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This paper describes a survey of the use of the electronic information floor (EIF) located in Glasgow Caledonian University's Caledonian Library and Information Centre. The survey used both observational and questionnaire based methods and builds on a previous study which used focus groups and semi-structured interviews. The study was divided into two parts: an observational study and a questionnaire based study. The observational study found word processing, sending and receiving e-mail, and Web browsing to be the most common activities. The more substantial part of the study was questionnaire based, the questionnaire being administered both on paper and electronically. The main findings were: most respondents were full-time students: most respondents were PC rather than Mac users; only 18 per cent use CD-ROMs and only about 13 per cent use online databases. About a third had problems in using the EIF. The main overall conclusions are that information searching is a minority activity and that the volume of non-curricular activity is substantial but unquantified. [ABSTRACT FROM AUTHOR]

Purpose: Cancer and Leukemia Group B (CALGB) 30105 tested two different concurrent chemoradiotherapy platforms with high-dose (74 Gy) three-dimensional conformal radiotherapy (3D-CRT) after two cycles of induction chemotherapy for Stage IIIA/IIIB non-small cell lung cancer (NSCLC) patients to determine if either could achieve a primary endpoint of >18-month median survival. Final results of 30105 demonstrated that induction carboplatin and gemcitabine and concurrent gemcitabine 3D-CRT was not feasible because of treatment-related toxicity. However, induction and concurrent carboplatin/paclitaxel with 74 Gy 3D-CRT had a median survival of 24 months, and is the basis for the experimental arm in CALGB 30610/RTOG 0617/N0628. We conducted a secondary analysis of all patients to determine predictors of treatment-related pulmonary toxicity.Methods and Materials: Patient, tumor, and treatment-related variables were analyzed to determine their relation with treatment-related pulmonary toxicity.Results: Older age, higher N stage, larger planning target volume (PTV)1, smaller total lung volume/PTV1 ratio, larger V20, and larger mean lung dose were associated with increasing pulmonary toxicity on univariate analysis. Multivariate analysis confirmed that V20 and nodal stage as well as treatment with concurrent gemcitabine were associated with treatment-related toxicity. A high-risk group comprising patients with N3 disease and V20 >38% was associated with 80% of Grades 3-5 pulmonary toxicity cases.Conclusions: Elevated V20 and N3 disease status are important predictors of treatment related pulmonary toxicity in patients treated with high-dose 3D-CRT and concurrent chemotherapy. Further studies may use these metrics in considering patients for these treatments. [ABSTRACT FROM AUTHOR]

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 (T FH ) cells exhibited heterogeneous phenotypes, including germinal center CD4 + T FH cells and CD4 + IL-10 + T FH 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., (© 2024. Springer Nature America, Inc.)

The differentiation and specificity of human CD4 + T follicular helper cells (T FH 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 + T FH cell response. The first vaccination induced an increase in the frequency of circulating T FH (cT FH ) and LN T FH cells at week 1 postvaccination. This increase was transient for cT FH cells, whereas the LN T FH cells further expanded during week 2 and remained elevated in frequency for at least 3 months. We observed several distinct subsets of T FH cells in the LN, including pre-T FH cells, memory T FH cells, germinal center (GC) T FH cells and interleukin-10 + T FH cell subsets beginning at baseline and at all time points postvaccination. The shift toward a GC T FH cell phenotype occurred with faster kinetics after the second vaccine compared to the first vaccine. We identified several influenza-specific T FH cell clonal lineages, including multiple responses targeting internal influenza virus proteins, and found that each T FH cell state was attainable within a clonal lineage. Thus, human T FH cells form a durable and dynamic multitissue network., (© 2024. The Author(s).)

