Top-down control of hippocampal signal-to-noise by prefrontal long-range inhibition.

Prefrontal cortex (PFC) is postulated to exert "top-down control" on information processing throughout the brain to promote specific behaviors. However, pathways mediating top-down control remain poorly understood. In particular, knowledge about direct prefrontal connections that might facilitate top-down control of hippocampal information processing remains sparse. Here we describe monosynaptic long-range GABAergic projections from PFC to hippocampus. These preferentially inhibit vasoactive intestinal polypeptide-expressing interneurons, which are known to disinhibit hippocampal microcircuits. Indeed, stimulating prefrontal-hippocampal GABAergic projections increases hippocampal feedforward inhibition and reduces hippocampal activity in vivo. The net effect of these actions is to specifically enhance the signal-to-noise ratio for hippocampal encoding of object locations and augment object-induced increases in spatial information. Correspondingly, activating or inhibiting these projections promotes or suppresses object exploration, respectively. Together, these results elucidate a top-down prefrontal pathway in which long-range GABAergic projections target disinhibitory microcircuits, thereby enhancing signals and network dynamics underlying exploratory behavior.

Spatial CRISPR genomics identifies regulators of the tumor microenvironment.

While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying extracellular gene functions, particularly in the tissue context. Here, we developed an approach for spatial functional genomics called Perturb-map. We applied Perturb-map to knock out dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Moreover, we paired Perturb-map and spatial transcriptomics for unbiased analysis of CRISPR-edited tumors. We found that in Tgfbr2 knockout tumors, the tumor microenvironment (TME) was converted to a fibro-mucinous state, and T cells excluded, concomitant with upregulated TGFß and TGFß-mediated fibroblast activation, indicating that TGFß-receptor loss on cancer cells increased TGFß bioavailability and its immunosuppressive effects on the TME. These studies establish Perturb-map for functional genomics within the tissue at single-cell resolution with spatial architecture preserved and provide insight into how TGFß responsiveness of cancer cells can affect the TME.

Multi-sensory Gamma Stimulation Ameliorates Alzheimer's-Associated Pathology and Improves Cognition.

We previously reported that inducing gamma oscillations with a non-invasive light flicker (gamma entrainment using sensory stimulus or GENUS) impacted pathology in the visual cortex of Alzheimer's disease mouse models. Here, we designed auditory tone stimulation that drove gamma frequency neural activity in auditory cortex (AC) and hippocampal CA1. Seven days of auditory GENUS improved spatial and recognition memory and reduced amyloid in AC and hippocampus of 5XFAD mice. Changes in activation responses were evident in microglia, astrocytes, and vasculature. Auditory GENUS also reduced phosphorylated tau in the P301S tauopathy model. Furthermore, combined auditory and visual GENUS, but not either alone, produced microglial-clustering responses, and decreased amyloid in medial prefrontal cortex. Whole brain analysis using SHIELD revealed widespread reduction of amyloid plaques throughout neocortex after multi-sensory GENUS. Thus, GENUS can be achieved through multiple sensory modalities with wide-ranging effects across multiple brain areas to improve cognitive function.

A CTCF-binding site in the Mdm1-Il22-Ifng locus shapes cytokine expression profiles and plays a critical role in early Th1 cell fate specification.

Cytokine expression during T cell differentiation is a highly regulated process that involves long-range promoter-enhancer and CTCF-CTCF contacts at cytokine loci. Here, we investigated the impact of dynamic chromatin loop formation within the topologically associating domain (TAD) in regulating the expression of interferon gamma (IFN-γ) and interleukin-22 (IL-22); these cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. In situ Hi-C analyses revealed inducible TADs that insulated Ifng and Il22 enhancers during Th1 cell differentiation. Targeted deletion of a 17 bp boundary motif of these TADs imbalanced Th1- and Th17-associated immunity, both in vitro and in vivo, upon Toxoplasma gondii infection. In contrast, this boundary element was dispensable for cytokine regulation in natural killer cells. Our findings suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes.

The Gut Microbiota Mediates the Anti-Seizure Effects of the Ketogenic Diet.

