Autophagy shapes CD8 T cell fate; yet the timing, triggers and targets of this process are poorly defined. Herein, we show that naive CD8 T cells have high autophagic flux, and we identify an autophagy checkpoint whereby antigen receptor engagement and inflammatory cytokines acutely repress autophagy by regulating amino acid transporter expression and intracellular amino acid delivery. Activated T cells with high levels of amino acid transporters have low autophagic flux in amino-acid-replete conditions but rapidly reinduce autophagy when amino acids are restricted. A census of proteins degraded and fueled by autophagy shows how autophagy shapes CD8 T cell proteomes. In cytotoxic T cells, dominant autophagy substrates include cytolytic effector molecules, and amino acid and glucose transporters. In naive T cells, mitophagy dominates and selective mitochondrial pruning supports the expression of molecules that coordinate T cell migration and survival. Autophagy thus differentially prunes naive and effector T cell proteomes and is dynamically repressed by antigen receptors and inflammatory cytokines to shape T cell differentiation.

Obesity increases the risk of many cancers and impairs the anti-tumour immune response. However, little is known about whether the source or composition of dietary fat affects tumour growth or anti-tumour immunity in obesity. Here, we show that high-fat diets (HFDs) derived from lard, beef tallow or butter accelerate tumour growth in a syngeneic model of melanoma, but HFDs based on coconut oil, palm oil or olive oil do not, despite equivalent obesity. Using butter-based and palm oil-based HFDs as examples, we find that these dietary fat sources differentially regulate natural killer and CD8 T cell infiltration and function within the tumour microenvironment, governed by distinct effects on the plasma metabolome and intracellular metabolism. We identify diet-related lipid intermediates, namely long-chain acylcarnitine species, as immunosuppressive metabolites enriched in mice fed butter compared to palm oil HFD. Together, these results highlight the significance of diet in maintaining a healthy immune system and suggest that modifying dietary fat may improve cancer outcomes in obesity. This study shows that animal-based high-fat diets accelerate tumour growth and impair anti-tumour response to melanoma in obese mice, whereas plant-based high-fat diets do not.

AMPK acts downstream of the tumor suppressor LKB1, yet its role in cancer has been controversial. AMPK is activated by biguanides, such as metformin and phenformin, and metformin use in diabetics has been associated with reduced cancer risk. However, whether this is mediated by cell-autonomous AMPK activation within tumor progenitor cells has been unclear. We report that T-cell-specific loss of AMPK-α1 caused accelerated growth of T cell acute lymphoblastic leukemia/lymphoma (T-ALL) induced by PTEN loss in thymic T cell progenitors. Oral administration of phenformin, but not metformin, delayed onset and growth of lymphomas, but only when T cells expressed AMPK-α1. This differential effect of biguanides correlated with detection of phenformin, but not metformin, in thymus. Phenformin also enhanced apoptosis in T-ALL cells both in vivo and in vitro. Thus, AMPK-α1 can be a cell-autonomous tumor suppressor in the context of T-ALL, and phenformin may have potential for the prevention of some cancers.

Integration of kinase signalling networks co-ordinates the transcriptional, translational, and metabolic changes required for T cell activation and differentiation. This study explores the role of the Serine/Threonine kinases PIM1 and PIM2 in controlling mouse CD8 T lymphocyte antigen receptor-mediated activation and differentiation in response to the cytokines Interleukin-2 (IL-2) or IL-15. We show that the PIM kinases are dispensable for antigen-receptor and IL-15 controlled differentiation programs, but that they play a selective role in IL-2 regulated CD8 T cell fate. One key insight was that PIM kinases controlled the migratory capabilities of effector CD8 T cells, with Pim1/Pim2-deficient CD8 T cells unable to fully switch off the naive T cell chemokine and adhesion receptor program during effector differentiation. PIM kinases were also needed for IL-2 to sustain high expression of the glucose transporters SLC2A1 and SLC2A3 and to maintain activity of the nutrient-sensing kinase mTORc1. Strikingly, PIM kinases did not have a dominant impact on IL-2-driven transcriptional programs but rather selectively modulated protein synthesis to shape cytotoxic T cell proteomes. This study reveals a selective role of PIM kinases in IL-2 control of CD8 T cells and highlights how regulated changes in protein synthesis can impact T cell phenotypes.

