Non-hematopoietic Stromal Cells Research Articles

Age-related epithelial defects limit thymic function and regeneration

The thymus is essential for establishing adaptive immunity yet undergoes age-related involution that leads to compromised immune responsiveness. The thymus is also extremely sensitive to acute insult and although capable of regeneration, this capacity declines with age for unknown reasons. We applied single-cell and spatial transcriptomics, lineage-tracing and advanced imaging to define age-related changes in nonhematopoietic stromal cells and discovered the emergence of two atypical thymic epithelial cell (TEC) states. These age-associated TECs (aaTECs) formed high-density peri-medullary epithelial clusters that were devoid of thymocytes; an accretion of nonproductive thymic tissue that worsened with age, exhibited features of epithelial-to-mesenchymal transition and was associated with downregulation of FOXN1. Interaction analysis revealed that the emergence of aaTECs drew tonic signals from other functional TEC populations at baseline acting as a sink for TEC growth factors. Following acute injury, aaTECs expanded substantially, further perturbing trophic regeneration pathways and correlating with defective repair of the involuted thymus. These findings therefore define a unique feature of thymic involution linked to immune aging and could have implications for developing immune-boosting therapies in older individuals.

Mesenchymal stem cells and their derived exosomes in multiple sclerosis disease: from paper to practice.

Multiple sclerosis (MS) is a chronic neurodegenerative, inflammatory, and demyelinating disease of the central nervous system (CNS). Current medicines are not sufficient to control the inflammation and progressive damage to the CNS that is known in MS. These drawbacks highlight the need for novel treatment options. Cell therapy can now be used to treat complex diseases when conventional therapies are ineffective. Mesenchymal stem cells (MSCs) are a diverse group of multipotential non-hematopoietic stromal cells which have immunomodulatory, neurogenesis, and remyelinating capacity. Their advantageous effects mainly rely on paracrine, cell-cell communication and differentiation properties which introduced them as excellent candidates for MS therapy. Exosomes, as one of the MSCs secretomes, have unique properties that make them highly promising candidates for innovative approach in regenerative medicine. This review discusses the therapeutic potential of MSCs and their derived exosomes as a novel treatment for MS, highlighting the differences between these two approaches.

From Marrow to Bone and Fat: Exploring the Multifaceted Roles of Leptin Receptor Positive Bone Marrow Mesenchymal Stromal Cells.

The bone marrow (BM) stromal cell microenvironment contains non-hematopoietic stromal cells called mesenchymal stromal cells (MSCs). MSCs are plastic adherent, form CFU-Fs, and give rise to osteogenic, adipogenic, chondrogenic progenitors, and most importantly provide HSC niche factor chemokine C-X-C motif ligand 12 (CXCL12) and stem cell factor (SCF). Different authors have defined different markers for mouse MSC identification like PDGFR+Sca-1+ subsets, Nestin+, or LepR+ cells. Of these, the LepR+ cells are the major source of SCF and CXCL12 in the BM microenvironment and play a major role in HSC maintenance and hematopoiesis. LepR+ cells give rise to most of the bones and BM adipocytes, further regulating the microenvironment. In adult BM, LepR+ cells are quiescent but after fracture or irradiation, they proliferate and differentiate into mesenchymal lineage osteogenic, adipogenic and/or chondrogenic cells. They also play a crucial role in the steady-state hematopoiesis process, as well as hematopoietic regeneration and the homing of hematopoietic stem cells (HSCs) after myeloablative injury and/or HSC transplantation. They line the sinusoidal cavities, maintain the trabeculae formation, and provide the space for HSC homing and retention. However, the LepR+ cell subset is heterogeneous; some subsets have higher adipogenic potential, while others express osteollineage-biased genes. Different transcription factors like Early B cell factor 3 (EBF3) or RunX2 help maintain this balance between the self-renewing and committed states, whether osteogenic or adipogenic. The study of LepR+ MSCs holds immense promise for advancing our understanding of HSC biology, tissue regeneration, metabolic disorders, and immune responses. In this review, we will discuss the origin of the BM resident LepR+ cells, different subtypes, and the role of LepR+ cells in maintaining hematopoiesis, osteogenesis, and BM adipogenesis following their multifaceted impact.

