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      Single‐cell transcriptomes of murine bone marrow stromal cells reveal niche‐associated heterogeneity

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          Abstract

          Bone marrow (BM) stromal cells are important in the development and maintenance of cells of the immune system. Using single cell RNA sequencing, we here explore the functional and phenotypic heterogeneity of individual transcriptomes of 1167 murine BM mesenchymal stromal cells. These cells exhibit a tremendous heterogeneity of gene expression, which precludes the identification of defined subpopulations. However, according to the expression of 108 genes involved in the communication of stromal cells with hematopoietic cells, we have identified 14 non‐overlapping subpopulations, with distinct cytokine or chemokine gene expression signatures. With respect to the maintenance of subsets of immune memory cells by stromal cells, we identified distinct subpopulations expressing Il7, Il15 and Tnfsf13b. Together, this study provides a comprehensive dissection of the BM stromal heterogeneity at the single cell transcriptome level and provides a basis to understand their lifestyle and their role as organizers of niches for the long‐term maintenance of immune cells.

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          Signalling through C-type lectin receptors: shaping immune responses

          Key Points Crosstalk between pattern recognition receptors (PRRs) expressed by dendritic cells orchestrates T helper (TH) cell differentiation through the induction of specific cytokine expression profiles, tailored to invading pathogens. C-type lectin receptors (CLRs) have an important role in orchestrating the induction of signalling pathways that regulate adaptive immune responses. CLRs can control adaptive immunity at various levels by inducing signalling on their own, through crosstalk with other PRRs or by inducing carbohydrate-specific signalling pathways. DC-specific ICAM3-grabbing non-integrin (DC-SIGN) interacts with mannose-carrying pathogens including Mycobacterium tuberculosis, HIV-1, measles virus and Candida albicans to activate the serine/threonine protein kinase RAF1. RAF1 signalling leads to the acetylation of Toll-like receptor (TLR)-activated nuclear factor-κB (NF-κB) subunit p65 and affects cytokine expression, such as inducing the upregulation of interleukin-10 (IL-10). DC-associated C-type lectin 1 (dectin 1) triggering by a broad range of fungal pathogens, such as C. albicans, Aspergillus fumigatus and Pneumocystis carinii, results in protective antifungal immunity through the crosstalk of two independent signalling pathways — one through spleen tyrosine kinase (SYK) and one through RAF1 — that are essential for the expression of TH1 and TH17 cell polarizing cytokines. Crosstalk between the SYK and RAF1 pathways is both synergistic and antagonizing to fine-tune NF-κB activity: although Ser276 phosphorylation of p65 leads to enhanced transcriptional activity of p65 itself through acetylation, it also inhibits the transcriptional activity of the NF-κB subunit RELB by sequestering it in p65–RELB dimers, which are transcriptionally inactive. The diversity in CLR-mediated signalling provides some major challenges for the researches to elucidate and manipulate the signalling properties of this exciting family of receptors. However, the recent advances strongly support the use of CLR targeting vaccination strategies using dendritic cells to induce or redirect adaptive immune responses as well as improve antigen delivery.
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            Cutting edge: the dependence of plasma cells and independence of memory B cells on BAFF and APRIL.

            Memory B (B(MEM)) cells and long-lived bone marrow plasma cells (BM-PCs) persist within local environmental survival niches that afford cellular longevity. However, the factors supporting B(MEM) cell survival within the secondary lymphoid organs and allowing BM-PC persistence in the bone marrow remain poorly characterized. We report herein that long-lived B(MEM) cell survival and function are completely independent of BAFF (B cell-activating factor of the TNF family) or APRIL (a proliferation-inducing ligand). Thus, B(MEM) cells represent the only mature B2 lineage subset whose survival is independent of these ligands. We have previously shown that the TNFR family member receptor BCMA (B cell maturation Ag) is a critical survival receptor for BM-PC survival in vivo. We identify in this study the ligands critical for BM-PC survival and show that either BAFF or APRIL supports the survival of BM-PCs in vivo. These data define the BAFF/APRIL-dependent and -independent components of long-lived humoral immunity.
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              Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-α.

