A myeloid program associated with COVID-19 severity is decreased by therapeutic blockade of IL-6 signaling.

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGEhi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS.

Cyclone: an accessible pipeline to analyze, evaluate, and optimize multiparametric cytometry data.

In the past decade, high-dimensional single-cell technologies have revolutionized basic and translational immunology research and are now a key element of the toolbox used by scientists to study the immune system. However, analysis of the data generated by these approaches often requires clustering algorithms and dimensionality reduction representation, which are computationally intense and difficult to evaluate and optimize. Here, we present Cytometry Clustering Optimization and Evaluation (Cyclone), an analysis pipeline integrating dimensionality reduction, clustering, evaluation, and optimization of clustering resolution, and downstream visualization tools facilitating the analysis of a wide range of cytometry data. We benchmarked and validated Cyclone on mass cytometry (CyTOF), full-spectrum fluorescence-based cytometry, and multiplexed immunofluorescence (IF) in a variety of biological contexts, including infectious diseases and cancer. In each instance, Cyclone not only recapitulates gold standard immune cell identification but also enables the unsupervised identification of lymphocytes and mononuclear phagocyte subsets that are associated with distinct biological features. Altogether, the Cyclone pipeline is a versatile and accessible pipeline for performing, optimizing, and evaluating clustering on a variety of cytometry datasets, which will further power immunology research and provide a scaffold for biological discovery.

Transcriptional space-time mapping identifies concerted immune and stromal cell patterns and gene programs in wound healing and cancer.

Tissue repair responses in metazoans are highly coordinated by different cell types over space and time. However, comprehensive single-cell-based characterization covering this coordination is lacking. Here, we captured transcriptional states of single cells over space and time during skin wound closure, revealing choreographed gene-expression profiles. We identified shared space-time patterns of cellular and gene program enrichment, which we call multicellular "movements" spanning multiple cell types. We validated some of the discovered space-time movements using large-volume imaging of cleared wounds and demonstrated the value of this analysis to predict "sender" and "receiver" gene programs in macrophages and fibroblasts. Finally, we tested the hypothesis that tumors are like "wounds that never heal" and found conserved wound healing movements in mouse melanoma and colorectal tumor models, as well as human tumor samples, revealing fundamental multicellular units of tissue biology for integrative studies.

Cyclone: an accessible pipeline to analyze, evaluate and optimize multiparametric cytometry data.

In the past decade, high-dimensional single cell technologies have revolutionized basic and translational immunology research and are now a key element of the toolbox used by scientists to study the immune system. However, analysis of the data generated by these approaches often requires clustering algorithms and dimensionality reduction representation which are computationally intense and difficult to evaluate and optimize. Here we present Cyclone, an analysis pipeline integrating dimensionality reduction, clustering, evaluation and optimization of clustering resolution, and downstream visualization tools facilitating the analysis of a wide range of cytometry data. We benchmarked and validated Cyclone on mass cytometry (CyTOF), full spectrum fluorescence-based cytometry, and multiplexed immunofluorescence (IF) in a variety of biological contexts, including infectious diseases and cancer. In each instance, Cyclone not only recapitulates gold standard immune cell identification, but also enables the unsupervised identification of lymphocytes and mononuclear phagocytes subsets that are associated with distinct biological features. Altogether, the Cyclone pipeline is a versatile and accessible pipeline for performing, optimizing, and evaluating clustering on variety of cytometry datasets which will further power immunology research and provide a scaffold for biological discovery.

Autologous T cell responses to primary human colorectal cancer spheroids are enhanced by ectonucleotidase inhibition.

