The WDR11 complex is a receptor for acidic-cluster-containing cargo proteins.

Vesicle trafficking is a fundamental process that allows for the sorting and transport of specific proteins (i.e., "cargoes") to different compartments of eukaryotic cells. Cargo recognition primarily occurs through coats and the associated proteins at the donor membrane. However, it remains unclear whether cargoes can also be selected at other stages of vesicle trafficking to further enhance the fidelity of the process. The WDR11-FAM91A1 complex functions downstream of the clathrin-associated AP-1 complex to facilitate protein transport from endosomes to the TGN. Here, we report the cryo-EM structure of human WDR11-FAM91A1 complex. WDR11 directly and specifically recognizes a subset of acidic clusters, which we term super acidic clusters (SACs). WDR11 complex assembly and its binding to SAC-containing proteins are indispensable for the trafficking of SAC-containing proteins and proper neuronal development in zebrafish. Our studies thus uncover that cargo proteins could be recognized in a sequence-specific manner downstream of a protein coat.

An ancestral mycobacterial effector promotes dissemination of infection.

The human pathogen Mycobacterium tuberculosis typically causes lung disease but can also disseminate to other tissues. We identified a M. tuberculosis (Mtb) outbreak presenting with unusually high rates of extrapulmonary dissemination and bone disease. We found that the causal strain carried an ancestral full-length version of the type VII-secreted effector EsxM rather than the truncated version present in other modern Mtb lineages. The ancestral EsxM variant exacerbated dissemination through enhancement of macrophage motility, increased egress of macrophages from established granulomas, and alterations in macrophage actin dynamics. Reconstitution of the ancestral version of EsxM in an attenuated modern strain of Mtb altered the migratory mode of infected macrophages, enhancing their motility. In a zebrafish model, full-length EsxM promoted bone disease. The presence of a derived nonsense variant in EsxM throughout the major Mtb lineages 2, 3, and 4 is consistent with a role for EsxM in regulating the extent of dissemination.

A type 1 immunity-restricted promoter of the IL-33 receptor gene directs antiviral T-cell responses.

The pleiotropic alarmin interleukin-33 (IL-33) drives type 1, type 2 and regulatory T-cell responses via its receptor ST2. Subset-specific differences in ST2 expression intensity and dynamics suggest that transcriptional regulation is key in orchestrating the context-dependent activity of IL-33-ST2 signaling in T-cell immunity. Here, we identify a previously unrecognized alternative promoter in mice and humans that is located far upstream of the curated ST2-coding gene and drives ST2 expression in type 1 immunity. Mice lacking this promoter exhibit a selective loss of ST2 expression in type 1- but not type 2-biased T cells, resulting in impaired expansion of cytotoxic T cells (CTLs) and T-helper 1 cells upon viral infection. T-cell-intrinsic IL-33 signaling via type 1 promoter-driven ST2 is critical to generate a clonally diverse population of antiviral short-lived effector CTLs. Thus, lineage-specific alternative promoter usage directs alarmin responsiveness in T-cell subsets and offers opportunities for immune cell-specific targeting of the IL-33-ST2 axis in infections and inflammatory diseases.

Human serous cavity macrophages and dendritic cells possess counterparts in the mouse with a distinct distribution between species.

In mouse peritoneal and other serous cavities, the transcription factor GATA6 drives the identity of the major cavity resident population of macrophages, with a smaller subset of cavity-resident macrophages dependent on the transcription factor IRF4. Here we showed that GATA6+ macrophages in the human peritoneum were rare, regardless of age. Instead, more human peritoneal macrophages aligned with mouse CD206+ LYVE1+ cavity macrophages that represent a differentiation stage just preceding expression of GATA6. A low abundance of CD206+ macrophages was retained in C57BL/6J mice fed a high-fat diet and in wild-captured mice, suggesting that differences between serous cavity-resident macrophages in humans and mice were not environmental. IRF4-dependent mouse serous cavity macrophages aligned closely with human CD1c+CD14+CD64+ peritoneal cells, which, in turn, resembled human peritoneal CD1c+CD14-CD64- cDC2. Thus, major populations of serous cavity-resident mononuclear phagocytes in humans and mice shared common features, but the proportions of different macrophage differentiation stages greatly differ between the two species, and dendritic cell (DC2)-like cells were especially prominent in humans.

Therapy-Induced Evolution of Human Lung Cancer Revealed by Single-Cell RNA Sequencing.

