APOE3ch alters microglial response and suppresses Aß-induced tau seeding and spread.

A recent case report described an individual who was a homozygous carrier of the APOE3 Christchurch (APOE3ch) mutation and resistant to autosomal dominant Alzheimer's Disease (AD) caused by a PSEN1-E280A mutation. Whether APOE3ch contributed to the protective effect remains unclear. We generated a humanized APOE3ch knock-in mouse and crossed it to an amyloid-ß (Aß) plaque-depositing model. We injected AD-tau brain extract to investigate tau seeding and spreading in the presence or absence of amyloid. Similar to the case report, APOE3ch expression resulted in peripheral dyslipidemia and a marked reduction in plaque-associated tau pathology. Additionally, we observed decreased amyloid response and enhanced microglial response around plaques. We also demonstrate increased myeloid cell phagocytosis and degradation of tau aggregates linked to weaker APOE3ch binding to heparin sulfate proteoglycans. APOE3ch influences the microglial response to Aß plaques, which suppresses Aß-induced tau seeding and spreading. The results reveal new possibilities to target Aß-induced tauopathy.

Co-opting templated aggregation to degrade pathogenic tau assemblies and improve motor function.

Protein aggregation causes a wide range of neurodegenerative diseases. Targeting and removing aggregates, but not the functional protein, is a considerable therapeutic challenge. Here, we describe a therapeutic strategy called "RING-Bait," which employs an aggregating protein sequence combined with an E3 ubiquitin ligase. RING-Bait is recruited into aggregates, whereupon clustering dimerizes the RING domain and activates its E3 function, resulting in the degradation of the aggregate complex. We exemplify this concept by demonstrating the specific degradation of tau aggregates while sparing soluble tau. Unlike immunotherapy, RING-Bait is effective against both seeded and cell-autonomous aggregation. RING-Bait removed tau aggregates seeded from Alzheimer's disease (AD) and progressive supranuclear palsy (PSP) brain extracts and was also effective in primary neurons. We used a brain-penetrant adeno-associated virus (AAV) to treat P301S tau transgenic mice, reducing tau pathology and improving motor function. A RING-Bait strategy could be applied to other neurodegenerative proteinopathies by replacing the Bait sequence to match the target aggregate.

The proteomic landscape of synaptic diversity across brain regions and cell types.

Neurons build synaptic contacts using different protein combinations that define the specificity, function, and plasticity potential of synapses; however, the diversity of synaptic proteomes remains largely unexplored. We prepared synaptosomes from 7 different transgenic mouse lines with fluorescently labeled presynaptic terminals. Combining microdissection of 5 different brain regions with fluorescent-activated synaptosome sorting (FASS), we isolated and analyzed the proteomes of 18 different synapse types. We discovered ∼1,800 unique synapse-type-enriched proteins and allocated thousands of proteins to different types of synapses (https://syndive.org/). We identify shared synaptic protein modules and highlight the proteomic hotspots for synapse specialization. We reveal unique and common features of the striatal dopaminergic proteome and discover the proteome signatures that relate to the functional properties of different interneuron classes. This study provides a molecular systems-biology analysis of synapses and a framework to integrate proteomic information for synapse subtypes of interest with cellular or circuit-level experiments.

SYK coordinates neuroprotective microglial responses in neurodegenerative disease.

Recent studies have begun to reveal critical roles for the brain's professional phagocytes, microglia, and their receptors in the control of neurotoxic amyloid beta (Aß) and myelin debris accumulation in neurodegenerative disease. However, the critical intracellular molecules that orchestrate neuroprotective functions of microglia remain poorly understood. In our studies, we find that targeted deletion of SYK in microglia leads to exacerbated Aß deposition, aggravated neuropathology, and cognitive defects in the 5xFAD mouse model of Alzheimer's disease (AD). Disruption of SYK signaling in this AD model was further shown to impede the development of disease-associated microglia (DAM), alter AKT/GSK3ß-signaling, and restrict Aß phagocytosis by microglia. Conversely, receptor-mediated activation of SYK limits Aß load. We also found that SYK critically regulates microglial phagocytosis and DAM acquisition in demyelinating disease. Collectively, these results broaden our understanding of the key innate immune signaling molecules that instruct beneficial microglial functions in response to neurotoxic material.