OBJECTIVE: T cells are major effectors of the antitumoural immune response. Their activation by tumour-associated antigens can unleash their proliferation and cytotoxic functions, leading to tumour cell elimination. However, tumour-related immunosuppressive mechanisms including the overexpression of immune checkpoints like programmed cell death protein-1 (PD-1), are also engaged, promoting immune escape. Current immunotherapies targeting these pathways have demonstrated weak efficacy in colorectal cancer (CRC). It is thus crucial to find new targets for immunotherapy in this cancer type. DESIGN: In a prospective cohort of patients with CRC, we investigated the phenotype of tumour-related and non-tumour related intestinal T cells (n=44), particularly the adenosinergic pathway, correlating with clinical phenotype. An autologous coculture model was developed between patient-derived primary tumour spheroids and their autologous tumour-associated lymphocytes. We used this relevant model to assess the effects of CD39 blockade on the antitumour T cell response. RESULTS: We show the increased expression of CD39, and its co-expression with PD-1, on tumour infiltrating T cells compared with mucosal lymphocytes. CD39 expression was higher in the right colon and early-stage tumours, thus defining a subset of patients potentially responsive to CD39 blockade. Finally, we demonstrate in autologous conditions that CD39 blockade triggers T cell infiltration and tumour spheroid destruction in cocultures. CONCLUSION: In CRC, CD39 is strongly expressed on tumour infiltrating lymphocytes and its inhibition represents a promising therapeutic strategy for treating patients.

Spatiotemporal co-dependency between macrophages and exhausted CD8+ T cells in cancer.

T cell exhaustion is a major impediment to antitumor immunity. However, it remains elusive how other immune cells in the tumor microenvironment (TME) contribute to this dysfunctional state. Here, we show that the biology of tumor-associated macrophages (TAMs) and exhausted T cells (Tex) in the TME is extensively linked. We demonstrate that in vivo depletion of TAMs reduces exhaustion programs in tumor-infiltrating CD8+ T cells and reinvigorates their effector potential. Reciprocally, transcriptional and epigenetic profiling reveals that Tex express factors that actively recruit monocytes to the TME and shape their differentiation. Using lattice light sheet microscopy, we show that TAM and CD8+ T cells engage in unique, long-lasting, antigen-specific synaptic interactions that fail to activate T cells but prime them for exhaustion, which is then accelerated in hypoxic conditions. Spatially resolved sequencing supports a spatiotemporal self-enforcing positive feedback circuit that is aligned to protect rather than destroy a tumor.

Tumor-associated macrophage heterogeneity is driven by tissue territories in breast cancer.

Tissue-resident macrophages adapt to local signals within tissues to acquire specific functions. Neoplasia transforms the tissue, raising the question as to how the environmental perturbations contribute to tumor-associated macrophage (TAM) identity and functions. Combining single-cell RNA sequencing (scRNA-seq) with spatial localization of distinct TAM subsets by imaging, we discover that TAM transcriptomic programs follow two main differentiation paths according to their localization in the stroma or in the neoplastic epithelium of the mammary duct. Furthermore, this diversity is exclusively detected in a spontaneous tumor model and tracks the different tissue territories as well as the type of tumor lesion. These TAM subsets harbor distinct capacity to activate CD8+ T cells and phagocyte tumor cells, supporting that specific tumor regions, rather than defined activation states, are the major drivers of TAM plasticity and heterogeneity. The distinctions created here provide a framework to design cancer treatment targeting specific TAM niches.

Discovering dominant tumor immune archetypes in a pan-cancer census.

Cancers display significant heterogeneity with respect to tissue of origin, driver mutations, and other features of the surrounding tissue. It is likely that individual tumors engage common patterns of the immune system-here "archetypes"-creating prototypical non-destructive tumor immune microenvironments (TMEs) and modulating tumor-targeting. To discover the dominant immune system archetypes, the University of California, San Francisco (UCSF) Immunoprofiler Initiative (IPI) processed 364 individual tumors across 12 cancer types using standardized protocols. Computational clustering of flow cytometry and transcriptomic data obtained from cell sub-compartments uncovered dominant patterns of immune composition across cancers. These archetypes were profound insofar as they also differentiated tumors based upon unique immune and tumor gene-expression patterns. They also partitioned well-established classifications of tumor biology. The IPI resource provides a template for understanding cancer immunity as a collection of dominant patterns of immune organization and provides a rational path forward to learn how to modulate these to improve therapy.