Vascular Cell Adhesion Molecule-1 Regulates Bone Marrow Mesenchymal Stem Cell Maintenance and Niche Function

Bone marrow (BM) mesenchymal stem cells (MSCs) are rare non-hematopoietic perivascular stromal cells characterized by their unique ability to self-renew and differentiate into bone, cartilage, and fat, ensuring proper skeletal development and maintenance. Different subsets of BM MSCs associate with arteriolar and sinusoidal vessels to form specialized niches that regulate hematopoietic stem cell (HSC) function by secreting high levels of niche factors such as CXC-chemokine ligand 12 (CXCL12), stem cell factor (SCF), and Vascular Cell Adhesion Molecule-1 (VCAM1). VCAM1 is classically expressed on endothelial and stromal cells where it acts as an adhesion molecule that preferentially binds to α4β1 integrin on HSCs and progenitor cells (HSPCs). Deletion of Vcam1 in endothelial and hematopoietic cells induces HSPC mobilization into the peripheral blood without affecting endothelial cells homeostasis. Recent work from our lab further demonstrated that VCAM1 expression on HSCs and leukemic stem cells confers innate immune tolerance ( Nat Cell Biol 2022). While the contribution of HSC and endothelial-derived VCAM1 to homeostasis has been well studied, the role of MSC-derived VCAM1 on HSC and MSC maintenance and multilineage potency remains unknown. Our supporting data suggest that MSCs are the BM's main source of Vcam1 (p<0.0001) and that VCAM1 is critical for the maintenance, survival, and function of MSCs. Using a mouse model in which Vcam1 can be conditionally deleted in BM MSCs by a tamoxifen-inducible N-Cadherin-Cre ER line (referred to as Vcam1 N-CadCreER), we found that deletion of Vcam1 significantly reduces the number (4-fold, p=0.0030), frequency (4-fold, p=0.0042), and viability (2-fold, p=0.0039) of BM MSCs. Previous findings in breast cancer revealed that VCAM1 promotes PI3K/Akt survival signaling via Akt phosphorylation. Accordingly, Vcam1 deletion in N-Cadherin-Cre ER MSCs, demonstrated a significant reduction in phospho-Akt (Ser473) (p<0.05), supporting the role of VCAM1 in mediating PI3K/Akt survival signaling in MSCs. Intriguingly, the remaining viable VCAM1-null MSCs isolated from Vcam1 N-CadCreER mice exhibited reduced clonogenic potential (determined by CFU-F assays; 3-fold, p=0.0001). Furthermore, micro-CT analysis of 6-month-old Vcam1 N-CadCreER mice presented an altered bone phenotype compared to the control as evidenced by their significantly increased bone thickness and volume (p<0.05), yet significantly decreased tissue mineral density and medullary cavity area (p<0.05). Bulk RNA sequencing (RNAseq) of sorted viable MSCs revealed distinct grouping between Control and Vcam1 N-CadCreER MSCs. Gene ontology analysis demonstrated that deletion of Vcam1 caused an upregulation of pathways associated with inflammation and cytokine release while pathways associated with ossification, extracellular matrix organization, and skeletal development were all downregulated. Surprisingly, both bulk RNAseq and qPCR analysis confirmed a significant reduction in the expression of HSC niche factors ( Vcam1 Cxcl12 and Scf) following Vcam1 deletion, indicative of reduced niche activity. As expected, Vcam1 deletion in N-Cadherin-Cre ER MSCs, caused significant mobilization of HSCs into the blood (p<0.0001) and BM homing defects (p<0.05), however the number and repopulation capacity of BM HSCs remained unaltered, as seen by BM transplantation experiments. Surprisingly, imaging analysis of whole-mount bone marrow sternum of phenotypic HSCs revealed no differences in HSC niche localization between Vcam1-null and Control mice. Accordingly, ELISA analysis of BM extracellular fluid revealed no differences in overall CXCL12 and SCF levels suggesting that there may be some local or systemic niche compensation at play. Single-cell (sc)RNAseq of pooled BM and compact bone sorted non-hematopoietic stromal cells (DAPI -Lineage -CD45 -Ter119 -CD19 -CD71 -)from Vcam1 N-CadCreERand Control mice further confirmed the downregulation of HSC niche factors in MSCs, and overall remodeling of the BM stromal cell composition following Vcam1 deletion. Interestingly, our scRNAseq analysis revealed increased expression of Cxcl12 and Scf in specific vascular and stromal cell populations, supporting the notion of HSC niche compensation. Overall, these findings demonstrate that MSC-derived Vcam1 is critical for BM MSC survival and their HSC niche function.