X-linked ubiquitin-specific peptidase 11 increases tauopathy vulnerability in women.

Although women experience significantly higher tau burden and increased risk for Alzheimer's disease (AD) than men, the underlying mechanism for this vulnerability has not been explained. Here, we demonstrate through in vitro and in vivo models, as well as human AD brain tissue, that X-linked ubiquitin specific peptidase 11 (USP11) augments pathological tau aggregation via tau deubiquitination initiated at lysine-281. Removal of ubiquitin provides access for enzymatic tau acetylation at lysines 281 and 274. USP11 escapes complete X-inactivation, and female mice and people both exhibit higher USP11 levels than males. Genetic elimination of usp11 in a tauopathy mouse model preferentially protects females from acetylated tau accumulation, tau pathology, and cognitive impairment. USP11 levels also strongly associate positively with tau pathology in females but not males. Thus, inhibiting USP11-mediated tau deubiquitination may provide an effective therapeutic opportunity to protect women from increased vulnerability to AD and other tauopathies.

Bispecific antibodies promote natural killer cell-mediated elimination of HIV-1 reservoir cells.

The persistence of CD4+ T cells carrying latent human immunodeficiency virus-1 (HIV-1) proviruses is the main barrier to a cure. New therapeutics to enhance HIV-1-specific immune responses and clear infected cells will probably be necessary to achieve reduction of the latent reservoir. In the present study, we report two single-chain diabodies (scDbs) that target the HIV-1 envelope protein (Env) and the human type III Fcγ receptor (CD16). We show that the scDbs promoted robust and HIV-1-specific natural killer (NK) cell activation and NK cell-mediated lysis of infected cells. Cocultures of CD4+ T cells from people with HIV-1 on antiretroviral therapy (ART) with autologous NK cells and the scDbs resulted in marked elimination of reservoir cells that was dependent on latency reversal. Treatment of human interleukin-15 transgenic NSG mice with one of the scDbs after ART initiation enhanced NK cell activity and reduced reservoir size. Thus, HIV-1-specific scDbs merit further evaluation as potential therapeutics for clearance of the latent reservoir.

Reciprocal regulation of T follicular helper cells and dendritic cells drives colitis development.

The immunological mechanisms underlying chronic colitis are poorly understood. T follicular helper (TFH) cells are critical in helping B cells during germinal center reactions. In a T cell transfer colitis model, a lymphoid structure composed of mature dendritic cells (DCs) and TFH cells was found within T cell zones of colonic lymphoid follicles. TFH cells were required for mature DC accumulation, the formation of DC-T cell clusters and colitis development. Moreover, DCs promoted TFH cell differentiation, contributing to colitis development. A lineage-tracing analysis showed that, following migration to the lamina propria, TFH cells transdifferentiated into long-lived pathogenic TH1 cells, promoting colitis development. Our findings have therefore demonstrated the reciprocal regulation of TFH cells and DCs in colonic lymphoid follicles, which is critical in chronic colitis pathogenesis.

Temporal dynamics and genomic programming of plasma cell fates.

Affinity-matured plasma cells (PCs) of varying lifespans are generated through a germinal center (GC) response. The developmental dynamics and genomic programs of antigen-specific PC precursors remain to be elucidated. Here, using a model antigen in mice, we demonstrate biphasic generation of PC precursors, with those generating long-lived bone marrow PCs preferentially produced in the late phase of GC response. Clonal tracing using single-cell RNA sequencing and B cell antigen receptor sequencing in spleen and bone marrow compartments, coupled with adoptive transfer experiments, reveals a new PC transition state that gives rise to functionally competent PC precursors. The latter undergo clonal expansion, dependent on inducible expression of TIGIT. We propose a model for the proliferation and programming of precursors of long-lived PCs, based on extended antigen encounters in the GC.