The ketogenic diet (KD) is used to treat refractory epilepsy, but the mechanisms underlying its neuroprotective effects remain unclear. Here, we show that the gut microbiota is altered by the KD and required for protection against acute electrically induced seizures and spontaneous tonic-clonic seizures in two mouse models. Mice treated with antibiotics or reared germ free are resistant to KD-mediated seizure protection. Enrichment of, and gnotobiotic co-colonization with, KD-associated Akkermansia and Parabacteroides restores seizure protection. Moreover, transplantation of the KD gut microbiota and treatment with Akkermansia and Parabacteroides each confer seizure protection to mice fed a control diet. Alterations in colonic lumenal, serum, and hippocampal metabolomic profiles correlate with seizure protection, including reductions in systemic gamma-glutamylated amino acids and elevated hippocampal GABA/glutamate levels. Bacterial cross-feeding decreases gamma-glutamyltranspeptidase activity, and inhibiting gamma-glutamylation promotes seizure protection in vivo. Overall, this study reveals that the gut microbiota modulates host metabolism and seizure susceptibility in mice.

Protein Barcodes Enable High-Dimensional Single-Cell CRISPR Screens.

CRISPR pools are being widely employed to identify gene functions. However, current technology, which utilizes DNA as barcodes, permits limited phenotyping and bulk-cell resolution. To enable novel screening capabilities, we developed a barcoding system operating at the protein level. We synthesized modules encoding triplet combinations of linear epitopes to generate >100 unique protein barcodes (Pro-Codes). Pro-Code-expressing vectors were introduced into cells and analyzed by CyTOF mass cytometry. Using just 14 antibodies, we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR, we simultaneously analyzed multiple phenotypic markers, including phospho-signaling, on dozens of knockouts. Pro-Code/CRISPR screens found two interferon-stimulated genes, the immunoproteasome component Psmb8 and a chaperone Rtp4, are important for antigen-dependent immune editing of cancer cells and identified Socs1 as a negative regulator of Pd-l1. The Pro-Code technology enables simultaneous high-dimensional protein-level phenotyping of 100s of genes with single-cell resolution.

Dysregulated lung stroma drives emphysema exacerbation by potentiating resident lymphocytes to suppress an epithelial stem cell reservoir.

Aberrant tissue-immune interactions are the hallmark of diverse chronic lung diseases. Here, we sought to define these interactions in emphysema, a progressive disease characterized by infectious exacerbations and loss of alveolar epithelium. Single-cell analysis of human emphysema lungs revealed the expansion of tissue-resident lymphocytes (TRLs). Murine studies identified a stromal niche for TRLs that expresses Hhip, a disease-variant gene downregulated in emphysema. Stromal-specific deletion of Hhip induced the topographic expansion of TRLs in the lung that was mediated by a hyperactive hedgehog-IL-7 axis. 3D immune-stem cell organoids and animal models of viral exacerbations demonstrated that expanded TRLs suppressed alveolar stem cell growth through interferon gamma (IFNγ). Finally, we uncovered an IFNγ-sensitive subset of human alveolar stem cells that was preferentially lost in emphysema. Thus, we delineate a stromal-lymphocyte-epithelial stem cell axis in the lung that is modified by a disease-variant gene and confers host susceptibility to emphysema.

Interplay between liver and blood stages of Plasmodium infection dictates malaria severity via γδ T cells and IL-17-promoted stress erythropoiesis.

Plasmodium replicates within the liver prior to reaching the bloodstream and infecting red blood cells. Because clinical manifestations of malaria only arise during the blood stage of infection, a perception exists that liver infection does not impact disease pathology. By developing a murine model where the liver and blood stages of infection are uncoupled, we showed that the integration of signals from both stages dictated mortality outcomes. This dichotomy relied on liver stage-dependent activation of Vγ4+ γδ T cells. Subsequent blood stage parasite loads dictated their cytokine profiles, where low parasite loads preferentially expanded IL-17-producing γδ T cells. IL-17 drove extra-medullary erythropoiesis and concomitant reticulocytosis, which protected mice from lethal experimental cerebral malaria (ECM). Adoptive transfer of erythroid precursors could rescue mice from ECM. Modeling of γδ T cell dynamics suggests that this protective mechanism may be key for the establishment of naturally acquired malaria immunity among frequently exposed individuals.

