The immunological features that distinguish COVID-19-associated acute respiratory distress syndrome (ARDS) from other causes of ARDS are incompletely understood. Here, we report the results of comparative lower respiratory tract transcriptional profiling of tracheal aspirate from 52 critically ill patients with ARDS from COVID-19 or from other etiologies, as well as controls without ARDS. In contrast to a “cytokine storm,” we observe reduced proinflammatory gene expression in COVID-19 ARDS when compared to ARDS due to other causes. COVID-19 ARDS is characterized by a dysregulated host response with increased PTEN signaling and elevated expression of genes with non-canonical roles in inflammation and immunity. In silico analysis of gene expression identifies several candidate drugs that may modulate gene expression in COVID-19 ARDS, including dexamethasone and granulocyte colony stimulating factor. Compared to ARDS due to other types of viral pneumonia, COVID-19 is characterized by impaired interferon-stimulated gene (ISG) expression. The relationship between SARS-CoV-2 viral load and expression of ISGs is decoupled in patients with COVID-19 ARDS when compared to patients with mild COVID-19. In summary, assessment of host gene expression in the lower airways of patients reveals distinct immunological features of COVID-19 ARDS.

30The gut microbiota produce hundreds of molecules that are present at high concentrations 31 in circulation and whose levels vary widely among humans. In most cases, molecule production 32 has not been linked to specific bacterial strains or metabolic pathways, and unraveling the 33 contribution of each molecule to host biology remains difficult. A general system to 'toggle' 34 molecules in this pool on/off in the host would enable interrogation of the mechanisms by which 35 they modulate host biology and disease processes. Such a system has been elusive due to 36 limitations in the genetic manipulability of Clostridium and its relatives, the source of many 37 molecules in this pool. Here, we describe a method for reliably constructing clean deletions in a 38 model commensal Clostridium, C. sporogenes (Cs), including multiply mutated strains. We 39 demonstrate the utility of this method by using it to 'toggle' off the production of ten Cs-derived 40 molecules that accumulate in host tissues. By comparing mice colonized by wild-type Cs versus a 41 mutant deficient in the production of branched short-chain fatty acids, we discover a previously 42 unknown IgA-modulatory activity of these abundant microbiome-derived molecules. Our method 43 opens the door to interrogating and sculpting a highly concentrated pool of chemicals from the 44 microbiome. 45 3 MAIN TEXT 46Gut bacteria produce hundreds of diffusible molecules that are notable for four reasons: 1) Most 47 have no host source, so their levels are determined predominantly or exclusively by the microbiome. 2) 48Many get into the bloodstream, so they can access peripheral tissues. 3) They often reach concentrations 49 that approach or exceed what a typical drug reaches, and the concentration range can be large -more 50 than an order of magnitude in many cases -so they have the potential to underlie biological differences 51 among humans. 4) Several of these molecules are known to be ligands for key host receptors; additional 52 compounds from this category are candidate ligands for, e.g., GPCRs and nuclear hormone receptors that 53 play an important role in the host immune and metabolic systems (1). Thus, the gut microbiome is a prolific 54 endocrine organ, but its output is not well understood. 55The biological activities of most of these molecules remain unknown. One reason is that there has 56 not been a general method for 'toggling' one or more of them on/off in the host, akin to a gene knockout 57 experiment in a model organism. Such a method would open the door to interrogating -and ultimately 58 controlling -one of the most concrete contributions gut bacteria make to host biology. 59Previous efforts that have sought to study an individual microbiome-derived molecule in the setting 60 of host colonization have used two main strategies: 1) Administering a compound by injection or gavage, 61 which can offer insights into mechanism of action but suffers from the lack of a clean background (i.e., 62 existing physiologic levels of the molecule of interest) and ...

Mass cytometry permits high-dimensional analysis of diverse aspects of cellular behavior. Here, we adapted this platform to simultaneously profile metabolism, signaling, cell cycle, and effector function with single-cell resolution. Using this approach, we measured enzymes characterizing glycolysis, the TCA cycle, fatty acid oxidation, oxidative phosphorylation, and nutrient transport.In conjunction, we measured downstream targets of TCR signaling regulating translation, proliferation and cytotoxicity as well as surface markers and transcription factors delineating CD8 T cell differentiation. High-dimensional single-cell metabolic analysis by mass cytometry permits identification of unique metabolic and differentiation states of extremely rare cell populations, such as antigen-specific T cells. We interrogated antigen-specific CD8 T cell activation in vitro as well as the trajectory of CD8 T cells responding to Listeria monocytogenes infection, a well-characterized model for studies of T cell differentiation. This integrated, highdimensional approach revealed a novel, activated, and highly metabolically active transitional T cell subset emerging at four days post-infection. These cells simultaneously exhibit glycolytic and oxidative functional programs, which we propose represents a key metabolic inflection point in CD8+ T cell differentiation. This approach should be useful for mechanistic investigations of metabolic regulation of immune responses in vivo.Recombinant human IL-2 (IL-2; TECIN (Teceleukin)) was provided by the National Cancer Institute.

28Harnessing immune defense mechanisms has revolutionized cancer therapy, but our 29 understanding of the factors governing immune responses in cancer remains incomplete, limiting patient 30 benefit. Here, we use mass cytometry to define the organism-wide immune landscape in response to 31 tumor development across five tissues in eight tumor models. Systemic immunity was dramatically 32 altered across mouse models and cancer patients, with changes in peripheral tissues differing from those 33 in the tumor microenvironment and taking place in phases during tumor growth. This tumor-experienced 34 immune system mounted dampened responses to orthogonal challenges, including reduced T cell 35 activation during viral or bacterial infection. Disruptions in T cell responses were not cell-intrinsic but 36 rather due to reduced responses in antigen-presenting cells (APCs). Promoting APC activation was 37 sufficient to restore T cell responses to orthogonal infection. All systemic immune changes were reversed 38 with surgical tumor resection, revealing remarkable plasticity in the systemic immune state, which 39 contrasts with terminal immune dysfunction in the tumor microenvironment. These results demonstrate 40 that tumor development dynamically reshapes the composition and function of the immune 41 macroenvironment. 42 43 44 45 46 47 48 MAIN TEXT 51Exploiting the mechanisms of immune activation and suppression has rapidly expanded our 52 toolkit against cancer, leading to diverse immunotherapeutic strategies and some impressive clinical 53 results. The efficacy of immunotherapies is currently limited, however, to select cancer types and patient 54 subsets, begging for a more thorough understanding of the factors that govern immune responses in 55 cancer patients. The field has garnered a robust understanding of the changes within the tumor 56 microenvironment (TME) that subvert immune surveillance and promote tumor growth. Heterogeneous 57 populations of immunosuppressive myeloid cells dominate many local immune landscapes, largely acting 58 to impede cytotoxic lymphocyte activity and survival 1-4 . Intratumoral cytotoxic CD8 T cells have been the 59 focus of the vast majority of immunomodulatory strategies in cancer therapy. However, recent studies 60 have demonstrated that cytotoxic T cells within the TME are highly and irreversibly dysfunctional, 61 acquiring epigenetic programs that render them incapable of normal effector functions, such as 62 proliferation, cytokine production, and cytolysis 5 . In parallel, we and others have found that systemic 63 immune responses are an essential component of tumor-eradicating immunity 6-10 . Consistent with these 64 results, activated T cells in human tumors after checkpoint blockade consist of clones not observed in the 65 tumor before the onset of therapy 11 . These findings argue that initiating a de novo systemic anti-tumor 66 immune response may be essential to achieving immunotherapeutic efficacy, especially in patients who 67 lack a strong pre-existing T cell response to...