominant patterns of immune organization and provides a rational path forward to learn how to modulate these to improve therapy.

Glioma progression is shaped by genetic evolution and microenvironment interactions.

The factors driving therapy resistance in diffuse glioma remain poorly understood. To identify treatment-associated cellular and genetic changes, we analyzed RNA and/or DNA sequencing data from the temporally separated tumor pairs of 304 adult patients with isocitrate dehydrogenase (IDH)-wild-type and IDH-mutant glioma. Tumors recurred in distinct manners that were dependent on IDH mutation status and attributable to changes in histological feature composition, somatic alterations, and microenvironment interactions. Hypermutation and acquired CDKN2A deletions were associated with an increase in proliferating neoplastic cells at recurrence in both glioma subtypes, reflecting active tumor growth. IDH-wild-type tumors were more invasive at recurrence, and their neoplastic cells exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. Mesenchymal transition was associated with the presence of a myeloid cell state defined by specific ligand-receptor interactions with neoplastic cells. Collectively, these recurrence-associated phenotypes represent potential targets to alter disease progression.

A dietary commensal microbe enhances antitumor immunity by activating tumor macrophages to sequester iron.

Innate immune cells generate a multifaceted antitumor immune response, including the conservation of essential nutrients such as iron. These cells can be modulated by commensal bacteria; however, identifying and understanding how this occurs is a challenge. Here we show that the food commensal Lactiplantibacillus plantarum IMB19 augments antitumor immunity in syngeneic and xenograft mouse tumor models. Its capsular heteropolysaccharide is the major effector molecule, functioning as a ligand for TLR2. In a two-pronged manner, it skews tumor-associated macrophages to a classically active phenotype, leading to generation of a sustained CD8+ T cell response, and triggers macrophage 'nutritional immunity' to deploy the high-affinity iron transporter lipocalin-2 for capturing and sequestering iron in the tumor microenvironment. This process induces a cycle of tumor cell death, epitope expansion and subsequent tumor clearance. Together these data indicate that food commensals might be identified and developed into 'oncobiotics' for a multi-layered approach to cancer therapy.

Deletion of PD-1 destabilizes the lineage identity and metabolic fitness of tumor-infiltrating regulatory T cells.

Regulatory T (Treg) cells have an immunosuppressive function and highly express the immune checkpoint receptor PD-1 in the tumor microenvironment; however, the function of PD-1 in tumor-infiltrating (TI) Treg cells remains controversial. Here, we showed that conditional deletion of PD-1 in Treg cells delayed tumor progression. In Pdcd1fl/flFoxp3eGFP-Cre-ERT2(+/-) mice, in which both PD-1-expressing and PD-1-deficient Treg cells coexisted in the same tissue environment, conditional deletion of PD-1 in Treg cells resulted in impairment of the proliferative and suppressive capacity of TI Treg cells. PD-1 antibody therapy reduced the TI Treg cell numbers, but did not directly restore the cytokine production of TI CD8+ T cells in TC-1 lung cancer. Single-cell analysis indicated that PD-1 signaling promoted lipid metabolism, proliferation and suppressive pathways in TI Treg cells. These results suggest that PD-1 ablation or inhibition can enhance antitumor immunity by weakening Treg cell lineage stability and metabolic fitness in the tumor microenvironment.

A virtual memory CD8+ T cell-originated subset causes alopecia areata through innate-like cytotoxicity.

Virtual memory T (TVM) cells are a T cell subtype with a memory phenotype but no prior exposure to foreign antigen. Although TVM cells have antiviral and antibacterial functions, whether these cells can be pathogenic effectors of inflammatory disease is unclear. Here we identified a TVM cell-originated CD44super-high(s-hi)CD49dlo CD8+ T cell subset with features of tissue residency. These cells are transcriptionally, phenotypically and functionally distinct from conventional CD8+ TVM cells and can cause alopecia areata. Mechanistically, CD44s-hiCD49dlo CD8+ T cells could be induced from conventional TVM cells by interleukin (IL)-12, IL-15 and IL-18 stimulation. Pathogenic activity of CD44s-hiCD49dlo CD8+ T cells was mediated by NKG2D-dependent innate-like cytotoxicity, which was further augmented by IL-15 stimulation and triggered disease onset. Collectively, these data suggest an immunological mechanism through which TVM cells can cause chronic inflammatory disease by innate-like cytotoxicity.

All-Optical Electrophysiology Reveals the Role of Lateral Inhibition in Sensory Processing in Cortical Layer 1.

Cortical layer 1 (L1) interneurons have been proposed as a hub for attentional modulation of underlying cortex, but the transformations that this circuit implements are not known. We combined genetically targeted voltage imaging with optogenetic activation and silencing to study the mechanisms underlying sensory processing in mouse barrel cortex L1. Whisker stimuli evoked precisely timed single spikes in L1 interneurons, followed by strong lateral inhibition. A mild aversive stimulus activated cholinergic inputs and evoked a bimodal distribution of spiking responses in L1. A simple conductance-based model that only contained lateral inhibition within L1 recapitulated the sensory responses and the winner-takes-all cholinergic responses, and the model correctly predicted that the network would function as a spatial and temporal high-pass filter for excitatory inputs. Our results demonstrate that all-optical electrophysiology can reveal basic principles of neural circuit function in vivo and suggest an intuitive picture for how L1 transforms sensory and modulatory inputs. VIDEO ABSTRACT.