Respiratory infections cause significant morbidity and mortality, yet it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning fatal disease. Transcriptomics strongly linked oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating fatty acid production, with fatal A(H7N9) early after hospital admission, persisting until death. Recovered patients had low OLAH expression throughout hospitalization. High OLAH levels were also detected in patients hospitalized with life-threatening seasonal influenza, COVID-19, respiratory syncytial virus (RSV), and multisystem inflammatory syndrome in children (MIS-C) but not during mild disease. In olah -/- mice, lethal influenza infection led to survival and mild disease as well as reduced lung viral loads, tissue damage, infection-driven pulmonary cell infiltration, and inflammation. This was underpinned by differential lipid droplet dynamics as well as reduced viral replication and virus-induced inflammation in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza replication in macrophages and their inflammatory potential. Our findings define how the expression of OLAH drives life-threatening viral disease., Competing Interests: Declaration of interests H.A.M. and B.Y.C. consult for Ena Respiratory. A.G.R. received research support from Illumina. P.G.T. is on the SAB of Immunoscape and Cytoagents, consulted for JNJ, received travel support/honoraria from Illumina and 10× Genomics, and has patents related to TCR discovery. J.C.C. and P.G.T. have patents related to treating or reducing severity of viral infections, including SARS-CoV-2., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intraocular pressure (IOP) and cerebrospinal fluid pressure (CSFP) telemetry in large animal models can be used to determine the exact IOP, CSFP, translaminar pressure, and translaminar pressure gradient exposure that each normal and treated eye is subjected to relative to its fellow eye. In this way, it is possible to determine the independent contributions of each of these parameters (mean and/or transient fluctuations) to the risk of both the onset and rate of progression of glaucoma. Importantly, we have shown that IOP and CSFP fluctuate continuously by up to 100% over the course of the day, so snapshot cage-side IOP measurements are unable to adequately capture the pressure in the eye; CSFP is not measurable noninvasively at all. Implementation of IOP and CSFP telemetry will allow us to precisely determine the pressure insult in each eye of each animal and thereby unravel the true mechanisms underlying pressure-induced damage to the retinal ganglion cells in glaucoma., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Perturbation of the apoptosis and necroptosis pathways critically influences embryogenesis. Receptor-associated protein kinase-1 (RIPK1) interacts with Fas-associated via death domain (FADD)-caspase-8-cellular Flice-like inhibitory protein long (cFLIP L ) to regulate both extrinsic apoptosis and necroptosis. Here, we describe Ripk1-mutant animals (Ripk1 R588E [RE]) in which the interaction between FADD and RIPK1 is disrupted, leading to embryonic lethality. This lethality is not prevented by further removal of the kinase activity of Ripk1 (Ripk1 R588E K45A [REKA]). Both Ripk1 RE and Ripk1 REKA animals survive to adulthood upon ablation of Ripk3. While embryonic lethality of Ripk1 RE mice is prevented by ablation of the necroptosis effector mixed lineage kinase-like (MLKL), animals succumb to inflammation after birth. In contrast, Mlkl ablation does not prevent the death of Ripk1 REKA embryos, but animals reach adulthood when both MLKL and caspase-8 are removed. Ablation of the nucleic acid sensor Zbp1 largely prevents lethality in both Ripk1 RE and Ripk1 REKA embryos. Thus, the RIPK1-FADD interaction prevents Z-DNA binding protein-1 (ZBP1)-induced, RIPK3-caspase-8-mediated embryonic lethality, affected by the kinase activity of RIPK1., Competing Interests: Declaration of interests D.R.G. consulted for Sonata Pharmaceuticals, Ventus Pharmaceuticals, and ASHA Therapeutics and received grant support from Amgen during this research., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome 1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection 6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Fibrolamellar carcinoma (FLC) is a liver tumor with a high mortality burden and few treatment options. A promising therapeutic vulnerability in FLC is its driver mutation, a conserved DNAJB1-PRKACA gene fusion that could be an ideal target neoantigen for immunotherapy. In this study, we aim to define endogenous CD8 T cell responses to this fusion in FLC patients and evaluate fusion-specific T cell receptors (TCRs) for use in cellular immunotherapies. We observe that fusion-specific CD8 T cells are rare and that FLC patient TCR repertoires lack large clusters of related TCR sequences characteristic of potent antigen-specific responses, potentially explaining why endogenous immune responses are insufficient to clear FLC tumors. Nevertheless, we define two functional fusion-specific TCRs, one of which has strong anti-tumor activity in vivo. Together, our results provide insights into the fragmented nature of neoantigen-specific repertoires in humans and indicate routes for clinical development of successful immunotherapies for FLC., Competing Interests: Declaration of interests J.C.C., A.M.K., A.E.Z., S.E.S., and P.G.T. have a patent application for TCRs for treating FLC. J.C.C. has additional patent applications in the field of immunotherapy. A.W.P. is a member of the scientific advisory board (SAB) of Bioinformatics Solutions Inc. (Canada), shareholder and SAB member of Evaxion Biotech (Denmark), consultant for Grey Wolf Therapeutics (UK), and cofounder of Resseptor Therapeutics (Melbourne). S.G. is a co-inventor on patent applications in the fields of cell/gene therapy for cancer, consultant of TESSA Therapeutics, member of the Data and Safety Monitoring Board of Immatics, and SAB member of Be Biopharma and has received honoraria from Tidal, Catamaran Bio, and Sanofi within the last 2 years. S.E.S. is a cofounder, stockholder, and paid consultant for Gliknik Inc., serves on the SAB and holds stock options for Virion Inc., and receives royalties from the Mayo Clinic for licensed IP surrounding manipulation of the PD-1:PD-L1 pathway for cancer treatment. P.G.T. is on the SAB of Immunoscape and Shennon Bio, received personal fees and research support from Elevate Bio, and consulted for 10×, Illumina, Pfizer, Cytoagents, and JNJ., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Understanding the intricate dynamics between adoptively transferred immune cells and the brain tumor immune microenvironment (TIME) is crucial for the development of effective T cell-based immunotherapies. In this study, we investigated the influence of the TIME and chimeric antigen receptor (CAR) design on the anti-glioma activity of B7-H3-specific CAR T-cells. Using an immunocompetent glioma model, we evaluated a panel of seven fully murine B7-H3 CARs with variations in transmembrane, costimulatory, and activation domains. We then investigated changes in the TIME following CAR T-cell therapy using high-dimensional flow cytometry and single-cell RNA sequencing. Our results show that five out of six B7-H3 CARs with single costimulatory domains demonstrated robust functionality in vitro. However, these CARs had significantly varied levels of antitumor activity in vivo. To enhance therapeutic effectiveness and persistence, we incorporated 41BB and CD28 costimulation through transgenic expression of 41BBL on CD28-based CAR T-cells. This CAR design was associated with significantly improved anti-glioma efficacy in vitro but did not result in similar improvements in vivo. Analysis of the TIME revealed that CAR T-cell therapy influenced the composition of the TIME, with the recruitment and activation of distinct macrophage and endogenous T-cell subsets crucial for successful antitumor responses. Indeed, complete brain macrophage depletion using a CSF1R inhibitor abrogated CAR T-cell antitumor activity. In sum, our study highlights the critical role of CAR design and its modulation of the TIME in mediating the efficacy of adoptive immunotherapy for high-grade glioma., Significance: CAR T-cell immunotherapies hold great potential for treating brain cancers; however, they are hindered by a challenging immune environment that dampens their effectiveness. In this study, we show that the CAR design influences the makeup of the immune environment in brain tumors, underscoring the need to target specific immune components to improve CAR T-cell performance, and highlighting the significance of using models with functional immune systems to optimize this therapy., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are understudied. For example, we lack a comprehensive understanding of the mechanisms of toxicities observed in patients receiving T cell therapies, including recent reports of encephalitis caused by reactivation of human herpesvirus 6 (HHV-6) 1 . Here, through petabase-scale viral genomics mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in cultures of human CD4 + T cells. Using single-cell sequencing, we identify a rare population of HHV-6 'super-expressors' (about 1 in 300-10,000 cells) that possess high viral transcriptional activity, among research-grade allogeneic chimeric antigen receptor (CAR) T cells. By analysing single-cell sequencing data from patients receiving cell therapy products that are approved by the US Food and Drug Administration 2 or are in clinical studies 3-5 , we identify the presence of HHV-6-super-expressor CAR T cells in patients in vivo. Together, the findings of our study demonstrate the utility of comprehensive genomics analyses in implicating cell therapy products as a potential source contributing to the lytic HHV-6 infection that has been reported in clinical trials 1,6-8 and may influence the design and production of autologous and allogeneic cell therapies., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

CD8 + T cells provide robust antiviral immunity, but how epitope-specific T cells evolve across the human lifespan is unclear. Here we defined CD8 + T cell immunity directed at the prominent influenza epitope HLA-A*02:01-M1 58-66 (A2/M1 58 ) across four age groups at phenotypic, transcriptomic, clonal and functional levels. We identify a linear differentiation trajectory from newborns to children then adults, followed by divergence and a clonal reset in older adults. Gene profiles in older adults closely resemble those of newborns and children, despite being clonally distinct. Only child-derived and adult-derived A2/M1 58 + CD8 + T cells had the potential to differentiate into highly cytotoxic epitope-specific CD8 + T cells, which was linked to highly functional public T cell receptor (TCR)αβ signatures. Suboptimal TCRαβ signatures in older adults led to less proliferation, polyfunctionality, avidity and recognition of peptide mutants, although displayed no signs of exhaustion. These data suggest that priming T cells at different stages of life might greatly affect CD8 + T cell responses toward viral infections., (© 2023. The Author(s).)