Background Neutrophils are important in the pathophysiology of coronavirus disease 2019 (COVID-19), but the molecular changes contributing to altered neutrophil phenotypes following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are not fully understood. We used quantitative mass spectrometry-based proteomics to explore neutrophil phenotypes immediately following acute SARS-CoV-2 infection and during recovery. Methods Prospective observational study of hospitalised patients with PCR-confirmed SARS-CoV-2 infection (May to December 2020). Patients were enrolled within 96 h of admission, with longitudinal sampling up to 29 days. Control groups comprised non-COVID-19 acute lower respiratory tract infection (LRTI) and age-matched noninfected controls. Neutrophils were isolated from peripheral blood and analysed using mass spectrometry. COVID-19 severity and recovery were defined using the World Health Organization ordinal scale. Results Neutrophil proteomes from 84 COVID-19 patients were compared to those from 91 LRTI and 42 control participants. 5800 neutrophil proteins were identified, with >1700 proteins significantly changed in neutrophils from COVID-19 patients compared to noninfected controls. Neutrophils from COVID-19 patients initially all demonstrated a strong interferon signature, but this signature rapidly declined in patients with severe disease. Severe disease was associated with increased abundance of proteins involved in metabolism, immunosuppression and pattern recognition, while delayed recovery from COVID-19 was associated with decreased granule components and reduced abundance of metabolic proteins, chemokine and leukotriene receptors, integrins and inhibitory receptors. Conclusions SARS-CoV-2 infection results in the sustained presence of circulating neutrophils with distinct proteomes suggesting altered metabolic and immunosuppressive profiles and altered capacities to respond to migratory signals and cues from other immune cells, pathogens or cytokines.

T cell immunity is impaired during ageing, particularly in memory responses needed for efficient vaccination. Autophagy and asymmetric cell division (ACD) are cell biological mechanisms key to memory formation, which undergo a decline upon ageing. However, despite the fundamental importance of these processes in cellular function, the link between ACD and in vivo fate decisions has remained highly correlative in T cells and in the field of mammalian ACD overall. Here we provide robust causal evidence linking ACD to in vivo T cell fate decisions and our data are consistent with the concept that initiation of asymmetric T cell fates is regulated by autophagy. Analysing the proteome of first-daughter CD8+ T cells following TCR-triggered activation, we reveal that mitochondrial proteins rely on autophagy for their asymmetric inheritance and that damaged mitochondria are polarized upon first division. These results led us to evaluate whether mitochondria were asymmetrically inherited and to functionally address their impact on T cell fate. For this we used a novel mouse model that allows sequential tagging of mitochondria in mother and daughter cells, enabling their isolation and subsequent in vivo analysis of CD8+ T cell progenies based on pre-mitotic cell cargo. Autophagy-deficient CD8+ T cells showed impaired clearance and symmetric inheritance of old mitochondria, suggesting that degradation events promote asymmetry and are needed to generate T cells devoid of old organelles. Daughter cells inheriting old mitochondria are more glycolytic and upon adoptive transfer show reduced memory potential, whereas daughter cells that have not inherited old mitochondria from the mother cell are long-lived and expand upon cognate-antigen challenge. Proteomic and single-cell transcriptomic analysis of cells inheriting aged mitochondria suggest that their early fate divergence relies on one carbon metabolism as a consequence of poor mitochondrial quality and function. These findings increase our understanding of how T cell diversity is early-imprinted during division and will help foster the development of strategies to modulate T cell function. ![Figure][1] The MitoSnap model allows tracking of pre-mitotic and post-mitotic cell cargoes. Both segregation and degradation (autophagy) contribute to the asymmetric inheritance of old mitochondria. Old mitochondria impact cell metabolism and function. Cells devoid of old mitochondria exhibit better memory potential in vivo . ### Competing Interest Statement The authors have declared no competing interest. The datasets generated or analyzed in this study are available from the corresponding lead author on reasonable request. [1]: pending:yes

Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors and find that while they express a near-identical set of proteins, they show consistent quantitative differences in the abundance of a subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs, with increased abundance of cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins. Prominent changes detected in proteins involved in mitochondrial metabolism correlated with enhanced mitochondrial potential, shown using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including growth factors and proteins involved in the inhibition of the immune system. The data indicate that reprogramming of fibroblasts to hiPSCs produces important differences in cytoplasmic and mitochondrial proteins compared to hESCs, with consequences affecting growth and metabolism. This study improves our understanding of the molecular differences between hiPSCs and hESCs, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications.