Skin γδ T cell inflammatory responses are hardwired in the thymus by oxysterol sensing via GPR183 and calibrated by dietary cholesterol.

Dietary components and metabolites have a profound impact on immunity and inflammation. Here, we investigated how sensing of cholesterol metabolite oxysterols by γδ T cells impacts their tissue residency and function. We show that dermal IL-17-producing γδ T (Tγδ17) cells essential for skin-barrier homeostasis require oxysterols sensing through G protein receptor 183 (GPR183) for their development and inflammatory responses. Single-cell transcriptomics and murine reporter strains revealed that GPR183 on developing γδ thymocytes is needed for their maturation by sensing medullary thymic epithelial-cell-derived oxysterols. In the skin, basal keratinocytes expressing the oxysterol enzyme cholesterol 25-hydroxylase (CH25H) maintain dermal Tγδ17 cells. Diet-driven increases in oxysterols exacerbate Tγδ17-cell-mediated psoriatic inflammation, dependent on GPR183 on γδ T cells. Hence, cholesterol-derived oxysterols control spatially distinct but biologically linked processes of thymic education and peripheral function of dermal T cells, implicating diet as a focal parameter of dermal Tγδ17 cells.

Restraint of IFN-γ expression through a distal silencer CNS-28 for tissue homeostasis.

Interferon-γ (IFN-γ) is a key cytokine in response to viral or intracellular bacterial infection in mammals. While a number of enhancers are described to promote IFN-γ responses, to the best of our knowledge, no silencers for the Ifng gene have been identified. By examining H3K4me1 histone modification in naive CD4+ T cells within Ifng locus, we identified a silencer (CNS-28) that restrains Ifng expression. Mechanistically, CNS-28 maintains Ifng silence by diminishing enhancer-promoter interactions within Ifng locus in a GATA3-dependent but T-bet-independent manner. Functionally, CNS-28 restrains Ifng transcription in NK cells, CD4+ cells, and CD8+ T cells during both innate and adaptive immune responses. Moreover, CNS-28 deficiency resulted in repressed type 2 responses due to elevated IFN-γ expression, shifting Th1 and Th2 paradigm. Thus, CNS-28 activity ensures immune cell quiescence by cooperating with other regulatory cis elements within the Ifng gene locus to minimize autoimmunity.

Retinoid X receptor gamma dictates the activation threshold of group 2 innate lymphoid cells and limits type 2 inflammation in the small intestine.

Group 2 innate lymphoid cells (ILC2s) are crucial in promoting type 2 inflammation that contributes to both anti-parasite immunity and allergic diseases. However, the molecular checkpoints in ILC2s that determine whether to immediately launch a proinflammatory response are unknown. Here, we found that retinoid X receptor gamma (Rxrg) was highly expressed in small intestinal ILC2s and rapidly suppressed by alarmin cytokines. Genetic deletion of Rxrg did not impact ILC2 development but facilitated ILC2 responses and the tissue inflammation induced by alarmins. Mechanistically, RXRγ maintained the expression of its target genes that support intracellular cholesterol efflux, which in turn reduce ILC2 proliferation. Furthermore, RXRγ expression prevented ILC2 response to mild stimulations, including low doses of alarmin cytokine and mechanical skin injury. Together, we propose that RXRγ expression and its mediated lipid metabolic states function as a cell-intrinsic checkpoint that confers the threshold of ILC2 activation in the small intestine.

Genetic variants of phospholipase C-γ2 alter the phenotype and function of microglia and confer differential risk for Alzheimer's disease.

Genetic association studies have demonstrated the critical involvement of the microglial immune response in Alzheimer's disease (AD) pathogenesis. Phospholipase C-gamma-2 (PLCG2) is selectively expressed by microglia and functions in many immune receptor signaling pathways. In AD, PLCG2 is induced uniquely in plaque-associated microglia. A genetic variant of PLCG2, PLCG2P522R, is a mild hypermorph that attenuates AD risk. Here, we identified a loss-of-function PLCG2 variant, PLCG2M28L, that confers an increased AD risk. PLCG2P522R attenuated disease in an amyloidogenic murine AD model, whereas PLCG2M28L exacerbated the plaque burden associated with altered phagocytosis and Aß clearance. The variants bidirectionally modulated disease pathology by inducing distinct transcriptional programs that identified microglial subpopulations associated with protective or detrimental phenotypes. These findings identify PLCG2M28L as a potential AD risk variant and demonstrate that PLCG2 variants can differentially orchestrate microglial responses in AD pathogenesis that can be therapeutically targeted.