Human astrovirus is a positive-sense, single-stranded RNA virus. Astrovirus infection causes gastrointestinal symptoms and can lead to encephalitis in immunocompromised patients. Positive-strand RNA viruses typically utilize host intracellular membranes to form replication organelles, which are potential antiviral targets. Many of these replication organelles are double-membrane vesicles (DMVs). Here, we show that astrovirus infection leads to an increase in DMV formation through a replication-dependent mechanism that requires some early components of the autophagy machinery. Results indicate that the upstream class III phosphatidylinositol 3-kinase (PI3K) complex, but not LC3 conjugation machinery, is utilized in DMV formation. Both chemical and genetic inhibition of the PI3K complex lead to significant reduction in DMVs, as well as viral replication. Elucidating the role of autophagy machinery in DMV formation during astrovirus infection reveals a potential target for therapeutic intervention for immunocompromised patients. IMPORTANCE These studies provide critical new evidence that astrovirus replication requires formation of double-membrane vesicles, which utilize class III phosphatidylinositol 3-kinase (PI3K), but not LC3 conjugation autophagy machinery, for biogenesis. These results are consistent with replication mechanisms for other positive-sense RNA viruses suggesting that targeting PI3K could be a promising therapeutic option for not only astrovirus, but other positive-sense RNA virus infections., Competing Interests: The authors declare no conflict of interest.

Astroviruses cause a spectrum of diseases spanning asymptomatic infections to severe diarrhea, but little is understood about their pathogenesis. We previously determined that small intestinal goblet cells were the main cell type infected by murine astrovirus-1. Here, we focused on the host immune response to infection and inadvertently discovered a role for indoleamine 2,3-dioxygenase 1 (Ido1), a host tryptophan catabolizing enzyme, in the cellular tropism of murine and human astroviruses. We identified that Ido1 expression was highly enriched among infected goblet cells, and spatially corresponded to the zonation of infection. Because Ido1 can act as a negative regulator of inflammation, we hypothesized it could dampen host antiviral responses. Despite robust interferon signaling in goblet cells, as well as tuft cell and enterocyte bystanders, we observed delayed cytokine induction and suppressed levels of fecal lipocalin-2. Although we found Ido -/- animals were more resistant to infection, this was not associated with fewer goblet cells nor could it be rescued by knocking out interferon responses, suggesting that IDO1 instead regulates cell permissivity. We characterized IDO1 -/- Caco-2 cells and observed significantly reduced human astrovirus-1 infection. Together this study highlights a role for Ido1 in astrovirus infection and epithelial cell maturation., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Influenza virus is responsible for a large global burden of disease, especially in children. Multiple Organ Dysfunction Syndrome (MODS) is a life-threatening and fatal complication of severe influenza infection., Methods: We measured RNA expression of 469 biologically plausible candidate genes in children admitted to North American pediatric intensive care units with severe influenza virus infection with and without MODS. Whole blood samples from 191 influenza-infected children (median age 6.4 years, IQR: 2.2, 11) were collected a median of 27 hours following admission; for 45 children a second blood sample was collected approximately seven days later. Extracted RNA was hybridized to NanoString mRNA probes, counts normalized, and analyzed using linear models controlling for age and bacterial co-infections (FDR q<0.05)., Results: Comparing pediatric samples collected near admission, children with Prolonged MODS for ≥7 days (n=38; 9 deaths) had significant upregulation of nine mRNA transcripts associated with neutrophil degranulation ( RETN, TCN1, OLFM4, MMP8, LCN2, BPI, LTF, S100A12, GUSB) compared to those who recovered more rapidly from MODS (n=27). These neutrophil transcripts present in early samples predicted Prolonged MODS or death when compared to patients who recovered, however in paired longitudinal samples, they were not differentially expressed over time. Instead, five genes involved in protein metabolism and/or adaptive immunity signaling pathways ( RPL3, MRPL3, HLA-DMB, EEF1G , CD8A ) were associated with MODS recovery within a week., Conclusion: Thus, early increased expression of neutrophil degranulation genes indicated worse clinical outcomes in children with influenza infection, consistent with reports in adult cohorts with influenza, sepsis, and acute respiratory distress syndrome., Competing Interests: AGR, TN, and MMN: NIH/NIAID as declared in the funding statement - grant support paid to Boston Children’s Hospital. Also current CDC grant for COVID-19 work unrelated to current manuscript also paid to Boston Children’s Hospital under PI AGR. AGR also receives Royalties from UpToDate, Inc. as Section editor of Pediatric Critical Care Medicine. Honoraria: Grand Rounds presentations on MIS-C not related to current manuscript. Participation on a Data Safety Monitoring/Advisory Board: NIH Grace Study. Leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid: Medical Advisory Board for Families Fight Flu and Chair of International Sepsis Forum. JCC: additionally declares support from ALSAC for the current work. Other entities not related to the current work: U01AI150747, R01AI136514, U01AI144616; American Association of Immunologists Abstract award 2021, Patent: Methods for Treating or Reducing the Severity of a Viral Infection Publication no. WO 2021/226174 A1. MWH declared Kiadis, Licensing income, unrelated to the content of this manuscript, American Board of Pediatrics Payment, Abbive payment. Partner Therapeutics Provision of study drug, unrelated to the current manuscript, Sobi Provision of study drug, unrelated to the current manuscript. JC: NIH, paid to institution not related to this work; GLG Group payment to her, Elsevier payment made to her for work done as Associate Editor; Patent pending for Methods and compositions for treating and preventing T cell-driven diseases. BMC: Not related to current work: receives grants paid to institution from American Lung Association. Sobi Participation on a Data Safety Monitoring Board/Advisory Board. SLW: Receives royalties from UpToDate, Inc. Has grants/contracts with Pennsylvania Dept of Health not related to current manuscript. PGT: declares support from ALSAC and NIAID R01 AI154470 for the current work. Has several grants not associated with current work: NIAID 5R01AI128805-05; 1R01AI154470; NIAID 75N93019C00052; 5R01AI136514; 5R01AI35025; NIAID 5U01AI144616; NIAID U01AI150747. Royalties/licenses paid to both PGT and his institution: TCR cloning technology—Miltenyi Biotec & TCR amplification technology—Shennon Bio. Consulting fees paid to him: Johnson and Johnson, Cytoagents, Immunoscape, Shennon Bio. Payment or honoraria for lectures, presentations, or other events: Illumina—Future Genomic Advances, Yale University, CZ Biohub, PACT Pharma, UCSD, Tufts, University of Arizona, Mt. Sinai, Umass, OSU, Korean Association of Immunology, SISMID, University of Washington, MSKCC, Washington University, University of Missouri, Fred Hutch, UNM Illumina Single Cell, Cincinnati Childrens, University of Toronto, Purdue University, Iowa State University, University of Georgia. Patent: Methods for Treating or Reducing the Severity of a Viral Infection Publication no. WO 2021/226174 A1. (continued disclosures for PGT:) Support for attending meetings and/or travel: NIH Study Section, Keystone Viral Immunity, NIAID CEIRR, CEIRS and CIVIC meetings, GRC, 10X Users Symposium, Illumina Symposia, ImmunOktoberfest, Options for the Control of Influenza XI, Carghese Workshop, FOCIS, Max Planck for Complex Systems, Santa Fe Institute, AAI, Weizmann Institute, APS Society, Banff Institute, ISIRV meeting, University of Melbourne, British Society of Immunology. Stock or stock options: Shennon Bio Scientific advisory board payments. (Additional patents:) US20170304293A1 Coordinated metabolic reprogramming in response to productive viral infections; Cloning and expression system for t-cell receptors; License payments made to me and institution; Method for detecting SARS-CoV-2 infection. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Novak, Crawford, Hahn, Hall, Thair, Newhams, Chou, Mourani, Tarquinio, Markovitz, Loftis, Weiss, Higgerson, Schwarz, Pinto, Thomas, Gedeit, Sanders, Mahapatra, Coates, Cvijanovich, Ackerman, Tellez, McQuillen, Kurachek, Shein, Lange, Thomas and Randolph.)