Activating Immune Recognition in Pancreatic Ductal Adenocarcinoma via Autophagy Inhibition, MEK Blockade, and CD40 Agonism.

BACKGROUND AND AIMS: Patients with pancreatic ductal adenocarcinoma (PDA) have not yet benefitted from the revolution in cancer immunotherapy due in large part to a dominantly immunosuppressive tumor microenvironment. MEK inhibition combined with autophagy inhibition leads to transient tumor responses in some patients with PDA. We examined the functional effects of combined MEK and autophagy inhibition on the PDA immune microenvironment and the synergy of combined inhibition of MEK and autophagy with CD40 agonism (aCD40) against PDA using immunocompetent model systems. METHODS: We implanted immunologically "cold" murine PDA cells orthotopically in wide type C57BL/6J mice. We administered combinations of inhibitors of MEK1/2, inhibitors of autophagy, and aCD40 and measured anticancer efficacy and immune sequelae using mass cytometry and multiplexed immunofluorescence imaging analysis to characterize the tumor microenvironment. We also used human and mouse PDA cell lines and human macrophages in vitro to perform functional assays to elucidate the cellular effects induced by the treatments. RESULTS: We find that coinhibition of MEK (using cobimetinib) and autophagy (using mefloquine), but not either treatment alone, activates the STING/type I interferon pathway in tumor cells that in turn activates paracrine tumor associated macrophages toward an immunogenic M1-like phenotype. This switch is further augmented by aCD40. Triple therapy (cobimetinib + mefloquine + aCD40) achieved cytotoxic T-cell activation in an immunologically "cold" mouse PDA model, leading to enhanced antitumor immunity. CONCLUSIONS: MEK and autophagy coinhibition coupled with aCD40 invokes immune repolarization and is an attractive therapeutic approach for PDA immunotherapy development.

Global absence and targeting of protective immune states in severe COVID-19.

Although infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has pleiotropic and systemic effects in some individuals1-3, many others experience milder symptoms. Here, to gain a more comprehensive understanding of the distinction between severe and mild phenotypes in the pathology of coronavirus disease 2019 (COVID-19) and its origins, we performed a whole-blood-preserving single-cell analysis protocol to integrate contributions from all major immune cell types of the blood-including neutrophils, monocytes, platelets, lymphocytes and the contents of the serum. Patients with mild COVID-19 exhibit a coordinated pattern of expression of interferon-stimulated genes (ISGs)3 across every cell population, whereas these ISG-expressing cells are systemically absent in patients with severe disease. Paradoxically, individuals with severe COVID-19 produce very high titres of anti-SARS-CoV-2 antibodies and have a lower viral load compared to individuals with mild disease. Examination of the serum from patients with severe COVID-19 shows that these patients uniquely produce antibodies that functionally block the production of the ISG-expressing cells associated with mild disease, by activating conserved signalling circuits that dampen cellular responses to interferons. Overzealous antibody responses pit the immune system against itself in many patients with COVID-19, and perhaps also in individuals with other viral infections. Our findings reveal potential targets for immunotherapies in patients with severe COVID-19 to re-engage viral defence.

Global Absence and Targeting of Protective Immune States in Severe COVID-19.

While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a wholeblood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferonstimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and autodirected antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense.

Global Absence and Targeting of Protective Immune States in Severe COVID-19.

While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a whole-blood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferon-stimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and auto-directed antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense. ONE SENTENCE SUMMARY: In severe COVID-19 patients, the immune system fails to generate cells that define mild disease; antibodies in their serum actively prevents the successful production of those cells.

KLRG1 and CD103 Expressions Define Distinct Intestinal Tissue-Resident Memory CD8 T Cell Subsets Modulated in Crohn's Disease.