Tumor editing suppresses innate and adaptive antitumor immunity and is reversed by inhibiting DNA methylation.

Cancer cells edit gene expression to evade immunosurveillance. However, genome-wide studies of gene editing during early tumorigenesis are lacking. Here we used single-cell RNA sequencing in a breast cancer genetically engineered mouse model (GEMM) to identify edited genes without bias. Late tumors repressed antitumor immunity genes, reducing infiltrating immune cells and tumor-immune cell communications. Innate immune genes, especially interferon-stimulated genes, dominated the list of downregulated tumor genes, while genes that regulate cell-intrinsic malignancy were mostly unedited. Naive and activated CD8+ T cells in early tumors were replaced with exhausted or precursor-exhausted cells in late tumors. Repression of immune genes was reversed by inhibiting DNA methylation using low-dose decitabine, which suppressed tumor growth and restored immune control, increasing the number, functionality and memory of tumor-infiltrating lymphocytes and reducing the number of myeloid suppressor cells. Decitabine induced important interferon, pyroptosis and necroptosis genes, inflammatory cell death and immune control in GEMM and implanted breast and melanoma tumors.

Affinity gaps among B cells in germinal centers drive the selection of MPER precursors.

Current prophylactic human immunodeficiency virus 1 (HIV-1) vaccine research aims to elicit broadly neutralizing antibodies (bnAbs). Membrane-proximal external region (MPER)-targeting bnAbs, such as 10E8, provide exceptionally broad neutralization, but some are autoreactive. Here, we generated humanized B cell antigen receptor knock-in mouse models to test whether a series of germline-targeting immunogens could drive MPER-specific precursors toward bnAbs. We found that recruitment of 10E8 precursors to germinal centers (GCs) required a minimum affinity for germline-targeting immunogens, but the GC residency of MPER precursors was brief due to displacement by higher-affinity endogenous B cell competitors. Higher-affinity germline-targeting immunogens extended the GC residency of MPER precursors, but robust long-term GC residency and maturation were only observed for MPER-HuGL18, an MPER precursor clonotype able to close the affinity gap with endogenous B cell competitors in the GC. Thus, germline-targeting immunogens could induce MPER-targeting antibodies, and B cell residency in the GC may be regulated by a precursor-competitor affinity gap.

Coordinated chemokine expression defines macrophage subsets across tissues.

Lung-resident macrophages, which include alveolar macrophages and interstitial macrophages (IMs), exhibit a high degree of diversity, generally attributed to different activation states, and often complicated by the influx of monocytes into the pool of tissue-resident macrophages. To gain a deeper insight into the functional diversity of IMs, here we perform comprehensive transcriptional profiling of resident IMs and reveal ten distinct chemokine-expressing IM subsets at steady state and during inflammation. Similar IM subsets that exhibited coordinated chemokine signatures and differentially expressed genes were observed across various tissues and species, indicating conserved specialized functional roles. Other macrophage types shared specific IM chemokine profiles, while also presenting their own unique chemokine signatures. Depletion of CD206hi IMs in Pf4creR26EYFP+DTR and Pf4creR26EYFPCx3cr1DTR mice led to diminished inflammatory cell recruitment, reduced tertiary lymphoid structure formation and fewer germinal center B cells in models of allergen- and infection-driven inflammation. These observations highlight the specialized roles of IMs, defined by their coordinated chemokine production, in regulating immune cell influx and organizing tertiary lymphoid tissue architecture.

Direct presentation of inflammation-associated self-antigens by thymic innate-like T cells induces elimination of autoreactive CD8+ thymocytes.