Acute respiratory distress syndrome (ARDS), termed pediatric ARDS (pARDS) in children, is a severe form of acute respiratory failure (ARF). Pathologic immune responses are implicated in pARDS pathogenesis. Here, we present a description of microbial sequencing and single cell gene expression in tracheal aspirates (TAs) obtained longitudinally from infants with ARF. We show reduced interferon stimulated gene (ISG) expression, altered mononuclear phagocyte (MNP) transcriptional programs, and progressive airway neutrophilia associated with unique transcriptional profiles in patients with moderate to severe pARDS compared to those with no or mild pARDS. We additionally show that an innate immune cell product, Folate Receptor 3 (FOLR3), is enriched in moderate or severe pARDS. Our findings demonstrate distinct inflammatory responses in pARDS that are dependent upon etiology and severity and specifically implicate reduced ISG expression, altered macrophage repair-associated transcriptional programs, and accumulation of aged neutrophils in the pathogenesis of moderate to severe pARDS caused by RSV., (© 2023. The Author(s).)

Microbial components have a range of direct effects on the fetal brain. However, little is known about the cellular targets and molecular mechanisms that mediate these effects. Neural progenitor cells (NPCs) control the size and architecture of the brain and understanding the mechanisms regulating NPCs is crucial to understanding brain developmental disorders. We identify ventricular radial glia (vRG), the primary NPC, as the target of bacterial cell wall (BCW) generated during the antibiotic treatment of maternal pneumonia. BCW enhanced proliferative potential of vRGs by shortening the cell cycle and increasing self-renewal. Expanded vRGs propagated to increase neuronal output in all cortical layers. Remarkably, Toll-like receptor 2 (TLR2), which recognizes BCW, localized at the base of primary cilia in vRGs and the BCW-TLR2 interaction suppressed ciliogenesis leading to derepression of Hedgehog (HH) signaling and expansion of vRGs. We also show that TLR6 is an essential partner of TLR2 in this process. Surprisingly, TLR6 alone was required to set the number of cortical neurons under healthy conditions. These findings suggest that an endogenous signal from TLRs suppresses cortical expansion during normal development of the neocortex and that BCW antagonizes that signal through the TLR2/cilia/HH signaling axis changing brain structure and function. IMPORTANCE Fetal brain development in early gestation can be impacted by transplacental infection, altered metabolites from the maternal microbiome, or maternal immune activation. It is less well understood how maternal microbial subcomponents that cross the placenta, such as bacterial cell wall (BCW), directly interact with fetal neural progenitors and neurons and affect development. This scenario plays out in the clinic when BCW debris released during antibiotic therapy of maternal infection traffics to the fetal brain. This study identifies the direct interaction of BCW with TLR2/6 present on the primary cilium, the signaling hub on fetal neural progenitor cells (NPCs). NPCs control the size and architecture of the brain and understanding the mechanisms regulating NPCs is crucial to understanding brain developmental disorders. Within a window of vulnerability before the appearance of fetal immune cells, the BCW-TLR2/6 interaction results in the inhibition of ciliogenesis, derepression of Sonic Hedgehog signaling, excess proliferation of neural progenitors, and abnormal cortical architecture. In the first example of TLR signaling linked to Sonic Hedgehog, BCW/TLR2/6 appears to act during fetal brain morphogenesis to play a role in setting the total cell number in the neocortex., Competing Interests: The authors declare a conflict of interest. PT serves on the SAB for Immunoscape and Cytoagents and has received travel support from 10X Genomics and honorarium and travel support from Illumina. The remaining authors have no conflicts of interest to disclose.

High-risk groups, including Indigenous people, are at risk of severe COVID-19. Here we found that Australian First Nations peoples elicit effective immune responses to COVID-19 BNT162b2 vaccination, including neutralizing antibodies, receptor-binding domain (RBD) antibodies, SARS-CoV-2 spike-specific B cells, and CD4 + and CD8 + T cells. In First Nations participants, RBD IgG antibody titers were correlated with body mass index and negatively correlated with age. Reduced RBD antibodies, spike-specific B cells and follicular helper T cells were found in vaccinated participants with chronic conditions (diabetes, renal disease) and were strongly associated with altered glycosylation of IgG and increased interleukin-18 levels in the plasma. These immune perturbations were also found in non-Indigenous people with comorbidities, indicating that they were related to comorbidities rather than ethnicity. However, our study is of a great importance to First Nations peoples who have disproportionate rates of chronic comorbidities and provides evidence of robust immune responses after COVID-19 vaccination in Indigenous people., (© 2023. The Author(s).)

We are interested in detecting a departure from the baseline in a longitudinal analysis in the context of multiple organ dysfunction syndrome (MODS). In particular, we are given gene expression reads at two time points for a fixed number of genes and individuals. The individuals can be subdivided into two groups, denoted as groups A and B . Using the two time points, we compute a contrast of gene expression reads per individual and gene. The age of each individual is known and it is used to compute, for each gene separately, a linear regression of the gene expression contrasts on the individual's age. Looking at the intercept of the linear regression to detect a departure from the baseline, we aim to reliably single out those genes for which there is a difference in the intercept among those individuals in group A and not in group B . In this work, we develop testing methodology for this setting based on two hypothesis tests-one under the null and one under an appropriately formulated alternative. We demonstrate the validity of our approach using a dataset created by bootstrapping from a real data application in the context of multiple organ dysfunction syndrome (MODS).

