Updates on radiotherapy-immunotherapy combinations: Proceedings of 6(th) annual ImmunoRad conference

Focal radiation therapy (RT) has attracted considerable attention as a combinatorial partner for immunotherapy (IT), largely reflecting a well-defined, predictable safety profile and at least some potential for immunostimulation. However, only a few RT-IT combinations have been tested successfully in patients with cancer, highlighting the urgent need for an improved understanding of the interaction between RT and IT in both preclinical and clinical scenarios. Every year since 2016, ImmunoRad gathers experts working at the interface between RT and IT to provide a forum for education and discussion, with the ultimate goal of fostering progress in the field at both preclinical and clinical levels. Here, we summarize the key concepts and findings presented at the Sixth Annual ImmunoRad conference

Construction d'un arbre de l'hématopoïèse dérivé des progéniteurs érythro-myéloïdes (EMP)

Erythro-myeloid progenitors (EMP) are developmentally restricted hematopoietic progenitors that produce the first definitive hematopoietic cells in the embryo. Importantly, it has been shown that they give rise to tissue-resident macrophages and mast cells that colonize organs during gestation and self-maintain during adult life without contribution from the bone marrow. These cells are specialized immune cells that contribute to the homeostasis of the tissues throughout steady state and tissue challenge (wounds or infections). This PhD project aimed to characterise the contribution of EMPs to the hematopoietic system of the embryo, with special focus to their niche of origin, the yolk sac. With the use of genetic mouse models, high parameter flow cytometry and single cell expression analysis, this project i) characterises the major definitive progenitor populations in the yolk sac, ii) identifies a novel pathway of direct megakaryopoiesis from EMPs and iii) uncovers two waves of EMP potential emerging at different stages. This work sheds light on the poorly characterised early definitive embryonic haematopoiesis and could have potential implications on macrophage ontogeny studies and early childhood myeloproliferative disorders.Les progéniteurs érythro-myéloïdes (EMP) sont des progéniteurs hématopoïétiques restreints au développement qui produisent les premières cellules hématopoïétiques définitives de l'embryon. Surtout, ils donnent naissance aux mastocytes et macrophages résidents qui colonisent les différents organes pendant la gestation et s’auto-renouvellent pendant la vie adulte sans apport de la moelle osseuse. Ces cellules sont des cellules immunitaires spécialisées qui contribuent à l'homéostasie des tissus aussi bien dans les tissus sains que suite à des blessures ou des infections. Ce projet de doctorat visait à caractériser la contribution des EMP au système hématopoïétique de l'embryon, avec un accent particulier sur leur niche d'origine, le sac vitellin. En utilisant des modèles génétiques de souris, la cytométrie de flux à paramètres élevés et l'analyse de l'expression au niveau unicellulaire, ce projet i) caractérise les principales populations de progéniteurs définitifs dans le sac vitellin, ii) identifie une nouvelle voie de mégacaryopoïèse directe à partir des EMP et iii) découvre deux vagues de potentiel EMP émergeant à des stades différents du développement. Ce projet a pour but de mieux comprendre la complexité de l'hématopoïèse embryonnaire et pourrait avoir des implications sur les études d'ontogenèse des macrophages et les troubles myéloprolifératifs infantiles

Overlapping Definitive Progenitor Waves Divide and Conquer to Build a Layered Hematopoietic System

We are indebted to Baptiste Saudemont and Yann Loe-Mie for critical advice, training, reagents and quality control for the MARS-Seq pipeline.We would like tothankRebecca Gentek, Marc Bajénoff (CIML in Marseille) and Kémy Adefor providing and acquiring exploratory data for Cdh5CreERT2experiments, Pascal Dardenne and Yvan Lallemand for handling and injections of mice, Sébastien Bastide and Caroline Kaiser for advice on scRNA-Seq analysis with Seurat, Tobias Weinberger and Rebeca Ponce Landete for isolation of adult tissues, Anne Dejean for transfer of the Cdh5CreERT2strainand other members of the Gomez Perdiguero and Cumano groups for critical discussions and feedback. We appreciate the support and advice of the Cytometry and Biomarkers (UTECHS CB) platform (Sophie Novault and Sandrine Schmutz), the Center of Bioinformatics, Biostatistics and Integrative Biology (C3BI), the Institute PasteurSingle Cell Initiative (Heather Marlow) and the Animalerie Centrale.ABSTRACT Adult innate immune cells are part of a layered hematopoietic system constructed from definitive hematopoietic stem and progenitor cells (HSPC) with diverse origins during development. One source of HSPC are fetal hematopoietic stem cells (HSC) that provide long-term reconstitution throughout life. However, the extent to which HSC produce mature cells in utero is only recently being uncovered. This is in part due to the added complexity of an overlapping wave of definitive progenitors that derive from yolk sac erythro-myeloid progenitors (EMP). HSC and EMP are generated from spatiotemporally distinct hemogenic endothelia, yet they both migrate to the fetal liver niche where they co-habitate and are presumed to reach their full potential. Delineation of the respective HSC and EMP pathways towards developmental immune cell differentiation has been confounded by challenges in ontogeny-specific cell labeling. In this study, in vivo inducible pulse chase labeling revealed that HSC contribute little to fetal myelopoiesis and that EMP are the predominant source of mature myeloid cells until birth. This is similar to what has been reported for the erythroid branch of hematopoiesis thereby establishing a developmentally-restricted privilege for erythro-myeloid differentiation from EMP compared to HSC. Tracing the origins of mature cells to the progenitor level by immunophenotyping and single cell RNA sequencing uncovered a dichotomy in the allocation of fetal liver EMP and HSC to myeloid progenitor subsets, both in timing and lineage bias. This has exposed an uncoupling between developmental granulopoiesis and monopoiesis from EMP and HSC pathways, and provides a framework for future studies of HSC-dependent and -independent hematopoiesis. HIGHLIGHTS EMP-to-HSC switch in fetal liver myelopoiesis occurs late in gestation EMP are efficient at producing early transit amplifying erythroid and myeloid intermediates scRNA-seq reveals three trajectories of EMP myelopoiesis Myeloid lineage commitment during development is cell type and ontogeny specifi