Lung cancer, the leading cause of cancer mortality, exhibits heterogeneity that enables adaptability, limits therapeutic success, and remains incompletely understood. Single-cell RNA sequencing (scRNA-seq) of metastatic lung cancer was performed using 49 clinical biopsies obtained from 30 patients before and during targeted therapy. Over 20,000 cancer and tumor microenvironment (TME) single-cell profiles exposed a rich and dynamic tumor ecosystem. scRNA-seq of cancer cells illuminated targetable oncogenes beyond those detected clinically. Cancer cells surviving therapy as residual disease (RD) expressed an alveolar-regenerative cell signature suggesting a therapy-induced primitive cell-state transition, whereas those present at on-therapy progressive disease (PD) upregulated kynurenine, plasminogen, and gap-junction pathways. Active T-lymphocytes and decreased macrophages were present at RD and immunosuppressive cell states characterized PD. Biological features revealed by scRNA-seq were biomarkers of clinical outcomes in independent cohorts. This study highlights how therapy-induced adaptation of the multi-cellular ecosystem of metastatic cancer shapes clinical outcomes.

LSD1 Ablation Stimulates Anti-tumor Immunity and Enables Checkpoint Blockade.

Chromatin regulators play a broad role in regulating gene expression and, when gone awry, can lead to cancer. Here, we demonstrate that ablation of the histone demethylase LSD1 in cancer cells increases repetitive element expression, including endogenous retroviral elements (ERVs), and decreases expression of RNA-induced silencing complex (RISC) components. Significantly, this leads to double-stranded RNA (dsRNA) stress and activation of type 1 interferon, which stimulates anti-tumor T cell immunity and restrains tumor growth. Furthermore, LSD1 depletion enhances tumor immunogenicity and T cell infiltration in poorly immunogenic tumors and elicits significant responses of checkpoint blockade-refractory mouse melanoma to anti-PD-1 therapy. Consistently, TCGA data analysis shows an inverse correlation between LSD1 expression and CD8+ T cell infiltration in various human cancers. Our study identifies LSD1 as a potent inhibitor of anti-tumor immunity and responsiveness to immunotherapy and suggests LSD1 inhibition combined with PD-(L)1 blockade as a novel cancer treatment strategy.

Neurons under T Cell Attack Coordinate Phagocyte-Mediated Synaptic Stripping.

Inflammatory disorders of the CNS are frequently accompanied by synaptic loss, which is thought to involve phagocytic microglia and complement components. However, the mechanisms accounting for aberrant synaptic connectivity in the context of CD8+ T cell-driven neuronal damage are poorly understood. Here, we profiled the neuronal translatome in a murine model of encephalitis caused by CD8+ T cells targeting antigenic neurons. Neuronal STAT1 signaling and downstream CCL2 expression were essential for apposition of phagocytes, ensuing synaptic loss and neurological disease. Analogous observations were made in the brains of Rasmussen's encephalitis patients. In this devastating CD8+ T cell-driven autoimmune disease, neuronal STAT1 phosphorylation and CCL2 expression co-clustered with infiltrating CD8+ T cells as well as phagocytes. Taken together, our findings uncover an active role of neurons in coordinating phagocyte-mediated synaptic loss and highlight neuronal STAT1 and CCL2 as critical steps in this process that are amenable to pharmacological interventions.

The alarmin interleukin-33 promotes the expansion and preserves the stemness of Tcf-1+ CD8+ T cells in chronic viral infection.

T cell factor 1 (Tcf-1) expressing CD8+ T cells exhibit stem-like self-renewing capacity, rendering them key for immune defense against chronic viral infection and cancer. Yet, the signals that promote the formation and maintenance of these stem-like CD8+ T cells (CD8+SL) remain poorly defined. Studying CD8+ T cell differentiation in mice with chronic viral infection, we identified the alarmin interleukin-33 (IL-33) as pivotal for the expansion and stem-like functioning of CD8+SL as well as for virus control. IL-33 receptor (ST2)-deficient CD8+ T cells exhibited biased end differentiation and premature loss of Tcf-1. ST2-deficient CD8+SL responses were restored by blockade of type I interferon signaling, suggesting that IL-33 balances IFN-I effects to control CD8+SL formation in chronic infection. IL-33 signals broadly augmented chromatin accessibility in CD8+SL and determined these cells' re-expansion potential. Our study identifies the IL-33-ST2 axis as an important CD8+SL-promoting pathway in the context of chronic viral infection.