Immunocompromised hematology patients are vulnerable to severe COVID-19 and respond poorly to vaccination. Relative deficits in immunity are, however, unclear, especially after 3 vaccine doses. We evaluated immune responses in hematology patients across three COVID-19 vaccination doses. Seropositivity was low after a first dose of BNT162b2 and ChAdOx1 (∼26%), increased to 59%-75% after a second dose, and increased to 85% after a third dose. While prototypical antibody-secreting cells (ASCs) and T follicular helper (Tfh) cell responses were elicited in healthy participants, hematology patients showed prolonged ASCs and skewed Tfh2/17 responses. Importantly, vaccine-induced expansions of spike-specific and peptide-HLA tetramer-specific CD4 + /CD8 + T cells, together with their T cell receptor (TCR) repertoires, were robust in hematology patients, irrespective of B cell numbers, and comparable to healthy participants. Vaccinated patients with breakthrough infections developed higher antibody responses, while T cell responses were comparable to healthy groups. COVID-19 vaccination induces robust T cell immunity in hematology patients of varying diseases and treatments irrespective of B cell numbers and antibody response., Competing Interests: Declaration of interests The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays and NDV-based SARS-CoV-2 vaccines, which list F.K. as co-inventor. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2. F.K. has consulted for Merck and Pfizer (before 2020) and is currently consulting for Pfizer, Seqirus, and Avimex. The Krammer laboratory is also collaborating with Pfizer on animal models of SARS-CoV-2. H.A.M. and B.Y.C. are currently consulting for Ena Respiratory. B.W.T. has received research funding from MSD, Seqirus, and Sanofi and is on the advisory board for Moderna, CSL-Behring, and Takeda., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Drak2-deficient (Drak2 -/- ) mice are resistant to multiple models of autoimmunity yet effectively eliminate pathogens and tumors. Thus, DRAK2 represents a potential target to treat autoimmune diseases. However, the mechanisms by which DRAK2 contributes to autoimmunity, particularly type 1 diabetes (T1D), remain unresolved. Here, we demonstrate that resistance to T1D in non-obese diabetic (NOD) mice is due to the absence of Drak2 in T cells and requires the presence of regulatory T cells (T regs ). Contrary to previous hypotheses, we show that DRAK2 does not limit TCR signaling. Rather, DRAK2 regulates IL-2 signaling by inhibiting STAT5A phosphorylation. We further demonstrate that enhanced sensitivity to IL-2 in the absence of Drak2 augments thymic T reg development. Overall, our data indicate that DRAK2 contributes to autoimmunity in multiple ways by regulating thymic T reg development and by impacting the sensitivity of conventional T cells to T reg -mediated suppression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Myelofibrosis (MF) is a disease associated with high unmet medical needs because allogeneic stem cell transplantation is not an option for most patients, and JAK inhibitors are generally effective for only 2 to 3 years and do not delay disease progression. MF is characterized by dysplastic megakaryocytic hyperplasia and progression to fulminant disease, which is associated with progressively increasing marrow fibrosis. Despite evidence that the inflammatory milieu in MF contributes to disease progression, the specific factors that promote megakaryocyte growth are poorly understood. Here, we analyzed changes in the cytokine profiles of MF mouse models before and after the development of fibrosis, coupled with the analysis of bone marrow populations using single-cell RNA sequencing. We found high interleukin 13 (IL-13) levels in the bone marrow of MF mice. IL-13 promoted the growth of mutant megakaryocytes and induced surface expression of transforming growth factor β and collagen biosynthesis. Similarly, analysis of samples from patients with MF revealed elevated levels of IL-13 in the plasma and increased IL-13 receptor expression in marrow megakaryocytes. In vivo, IL-13 overexpression promoted disease progression, whereas reducing IL-13/IL-4 signaling reduced several features of the disease, including fibrosis. Finally, we observed an increase in the number of marrow T cells and mast cells, which are known sources of IL-13. Together, our data demonstrate that IL-13 is involved in disease progression in MF and that inhibition of the IL-13/IL-4 signaling pathway might serve as a novel therapeutic target to treat MF., (© 2022 by The American Society of Hematology.)

Chimeric antigen receptor (CAR) T-cell therapy targeting T-cell acute lymphoblastic leukemia (T-ALL) faces limitations such as antigen selection and limited T-cell persistence. CD7 is an attractive antigen for targeting T-ALL, but overlapping expression on healthy T cells leads to fratricide of CD7-CAR T cells, requiring additional genetic modification. We took advantage of naturally occurring CD7- T cells to generate CD7-CAR (CD7-CARCD7-) T cells. CD7-CARCD7- T cells exhibited a predominantly CD4+ memory phenotype and had significant antitumor activity upon chronic antigen exposure in vitro and in xenograft mouse models. Based on these encouraging results, we next explored the utility of CD7- T cells for the immunotherapy of CD19+ hematological malignancies. Direct comparison of nonselected (bulk) CD19-CAR and CD19-CARCD7- T cells revealed that CD19-CARCD7- T cells had enhanced antitumor activity compared with their bulk counterparts in vitro and in vivo. Lastly, to gain insight into the behavior of CD19-CAR T cells with low levels of CD7 gene expression (CD7lo) in humans, we mined single-cell gene and T-cell receptor (TCR) expression data sets from our institutional CD19-CAR T-cell clinical study. CD19-CARCD7lo T cells were present in the initial CD19-CAR T-cell product and could be detected postinfusion. Intriguingly, the only functional CD4+ CD19-CAR T-cell cluster observed postinfusion exhibited CD7lo expression. Additionally, samples from patients responsive to therapy had a higher proportion of CD7lo T cells than nonresponders (NCT03573700). Thus, CARCD7- T cells have favorable biological characteristics and may present a promising T-cell subset for adoptive cell therapy of T-ALL and other hematological malignancies., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

A layer of proteoglycans and glycoproteins known as glycocalyx covers the surface of the trabecular meshwork (TM), juxtacanalicular tissue (JCT), and Schlemm's canal (SC) inner wall of the conventional aqueous outflow pathway in the eye. This has been shown to play a role in the mechanotransduction of fluid shear stress and in the regulation of the outflow resistance. The outflow resistance in the conventional outflow pathway is the main determinant of the intraocular pressure (IOP) through an active, two-way, fluid-structure interaction coupling between the outflow tissues and aqueous humor. A 3D microstructural finite element (FE) model of a healthy human eye TM/JCT/SC complex with interspersed aqueous humor was constructed. A very thin charged double layer that represents the endothelial glycocalyx layer covered the surface of the elastic outflow tissues. The aqueous humor was modeled as electroosmotic flow that is charged when it is in contact with the outflow tissues. The electrical-fluid-structure interaction (EFSI) method was used to couple the charged double layer (glycocalyx), fluid (aqueous humor), and solid (outflow tissues). When the IOP was elevated to 15 mmHg, the maximum aqueous humor velocity in the EFSI model was decreased by 2.35 mm/s (9%) compared to the fluid-structure interaction (FSI) model. The charge or electricity in the living human conventional outflow pathway generated by the charged endothelial glycocalyx layer plays a minor biomechanical role in the resultant stresses and strains as well as the hydrodynamics of the aqueous humor.

