Mucosal fungi promote gut barrier function and social behavior via Type 17 immunity.

Fungal communities (the mycobiota) are an integral part of the gut microbiota, and the disruption of their integrity contributes to local and gut-distal pathologies. Yet, the mechanisms by which intestinal fungi promote homeostasis remain unclear. We characterized the mycobiota biogeography along the gastrointestinal tract and identified a subset of fungi associated with the intestinal mucosa of mice and humans. Mucosa-associated fungi (MAF) reinforced intestinal epithelial function and protected mice against intestinal injury and bacterial infection. Notably, intestinal colonization with a defined consortium of MAF promoted social behavior in mice. The gut-local effects on barrier function were dependent on IL-22 production by CD4+ T helper cells, whereas the effects on social behavior were mediated through IL-17R-dependent signaling in neurons. Thus, the spatial organization of the gut mycobiota is associated with host-protective immunity and epithelial barrier function and might be a driver of the neuroimmune modulation of mouse behavior through complementary Type 17 immune mechanisms.

SARS-CoV-2 mRNA vaccination elicits a robust and persistent T follicular helper cell response in humans.

SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine-needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we evaluated the T cell receptor sequences and phenotype of lymph node TFH. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLA-DPB1∗04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.

Influenza vaccination stimulates maturation of the human T follicular helper cell response.

The differentiation and specificity of human CD4+ T follicular helper cells (TFH cells) after influenza vaccination have been poorly defined. Here we profiled blood and draining lymph node (LN) samples from human volunteers for over 2 years after two influenza vaccines were administered 1 year apart to define the evolution of the CD4+ TFH cell response. The first vaccination induced an increase in the frequency of circulating TFH (cTFH) and LN TFH cells at week 1 postvaccination. This increase was transient for cTFH cells, whereas the LN TFH cells further expanded during week 2 and remained elevated in frequency for at least 3 months. We observed several distinct subsets of TFH cells in the LN, including pre-TFH cells, memory TFH cells, germinal center (GC) TFH cells and interleukin-10+ TFH cell subsets beginning at baseline and at all time points postvaccination. The shift toward a GC TFH cell phenotype occurred with faster kinetics after the second vaccine compared to the first vaccine. We identified several influenza-specific TFH cell clonal lineages, including multiple responses targeting internal influenza virus proteins, and found that each TFH cell state was attainable within a clonal lineage. Thus, human TFH cells form a durable and dynamic multitissue network.

CD4+ T cells exhibit distinct transcriptional phenotypes in the lymph nodes and blood following mRNA vaccination in humans.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and mRNA vaccination induce robust CD4+ T cell responses. Using single-cell transcriptomics, here, we evaluated CD4+ T cells specific for the SARS-CoV-2 spike protein in the blood and draining lymph nodes (dLNs) of individuals 3 months and 6 months after vaccination with the BNT162b2 mRNA vaccine. We analyzed 1,277 spike-specific CD4+ T cells, including 238 defined using Trex, a deep learning-based reverse epitope mapping method to predict antigen specificity. Human dLN spike-specific CD4+ follicular helper T (TFH) cells exhibited heterogeneous phenotypes, including germinal center CD4+ TFH cells and CD4+IL-10+ TFH cells. Analysis of an independent cohort of SARS-CoV-2-infected individuals 3 months and 6 months after infection found spike-specific CD4+ T cell profiles in blood that were distinct from those detected in blood 3 months and 6 months after BNT162b2 vaccination. Our findings provide an atlas of human spike-specific CD4+ T cell transcriptional phenotypes in the dLNs and blood following SARS-CoV-2 vaccination or infection.

Human gut mycobiota tune immunity via CARD9-dependent induction of anti-fungal IgG antibodies.

Antibodies mediate natural and vaccine-induced immunity against viral and bacterial pathogens, whereas fungi represent a widespread kingdom of pathogenic species for which neither vaccine nor neutralizing antibody therapies are clinically available. Here, using a multi-kingdom antibody profiling (multiKAP) approach, we explore the human antibody repertoires against gut commensal fungi (mycobiota). We identify species preferentially targeted by systemic antibodies in humans, with Candida albicans being the major inducer of antifungal immunoglobulin G (IgG). Fungal colonization of the gut induces germinal center (GC)-dependent B cell expansion in extraintestinal lymphoid tissues and generates systemic antibodies that confer protection against disseminated C. albicans or C. auris infection. Antifungal IgG production depends on the innate immunity regulator CARD9 and CARD9+CX3CR1+ macrophages. In individuals with invasive candidiasis, loss-of-function mutations in CARD9 are associated with impaired antifungal IgG responses. These results reveal an important role of gut commensal fungi in shaping the human antibody repertoire through CARD9-dependent induction of host-protective antifungal IgG.