An Organoid Biobank of Neuroendocrine Neoplasms Enables Genotype-Phenotype Mapping.

Gastroenteropancreatic (GEP) neuroendocrine neoplasm (NEN) that consists of neuroendocrine tumor and neuroendocrine carcinoma (NEC) is a lethal but under-investigated disease owing to its rarity. To fill the scarcity of clinically relevant models of GEP-NEN, we here established 25 lines of NEN organoids and performed their comprehensive molecular characterization. GEP-NEN organoids recapitulated pathohistological and functional phenotypes of the original tumors. Whole-genome sequencing revealed frequent genetic alterations in TP53 and RB1 in GEP-NECs, and characteristic chromosome-wide loss of heterozygosity in GEP-NENs. Transcriptome analysis identified molecular subtypes that are distinguished by the expression of distinct transcription factors. GEP-NEN organoids gained independence from the stem cell niche irrespective of genetic mutations. Compound knockout of TP53 and RB1, together with overexpression of key transcription factors, conferred on the normal colonic epithelium phenotypes that are compatible with GEP-NEN biology. Altogether, our study not only provides genetic understanding of GEP-NEN, but also connects its genetics and biological phenotypes.

Translocation of Viable Gut Microbiota to Mesenteric Adipose Drives Formation of Creeping Fat in Humans.

A mysterious feature of Crohn's disease (CD) is the extra-intestinal manifestation of "creeping fat" (CrF), defined as expansion of mesenteric adipose tissue around the inflamed and fibrotic intestine. In the current study, we explore whether microbial translocation in CD serves as a central cue for CrF development. We discovered a subset of mucosal-associated gut bacteria that consistently translocated and remained viable in CrF in CD ileal surgical resections, and identified Clostridium innocuum as a signature of this consortium with strain variation between mucosal and adipose isolates, suggesting preference for lipid-rich environments. Single-cell RNA sequencing characterized CrF as both pro-fibrotic and pro-adipogenic with a rich milieu of activated immune cells responding to microbial stimuli, which we confirm in gnotobiotic mice colonized with C. innocuum. Ex vivo validation of expression patterns suggests C. innocuum stimulates tissue remodeling via M2 macrophages, leading to an adipose tissue barrier that serves to prevent systemic dissemination of bacteria.

Synergistic Coordination of Chromatin Torsional Mechanics and Topoisomerase Activity.

DNA replication in eukaryotes generates DNA supercoiling, which may intertwine (braid) daughter chromatin fibers to form precatenanes, posing topological challenges during chromosome segregation. The mechanisms that limit precatenane formation remain unclear. By making direct torque measurements, we demonstrate that the intrinsic mechanical properties of chromatin play a fundamental role in dictating precatenane formation and regulating chromatin topology. Whereas a single chromatin fiber is torsionally soft, a braided fiber is torsionally stiff, indicating that supercoiling on chromatin substrates is preferentially directed in front of the fork during replication. We further show that topoisomerase II relaxation displays a strong preference for a single chromatin fiber over a braided fiber. These results suggest a synergistic coordination-the mechanical properties of chromatin inherently suppress precatenane formation during replication elongation by driving DNA supercoiling ahead of the fork, where supercoiling is more efficiently removed by topoisomerase II. VIDEO ABSTRACT.

Unleashing Type-2 Dendritic Cells to Drive Protective Antitumor CD4+ T Cell Immunity.

Differentiation of proinflammatory CD4+ conventional T cells (Tconv) is critical for productive antitumor responses yet their elicitation remains poorly understood. We comprehensively characterized myeloid cells in tumor draining lymph nodes (tdLN) of mice and identified two subsets of conventional type-2 dendritic cells (cDC2) that traffic from tumor to tdLN and present tumor-derived antigens to CD4+ Tconv, but then fail to support antitumor CD4+ Tconv differentiation. Regulatory T cell (Treg) depletion enhanced their capacity to elicit strong CD4+ Tconv responses and ensuing antitumor protection. Analogous cDC2 populations were identified in patients, and as in mice, their abundance relative to Treg predicts protective ICOS+ PD-1lo CD4+ Tconv phenotypes and survival. Further, in melanoma patients with low Treg abundance, intratumoral cDC2 density alone correlates with abundant CD4+ Tconv and with responsiveness to anti-PD-1 therapy. Together, this highlights a pathway that restrains cDC2 and whose reversal enhances CD4+ Tconv abundance and controls tumor growth.

Periodic Remodeling in a Neural Circuit Governs Timing of Female Sexual Behavior.

Behaviors are inextricably linked to internal state. We have identified a neural mechanism that links female sexual behavior with the estrus, the ovulatory phase of the estrous cycle. We find that progesterone-receptor (PR)-expressing neurons in the ventromedial hypothalamus (VMH) are active and required during this behavior. Activating these neurons, however, does not elicit sexual behavior in non-estrus females. We show that projections of PR+ VMH neurons to the anteroventral periventricular (AVPV) nucleus change across the 5-day mouse estrous cycle, with ∼3-fold more termini and functional connections during estrus. This cyclic increase in connectivity is found in adult females, but not males, and regulated by estrogen signaling in PR+ VMH neurons. We further show that these connections are essential for sexual behavior in receptive females. Thus, estrogen-regulated structural plasticity of behaviorally salient connections in the adult female brain links sexual behavior to the estrus phase of the estrous cycle.