ABSTRACT: Fibroblastic reticular cells (FRCs) (CD45- Ter119-MADCAM1- CD21/35- CD31- PDPN-) from skin draining lymph nodes of Grem1-Cre-ERT2.cki; Rosa26-LSL-EYFP mice were sorted and subjected to bulk RNA-seq of Grem1 reporter + and Grem1 reporter - cells.
Project description:Head and neck squamous cell carcinoma (HNSCC) patients have a very short survival. To improve patient survival, there is an emerging need for a better understanding of the obstacles of ionizing irradiation (IR), besides surgery, currently the best treatment option for HNSCC. A confounding factor is the immune suppressive tumor microenvironment (IS-TME) which is orchestrated by tenascin-C (TNC), a highly abundant extracellular matrix (ECM) molecule, getting upregulated by IR. Here, we investigated the roles of TNC on IR-induced tumor regression in a murine oral squamous cell carcinoma (OSCC) model expressing or lacking TNC. While tumors in a TNC-expressing host were radiosensitive, tumors in the TNC genetically-depleted mice were radioresistant pointing at a so far underexplored function of TNC in promoting anti-tumor defense. Upon IR, fibroblast reticular cells (FRCs), a known source of TNC, were more numerous and promoted the IS-TME only in the TNC-expressing tumors. We applied IR to FRCs isolated from lymph nodes and observed that whereas TNC-expressing FRCs were radiosensitive, the TNC-depleted FRCs were radioresistant mimicking the tumor phenotype. Irradiation enhanced the crosstalk of TNC-expressing FRCs with the OSCC causing enhanced tumor cell death but also tumor-promoting plasticity in the survivors as well as induction of Rad51, indicative of enhanced double strand DNA break repair and radioresistance. On the other hand, IR also increased TNC and CCL21 expression in the cocultured FRCs, enforcing an IS-TME. These results demonstrate that TNC determines the FRC tumor promoting phenotype counteracting IR-induced tumor regression, which is relevant in human cancer as a high FRC signature in conjunction with high TNC levels correlates with shorter survival in the irradiated HNSCC patients. We propose that tumor FRCs highly expressing TNC may be an excellent novel target to improve radiotherapy-induced tumor eradication.
Project description:Peyer`s patches (PP) are underpinned by a dense network of FRCs. To elaborate the subset composition and topological organization of PP FRCs, we employed well-characterized Ccl19-Cre and Col6a1-Cre mouse models that permit FRC targeting in secondary lymphoid organs. To define PP FRC subsets and their relation to the observed FRC lineages, we isolated fibroblasts from Col6a1Eyfp and Ccl19Eyfp PPs and performed droplet-based single cell transcriptomics analysis. Taken together, the molecular definition of the PP FRC landscape reveals two FRC lineages furnishing distinct microenvironmental niches.
Project description:Lymph node (LN) stromal cells, particularly fibroblastic reticular cells (FRCs), provide critical structural support and regulate immunity, tolerance and transport properties of LNs. In many tumors, LN metastasis is predictive of poor prognosis, however, stromal contribution to the evolving microenvironment of tumor draining LNs (TDLN) remains poorly understood. Here we present comparative transcriptional data of resting and TDLN FRCs after different time points of tumor drainage. FRCs were isolated from lymph nodes and FACS sorted based on the expression of CD45-, CD31- and PDPN+
Project description:Immune protection of the body cavities depends on the swift activation of innate and adaptive immune responses in non-classical secondary lymphoid organs known as fat-associated lymphoid clusters (FALCs). While it is well-established that fibroblastic reticular cells (FRCs) are an integral component of the immune-stimulating infrastructure of lymph nodes and other classical secondary lymphoid organs, it has remained elusive whether and how FRCs in FALCs contribute to peritoneal immunity. Using FRC-specific gene targeting, we found that FALCs are underpinned by an elaborated FRC network and that initiation of peritoneal immunity was governed through FRC activation via MyD88-dependent innate immunological sensing. FRC-specific ablation of Myd88 expression blocked recruitment of inflammatory monocytes into FALCs and subsequent CD4+ T cell-dependent B-cell activation. Moreover, containment of Salmonella infection was compromised in conditionally Myd88-deficient mice indicating that FRCs in FALCs function as initial checkpoint in the orchestration of protective immune responses in the peritoneal cavity.