Identification of Near-Pan-neutralizing Antibodies against HIV-1 by Deconvolution of Plasma Humoral Responses.

Anti-HIV-1 envelope broadly neutralizing monoclonal antibodies (bNAbs) isolated from memory B cells may not fully represent HIV-1-neutralizing profiles measured in plasma. Accordingly, we characterized near-pan-neutralizing antibodies extracted directly from the plasma of two "elite neutralizers." Circulating anti-gp120 polyclonal antibodies were deconvoluted using proteomics to guide lineage analysis of bone marrow plasma cells. In both subjects, a single lineage of anti-CD4-binding site (CD4bs) antibodies explained the plasma-neutralizing activity. Importantly, members of these lineages potently neutralized 89%-100% of a multi-tier 117 pseudovirus panel, closely matching the specificity and breadth of the circulating antibodies. X-ray crystallographic analysis of one monoclonal, N49P7, suggested a unique ability to bypass the CD4bs Phe43 cavity, while reaching deep into highly conserved residues of Layer 3 of the gp120 inner domain, likely explaining its extreme potency and breadth. Further direct analyses of plasma anti-HIV-1 bNAbs should provide new insights for developing antibody-based antiviral agents and vaccines.

Immunizations with diverse sarbecovirus receptor-binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.

An Autism-Linked Mutation Disables Phosphorylation Control of UBE3A.

Deletion of UBE3A causes the neurodevelopmental disorder Angelman syndrome (AS), while duplication or triplication of UBE3A is linked to autism. These genetic findings suggest that the ubiquitin ligase activity of UBE3A must be tightly maintained to promote normal brain development. Here, we found that protein kinase A (PKA) phosphorylates UBE3A in a region outside of the catalytic domain at residue T485 and inhibits UBE3A activity toward itself and other substrates. A de novo autism-linked missense mutation disrupts this phosphorylation site, causing enhanced UBE3A activity in vitro, enhanced substrate turnover in patient-derived cells, and excessive dendritic spine development in the brain. Our study identifies PKA as an upstream regulator of UBE3A activity and shows that an autism-linked mutation disrupts this phosphorylation control. Moreover, our findings implicate excessive UBE3A activity and the resulting synaptic dysfunction to autism pathogenesis.

Targeting the adaptability of heterogeneous aneuploids.

Aneuploid genomes, characterized by unbalanced chromosome stoichiometry (karyotype), are associated with cancer malignancy and drug resistance of pathogenic fungi. The phenotypic diversity resulting from karyotypic diversity endows the cell population with superior adaptability. We show here, using a combination of experimental data and a general stochastic model, that the degree of phenotypic variation, thus evolvability, escalates with the degree of overall growth suppression. Such scaling likely explains the challenge of treating aneuploidy diseases with a single stress-inducing agent. Instead, we propose the design of an "evolutionary trap" (ET) targeting both karyotypic diversity and fitness. This strategy entails a selective condition "channeling" a karyotypically divergent population into one with a predominant and predictably drugable karyotypic feature. We provide a proof-of-principle case in budding yeast and demonstrate the potential efficacy of this strategy toward aneuploidy-based azole resistance in Candida albicans. By analyzing existing pharmacogenomics data, we propose the potential design of an ET against glioblastoma.

Optical clocks at sea.

Deployed optical clocks will improve positioning for navigational autonomy1, provide remote time standards for geophysical monitoring2 and distributed coherent sensing3, allow time synchronization of remote quantum networks4,5 and provide operational redundancy for national time standards. Although laboratory optical clocks now reach fractional inaccuracies below 10-18 (refs. 6,7), transportable versions of these high-performing clocks8,9 have limited utility because of their size, environmental sensitivity and cost10. Here we report the development of optical clocks with the requisite combination of size, performance and environmental insensitivity for operation on mobile platforms. The 35 l clock combines a molecular iodine spectrometer, fibre frequency comb and control electronics. Three of these clocks operated continuously aboard a naval ship in the Pacific Ocean for 20 days while accruing timing errors below 300 ps per day. The clocks have comparable performance to active hydrogen masers in one-tenth the volume. Operating high-performance clocks at sea has been historically challenging and continues to be critical for navigation. This demonstration marks a significant technological advancement that heralds the arrival of future optical timekeeping networks.