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

International audienceMacrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry

Megakaryocyte production is sustained by direct differentiation from erythromyeloid progenitors in the yolk sac until midgestation

International audienceThe extra-embryonic yolk sac contains the first definitive multipotent hematopoietic cells, denominated erythromyeloid progenitors. They originate in situ prior to the emergence of hematopoietic stem cells and give rise to erythroid, monocytes, granulocytes, mast cells and macrophages, the latter in a Myb transcription factor-independent manner. We uncovered here the heterogeneity of yolk sac erythromyeloid progenitors, at the single cell level, and discriminated multipotent from committed progenitors, prior to fetal liver colonization. We identified two temporally distinct megakaryocyte differentiation pathways. The first occurs in the yolk sac, bypasses intermediate bipotent megakaryocyte-erythroid progenitors and, similar to the differentiation of macrophages, is Myb-independent. By contrast, the second originates later, from Myb-dependent bipotent progenitors expressing Csf2rb and colonize the fetal liver, where they give rise to megakaryocytes and to large numbers of erythrocytes. Understanding megakaryocyte development is crucial as they play key functions during vascular development, in particular in separating blood and lymphatic networks

Evaluation of the role of the immune system response following minibeam radiation therapy

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Combining FLASH and spatially fractionated radiation therapy: The best of both worlds

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Evaluation of the Role of the Immune System Response After Minibeam Radiation Therapy

International audiencePurpose: Minibeam radiation therapy (MBRT) is an innovative technique that uses a spatial dose modulation. The dose distribution consists of high doses (peaks) in the path of the minibeam and low doses (valleys). The underlying biological mechanism associated with MBRT efficacy remains currently unclear and thus we investigated the potential role of the immune system after treatment with MBRT.Methods and materials: Rats bearing an orthotopic glioblastoma cell line were treated with 1 fraction of high dose conventional radiation therapy (30 Gy) or 1 fraction of the same mean dose in MBRT. Both immunocompetent (F344) and immunodeficient (Nude) rats were analyzed in survival studies. Systemic and intratumoral immune cell population changes were studied with flow cytometry and immunohistochemistry (IHC) 2 and 7 days after the irradiation.Results: The absence of response of Nude rats after MBRT suggested that T cells were key in the mode of action of MBRT. An inflammatory phenotype was observed in the blood 1 week after irradiation compared with conventional irradiation. Tumor immune cell analysis by flow cytometry showed a substantial infiltration of lymphocytes, specifically of CD8 T cells and B cells in both conventional and MBRT-treated animals. IHC revealed that MBRT induced a faster recruitment of CD8 and CD4 T cells. Animals that were cured by radiation therapy did not suffer tumor growth after reimplantation of tumoral cells, proving the long-term immunity response generated after a high dose of radiation.Conclusions: Our findings show that MBRT can elicit a robust antitumor immune response in glioblastoma while avoiding the high toxicity of a high dose of conventional radiation therapy

Yolk sac, but not hematopoietic stem cell–derived progenitors, sustain erythropoiesis throughout murine embryonic life

International audienceIn the embryo, the first hematopoietic cells derive from the yolk sac and are thought to be rapidly replaced by the progeny of hematopoietic stem cells. We used three lineage-tracing mouse models to show that, contrary to what was previously assumed, hematopoietic stem cells do not contribute significantly to erythrocyte production up until birth. Lineage tracing of yolk sac erythromyeloid progenitors, which generate tissue resident macrophages, identified highly proliferative erythroid progenitors that rapidly differentiate after intra-embryonic injection, persisting as the major contributors to the embryonic erythroid compartment. We show that erythrocyte progenitors of yolk sac origin require 10-fold lower concentrations of erythropoietin than their hematopoietic stem cell-derived counterparts for efficient erythrocyte production. We propose that, in a low erythropoietin environment in the fetal liver, yolk sac-derived erythrocyte progenitors efficiently outcompete hematopoietic stem cell progeny, which fails to generate megakaryocyte and erythrocyte progenitors