Project description:Lymph node is a secondary lymphoid organ that has structural organization related to their immune function, which is highly ordered and compartmentalized. Lymph nodes shelter immune cells and also fibroblastic-like cells such as fibroblastic reticular cells (FRCs). FRCs have mesodermal origin and are classically characterized by the expression of podoplanin (PDPN/gp38) and lack of CD31 expression. FRCs are implicated in several immune processes but the pathways subjacent their function are still a matter of research. Another cell population found in LNs, the double negative cells (DNCs), are even less known. They do not express PDPN or CD31 markers and a specific marker is absent, their localization within the LN is undefined and their phenotype or function, remain to be clarified. Although these cells have been studied in murine models, studies on human FRCs and DNCs are limited and therefore our study should contribute to the understanding of biology and function of these cells and should promote knowledge of efficiency and disorders in the lymph node immune response. This study evaluates gene expression and secretion of cytokines and chemokines in human-derived FRCs and DNCs during homeostasis and following inflammatory stimuli. Our results demonstrate that cytokines and chemokines secreted by human FRCs and DNCs partially differ from those identified in murine models. In addition, our study demonstrates that DNCs expressed a more varied of cytokines when compared with FRCs, while FRCs expressed a wider chemokines pattern. Such differences maybe related with specific functional differences between those cell populations within LNs.
Project description:Although cross-species transcriptional analysis has been generated for DCs37, transcriptomic conservation between mouse and human FRCs at single-cell resolution has been unclear. To test whether GREM1+ FRCs might also play a role in DC homeostasis in humans, we performed scRNA-seq of CD45–PDPN+ stromal cells, as well as CD45+CD11c+ immune cells from healthy human LNs of three donors.
Project description:Age-associated B cells (ABCs) accumulate in systemic autoimmunity, and contributes to pathogenesis. ABCs are characterized by high expression of CD11c and T-bet. As a transcription factor, T-bet cannot be labeled in live cells with antibodies, so we constructed a reporter mouse strain in which fluorescent protein tdTomato fused to T-bet. Then we sorted ABCs (CD11c+T-bet+CD21- B cell) and non-ABCs (CD11c-T- bet- B cell) from the Bm12-induced lupus model for RNA sequencing.
Project description:B cell tolerance is established using deletion, anergy and receptor editing mediated by secondary recombination but it is unclear why autoreactive B cells choose a tolerance mechanism over another. We identified subgroups of patients with either rheumatoid arthritis or common variable immunodeficiency who presented defects in secondary recombination, which correlated with unusual CD21-/lo naïve B cells in their blood. CD21-/lo B cells were unable to induce calcium flux, get activated or proliferate in response to B cell receptor and/or CD40 triggering, suggesting that these B cells are anergic. Moreover, CD21-/lo B cells are often autoreactive and may express anti-nuclear antibodies. Thus, anergy can be a default tolerance mechanism mainly induced when receptor editing fails to silence developing autoreactive B cells. Experiment Overall Design: RNA was extracted from batch sorted CD19+CD21+CD10-CD27- and CD19+CD21-CD10-CD27- naïve B cells isolated from donors using the Absolutely RNA microprep kit (Stratagene). 100-200 ng of RNA was obtained per sample, and the quality of the purified RNA was assessed by the Bioanalyzer from Agilent. Using the Ovation biotin system kit from Nugen, 30-50ng of RNA was amplified and labeled to produce cDNA. Labeled cDNA was hybridized on chips containing the whole human genome (Human Genome U133 2.0 from Affymetrix). Data from CD21+ and CD21- B cell populations were compared in order to determine the gene signature of the newly described CD21- B cells.
Project description:In this study we focussed our investigations on ECM remodelling by FRCs during lymph node (LN) expansion, and the interconnection between the cellular and ECM components of the conduit network. We demonstrate a loss of ECM components of the conduit during acute LN expansion
Project description:Although cross-species transcriptional analysis has been generated for DCs, transcriptomic conservation between mouse and human FRCs at single-cell resolution has been unclear. To test whether GREM1+ FRCs might also play a role in DC homeostasis in humans, we performed scRNA-seq of CD45–PDPN+ stromal cells, as well as CD45+CD11c+ immune cells from healthy human LNs of three human donors. Data was generated using the 10x platform.