Probing entanglement in a 2D hard-core Bose-Hubbard lattice.

Entanglement and its propagation are central to understanding many physical properties of quantum systems1-3. Notably, within closed quantum many-body systems, entanglement is believed to yield emergent thermodynamic behaviour4-7. However, a universal understanding remains challenging owing to the non-integrability and computational intractability of most large-scale quantum systems. Quantum hardware platforms provide a means to study the formation and scaling of entanglement in interacting many-body systems8-14. Here we use a controllable 4 × 4 array of superconducting qubits to emulate a 2D hard-core Bose-Hubbard (HCBH) lattice. We generate superposition states by simultaneously driving all lattice sites and extract correlation lengths and entanglement entropy across its many-body energy spectrum. We observe volume-law entanglement scaling for states at the centre of the spectrum and a crossover to the onset of area-law scaling near its edges.

A hot-Jupiter progenitor on a super-eccentric retrograde orbit.

Giant exoplanets orbiting close to their host stars are unlikely to have formed in their present configurations1. These 'hot Jupiter' planets are instead thought to have migrated inward from beyond the ice line and several viable migration channels have been proposed, including eccentricity excitation through angular-momentum exchange with a third body followed by tidally driven orbital circularization2,3. The discovery of the extremely eccentric (e = 0.93) giant exoplanet HD 80606 b (ref. 4) provided observational evidence that hot Jupiters may have formed through this high-eccentricity tidal-migration pathway5. However, no similar hot-Jupiter progenitors have been found and simulations predict that one factor affecting the efficacy of this mechanism is exoplanet mass, as low-mass planets are more likely to be tidally disrupted during periastron passage6-8. Here we present spectroscopic and photometric observations of TIC 241249530 b, a high-mass, transiting warm Jupiter with an extreme orbital eccentricity of e = 0.94. The orbit of TIC 241249530 b is consistent with a history of eccentricity oscillations and a future tidal circularization trajectory. Our analysis of the mass and eccentricity distributions of the transiting-warm-Jupiter population further reveals a correlation between high mass and high eccentricity.

Thresholds for adding degraded tropical forest to the conservation estate.

Logged and disturbed forests are often viewed as degraded and depauperate environments compared with primary forest. However, they are dynamic ecosystems1 that provide refugia for large amounts of biodiversity2,3, so we cannot afford to underestimate their conservation value4. Here we present empirically defined thresholds for categorizing the conservation value of logged forests, using one of the most comprehensive assessments of taxon responses to habitat degradation in any tropical forest environment. We analysed the impact of logging intensity on the individual occurrence patterns of 1,681 taxa belonging to 86 taxonomic orders and 126 functional groups in Sabah, Malaysia. Our results demonstrate the existence of two conservation-relevant thresholds. First, lightly logged forests (<29% biomass removal) retain high conservation value and a largely intact functional composition, and are therefore likely to recover their pre-logging values if allowed to undergo natural regeneration. Second, the most extreme impacts occur in heavily degraded forests with more than two-thirds (>68%) of their biomass removed, and these are likely to require more expensive measures to recover their biodiversity value. Overall, our data confirm that primary forests are irreplaceable5, but they also reinforce the message that logged forests retain considerable conservation value that should not be overlooked.

A FBXO7/EYA2-SCFFBXW7 axis promotes AXL-mediated maintenance of mesenchymal and immune evasion phenotypes of cancer cells.