Intestinal tissue-resident memory CD8 T cells (Trm) are non-recirculating effector cells ideally positioned to detect and react to microbial infections in the gut mucosa. There is an emerging understanding of Trm cell differentiation and functions, but their implication in inflammatory bowel diseases, such as Crohn's disease (CD), is still unknown. Here, we describe CD8 cells in the human intestine expressing KLRG1 or CD103, two receptors of E-cadherin. While CD103 CD8 T cells are present in high numbers in the mucosa of CD patients and controls, KLRG1 CD8 T cells are increased in inflammatory conditions. Mucosal CD103 CD8 T cells are more responsive to TCR restimulation, but KLRG1 CD8 T cells show increased cytotoxic and proliferative potential. CD103 CD8 T cells originate mostly from KLRG1 negative cells after TCR triggering and TGFß stimulation. Interestingly, mucosal CD103 CD8 T cells from CD patients display major changes in their transcriptomic landscape compared to controls. They express Th17 related genes including CCL20, IL22, and IL26, which could contribute to the pathogenesis of CD. Overall, these findings suggest that CD103 CD8 T cells in CD induce a tissue-wide alert increasing innate immune responses and recruitment of effector cells such as KLRG1 CD8 T cells.

Cocultures of human colorectal tumor spheroids with immune cells reveal the therapeutic potential of MICA/B and NKG2A targeting for cancer treatment.

BACKGROUND: Immunotherapies still fail to benefit colorectal cancer (CRC) patients. Relevant functional assays aimed at studying these failures and the efficacy of cancer immunotherapy in human are scarce. 3D tumor cultures, called tumor organoids or spheroids, represent interesting models to study cancer treatments and could help to challenge these issues. METHODS: We analyzed heterotypic cocultures of human colon tumor-derived spheroids with immune cells to assess the infiltration, activation and function of T and NK cells toward human colorectal tumors in vitro. RESULTS: We showed that allogeneic T and NK cells rapidly infiltrated cell line-derived spheroids, inducing immune-mediated tumor cell apoptosis and spheroid destruction. NKG2D, a key activator of cytotoxic responses, was engaged on infiltrating cells. We thus assessed the therapeutic potential of an antibody targeting the specific ligands of NKG2D, MICA and MICB, in this system. Anti-MICA/B enhanced immune-dependent destruction of tumor spheroid by driving an increased NK cells infiltration and activation. Interestingly, tumor cells reacted to immune infiltration by upregulating HLA-E, ligand of the inhibitory receptor NKG2A expressed by CD8 and NK cells. NKG2A was increased after anti-MICA/B treatment and, accordingly, combination of anti-MICA/B and anti-NKG2A was synergistic. These observations were ultimately confirmed in a clinical relevant model of coculture between CRC patients-derived spheroids and autologous tumor-infiltrating lymphocytes. CONCLUSIONS: Altogether, we show that tumor spheroids represent a relevant tool to study tumor-lymphocyte interactions on human tissues and revealed the antitumor potential of immunomodulatory antibodies targeting MICA/B and NKG2A.

Role of Chemokine Receptor CCR4 and Regulatory T Cells in Wound Healing of Diabetic Mice.

Wound healing is a well-coordinated process that involves inflammatory mediators and cellular responses; however, if any disturbances are present during this process, tissue repair is impaired. Chronic wounds are one of the serious long-term complications associated with diabetes mellitus. The chemokine receptor CCR4 and its respective ligands, CCL17 and CCL22, are involved in regulatory T cell recruitment and activation in inflamed skin; however, the role of regulatory T cells in wounds is still not clear. Our aim was to investigate the role of CCR4 and regulatory T cells in cutaneous wound healing in diabetic mice. Alloxan-induced diabetic wild- type mice (diabetic) developed wounds that were difficult to heal, differently from CCR4-/- diabetic mice (CCR4-/- diabetic), and also from anti-CCL17/22 or anti-CD25-injected diabetic mice that presented with accelerated wound healing and fewer regulatory T cells in the wound bed. Consequently, CCR4-/- diabetic mice also presented with alteration on T cells population in the wound and draining lymph nodes; on day 14, these mice also displayed an increase of collagen fiber deposition. Still, cytokine levels were decreased in the wounds of CCR4-/- diabetic mice on day 2. Our data suggest that the receptor CCR4 and regulatory T cells negatively affect wound healing in diabetic mice.