Upregulation of diverse self-antigens that constitute components of the inflammatory response overlaps spatially and temporally with the emergence of pathogen-derived foreign antigens. Therefore, discrimination between these inflammation-associated self-antigens and pathogen-derived molecules represents a unique challenge for the adaptive immune system. Here, we demonstrate that CD8+ T cell tolerance to T cell-derived inflammation-associated self-antigens is efficiently induced in the thymus and supported by redundancy in cell types expressing these molecules. In addition to thymic epithelial cells, this included thymic eosinophils and innate-like T cells, a population that expressed molecules characteristic for all major activated T cell subsets. We show that direct T cell-to-T cell antigen presentation by minute numbers of innate-like T cells was sufficient to eliminate autoreactive CD8+ thymocytes. Tolerance to such effector molecules was of critical importance, as its breach caused by decreased thymic abundance of a single model inflammation-associated self-antigen resulted in autoimmune elimination of an entire class of effector T cells.

Antitumor progenitor exhausted CD8+ T cells are sustained by TCR engagement.

The durability of an antitumor immune response is mediated in part by the persistence of progenitor exhausted CD8+ T cells (Tpex). Tpex serve as a resource for replenishing effector T cells and preserve their quantity through self-renewal. However, it is unknown how T cell receptor (TCR) engagement affects the self-renewal capacity of Tpex in settings of continued antigen exposure. Here we use a Lewis lung carcinoma model that elicits either optimal or attenuated TCR signaling in CD8+ T cells to show that formation of Tpex in tumor-draining lymph nodes and their intratumoral persistence is dependent on optimal TCR engagement. Notably, attenuated TCR stimulation accelerates the terminal differentiation of optimally primed Tpex. This TCR-reinforced Tpex development and self-renewal is coupled to proximal positioning to dendritic cells and epigenetic imprinting involving increased chromatin accessibility at Egr2 and Tcf1 target loci. Collectively, this study highlights the critical function of TCR engagement in sustaining Tpex during tumor progression.

An intestinal TH17 cell-derived subset can initiate cancer.

Approximately 25% of cancers are preceded by chronic inflammation that occurs at the site of tumor development. However, whether this multifactorial oncogenic process, which commonly occurs in the intestines, can be initiated by a specific immune cell population is unclear. Here, we show that an intestinal T cell subset, derived from interleukin-17 (IL-17)-producing helper T (TH17) cells, induces the spontaneous transformation of the intestinal epithelium. This subset produces inflammatory cytokines, and its tumorigenic potential is not dependent on IL-17 production but on the transcription factors KLF6 and T-BET and interferon-γ. The development of this cell type is inhibited by transforming growth factor-ß1 (TGFß1) produced by intestinal epithelial cells. TGFß signaling acts on the pretumorigenic TH17 cell subset, preventing its progression to the tumorigenic stage by inhibiting KLF6-dependent T-BET expression. This study therefore identifies an intestinal T cell subset initiating cancer.

A taxonomy of transcriptomic cell types across the isocortex and hippocampal formation.

The isocortex and hippocampal formation (HPF) in the mammalian brain play critical roles in perception, cognition, emotion, and learning. We profiled ∼1.3 million cells covering the entire adult mouse isocortex and HPF and derived a transcriptomic cell-type taxonomy revealing a comprehensive repertoire of glutamatergic and GABAergic neuron types. Contrary to the traditional view of HPF as having a simpler cellular organization, we discover a complete set of glutamatergic types in HPF homologous to all major subclasses found in the six-layered isocortex, suggesting that HPF and the isocortex share a common circuit organization. We also identify large-scale continuous and graded variations of cell types along isocortical depth, across the isocortical sheet, and in multiple dimensions in hippocampus and subiculum. Overall, our study establishes a molecular architecture of the mammalian isocortex and hippocampal formation and begins to shed light on its underlying relationship with the development, evolution, connectivity, and function of these two brain structures.

In vivo CRISPR screening reveals nutrient signaling processes underpinning CD8+ T cell fate decisions.