The Immunological Proteome Resource (ImmPRes ; ) is an open access public resource integrating proteomic data generated by large-scale mass-spectrometry analysis of murine hematopoietic populations. The initial focus is T lymphocytes and how their proteomes are shaped by immune activation, environment, and intracellular signalling pathways with an aim to expand it to B cells and innate immune cells. It is a multidisciplinary effort between immunology and mass spectrometry-based labs with the objective to help define an in-depth high-quality map of immune cell proteomes. Maintaining data reproducibility and integrity are a priority within the resource, thus there is an in-depth protocols section explaining in detail the sample processing and the mass spectrometry-based analysis. ImmPRes provides open access to proteomic datasets covering a wide range of murine leukocyte populations with analysis of copy numbers per cell of > 10,000 proteins, enabling new understanding of lymphocyte phenotypes. All data is accessible via a simple graphical interface that supports easy interrogation of the data and options to download data summaries and raw data files. ### Competing Interest Statement The authors have declared no competing interest.

Rationale: Chronic obstructive pulmonary disease (COPD) is a disease characterized by persistent airway inflammation and disordered macrophage function. The extent to which alterations in macrophage bioenergetics contribute to impaired antioxidant responses and disease pathogenesis has yet to be fully delineated. Objectives: Through the study of COPD alveolar macrophages (AMs) and peripheral monocyte-derived macrophages (MDMs), we sought to establish if intrinsic defects in core metabolic processes drive macrophage dysfunction and redox imbalance. Methods: AMs and MDMs from donors with COPD and healthy donors underwent functional, metabolic, and transcriptional profiling. Measurements and Main Results: We observed that AMs and MDMs from donors with COPD display a critical depletion in glycolytic- and mitochondrial respiration-derived energy reserves and an overreliance on glycolysis as a source for ATP, resulting in reduced energy status. Defects in oxidative metabolism extend to an impaired redox balance associated with defective expression of the NADPH-generating enzyme, ME1 (malic enzyme 1), a known target of the antioxidant transcription factor NRF2 (nuclear factor erythroid 2-related factor 2). Consequently, selective activation of NRF2 resets the COPD transcriptome, resulting in increased generation of TCA cycle intermediaries, improved energetic status, favorable redox balance, and recovery of macrophage function. Conclusions: In COPD, an inherent loss of metabolic plasticity leads to metabolic exhaustion and reduced redox capacity, which can be rescued by activation of the NRF2 pathway. Targeting these defects, via NRF2 augmentation, may therefore present an attractive therapeutic strategy for the treatment of the aberrant airway inflammation described in COPD.

Introduction: COVID19 can cause profound systemic inflammation. Understanding inflammation and immune cell function may allow prediction of outcomes, longer-term effects and identification of further therapeutic targets. Methods: Prospective observational study of patients with PCR-confirmed SARS-CoV-2 enrolled within 96 hours of admission at two UK hospitals(May2020–March2021), with longitudinal sampling up to 29 days. mRNAseq was performed in peripheral blood cells, 45 serum cytokines were measured with Olink Target48 panel, isolated neutrophils were processed for proteomic and functional analyses. Severe disease was defined as requirement for ventilatory support or death within 28 days of admission. Results: 176 COVID19 patients were included (mean age 64.9years, SD13.6), 101 were male (57.4%). 56 patients developed severe disease (31.8%), mortality was 16.5%. A subset underwent neutrophil proteomics (n=81). 19 serum cytokines were significantly associated with severity (p<0.05; ROC analysis), CCL7 had the strongest discriminatory value (AUC 0.78, 95%CI 0.70-0.85). There were 317 significant differentially expressed genes (Wald test with Benjanini-Hochberg correction; p<0.05) at baseline comparing those discharged or still hospitalised at 4 weeks; pathways included complement and B cell activation. Neutrophil proteomics showed upregulation of interferon signalling, which decreased over time but was not linked to severity. Cytokine and toll like receptor levels, plus the eosinophil-associated protein PRG2, were associated with disease severity and outcome. Conclusion: Proteomic and transcriptomic approaches identified changes associated with patient outcomes.