Background: Aqueous humor outflow resistance in the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), and Schlemm's canal (SC) endothelium of the conventional outflow pathway actively contribute to intraocular pressure (IOP) regulation. Outflow resistance is actively affected by the dynamic outflow pressure gradient across the TM, JCT, and SC inner wall tissues. The resistance effect implies the presence of a fluid-structure interaction (FSI) coupling between the outflow tissues and the aqueous humor. However, the biomechanical interactions between viscoelastic outflow tissues and aqueous humor dynamics are largely unknown., Methods: A 3D microstructural finite element (FE) model of a healthy human eye TM/JCT/SC complex was constructed with elastic and viscoelastic material properties for the bulk extracellular matrix and embedded elastic cable elements. The FE models were subjected to both idealized and a physiologic IOP load boundary using the FSI method., Results: The elastic material model for both the idealized and physiologic IOP load boundary at equal IOPs showed similar stresses and strains in the outflow tissues as well as pressure in the aqueous humor. However, outflow tissues with viscoelastic material properties were sensitive to the IOP load rate, resulting in different mechanical and hydrodynamic responses in the tissues and aqueous humor., Conclusions: Transient IOP fluctuations may cause a relatively large IOP difference of ~20 mmHg in a very short time frame of ~0.1 s, resulting in a rate stiffening in the outflow tissues. Rate stiffening reduces strains and causes a rate-dependent pressure gradient across the outflow tissues. Thus, the results suggest it is necessary to use a viscoelastic material model in outflow tissues that includes the important role of IOP load rate.

T cells expressing CD19-specific chimeric antigen receptors (CD19-CARs) have potent antileukemia activity in pediatric and adult patients with relapsed and/or refractory B-cell acute lymphoblastic leukemia (B-ALL). However, not all patients achieve a complete response (CR), and a significant percentage relapse after CD19-CAR T-cell therapy due to T-cell intrinsic and/or extrinsic mechanisms. Thus, there is a need to evaluate new CD19-CAR T-cell products in patients to improve efficacy. We developed a phase 1/2 clinical study to evaluate an institutional autologous CD19-CAR T-cell product in pediatric patients with relapsed/refractory B-ALL. Here we report the outcome of the phase 1 study participants (n = 12). Treatment was well tolerated, with a low incidence of both cytokine release syndrome (any grade, n = 6) and neurotoxicity (any grade, n = 3). Nine out of 12 patients (75%) achieved a minimal residual disease-negative CR in the bone marrow (BM). High disease burden (≥40% morphologic blasts) before CAR T-cell infusion correlated with increased side effects and lower response rate, but not with CD19-CAR T-cell expansion. After infusion, CD8+ CAR T cells had a proliferative advantage over CD4+ CAR T cells and at peak expansion, had an effector memory phenotype with evidence of antigen-driven differentiation. Patients that proceeded to allogeneic hematopoietic cell transplantation (AlloHCT) had sustained, durable responses. In summary, the initial evaluation of our institutional CD19-CAR T-cell product demonstrates safety and efficacy while highlighting the impact of pre-infusion disease burden on outcomes. This trial was registered at www.clinicaltrials.gov as #NCT03573700., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Although the tissues comprising the ocular conventional outflow pathway have shown strong viscoelastic mechanical response to aqueous humor pressure dynamics, the viscoelastic mechanical properties of the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), and Schlemm's canal (SC) inner wall are largely unknown., Methods: A quadrant of the anterior segment from two human donor eyes at low- and high-flow (LF and HF) outflow regions was pressurized and imaged using optical coherence tomography (OCT). A finite element (FE) model of the TM, the adjacent JCT, and the SC inner wall was constructed and viscoelastic beam elements were distributed in the extracellular matrix (ECM) of the TM and JCT to represent anisotropic collagen. An inverse FE-optimization algorithm was used to calculate the viscoelastic properties of the ECM/beam elements such that the TM/JCT/SC model and OCT imaging data best matched over time., Results: The ECM of the glaucoma tissues showed significantly larger time-dependent shear moduli compared to the heathy tissues. Significantly larger shear moduli were also observed in the LF regions of both the healthy and glaucoma eyes compared to the HF regions., Conclusions: The outflow tissues in both glaucoma eyes and HF regions are stiffer and less able to respond to dynamic IOP.

The absence of Caspase-8 or its adapter, Fas-associated death domain (FADD), results in activation of receptor interacting protein kinase-3 (RIPK3)- and mixed-lineage kinase-like (MLKL)-dependent necroptosis in vivo. Here, we show that spontaneous activation of RIPK3, phosphorylation of MLKL, and necroptosis in Caspase-8- or FADD-deficient cells was dependent on the nucleic acid sensor, Z-DNA binding protein-1 (ZBP1). We genetically engineered a mouse model by a single insertion of FLAG tag onto the N terminus of endogenous MLKL ( Mlkl FLAG/FLAG ), creating an inactive form of MLKL that permits monitoring of phosphorylated MLKL without activating necroptotic cell death. Casp8 -/- Mlkl FLAG/FLAG mice were viable and displayed phosphorylated MLKL in a variety of tissues, together with dramatically increased expression of ZBP1 compared to Casp8 +/+ mice. Studies in vitro revealed an increased expression of ZBP1 in cells lacking FADD or Caspase-8, which was suppressed by reconstitution of Caspase-8 or FADD. Ablation of ZBP1 in Casp8 -/- Mlkl FLAG/FLAG mice suppressed spontaneous MLKL phosphorylation in vivo. ZBP1 expression and downstream activation of RIPK3 and MLKL in cells lacking Caspase-8 or FADD relied on a positive feedback mechanism requiring the nucleic acid sensors cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and TBK1 signaling pathways. Our study identifies a molecular mechanism whereby Caspase-8 and FADD suppress spontaneous necroptotic cell death.