A novel cryopreservation and biobanking strategy to study lymphoid tissue stromal cells in human disease.

Nonhematopoietic lymph node stromal cells (LNSCs) regulate lymphocyte trafficking, survival, and function for key roles in host defense, autoimmunity, alloimmunity, and lymphoproliferative disorders. However, the study of LNSCs in human diseases is complicated by a dependence on viable lymphoid tissues, which are most often excised prior to establishment of a specific diagnosis. Here, we demonstrate that cryopreservation can be used to bank lymphoid tissue for the study of LNSCs in human disease. Using human tonsils and lymph nodes (LN), lymphoid tissue fragments were cryopreserved for subsequent enzymatic digestion and recovery of viable nonhematopoietic cells. Flow cytometry and single-cell transcriptomics identified comparable proportions of LN stromal cell types in fresh and cryopreserved tissue. Moreover, cryopreservation had little effect on transcriptional profiles, which showed significant overlap between tonsils and LN. The presence and spatial distribution of transcriptionally defined cell types were confirmed by in situ analyses. Our broadly applicable approach promises to greatly enable research into the roles of LNSCs in human disease.

Reconstructing the cell-cell interaction network among mouse immune cells.

Intercellular interactions and cell-cell communication are critical to regulating cell functions, especially in normal immune cells and immunotherapies. Ligand-receptor pairs mediating these cell-cell interactions can be identified using diverse experimental and computational approaches. Here, we reconstructed the intercellular interaction network between Mus musculus immune cells using publicly available receptor-ligand interaction databases and gene expression data from the immunological genome project. This reconstructed network accounts for 50,317 unique interactions between 16 cell types between 731 receptor-ligand pairs. Analysis of this network shows that cells of hematopoietic lineages use fewer communication pathways for interacting with each other, while nonhematopoietic stromal cells use the most network communications. We further observe that the WNT, BMP, and LAMININ pathways are the most significant contributors to the overall number of cell-cell interactions among the various pathways in the reconstructed communication network. This resource will enable the systematic analysis of normal and pathologic immune cell interactions, along with the study of emerging immunotherapies.

Type I IFN stimulates lymph node stromal cells from adult and old mice during a West Nile virus infection.

Advanced age is a significant risk factor during viral infection due to an age-associated decline in the immune response. Older individuals are especially susceptible to severe neuroinvasive disease after West Nile virus (WNV) infection. Previous studies have characterized age-associated defects in hematopoietic immune cells during WNV infection that culminate in diminished antiviral immunity. Situated amongst immune cells in the draining lymph node (DLN) are structural networks of nonhematopoietic lymph node stromal cells (LNSCs). LNSCs are comprised of numerous, diverse subsets, with critical roles in the coordination of robust immune responses. The contributions of LNSCs to WNV immunity and immune senescence are unclear. Here, we examine LNSC responses to WNV within adult and old DLNs. Acute WNV infection triggered cellular infiltration and LNSC expansion in adults. Comparatively, aged DLNs exhibited diminished leukocyte accumulation, delayed LNSC expansion, and altered fibroblast and endothelial cell subset composition, signified by fewer LECs. We established an ex vivo culture system to probe LNSC function. Adult and old LNSCs both recognized an ongoing viral infection primarily through type I IFN signaling. Gene expression signatures were similar between adult and old LNSCs. Aged LNSCs were found to constitutively upregulate immediate early response genes. Collectively, these data suggest LNSCs uniquely respond to WNV infection. We are the first to report age-associated differences in LNSCs on the population and gene expression level during WNV infection. These changes may compromise antiviral immunity, leading to increased WNV disease in older individuals.