Abstract After exposure to chronic inflammatory stimuli, the immune system can switch from a functional state where it acts to reestablish homeostasis to a dysfunctional state. In the context of cancer, T cells that become exposed to continuous stimulation eventually reach a state of exhaustion, characterized by poor effector function and expression of inhibitory receptors, such as PD-1. Although PD-1 signaling inhibition leads to T cell reinvigoration and has been applied as an effective treatment versus a wide range of tumors, the signaling pathway downstream of this receptor is still poorly understood. Recent work from others and us challenged the notion that the phosphatase shp-2 is essential for activation of the molecular cascade downstream PD-1 receptor engagement. The shp-2 homologue (shp-1) has also been associated with PD-1 signaling in T cells and functional redundancy between these phosphatases might occur downstream of this receptor. Therefore, we investigated the effect of shp-1 and the combination of both (shp-1/2) downstream of PD-1 by knocking out these phosphatases in T cells in a mouse model. In vivo results after tumor engraftment suggest that shp-1 as well as shp-1/2 deletion in T cells are not sufficient to ameliorate tumor control. Furthermore, ablation of shp-1 and shp-1/2 impair the beneficial effects of the anti-PD1 treatment. In fact, deletion of both phosphatases leads to decrease CD8+ T cell presence in the tumor microenvironment and in vitro results show that these cells have impaired survival. This data implies that elimination or inhibition of shp-1/2 is not a suitable strategy for effective immunotherapeutic approaches as well as highlights the importance of further elucidating the mechanisms behind this important inhibitory pathway. Citation Format: Pedro Ventura, Milica Gakovic, Berenice Fischer, Sarah Thomson, Hanif J Khameneh, Alessandro Zenobi, Giorgia Rota, Eric Vivier, Walter Birchmeier, Doreen Cantrell, Greta Guarda. Function of shp-1 and shp-2 phosphatases in T cell-mediated anti-tumor response [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P051.

T cell activation, proliferation, and differentiation into effector and memory states involve massive remodeling of T cell size and molecular content and create a massive increase in demand for energy and amino acids. Protein synthesis is an energy- and resource-demanding process; as such, changes in T cell energy production are intrinsically linked to proteome remodeling. In this review, we discuss how protein synthesis and degradation change over the course of a T cell immune response and the crosstalk between these processes and T cell energy metabolism. We highlight how the use of high-resolution mass spectrometry to analyze T cell proteomes can improve our understanding of how these processes are regulated.