Activated dendritic cells modulate proliferation and differentiation of human myoblasts.

Idiopathic Inflammatory Myopathies (IIMs) are a heterogeneous group of autoimmune diseases affecting skeletal muscle tissue homeostasis. They are characterized by muscle weakness and inflammatory infiltration with tissue damage. Amongst the cells in the muscle inflammatory infiltration, dendritic cells (DCs) are potent antigen-presenting and key components in autoimmunity exhibiting an increased activation in inflamed tissues. Since, the IIMs are characterized by the focal necrosis/regeneration and muscle atrophy, we hypothesized that DCs may play a role in these processes. Due to the absence of a reliable in vivo model for IIMs, we first performed co-culture experiments with immature DCs (iDC) or LPS-activated DCs (actDC) and proliferating myoblasts or differentiating myotubes. We demonstrated that both iDC or actDCs tightly interact with myoblasts and myotubes, increased myoblast proliferation and migration, but inhibited myotube differentiation. We also observed that actDCs increased HLA-ABC, HLA-DR, VLA-5, and VLA-6 expression and induced cytokine secretion on myoblasts. In an in vivo regeneration model, the co-injection of human myoblasts and DCs enhanced human myoblast migration, whereas the absolute number of human myofibres was unchanged. In conclusion, we suggest that in the early stages of myositis, DCs may play a crucial role in inducing muscle-damage through cell-cell contact and inflammatory cytokine secretion, leading to muscle regeneration impairment.

TGF-ß and VEGF cooperatively control the immunotolerant tumor environment and the efficacy of cancer immunotherapies.

Tregs imprint an early immunotolerant tumor environment that prevents effective antitumor immune responses. Using transcriptomics of tumor tissues, we identified early upregulation of VEGF and TGF-ß pathways compatible with tolerance imprinting. Silencing of VEGF or TGF-ß in tumor cells induced early and pleiotropic modulation of immune-related transcriptome signatures in tumor tissues. These were surprisingly similar for both silenced tumors and related to common downstream effects on Tregs. Silencing of VEGF or TGF-ß resulted in dramatically delayed tumor growth, associated with decreased Tregs and myeloid-derived suppressor cells and increased effector T cell activation in tumor infiltrates. Strikingly, co-silencing of TGF-ß and VEGF led to a substantial spontaneous tumor eradication rate and the combination of their respective inhibitory drugs was synergistic. VEGF and/or TGF-ß silencing also restored tumor sensitivity to tumor-specific cell therapies and markedly improved the efficacy of anti-PD-1/anti-CTLA-4 treatment. Thus, TGF-ß and VEGF cooperatively control the tolerant environment of tumors and are targets for improved cancer immunotherapies.

Regulatory T Cells Orchestrate Similar Immune Evasion of Fetuses and Tumors in Mice.

Embryos and tumors are both masses of dividing cells expressing foreign Ags, but they are not rejected by the immune system. We hypothesized that similar tolerogenic mechanisms prevent their rejection. Global comparison of fetal and tumor microenvironments through transcriptomics in mice revealed strikingly similar and dramatic decreases in expression of numerous immune-related pathways, including Ag presentation and T cell signaling. Unsupervised analyses highlighted the parallel kinetics and similarities of immune signature downregulation, from the very first days after tumor or embryo implantation. Besides upregulated signatures related to cell proliferation, the only significant signatures shared by the two conditions across all biological processes and all time points studied were downmodulated immune response signatures. Regulatory T cell depletion completely reverses this immune downmodulation to an immune upregulation that leads to fetal or tumor immune rejection. We propose that evolutionarily selected mechanisms that protect mammalian fetuses from immune attack are hijacked to license tumor development.