Purpose: The laminar region of the optic nerve head (ONH), thought to be the site of damage to the retinal ganglion cell axons in glaucoma, is continuously loaded on its anterior and posterior surfaces by dynamic intraocular pressure (IOP) and orbital cerebrospinal fluid pressure (CSFP), respectively. Thus, translaminar pressure (TLP; TLP = IOP-CSFP) has been proposed as a glaucoma risk factor., Methods: Three eye-specific finite element models of the posterior human eye were constructed, including full 3D microstructures of the load-bearing lamina cribrosa (LC) with interspersed laminar neural tissues (NTs), and heterogeneous, anisotropic, hyperelastic material formulations for the surrounding peripapillary sclera and adjacent pia. ONH biomechanical responses were simulated using three combinations of IOP and CSFP loadings consistent with posture change from sitting to supine., Results: Results show that tensile, compressive, and shear stresses and strains in the ONH were higher in the supine position compared to the sitting position (P < 0.05). In addition, LC beams bear three to five times more TLP-driven stress than interspersed laminar NT, whereas laminar NT exhibit three to five times greater strain than supporting LC (P < 0.05). Compared with CSFP, IOP drove approximately four times greater stress and strain in the LC, NT, and peripapillary sclera, normalized per mm Hg pressure change. In addition, IOP drove approximately three-fold greater scleral canal expansion and anterior-posterior laminar deformation than CSFP per mm Hg (P < 0.05)., Conclusions: Whereas TLP has been hypothesized to play a prominent role in ONH biomechanics, the IOP and CSFP effects are not equivalent, as IOP-driven stress, strain, and deformation play a more dominant role than CSFP effects.

While adult bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) and their extrinsic regulation is well studied, little is known about the composition, function, and extrinsic regulation of the first HSPCs to enter the BM during development. Here, we functionally interrogate murine BM HSPCs from E15.5 through P0. Our work reveals that fetal BM HSPCs are present by E15.5, but distinct from the HSPC pool seen in fetal liver, both phenotypically and functionally, until near birth. We also generate a transcriptional atlas of perinatal BM HSPCs and the BM niche in mice across ontogeny, revealing that fetal BM lacks HSPCs with robust intrinsic stem cell programs, as well as niche cells supportive of HSPCs. In contrast, stem cell programs are preserved in neonatal BM HSPCs, which reside in a niche expressing HSC supportive factors distinct from those seen in adults. Collectively, our results provide important insights into the factors shaping hematopoiesis during this understudied window of hematopoietic development., (© 2022. The Author(s).)

Current chimeric antigen receptor-modified (CAR) T-cell products are evaluated in bulk, without assessing functional heterogeneity. We therefore generated a comprehensive single-cell gene expression and T-cell receptor (TCR) sequencing data set using pre- and postinfusion CD19-CAR T cells from blood and bone marrow samples of pediatric patients with B-cell acute lymphoblastic leukemia. We identified cytotoxic postinfusion cells with identical TCRs to a subset of preinfusion CAR T cells. These effector precursor cells exhibited a unique transcriptional profile compared with other preinfusion cells, corresponding to an unexpected surface phenotype (TIGIT+, CD62Llo, CD27-). Upon stimulation, these cells showed functional superiority and decreased expression of the exhaustion-associated transcription factor TOX. Collectively, these results demonstrate diverse effector potentials within preinfusion CAR T-cell products, which can be exploited for therapeutic applications. Furthermore, we provide an integrative experimental and analytic framework for elucidating the mechanisms underlying effector development in CAR T-cell products., Significance: Utilizing clonal trajectories to define transcriptional potential, we find a unique signature of CAR T-cell effector precursors present in preinfusion cell products. Functional assessment of cells with this signature indicated early effector potential and resistance to exhaustion, consistent with postinfusion cellular patterns observed in patients. This article is highlighted in the In This Issue feature, p. 2007., (©2022 The Authors; Published by the American Association for Cancer Research.)

Purpose: The purpose of this study was to assess ocular coat mechanical behavior using controlled ocular microvolumetric injections (MVI) of 15 µL of balanced salt solution (BSS) infused over 1 second into the anterior chamber (AC) via a syringe pump., Methods: Intraocular pressure (IOP) was continuously recorded at 200 Hz with a validated implantable IOP telemetry system in 7 eyes of 7 male rhesus macaques (nonhuman primates [NHPs]) during 5 MVIs in a series at native (3 trials), 15 and 20 mm Hg baseline IOPs, repeated in 2 to 5 sessions at least 2 weeks apart. Ocular rigidity coefficients (K) and ocular pulse volume (PV) were calculated for each trial. Data were averaged across sessions within eyes; PV was analyzed with a three-level nested ANOVA, and parameter relationships were analyzed with Pearson Correlation and linear regression., Results: After MVI at native baseline IOP of 10.4 ± 1.6 mm Hg, IOP increased by 9.1 ± 2.8 mm Hg (∆IOP) at a 9.6 ± 2.7 mm Hg/s slope, ocular pulse amplitude (OPA) was 0.70 ± 0.13 mm Hg on average; the average K was 0.042 ± 0.010 µL-1 and average PV was 1.16 ± 0.43 µL. PV varied significantly between trials, days, and eyes (P ≤ 0.05). OPA was significantly correlated with K at native IOP: Pearson coefficients ranged from 0.71 to 0.83 (P ≤ 0.05) and R2 ranged from 0.50 to 0.69 (P ≤ 0.05) during the first trial., Conclusions: The MVI-driven ∆IOP and slope can be used to assess ocular coat mechanical behavior and measure ocular rigidity., Translational Relevance: Importantly, OPA at native IOP is correlated with ocular rigidity despite the significant variability in PV between heartbeats.

Drug-tolerant persister cells (persisters) evade apoptosis upon targeted and conventional cancer therapies and represent a major non-genetic barrier to effective cancer treatment. Here, we show that cells that survive treatment with pro-apoptotic BH3 mimetics display a persister phenotype that includes colonization and metastasis in vivo and increased sensitivity toward ferroptosis by GPX4 inhibition. We found that sublethal mitochondrial outer membrane permeabilization (MOMP) and holocytochrome c release are key requirements for the generation of the persister phenotype. The generation of persisters is independent of apoptosome formation and caspase activation, but instead, cytosolic cytochrome c induces the activation of heme-regulated inhibitor (HRI) kinase and engagement of the integrated stress response (ISR) with the consequent synthesis of ATF4, all of which are required for the persister phenotype. Our results reveal that sublethal cytochrome c release couples sublethal MOMP to caspase-independent initiation of an ATF4-dependent, drug-tolerant persister phenotype., Competing Interests: Declaration of interests D.R.G. consults for Ventus Therapeutics, Inzen Therapeutics, and Horizon Therapeutics., (Copyright © 2022 Elsevier Inc. All rights reserved.)