Tissue-specific abundance of interferon-gamma drives regulatory T cells to restrain DC1-mediated priming of cytotoxic T cells against lung cancer.

Local environmental factors influence CD8+ T cell priming in lymph nodes (LNs). Here, we sought to understand how factors unique to the tumor-draining mediastinal LN (mLN) impact CD8+ T cell responses toward lung cancer. Type 1 conventional dendritic cells (DC1s) showed a mLN-specific failure to induce robust cytotoxic T cells responses. Using regulatory T (Treg) cell depletion strategies, we found that Treg cells suppressed DC1s in a spatially coordinated manner within tissue-specific microniches within the mLN. Treg cell suppression required MHC II-dependent contact between DC1s and Treg cells. Elevated levels of IFN-γ drove differentiation Treg cells into Th1-like effector Treg cells in the mLN. In patients with cancer, Treg cell Th1 polarization, but not CD8+/Treg cell ratios, correlated with poor responses to checkpoint blockade immunotherapy. Thus, IFN-γ in the mLN skews Treg cells to be Th1-like effector Treg cells, driving their close interaction with DC1s and subsequent suppression of cytotoxic T cell responses.

Oncolytic Virotherapy Promotes Intratumoral T Cell Infiltration and Improves Anti-PD-1 Immunotherapy.

Here we report a phase 1b clinical trial testing the impact of oncolytic virotherapy with talimogene laherparepvec on cytotoxic T cell infiltration and therapeutic efficacy of the anti-PD-1 antibody pembrolizumab. Twenty-one patients with advanced melanoma were treated with talimogene laherparepvec followed by combination therapy with pembrolizumab. Therapy was generally well tolerated, with fatigue, fevers, and chills as the most common adverse events. No dose-limiting toxicities occurred. Confirmed objective response rate was 62%, with a complete response rate of 33% per immune-related response criteria. Patients who responded to combination therapy had increased CD8+ T cells, elevated PD-L1 protein expression, as well as IFN-γ gene expression on several cell subsets in tumors after talimogene laherparepvec treatment. Response to combination therapy did not appear to be associated with baseline CD8+ T cell infiltration or baseline IFN-γ signature. These findings suggest that oncolytic virotherapy may improve the efficacy of anti-PD-1 therapy by changing the tumor microenvironment. VIDEO ABSTRACT.

Interferon gamma constrains type 2 lymphocyte niche boundaries during mixed inflammation.

Allergic immunity is orchestrated by group 2 innate lymphoid cells (ILC2s) and type 2 helper T (Th2) cells prominently arrayed at epithelial- and microbial-rich barriers. However, ILC2s and Th2 cells are also present in fibroblast-rich niches within the adventitial layer of larger vessels and similar boundary structures in sterile deep tissues, and it remains unclear whether they undergo dynamic repositioning during immune perturbations. Here, we used thick-section quantitative imaging to show that allergic inflammation drives invasion of lung and liver non-adventitial parenchyma by ILC2s and Th2 cells. However, during concurrent type 1 and type 2 mixed inflammation, IFNγ from broadly distributed type 1 lymphocytes directly blocked both ILC2 parenchymal trafficking and subsequent cell survival. ILC2 and Th2 cell confinement to adventitia limited mortality by the type 1 pathogen Listeria monocytogenes. Our results suggest that the topography of tissue lymphocyte subsets is tightly regulated to promote appropriately timed and balanced immunity.

The transcription factor IRF2 drives interferon-mediated CD8+ T cell exhaustion to restrict anti-tumor immunity.