A mesenchymal tumor phenotype associates with immunotherapy resistance, although the mechanism is unclear. Here, we identified FBXO7 as a maintenance regulator of mesenchymal and immune evasion phenotypes of cancer cells. FBXO7 bound and stabilized SIX1 co-transcriptional regulator EYA2, stimulating mesenchymal gene expression and suppressing IFNα/ß, chemokines CXCL9/10, and antigen presentation machinery, driven by AXL extracellular ligand GAS6. Ubiquitin ligase SCFFBXW7 antagonized this pathway by promoting EYA2 degradation. Targeting EYA2 Tyr phosphatase activity decreased mesenchymal phenotypes and enhanced cancer cell immunogenicity, resulting in attenuated tumor growth and metastasis, increased infiltration of cytotoxic T and NK cells, and enhanced anti-PD-1 therapy response in mouse tumor models. FBXO7 expression correlated with mesenchymal and immune-suppressive signatures in patients with cancer. An FBXO7-immune gene signature predicted immunotherapy responses. Collectively, the FBXO7/EYA2-SCFFBXW7 axis maintains mesenchymal and immune evasion phenotypes of cancer cells, providing rationale to evaluate FBXO7/EYA2 inhibitors in combination with immune-based therapies to enhance onco-immunotherapy responses.

SARS-CoV-2 Omicron boosting induces de novo B cell response in humans.

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses and the development of vaccines aimed at the new variants1-4. SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells5-9. However, it remains unclear whether the additional doses induce germinal centre reactions whereby re-engaged B cells can further mature, and whether variant-derived vaccines can elicit responses to variant-specific epitopes. Here we show that boosting with an mRNA vaccine against the original monovalent SARS-CoV-2 mRNA vaccine or the bivalent B.1.351 and B.1.617.2 (Beta/Delta) mRNA vaccine induced robust spike-specific germinal centre B cell responses in humans. The germinal centre response persisted for at least eight weeks, leading to significantly more mutated antigen-specific bone marrow plasma cell and memory B cell compartments. Spike-binding monoclonal antibodies derived from memory B cells isolated from individuals boosted with either the original SARS-CoV-2 spike protein, bivalent Beta/Delta vaccine or a monovalent Omicron BA.1-based vaccine predominantly recognized the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted sorting approach, we isolated monoclonal antibodies that recognized the BA.1 spike protein but not the original SARS-CoV-2 spike protein from individuals who received the mRNA-1273.529 booster; these antibodies were less mutated and recognized novel epitopes within the spike protein, suggesting that they originated from naive B cells. Thus, SARS-CoV-2 booster immunizations in humans induce robust germinal centre B cell responses and can generate de novo B cell responses targeting variant-specific epitopes.

Immune regulation by fungal strain diversity in inflammatory bowel disease.

The fungal microbiota (mycobiota) is an integral part of the complex multikingdom microbial community colonizing the mammalian gastrointestinal tract and has an important role in immune regulation1-6. Although aberrant changes in the mycobiota have been linked to several diseases, including inflammatory bowel disease3-9, it is currently unknown whether fungal species captured by deep sequencing represent living organisms and whether specific fungi have functional consequences for disease development in affected individuals. Here we developed a translational platform for the functional analysis of the mycobiome at the fungal-strain- and patient-specific level. Combining high-resolution mycobiota sequencing, fungal culturomics and genomics, a CRISPR-Cas9-based fungal strain editing system, in vitro functional immunoreactivity assays and in vivo models, this platform enables the examination of host-fungal crosstalk in the human gut. We discovered a rich genetic diversity of opportunistic Candida albicans strains that dominate the colonic mucosa of patients with inflammatory bowel disease. Among these human-gut-derived isolates, strains with high immune-cell-damaging capacity (HD strains) reflect the disease features of individual patients with ulcerative colitis and aggravated intestinal inflammation in vivo through IL-1ß-dependent mechanisms. Niche-specific inflammatory immunity and interleukin-17A-producing T helper cell (TH17 cell) antifungal responses by HD strains in the gut were dependent on the C. albicans-secreted peptide toxin candidalysin during the transition from a benign commensal to a pathobiont state. These findings reveal the strain-specific nature of host-fungal interactions in the human gut and highlight new diagnostic and therapeutic targets for diseases of inflammatory origin.

Germinal centre-driven maturation of B cell response to mRNA vaccination.