Mesenchymal Stem Cells and Psoriasis: Systematic Review.

Mesenchymal Stem Cells (MSCs) are multipotent non-hematopoietic stromal cells found in different body tissues such as bone marrow, adipose tissue, periosteum, Wharton's jelly, umbilical cord, blood, placenta, amniotic fluid, and skin. The biological behavior of MSCs depends mainly on their interaction with the microenvironment in which they are found, whose quality deeply influences the regenerative and immunomodulatory properties of these cells. Several studies confirm the interaction between MSCs and inflammatory microenvironment in the pathogenesis of psoriasis, designating MSCs as an important factor driving psoriasis development. This review aims to describe the most recent evidence on how the inflammatory microenvironment that characterizes psoriasis influences the homeostasis of MSCs and how they can be used to treat the disease.

Fibroblastic Stromal Cells Expressing Delta-like Notch Ligands Control Extrathymic T Cell Development in Mesenteric Lymph Nodes

Early T cell development is supported by signals mediated by the Notch1 receptor in T lineage progenitors and Delta-like4 (Dll4) ligand in thymic epithelial cells. Although T cell development is normally restricted to the thymus, extrathymic T cell development has been reported in athymic Foxn1nu/nu nude mice as well as during times of thymopoietic stress such as post-bone marrow transplantation (BMT). A population of CD4+CD8a double positive T cell progenitors can be found in the mesenteric lymph nodes (MLN) in both athymic mice and early post-BMT, suggesting the presence of an extrathymic niche conducive to T cell development. However, whether Notch signaling is required, as well as the cellular source(s) of Notch ligand throughout the process in the MLN, remains unknown. We hypothesize that MLNs harbor a unique environment in which Notch ligands are available to circulating progenitors and critical to sustain extrathymic T cell development in these contexts. To test this, we utilized systemic neutralizing antibodiesas well as loss-of-function genetic models to assess whether the Notch ligands Dll4 or Dll1 are important for extrathymic T cell development. We found that, like homeostatic thymocyte development, Dll4 appears to play an essential role in generation of early T cell progenitors found in the MLN. Furthermore, the source of Dll4 appears to reside within subsets of non-hematopoietic fibroblastic stromal cells lineage traced by a Ccl19-Cre transgene. In sum, these findings shed new light on the cellular and molecular cues regulating T cell development outside of the thymus.

Abstract 1653: Toward developing a humanized mouse model of the microenvironment associated with the malignant cells of Hodgkin lymphoma

Abstract The tumor microenvironment (TME) represents a highly complex compartment that includes cancer cells, non-hematopoietic stromal cells, extracellular matrix, lymphocytes, and myeloid cells. Interactions between these populations and the factors they secrete play critical roles in determining the host response to cancer. Conventional mouse xenograft models of human cancer lack a competent immune system. Indeed, the use of xenograft models often fail to translate into effective human treatments. Classical Hodgkin Lymphoma (HL) is characterized by the presence in the lymphonodes of abnormal Reed-Sternberg cells, and many cell types beyond them, in particular Treg. To reconstitute a more complete TME, human PBMCs were injected into NSG mice bearing L540 HL cells. Then over time the presence of various human CD45+ cell populations were monitored within the tumor mass, in the spleen and in circulation. The goal of our study is to gain a better understanding of the interaction of the immune cell infiltrates into the HL microenvironment, leading ultimately to improved treatment outcomes. Lymphocyte infiltration into the tumor showed a predominance of human CD3+ cells (T lymphocytes), even if in the spleen we found both human T and B cells, indicating that this model might help us design more effective treatments. To reflect the immune reconstitution of these animals and its interaction with tumor and to better understand the capacity of T lymphocytes to kill the cancer cells, we evaluated the production of human IFN- gamma. We found an increase of IFN- gamma in L540-bearing mice reconstituted with hPBMCs compared to the mice without them, indicating a certain functionality of the T cells in these tumors. Moreover, in this model, the immune reconstitution did not decrease tumor growth, rendering this model useful for the study of biotherapeutic and immunotherapeutic studies. The model might be useful to study the anticancer therapy at a preclinical level because a remodeling of the anti-tumor immune response can be evaluated. Citation Format: Maria Teresa Bilotta, Antonella Antignani, David J. Fitzgerald. Toward developing a humanized mouse model of the microenvironment associated with the malignant cells of Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1653.