How early events in effector T cell (TEFF) subsets tune memory T cell (TMEM) responses remains incompletely understood. Here, we systematically investigated metabolic factors in fate determination of TEFF and TMEM cells using in vivo pooled CRISPR screening, focusing on negative regulators of TMEM responses. We found that amino acid transporters Slc7a1 and Slc38a2 dampened the magnitude of TMEM differentiation, in part through modulating mTORC1 signaling. By integrating genetic and systems approaches, we identified cellular and metabolic heterogeneity among TEFF cells, with terminal effector differentiation associated with establishment of metabolic quiescence and exit from the cell cycle. Importantly, Pofut1 (protein-O-fucosyltransferase-1) linked GDP-fucose availability to downstream Notch-Rbpj signaling, and perturbation of this nutrient signaling axis blocked terminal effector differentiation but drove context-dependent TEFF proliferation and TMEM development. Our study establishes that nutrient uptake and signaling are key determinants of T cell fate and shape the quantity and quality of TMEM responses.

A neural circuit state change underlying skilled movements.

In motor neuroscience, state changes are hypothesized to time-lock neural assemblies coordinating complex movements, but evidence for this remains slender. We tested whether a discrete change from more autonomous to coherent spiking underlies skilled movement by imaging cerebellar Purkinje neuron complex spikes in mice making targeted forelimb-reaches. As mice learned the task, millimeter-scale spatiotemporally coherent spiking emerged ipsilateral to the reaching forelimb, and consistent neural synchronization became predictive of kinematic stereotypy. Before reach onset, spiking switched from more disordered to internally time-locked concerted spiking and silence. Optogenetic manipulations of cerebellar feedback to the inferior olive bi-directionally modulated neural synchronization and reaching direction. A simple model explained the reorganization of spiking during reaching as reflecting a discrete bifurcation in olivary network dynamics. These findings argue that to prepare learned movements, olivo-cerebellar circuits enter a self-regulated, synchronized state promoting motor coordination. State changes facilitating behavioral transitions may generalize across neural systems.

Functional diversity for body actions in the mesencephalic locomotor region.

The mesencephalic locomotor region (MLR) is a key midbrain center with roles in locomotion. Despite extensive studies and clinical trials aimed at therapy-resistant Parkinson's disease (PD), debate on its function remains. Here, we reveal the existence of functionally diverse neuronal populations with distinct roles in control of body movements. We identify two spatially intermingled glutamatergic populations separable by axonal projections, mouse genetics, neuronal activity profiles, and motor functions. Most spinally projecting MLR neurons encoded the full-body behavior rearing. Loss- and gain-of-function optogenetic perturbation experiments establish a function for these neurons in controlling body extension. In contrast, Rbp4-transgene-positive MLR neurons project in an ascending direction to basal ganglia, preferentially encode the forelimb behaviors handling and grooming, and exhibit a role in modulating movement. Thus, the MLR contains glutamatergic neuronal subpopulations stratified by projection target exhibiting roles in action control not restricted to locomotion.

The antigenic anatomy of SARS-CoV-2 receptor binding domain.

Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 µg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.

Pathogenic ubiquitination of GSDMB inhibits NK cell bactericidal functions.

Gasdermin B (GSDMB) belongs to a large family of pore-forming cytolysins that execute inflammatory cell death programs. While genetic studies have linked GSDMB polymorphisms to human disease, its function in the immunological response to pathogens remains poorly understood. Here, we report a dynamic host-pathogen conflict between GSDMB and the IpaH7.8 effector protein secreted by enteroinvasive Shigella flexneri. We show that IpaH7.8 ubiquitinates and targets GSDMB for 26S proteasome destruction. This virulence strategy protects Shigella from the bacteriocidic activity of natural killer cells by suppressing granzyme-A-mediated activation of GSDMB. In contrast to the canonical function of most gasdermin family members, GSDMB does not inhibit Shigella by lysing host cells. Rather, it exhibits direct microbiocidal activity through recognition of phospholipids found on Gram-negative bacterial membranes. These findings place GSDMB as a central executioner of intracellular bacterial killing and reveal a mechanism employed by pathogens to counteract this host defense system.