              Platelet-derived growth factor receptor α (PDGFR-α) and stem cell antigen 1 (Sca-1) have recently been identified as selective markers of mouse mesenchymal stem cells (MSCs). PDGFR-α(+)Sca-1(+) (PαS) MSCs have augmented growth potential and robust tri-lineage differentiation compared with standard culture-selected MSCs. In addition, the selective isolation of PαS MSCs avoids cellular contamination that can complicate other methods. Here we describe in detail our protocol to isolate PαS MSCs using flow cytometry. In brief, the tibia and femora are isolated and crushed using a pestle and mortar. The crushed bones are then chopped and incubated for 1 h at 37 °C in 20 ml of DMEM containing 0.2% (wt/vol) collagenase. The cell suspension is filtered before red blood cell lysis and incubated with the following antibodies: allophycocyanin (APC)-conjugated PDGFR-α, FITC-conjugated Sca-1, phycoerythrin (PE)-conjugated CD45 and Ter119. Appropriate gates are constructed on a cell sorter to exclude dead cells and lineage (CD45(+)Ter-119(+))-positive cells. Approximately 10,000 PαS MSCs may then be isolated per mouse. The total protocol takes ~7 h to complete.
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                Author and article information

                Contributors
                pawel.durek@drfz.de
                mashregi@drfz.de
                Journal
                Eur J Immunol
                Eur. J. Immunol
                10.1002/(ISSN)1521-4141
                EJI
                European Journal of Immunology
                John Wiley and Sons Inc. (Hoboken )
                0014-2980
                1521-4141
                07 June 2019
                September 2019
                : 49
                : 9 ( doiID: 10.1002/eji.v49.9 )
                : 1372-1379
                Affiliations
                [ 1 ] Deutsches Rheuma‐Forschungszentrum (DRFZ) an Institute of the Leibniz Association Berlin Germany
                [ 2 ] Sanofi‐Aventis Germany Frankfurt am Main Germany
                [ 3 ] Division of Nephrology and Internal Intensive Care Medicine Charité‐Universitätsmedizin Berlin Germany
                [ 4 ] Department of Rheumatology and Clinical Immunology Charité‐Universitätsmedizin Berlin Germany
                Author notes
                [*] [* ] Full correspondence : Dr. Mir‐Farzin Mashreghi, Deutsches Rheuma‐Forschungszentrum Berlin, Charitéplatz 1, 10117 Berlin, Germany

                E‐mail: mashregi@ 123456drfz.de

                Additional correspondence : Dr. Pawel Durek

                E‐mail: pawel.durek@ 123456drfz.de

                Author information
                https://orcid.org/0000-0001-5753-0000
                https://orcid.org/0000-0002-8015-6907
                Article
                EJI4595
                10.1002/eji.201848053
                6771914
                31149730
                bbc19667-db2f-4c2c-875f-2fcdaa9201cb
                © 2019 The Authors. European Journal of Immunology published by WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 13 December 2018
                : 11 April 2019
                : 29 May 2019
                Page count
                Figures: 4, Tables: 0, Pages: 8, Words: 4648
                Funding
                Funded by: H2020 European Research Council
                Award ID: Advanced Grant IMMEMO (ERC‐2010‐AdG.20100317 Grant
                Funded by: Deutsche Forschungsgemeinschaft
                Award ID: DFG (TRR130)
                Award ID: HA5354/6‐2
                Award ID: HA5354/8‐1
                Award ID: DFG priority program 1468 IMMUNOBONE
                Award ID: SFB 633
                Funded by: European Regional Development Fund
                Award ID: ERDF 2014–2020
                Award ID: EFRE 1.8/11
                Award ID: Deutsches Rheuma‐Fo
                Categories
                Short Communication|Basic
                Systems immunology
                Short Communication
                Basic
                Custom metadata
                2.0
                eji4595
                September 2019
                Converter:WILEY_ML3GV2_TO_NLMPMC version:5.6.9 mode:remove_FC converted:01.10.2019

                Immunology
                bone marrow,cytokines,hematopoietic cells,single cell sequencing,stromal cells
                Immunology
                bone marrow, cytokines, hematopoietic cells, single cell sequencing, stromal cells

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