As the establishment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell memory in children remains largely unexplored, we recruited convalescent COVID-19 children and adults to define their circulating memory SARS-CoV-2-specific CD4 + and CD8 + T cells prior to vaccination. We analyzed epitope-specific T cells directly ex vivo using seven HLA class I and class II tetramers presenting SARS-CoV-2 epitopes, together with Spike-specific B cells. Unvaccinated children who seroconverted had comparable Spike-specific but lower ORF1a- and N-specific memory T cell responses compared with adults. This agreed with our TCR sequencing data showing reduced clonal expansion in children. A strong stem cell memory phenotype and common T cell receptor motifs were detected within tetramer-specific T cells in seroconverted children. Conversely, children who did not seroconvert had tetramer-specific T cells of predominantly naive phenotypes and diverse TCRαβ repertoires. Our study demonstrates the generation of SARS-CoV-2-specific T cell memory with common TCRαβ motifs in unvaccinated seroconverted children after their first virus encounter., Competing Interests: Declaration of interests SARS-CoV-2 serological assays (US Provisional Application #62/994252, 63/018457, 63/020503, and 63/024436) and NDV-based SARS-CoV-2 vaccines (US Provisional Application #63/251020) list F.K. as a co-inventor. F.A. is also a co-inventor of the serological assay patents. Patent applications were submitted by the Icahn School of Medicine at Mount Sinai. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2. F.K. has consulted for Merck and Pfizer (before 2020) and is currently consulting for Pfizer, Third Rock Ventures, Seqirus, and Avimex. F.K. laboratory collaborates with Pfizer on the animal models of SARS-CoV-2. P.G.T. is on the SAB of Immunoscape and Cytoagents, has consulted for JNJ, received travel support and/or honoraria from Illumina, 10X Genomics, and has patents on TCR discovery and expression. P.S.P. received research grants from Astra Zeneca and Pfizer and has served on advisory boards for Sanofi Pasteur and Seqirus., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Activated CD8 + T lymphocytes differentiate into heterogeneous subsets. Using super-resolution imaging, we found that prior to the first division, dynein-dependent vesicular transport polarized active TORC1 toward the microtubule-organizing center (MTOC) at the proximal pole. This active TORC1 was physically associated with active eIF4F, required for the translation of c-myc mRNA. As a consequence, c-myc-translating polysomes polarized toward the cellular pole proximal to the immune synapse, resulting in localized c-myc translation. Upon division, the TORC1-eIF4A complex preferentially sorted to the proximal daughter cell, facilitating asymmetric c-Myc synthesis. Transient disruption of eIF4A activity at first division skewed long-term cell fate trajectories to memory-like function. Using a genetic barcoding approach, we found that first-division sister cells often displayed differences in transcriptional profiles that largely correlated with c-Myc and TORC1 target genes. Our findings provide mechanistic insights as to how distinct T cell fate trajectories can be established during the first division., Competing Interests: Declaration of interests The authors declare no competing interests. D.R.G. consults for Inzen Therapeutics and Ventus Therapeutics. D.R. is a shareholder of eFFECTOR Therapeutics. D.R.G. is a member of Molecular Cell’s Advisory Board., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The identification of mechanisms to promote memory T (T mem ) cells has important implications for vaccination and anti-cancer immunotherapy 1-4 . Using a CRISPR-based screen for negative regulators of T mem cell generation in vivo 5 , here we identify multiple components of the mammalian canonical BRG1/BRM-associated factor (cBAF) 6,7 . Several components of the cBAF complex are essential for the differentiation of activated CD8 + T cells into T effector (T eff ) cells, and their loss promotes T mem cell formation in vivo. During the first division of activated CD8 + T cells, cBAF and MYC 8 frequently co-assort asymmetrically to the two daughter cells. Daughter cells with high MYC and high cBAF display a cell fate trajectory towards T eff cells, whereas those with low MYC and low cBAF preferentially differentiate towards T mem cells. The cBAF complex and MYC physically interact to establish the chromatin landscape in activated CD8 + T cells. Treatment of naive CD8 + T cells with a putative cBAF inhibitor during the first 48 h of activation, before the generation of chimeric antigen receptor T (CAR-T) cells, markedly improves efficacy in a mouse solid tumour model. Our results establish cBAF as a negative determinant of T mem cell fate and suggest that manipulation of cBAF early in T cell differentiation can improve cancer immunotherapy., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Prior work by Michael R. Dougherty and colleagues (Yu et al., 2014) shows that when a scientist monitors the p value during data collection and uses a critical p as the signal to stop collecting data, the resulting p is distorted due to Type I error-rate inflation. They argued similarly that the use of a critical Bayes factor (BF (crit) ) for stopping distorts the obtained Bayes factor (BF), a position that has met with controversy. The present paper clarified that when BF (crit) is used as a stopping criterion, the sample becomes biased in that data consistent with large effects have a greater chance to be included than do other data, thus biasing the input to Bayesian inference. We report simulations of yoked pairs of scientists in which Scientist A uses BF (crit) to optionally stop, while Scientist B, sampling from the same population, stops when A stops. Thus, optional stopping is compared not to a hypothetical in which no stopping occurs, but to a situation in which B stops for reasons unrelated to the characteristics of B's sample. The results indicated that optional stopping biased the input for Bayesian inference. We also simulated the use of effect-size stabilization as a stopping criterion and found no bias in that case., (© 2021. The Psychonomic Society, Inc.)

Only a small proportion of patients with cancer show lasting responses to immune checkpoint blockade (ICB)-based monotherapies. The RNA-editing enzyme ADAR1 is an emerging determinant of resistance to ICB therapy and prevents ICB responsiveness by repressing immunogenic double-stranded RNAs (dsRNAs), such as those arising from the dysregulated expression of endogenous retroviral elements (EREs) 1-4 . These dsRNAs trigger an interferon-dependent antitumour response by activating A-form dsRNA (A-RNA)-sensing proteins such as MDA-5 and PKR 5 . Here we show that ADAR1 also prevents the accrual of endogenous Z-form dsRNA elements (Z-RNAs), which were enriched in the 3' untranslated regions of interferon-stimulated mRNAs. Depletion or mutation of ADAR1 resulted in Z-RNA accumulation and activation of the Z-RNA sensor ZBP1, which culminated in RIPK3-mediated necroptosis. As no clinically viable ADAR1 inhibitors currently exist, we searched for a compound that can override the requirement for ADAR1 inhibition and directly activate ZBP1. We identified a small molecule, the curaxin CBL0137, which potently activates ZBP1 by triggering Z-DNA formation in cells. CBL0137 induced ZBP1-dependent necroptosis in cancer-associated fibroblasts and reversed ICB unresponsiveness in mouse models of melanoma. Collectively, these results demonstrate that ADAR1 represses endogenous Z-RNAs and identifies ZBP1-mediated necroptosis as a new determinant of tumour immunogenicity masked by ADAR1. Therapeutic activation of ZBP1-induced necroptosis provides a readily translatable avenue for rekindling the immune responsiveness of ICB-resistant human cancers., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)