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

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.</p

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-RAGE hi 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. </p

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.</p

Impact of VEGF and TGF beta production by tumor cells on anti-tumor immune response and dominant tolerance installation by regulatory T cells

Ma thèse a pour but d'étudier l'impact des molécules immunosuppressives TGFβ et VEGF exprimées par les cellules tumorales dans la tolérance mise en place contre les tumeurs par les lymphocytes T régulateurs (Tregs). Pour cela, j'ai utilisé des lignées tumorales murines de mélanome B16 invalidées pour l'expression de TGFβ ou de VEGF par shRNA. J'ai pu observer que les invalidations induisent de profondes modifications de la réponse immunitaire contre les tumeurs, qui se traduisent par une forte diminution de son bras régulateur et une forte augmentation de son bras effecteur. Ces modifications sont le fait d'évènements très précoces et différents entre le VEGF et le TGFβ. Le ciblage simultané de TGFβ et de VEGF induit alors un rejet spontané des tumeurs chez 40% des animaux inoculés, et leur ciblage additionnel dans la stratégie thérapeutique utilisant les anticorps anti-PD-1 et anti-CTLA-4 induit un effet additif important. Nos résultats montrent donc que le VEGF et le TGFβ exprimés par les tumeurs sont des facteurs importants pour la tolérance immunitaire aux tumeurs et particulièrement pour la mobilisation des Tregs, et qu'il y a un fort rationnel à chercher à combiner leur ciblage avec les différentes stratégies thérapeutiques anti-tumorales existantes.My thesis aims at studying the impact tumor-derived immunosuppressive molecules TGFβ and VEGF on the dominant tolerance establishment by regulatory T cells (Tregs). For this I used murine B16 melanoma tumor cell lines knocked-down by shRNA for the expression of TGFβ or VEGF. I observed that these silencings induce dramatic changes in the immune response against tumors, which result in a large decrease of its regulatory arm and a strong increase of its effector arm. These changes result from very early mechanisms that differ between VEGF and TGFβ silencings. Accordingly, simultaneous targeting of TGFβ and VEGF induces significant tumor rejection, and their additional targeting in the anti-PD-1 / anti-CTLA-4 therapeutic strategy brings obvious additive effect. Globally, our results show that tumor-derived VEGF and TGFβ are important factors for the mobilization of Tregs and more generally for immune tolerance to tumors, and that there is a strong rational to combine their targeting with the different existing anti-tumor therapies

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

International audienc

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

International audienc

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

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

Abstract 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