Rationale Neutrophils are important in the pathophysiology of COVID19 but the molecular changes contributing to altered neutrophil phenotypes following SARS-CoV-2 infection are not fully understood. Objectives To use quantitative mass spectrometry-based proteomics to explore neutrophil phenotypes following acute SARS-CoV-2 infection and during recovery. Methods Prospective observational study of hospitalised patients with PCR-confirmed SARS-CoV-2 infection (May 2020-December 2020). Patients were enrolled within 96 hours of admission, with longitudinal sampling up to 29 days. Control groups comprised non-COVID19 acute lower respiratory tract infection (LRTI) and age-matched non-infected controls. Neutrophils isolated from peripheral blood were processed for mass spectrometry. COVID19 severity and recovery were defined using the WHO ordinal scale. Measurements and Main Results 84 COVID19 patients were included and compared to 91 LRTI patients and 42 controls. 5,800 neutrophil proteins were identified and 1,748 proteins were significantly different (q-value<0.05) in neutrophils from COVID19 patients compared to those of non-infected controls, including a robust interferon response at baseline, which was lost in severe patients one week after enrolment. Neutrophil changes associated with COVID19 disease severity and prolonged illness were characterized and candidate targets for modulation of neutrophil function were identified. Delayed recovery from COVID19 was associated with changes in metabolic and signalling proteins, complement, chemokine and leukotriene receptors, integrins and inhibitory receptors. Conclusions SARS-CoV-2 infection results in the sustained presence of recirculating neutrophils with distinct metabolic profiles and altered capacities to respond to migratory signals and cues from other immune cells, pathogens or cytokines. Scientific Knowledge on the Subject Inflammation is the primary driver of morbidity and mortality in severe COVID19. Type I interferon responses, T-cell exhaustion, cytokine storm, emergency myelopoiesis, myeloid compartment dysregulation and procoagulant pathway activation are well established contributors to COVID19 disease severity. Neutrophils play an important role in COVID19, with elevated neutrophil-to-lymphocyte ratios and the emergence of a circulating immature neutrophil population in individuals with severe symptoms. Neutrophil infiltration in the lungs coupled with the release of neutrophil extracellular traps has also been reported in severe and fatal COVID19. The aim of this study was to quantitatively map the proteomes of peripheral blood neutrophils from a cohort of hospitalised COVID19 patients to understand how SARS-CoV-2 infection changes neutrophil phenotypes and functional capacity. What this study adds to the field High-resolution mass spectrometry was used to characterise the proteomes of peripheral blood neutrophils from >200 individuals at different stages of disease. This work has comprehensively mapped neutrophil molecular changes associated with mild and severe COVID19 and identified significant quantitative changes in more than 1700 proteins in neutrophils from patients hospitalised with COVID19 versus patients with non-COVID19 acute respiratory infections. The study identifies neutrophil protein signatures associated with COVID19 disease severity. The data also show that alterations in neutrophil proteomes can persist in fully recovered patients and identify distinct neutrophil proteomes in recovered versus non recovered patients. Our study provides novel insights into neutrophil responses during acute COVID19 and reveals that altered neutrophil phenotypes persist in convalescent COVID19 patients. ### Competing Interest Statement James D. Chalmers has received research grants from AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Gilead Sciences, Novartis and Insmed; and received consultancy or speaker fees from AstraZeneca, Boehringer Ingelheim, Chiesi, GlaxoSmithKline, Insmed, Janssen, Novartis and Zambon. ### Funding Statement This work was funded by the Chief Scientist Office Rapid Response COVID19 Research Grant (COV/DUND/20/01) and the UK Coronavirus Immunology Consortium (MR/V028448) and the Wellcome Trust (205023/Z/16/Z). DAC is supported by a Wellcome Trust Principal Research fellowship (097418/Z/11/Z). JDC is supported by the GSK/Asthma and Lung UK Chair of Respiratory Research (C17-2) and a Scottish Senior Fellowship from the Chief Scientist Office (SCAF17/03). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The East of Scotland Research Ethics Committee gave ethical approval for this work (reference number 20/ES/0055) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All the mass spectrometry files, as well as the processed search result files are available at PRIDE

Bach2 codes for a transcriptional regulator exerting major influences on T cell-mediated immune regulation. Effector CTLs derived from in vitro activation of murine CD8(+) T cells showed increased proliferative and cytolytic capacity in the absence of BACH2. Before activation, BACH2-deficient splenic CD8(+) T cells had a higher abundance of memory and reduced abundance of naive cells compared to wild-type. CTLs derived from central memory T cells were more potently cytotoxic than those derived from naive T cells, but even within separated subsets, BACH2-deficiency conferred a cytotoxic advantage. Immunofluorescence and electron microscopy revealed larger granules in BACH2-deficient compared to wild-type CTLs, and proteomic analysis showed an increase in granule content, including perforin and granzymes. Thus, the enhanced cytotoxicity observed in effector CTLs lacking BACH2 arises not only from differences in their initial differentiation state but also inherent production of enlarged cytolytic granules. These results demonstrate how a single gene deletion can produce a CTL super-killer.

Summary: To overcome oxidative, inflammatory, and metabolic stress, cells have evolved cytoprotective protein networks controlled by nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) and its negative regulator, Kelch-like ECH associated protein 1 (Keap1). Here, using high-resolution mass spectrometry we characterize the proteomes of macrophages with altered Nrf2 status revealing significant differences among the genotypes in metabolism and redox homeostasis, which were validated with respirometry and metabolomics. Nrf2 affected the proteome following lipopolysaccharide (LPS) stimulation, with alterations in redox, carbohydrate and lipid metabolism, and innate immunity. Notably, Nrf2 activation promoted mitochondrial fusion. The Keap1 inhibitor, 4-octyl itaconate remodeled the inflammatory macrophage proteome, increasing redox and suppressing type I interferon (IFN) response. Similarly, pharmacologic or genetic Nrf2 activation inhibited the transcription of IFN-β and its downstream effector IFIT2 during LPS stimulation. These data suggest that Nrf2 activation facilitates metabolic reprogramming and mitochondrial adaptation, and finetunes the innate immune response in macrophages.