Germinal centres (GC) are lymphoid structures in which B cells acquire affinity-enhancing somatic hypermutations (SHM), with surviving clones differentiating into memory B cells (MBCs) and long-lived bone marrow plasma cells1-5 (BMPCs). SARS-CoV-2 mRNA vaccination induces a persistent GC response that lasts for at least six months in humans6-8. The fate of responding GC B cells as well as the functional consequences of such persistence remain unknown. Here, we detected SARS-CoV-2 spike protein-specific MBCs in 42 individuals who had received two doses of the SARS-CoV-2 mRNA vaccine BNT162b2 six month earlier. Spike-specific IgG-secreting BMPCs were detected in 9 out of 11 participants. Using a combined approach of sequencing the B cell receptors of responding blood plasmablasts and MBCs, lymph node GC B cells and plasma cells and BMPCs from eight individuals and expression of the corresponding monoclonal antibodies, we tracked the evolution of 1,540 spike-specific B cell clones. On average, early blood spike-specific plasmablasts exhibited the lowest SHM frequencies. By contrast, SHM frequencies of spike-specific GC B cells increased by 3.5-fold within six months after vaccination. Spike-specific MBCs and BMPCs accumulated high levels of SHM, which corresponded with enhanced anti-spike antibody avidity in blood and enhanced affinity as well as neutralization capacity of BMPC-derived monoclonal antibodies. We report how the notable persistence of the GC reaction induced by SARS-CoV-2 mRNA vaccination in humans culminates in affinity-matured long-term antibody responses that potently neutralize the virus.

A TMPRSS2 inhibitor acts as a pan-SARS-CoV-2 prophylactic and therapeutic.

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced owing to emerging variants of concern1,2. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against variants of concern3,4. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs) such as TMPRSS2; these proteases cleave the viral spike protein to expose the fusion peptide for cell entry, and thus have an essential role in the virus lifecycle5,6. Here we identify and characterize a small-molecule compound, N-0385, which exhibits low nanomolar potency and a selectivity index of higher than 106 in inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids7. In Calu-3 cells it inhibits the entry of the SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Notably, in the K18-human ACE2 transgenic mouse model of severe COVID-19, we found that N-0385 affords a high level of prophylactic and therapeutic benefit after multiple administrations or even after a single administration. Together, our findings show that TTSP-mediated proteolytic maturation of the spike protein is critical for SARS-CoV-2 infection in vivo, and suggest that N-0385 provides an effective early treatment option against COVID-19 and emerging SARS-CoV-2 variants of concern.

Combination Targeted Therapy in Relapsed Diffuse Large B-Cell Lymphoma.

BACKGROUND: The identification of oncogenic mutations in diffuse large B-cell lymphoma (DLBCL) has led to the development of drugs that target essential survival pathways, but whether targeting multiple survival pathways may be curative in DLBCL is unknown. METHODS: We performed a single-center, phase 1b-2 study of a regimen of venetoclax, ibrutinib, prednisone, obinutuzumab, and lenalidomide (ViPOR) in relapsed or refractory DLBCL. In phase 1b, which included patients with DLBCL and indolent lymphomas, four dose levels of venetoclax were evaluated to identify the recommended phase 2 dose, with fixed doses of the other four drugs. A phase 2 expansion in patients with germinal-center B-cell (GCB) and non-GCB DLBCL was performed. ViPOR was administered every 21 days for six cycles. RESULTS: In phase 1b of the study, involving 20 patients (10 with DLBCL), a single dose-limiting toxic effect of grade 3 intracranial hemorrhage occurred, a result that established venetoclax at a dose of 800 mg as the recommended phase 2 dose. Phase 2 included 40 patients with DLBCL. Toxic effects that were observed among all the patients included grade 3 or 4 neutropenia (in 24% of the cycles), thrombocytopenia (in 23%), anemia (in 7%), and febrile neutropenia (in 1%). Objective responses occurred in 54% of 48 evaluable patients with DLBCL, and complete responses occurred in 38%; complete responses were exclusively in patients with non-GCB DLBCL and high-grade B-cell lymphoma with rearrangements of MYC and BCL2 or BCL6 (or both). Circulating tumor DNA was undetectable in 33% of the patients at the end of ViPOR therapy. With a median follow-up of 40 months, 2-year progression-free survival and overall survival were 34% (95% confidence interval [CI], 21 to 47) and 36% (95% CI, 23 to 49), respectively. CONCLUSIONS: Treatment with ViPOR was associated with durable remissions in patients with specific molecular DLBCL subtypes and was associated with mainly reversible adverse events. (Funded by the Intramural Research Program of the National Cancer Institute and the National Center for Advancing Translational Sciences of the National Institutes of Health and others; ClinicalTrials.gov number, NCT03223610.).

Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing.

Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.