Type I and II interferons (IFNs) stimulate pro-inflammatory programs that are critical for immune activation, but also induce immune-suppressive feedback circuits that impede control of cancer growth. Here, we sought to determine how these opposing programs are differentially induced. We demonstrated that the transcription factor interferon regulatory factor 2 (IRF2) was expressed by many immune cells in the tumor in response to sustained IFN signaling. CD8+ T cell-specific deletion of IRF2 prevented acquisition of the T cell exhaustion program within the tumor and instead enabled sustained effector functions that promoted long-term tumor control and increased responsiveness to immune checkpoint and adoptive cell therapies. The long-term tumor control by IRF2-deficient CD8+ T cells required continuous integration of both IFN-I and IFN-II signals. Thus, IRF2 is a foundational feedback molecule that redirects IFN signals to suppress T cell responses and represents a potential target to enhance cancer control.

An Epstein-Barr virus protein interaction map reveals NLRP3 inflammasome evasion via MAVS UFMylation.

Epstein-Barr virus (EBV) causes infectious mononucleosis, triggers multiple sclerosis, and is associated with 200,000 cancers/year. EBV colonizes the human B cell compartment and periodically reactivates, inducing expression of 80 viral proteins. However, much remains unknown about how EBV remodels host cells and dismantles key antiviral responses. We therefore created a map of EBV-host and EBV-EBV interactions in B cells undergoing EBV replication, uncovering conserved herpesvirus versus EBV-specific host cell targets. The EBV-encoded G-protein-coupled receptor BILF1 associated with MAVS and the UFM1 E3 ligase UFL1. Although UFMylation of 14-3-3 proteins drives RIG-I/MAVS signaling, BILF1-directed MAVS UFMylation instead triggered MAVS packaging into mitochondrial-derived vesicles and lysosomal proteolysis. In the absence of BILF1, EBV replication activated the NLRP3 inflammasome, which impaired viral replication and triggered pyroptosis. Our results provide a viral protein interaction network resource, reveal a UFM1-dependent pathway for selective degradation of mitochondrial cargo, and highlight BILF1 as a novel therapeutic target.

Antagonistic Inflammatory Phenotypes Dictate Tumor Fate and Response to Immune Checkpoint Blockade.

Inflammation can support or restrain cancer progression and the response to therapy. Here, we searched for primary regulators of cancer-inhibitory inflammation through deep profiling of inflammatory tumor microenvironments (TMEs) linked to immune-dependent control in mice. We found that early intratumoral accumulation of interferon gamma (IFN-γ)-producing natural killer (NK) cells induced a profound remodeling of the TME and unleashed cytotoxic T cell (CTL)-mediated tumor eradication. Mechanistically, tumor-derived prostaglandin E2 (PGE2) acted selectively on EP2 and EP4 receptors on NK cells, hampered the TME switch, and enabled immune evasion. Analysis of patient datasets across human cancers revealed distinct inflammatory TME phenotypes resembling those associated with cancer immune control versus escape in mice. This allowed us to generate a gene-expression signature that integrated opposing inflammatory factors and predicted patient survival and response to immune checkpoint blockade. Our findings identify features of the tumor inflammatory milieu associated with immune control of cancer and establish a strategy to predict immunotherapy outcomes.

Butyrophilin-2A1 Directly Binds Germline-Encoded Regions of the Vγ9Vδ2 TCR and Is Essential for Phosphoantigen Sensing.

Vγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1's P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.

Interferon-γ induces tumor resistance to anti-PD-1 immunotherapy by promoting YAP phase separation.

Interferon-γ (IFN-γ)-mediated adaptive resistance is one major barrier to improving immunotherapy in solid tumors. However, the mechanisms are not completely understood. Here, we report that IFN-γ promotes nuclear translocation and phase separation of YAP after anti-PD-1 therapy in tumor cells. Hydrophobic interactions of the YAP coiled-coil domain mediate droplet initiation, and weak interactions of the intrinsically disordered region in the C terminus promote droplet formation. YAP partitions with the transcription factor TEAD4, the histone acetyltransferase EP300, and Mediator1 and forms transcriptional hubs for maximizing target gene transcriptions, independent of the canonical STAT1-IRF1 transcription program. Disruption of YAP phase separation reduced tumor growth, enhanced immune response, and sensitized tumor cells to anti-PD-1 therapy. YAP activity is negatively correlated with patient outcome. Our study indicates that YAP mediates the IFN-γ pro-tumor effect through its nuclear phase separation and suggests that YAP can be used as a predictive biomarker and target of anti-PD-1 combination therapy.