Phosphoinositide 3-kinase p110 delta (PI3K p110δ) is pivotal for CD8+ T cell immune responses. The current study explores PI3K p110δ induction and repression of antigen receptor and cytokine regulated programs to inform how PI3K p110δ directs CD8+ T cell fate. The studies force a revision of the concept that PI3K p110δ controls metabolic pathways in T cells and reveal major differences in PI3K p110δ regulated transcriptional programs between naïve and effector cytotoxic T cells (CTL). These differences include differential control of the expression of cytolytic effector molecules and costimulatory receptors. Key insights from the work include that PI3K p110δ signalling pathways repress expression of the critical inhibitory receptors CTLA4 and SLAMF6 in CTL. Moreover, in both naïve and effector T cells the dominant role for PI3K p110δ is to restrain the production of the chemokines that orchestrate communication between adaptive and innate immune cells. The study provides a comprehensive resource for understanding how PI3K p110δ uses multiple processes mediated by Protein Kinase B/AKT, FOXO1 dependent and independent mechanisms and mitogen-activated protein kinases (MAPK) to direct CD8+ T cell fate.

Metabolic and nutrient-sensing pathways play an important role in controlling the efficacy of effector T cells. Oxygen is a critical regulator of cellular metabolism. However, during immune responses T cells must function in oxygen-deficient, or hypoxic, environments. Here, we used high resolution mass spectrometry to investigate how the proteome of primary murine CD8(+) cytotoxic T lymphocytes (CTLs) is reconfigured in response to hypoxia in vitro. We identified and quantified over 7,600 proteins and discovered that hypoxia increased the abundance of a selected number of proteins in CTLs. This included glucose transporters, metabolic enzymes, transcription factors, cytolytic effector molecules, checkpoint receptors and adhesion molecules. While some of these proteins may augment the effector functions of CTLs, others may limit their cytotoxicity. Moreover, we determined that hypoxia could inhibit IL-2-induced proliferation cues and antigen-induced pro-inflammatory cytokine production in CTLs. These data provide a comprehensive resource for understanding the magnitude of the CTL response to hypoxia and emphasise the importance of oxygen-sensing pathways for controlling CD8(+) T cells. Additionally, this study provides new understanding about how hypoxia may promote the effector function of CTLs, while contributing to their dysfunction in some contexts.

Background: Acute respiratory distress syndrome (ARDS) is a severe critical condition with a high mortality that is currently in focus given that it is associated with mortality caused by coronavirus disease 2019 (COVID-19). Neutrophils play a key role in the lung injury characteristic of non-COVID-19 ARDS and there is also accumulating evidence of neutrophil mediated lung injury in patients who succumb to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: We undertook a functional proteomic and metabolomic survey of circulating neutrophil populations, comparing patients with COVID-19 ARDS and non-COVID-19 ARDS to understand the molecular basis of neutrophil dysregulation. Results: Expansion of the circulating neutrophil compartment and the presence of activated low and normal density mature and immature neutrophil populations occurs in ARDS, irrespective of cause. Release of neutrophil granule proteins, neutrophil activation of the clotting cascade and upregulation of the Mac-1 platelet binding complex with formation of neutrophil platelet aggregates is exaggerated in COVID-19 ARDS. Importantly, activation of components of the neutrophil type I interferon responses is seen in ARDS following infection with SARS-CoV-2, with associated rewiring of neutrophil metabolism, and the upregulation of antigen processing and presentation. Whilst dexamethasone treatment constricts the immature low density neutrophil population, it does not impact upon prothrombotic hyperinflammatory neutrophil signatures. Conclusions: Given the crucial role of neutrophils in ARDS and the evidence of a disordered myeloid response observed in COVID-19 patients, this work maps the molecular basis for neutrophil reprogramming in the distinct clinical entities of COVID-19 and non-COVID-19 ARDS.