Molecular and Circuit-Dynamical Identification of Top-Down Neural Mechanisms for Restraint of Reward Seeking.

Reward-seeking behavior is fundamental to survival, but suppression of this behavior can be essential as well, even for rewards of high value. In humans and rodents, the medial prefrontal cortex (mPFC) has been implicated in suppressing reward seeking; however, despite vital significance in health and disease, the neural circuitry through which mPFC regulates reward seeking remains incompletely understood. Here, we show that a specific subset of superficial mPFC projections to a subfield of nucleus accumbens (NAc) neurons naturally encodes the decision to initiate or suppress reward seeking when faced with risk of punishment. A highly resolved subpopulation of these top-down projecting neurons, identified by 2-photon Ca2+ imaging and activity-dependent labeling to recruit the relevant neurons, was found capable of suppressing reward seeking. This natural activity-resolved mPFC-to-NAc projection displayed unique molecular-genetic and microcircuit-level features concordant with a conserved role in the regulation of reward-seeking behavior, providing cellular and anatomical identifiers of behavioral and possible therapeutic significance.

Apoptotic cell-induced AhR activity is required for immunological tolerance and suppression of systemic lupus erythematosus in mice and humans.

The transcription factor AhR modulates immunity at multiple levels. Here we report that phagocytes exposed to apoptotic cells exhibited rapid activation of AhR, which drove production of the cytokine IL-10. Activation of AhR was dependent on interactions between apoptotic-cell DNA and the pattern-recognition receptor TLR9 that was required for the prevention of immune responses to DNA and histones in vivo. Moreover, disease progression in mouse systemic lupus erythematosus (SLE) correlated with strength of the AhR signal, and the disease course could be altered by modulation of AhR activity. Deletion of AhR in the myeloid lineage caused systemic autoimmunity in mice, and an enhanced AhR transcriptional signature correlated with disease in patients with SLE. Thus, AhR activity induced by apoptotic cell phagocytes maintains peripheral tolerance.

Spliceosome-Targeted Therapies Induce dsRNA Responses.

In a recent issue of Cell, Bowling et al. describe a mechanism by which spliceosome-targeted therapies result in intron-containing transcripts that form double-stranded RNAs (dsRNAs), thereby activating tumor antiviral signaling (viral mimicry) and downstream adaptive immunity.

DNA-Demethylating Agents Target Colorectal Cancer Cells by Inducing Viral Mimicry by Endogenous Transcripts.

DNA-demethylating agents have shown clinical anti-tumor efficacy via an unknown mechanism of action. Using a combination of experimental and bioinformatics analyses in colorectal cancer cells, we demonstrate that low-dose 5-AZA-CdR targets colorectal cancer-initiating cells (CICs) by inducing viral mimicry. This is associated with induction of dsRNAs derived at least in part from endogenous retroviral elements, activation of the MDA5/MAVS RNA recognition pathway, and downstream activation of IRF7. Indeed, disruption of virus recognition pathways, by individually knocking down MDA5, MAVS, or IRF7, inhibits the ability of 5-AZA-CdR to target colorectal CICs and significantly decreases 5-AZA-CdR long-term growth effects. Moreover, transfection of dsRNA into CICs can mimic the effects of 5-AZA-CdR. Together, our results represent a major shift in understanding the anti-tumor mechanisms of DNA-demethylating agents and highlight the MDA5/MAVS/IRF7 pathway as a potentially druggable target against CICs.

The FUS::DDIT3 fusion oncoprotein inhibits BAF complex targeting and activity in myxoid liposarcoma.

Mammalian SWI/SNF (mSWI/SNF or BAF) ATP-dependent chromatin remodeling complexes play critical roles in governing genomic architecture and gene expression and are frequently perturbed in human cancers. Transcription factors (TFs), including fusion oncoproteins, can bind to BAF complex surfaces to direct chromatin targeting and accessibility, often activating oncogenic gene loci. Here, we demonstrate that the FUS::DDIT3 fusion oncoprotein hallmark to myxoid liposarcoma (MLPS) inhibits BAF complex-mediated remodeling of adipogenic enhancer sites via sequestration of the adipogenic TF, CEBPB, from the genome. In mesenchymal stem cells, small-molecule inhibition of BAF complex ATPase activity attenuates adipogenesis via failure of BAF-mediated DNA accessibility and gene activation at CEBPB target sites. BAF chromatin occupancy and gene expression profiles of FUS::DDIT3-expressing cell lines and primary tumors exhibit similarity to SMARCB1-deficient tumor types. These data present a mechanism by which a fusion oncoprotein generates a BAF complex loss-of-function phenotype, independent of deleterious subunit mutations.

Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7R-Dependent STING Activation by Tumor-Derived cGAMP.

Clearance of apoptotic cells by macrophages prevents excessive inflammation and supports immune tolerance. Here, we examined the effect of blocking apoptotic cell clearance on anti-tumor immune response. We generated an antibody that selectively inhibited efferocytosis by phagocytic receptor MerTK. Blockade of MerTK resulted in accumulation of apoptotic cells within tumors and triggered a type I interferon response. Treatment of tumor-bearing mice with anti-MerTK antibody stimulated T cell activation and synergized with anti-PD-1 or anti-PD-L1 therapy. The anti-tumor effect induced by anti-MerTK treatment was lost in Stinggt/gt mice, but not in Cgas-/- mice. Abolishing cGAMP production in Cgas-/- tumor cells, depletion of extracellular ATP, or inactivation of the ATP-gated P2X7R channel also compromised the effects of MerTK blockade. Mechanistically, extracellular ATP acted via P2X7R to enhance the transport of extracellular cGAMP into macrophages and subsequent STING activation. Thus, MerTK blockade increases tumor immunogenicity and potentiates anti-tumor immunity, which has implications for cancer immunotherapy.

No thick carbon dioxide atmosphere on the rocky exoplanet TRAPPIST-1 c.

Seven rocky planets orbit the nearby dwarf star TRAPPIST-1, providing a unique opportunity to search for atmospheres on small planets outside the Solar System1. Thanks to the recent launch of the James Webb Space Telescope (JWST), possible atmospheric constituents such as carbon dioxide (CO2) are now detectable2,3. Recent JWST observations of the innermost planet TRAPPIST-1 b showed that it is most probably a bare rock without any CO2 in its atmosphere4. Here we report the detection of thermal emission from the dayside of TRAPPIST-1 c with the Mid-Infrared Instrument (MIRI) on JWST at 15 µm. We measure a planet-to-star flux ratio of fp/f⁎ = 421 ± 94 parts per million (ppm), which corresponds to an inferred dayside brightness temperature of 380 ± 31 K. This high dayside temperature disfavours a thick, CO2-rich atmosphere on the planet. The data rule out cloud-free O2/CO2 mixtures with surface pressures ranging from 10 bar (with 10 ppm CO2) to 0.1 bar (pure CO2). A Venus-analogue atmosphere with sulfuric acid clouds is also disfavoured at 2.6σ confidence. Thinner atmospheres or bare-rock surfaces are consistent with our measured planet-to-star flux ratio. The absence of a thick, CO2-rich atmosphere on TRAPPIST-1 c suggests a relatively volatile-poor formation history, with less than [Formula: see text] Earth oceans of water. If all planets in the system formed in the same way, this would indicate a limited reservoir of volatiles for the potentially habitable planets in the system.

Autonomous rhythmic activity in glioma networks drives brain tumour growth.

Diffuse gliomas, particularly glioblastomas, are incurable brain tumours1. They are characterized by networks of interconnected brain tumour cells that communicate via Ca2+ transients2-6. However, the networks' architecture and communication strategy and how these influence tumour biology remain unknown. Here we describe how glioblastoma cell networks include a small, plastic population of highly active glioblastoma cells that display rhythmic Ca2+ oscillations and are particularly connected to others. Their autonomous periodic Ca2+ transients preceded Ca2+ transients of other network-connected cells, activating the frequency-dependent MAPK and NF-κB pathways. Mathematical network analysis revealed that glioblastoma network topology follows scale-free and small-world properties, with periodic tumour cells frequently located in network hubs. This network design enabled resistance against random damage but was vulnerable to losing its key hubs. Targeting of autonomous rhythmic activity by selective physical ablation of periodic tumour cells or by genetic or pharmacological interference with the potassium channel KCa3.1 (also known as IK1, SK4 or KCNN4) strongly compromised global network communication. This led to a marked reduction of tumour cell viability within the entire network, reduced tumour growth in mice and extended animal survival. The dependency of glioblastoma networks on periodic Ca2+ activity generates a vulnerability7 that can be exploited for the development of novel therapies, such as with KCa3.1-inhibiting drugs.

DNA hypomethylating agents increase activation and cytolytic activity of CD8+ T cells.

We demonstrate that DNA hypomethylating agent (HMA) treatment can directly modulate the anti-tumor response and effector function of CD8+ T cells. In vivo HMA treatment promotes CD8+ T cell tumor infiltration and suppresses tumor growth via CD8+ T cell-dependent activity. Ex vivo, HMAs enhance primary human CD8+ T cell activation markers, effector cytokine production, and anti-tumor cytolytic activity. Epigenomic and transcriptomic profiling shows that HMAs vastly regulate T cell activation-related transcriptional networks, culminating with over-activation of NFATc1 short isoforms. Mechanistically, demethylation of an intragenic CpG island immediately downstream to the 3' UTR of the short isoform was associated with antisense transcription and alternative polyadenylation of NFATc1 short isoforms. High-dimensional single-cell mass cytometry analyses reveal a selective effect of HMAs on a subset of human CD8+ T cell subpopulations, increasing both the number and abundance of a granzyme Bhigh, perforinhigh effector subpopulation. Overall, our findings support the use of HMAs as a therapeutic strategy to boost anti-tumor immune response.

Yeast TLDc domain proteins regulate assembly state and subcellular localization of the V-ATPase.

Yeast vacuoles perform crucial cellular functions as acidic degradative organelles, storage compartments, and signaling hubs. These functions are mediated by important protein complexes, including the vacuolar-type H+-ATPase (V-ATPase), responsible for organelle acidification. To gain a more detailed understanding of vacuole function, we performed cross-linking mass spectrometry on isolated vacuoles, detecting many known as well as novel protein-protein interactions. Among these, we identified the uncharacterized TLDc-domain-containing protein Rtc5 as a novel interactor of the V-ATPase. We further analyzed the influence of Rtc5 and of Oxr1, the only other yeast TLDc-domain-containing protein, on V-ATPase function. We find that both Rtc5 and Oxr1 promote the disassembly of the vacuolar V-ATPase in vivo, counteracting the role of the RAVE complex, a V-ATPase assembly chaperone. Furthermore, Oxr1 is necessary for the retention of a Golgi-specific subunit of the V-ATPase in this compartment. Collectively, our results shed light on the in vivo roles of yeast TLDc-domain proteins as regulators of the V-ATPase, highlighting the multifaceted regulation of this crucial protein complex.

Regulation of WASH-dependent actin polymerization and protein trafficking by ubiquitination.

Endosomal protein trafficking is an essential cellular process that is deregulated in several diseases and targeted by pathogens. Here, we describe a role for ubiquitination in this process. We find that the E3 RING ubiquitin ligase, MAGE-L2-TRIM27, localizes to endosomes through interactions with the retromer complex. Knockdown of MAGE-L2-TRIM27 or the Ube2O E2 ubiquitin-conjugating enzyme significantly impaired retromer-mediated transport. We further demonstrate that MAGE-L2-TRIM27 ubiquitin ligase activity is required for nucleation of endosomal F-actin by the WASH regulatory complex, a known regulator of retromer-mediated transport. Mechanistic studies showed that MAGE-L2-TRIM27 facilitates K63-linked ubiquitination of WASH K220. Significantly, disruption of WASH ubiquitination impaired endosomal F-actin nucleation and retromer-dependent transport. These findings provide a cellular and molecular function for MAGE-L2-TRIM27 in retrograde transport, including an unappreciated role of K63-linked ubiquitination and identification of an activating signal of the WASH regulatory complex.

A population of ultraviolet-dim protoclusters detected in absorption.

Galaxy protoclusters, which will eventually grow into the massive clusters we see in the local Universe, are usually traced by locating overdensities of galaxies1. Large spectroscopic surveys of distant galaxies now exist, but their sensitivity depends mainly on a galaxy's star-formation activity and dust content rather than its mass. Tracers of massive protoclusters that do not rely on their galaxy constituents are therefore needed. Here we report observations of Lyman-α absorption in the spectra of a dense grid of background galaxies2,3, which we use to locate a substantial number of candidate protoclusters at redshifts 2.2 to 2.8 through their intergalactic gas. We find that the structures producing the most absorption, most of which were previously unknown, contain surprisingly few galaxies compared with the dark-matter content of their analogues in cosmological simulations4,5. Nearly all of the structures are expected to be protoclusters, and we infer that half of their expected galaxy members are missing from our survey because they are unusually dim at rest-frame ultraviolet wavelengths. We attribute this to an unexpectedly strong and early influence of the protocluster environment6,7 on the evolution of these galaxies that reduced their star formation or increased their dust content.