Expression of glucocorticoid and androgen receptors in bone marrow–derived hematopoietic and nonhematopoietic murine endometrial cells

To determine whether bone marrow (BM)-derived cells engrafting the murine endometrium express the glucocorticoid receptor (GR) and androgen receptor (AR). Recent data demonstrate that BM is a long-term source of multiple hematopoietic and nonhematopoietic endometrial cell types. Important roles for glucocorticoids and androgens in regulating endometrial functions, including decidualization and early embryo attachment/invasion, have very recently emerged. Whether endometrial cells of BM origin express glucocorticoid or ARs has not been previously studied. Animal study. Basic science laboratory. Wild-type C57BL/6J male mice expressing enhanced green fluorescent protein (GFP) and syngeneic wild-type C57BL/6J female mice aged 6-9 weeks. Murine bone marrow transplant. Bone marrow cells were harvested from adult wild-type C57BL/6 mice and subjected to flow cytometry to identify the percentage of hematopoietic and nonhematopoietic cells expressing GR or AR. Uterine tissue sections from lethally irradiated syngeneic adult female C57BL/6 mice that had been recipients of BM transplants from adult male transgenic donor mice ubiquitously expressing GFP were studied. Immunohistochemistry was performed in the uterine tissue sections of the recipient mice at 5, 9, and 12 months after transplant using specific anti-GR, anti-AR, anti-GFP, anti-CD45 (pan leukocyte marker), and anti-F4/80 (murine macrophage marker) primary antibodies. Confocal laser microscopy was used to localize and quantitate BM-derived (GFP+) cell types in the endometrial stromal and epithelial compartments and determine whether BM-derived cell types in the murine endometrium express GR or AR. Hematopoietic cells comprised 93.6%-96.6% of all cells in the BM, of which 98.1% ± 0.2% expressed GR and 92.2% ± 4.4% expressed AR. Nonhematopoietic cells comprised 0.4%-1.3% of BM, of which 52.8% ± 5.9% expressed GR and 48.9% ± 3.4% expressed AR. After BM transplant, the proportion of cells originating from BM in the endometrial stromal compartment increased over time, reaching 13.5% ± 2.3% at 12 months after transplant. In the epithelial compartments, <1% of the cells were of BM origin at 12 months after transplant. Most (60%-72%) GR+ and/or AR+ BM-derived cells in the stroma were hematopoietic (CD45+) cells, of which 37%-51% were macrophages. Nonetheless, 28%-33% of GR+ cells, and 28%-40% of AR+ BM-derived cells, were nonhematopoietic (CD45-) stromal cells of BM origin. A substantial number of BM-derived cells express GR and AR, suggesting a role for these cells in both glucocorticoid-regulated and androgen-regulated endometrial functions, such as proliferation and/or decidualization.

Lung-Resident Mesenchymal Stem Cell Fates within Lung Cancer

Simple SummaryLung cancer remains the leading cause of cancer-related deaths worldwide. Herein, the heterogeneous tumor stroma decisively impacts on tumor progression, therapy resistance, and, thus, poor clinical outcome. Among the numerous non-epithelial cells constructing the complex environment of lung carcinomas, mesenchymal stem cells (MSC) gained attraction being stromal precursor cells that could be recruited and ‘educated’ by lung cancer cells to adopt a tumor-associated MSC phenotype, serve as source for activated fibroblasts and presumably for vascular mural cells finally reinforcing tumor progression. Lung-resident MSCs should be considered as ‘local MSCs in stand by’ ready to be arranged within the cancer stroma.Lung-resident mesenchymal stem cells (LR-MSCs) are non-hematopoietic multipotent stromal cells that predominately reside adventitial within lung blood vessels. Based on their self-renewal and differentiation properties, LR-MSCs turned out to be important regulators of normal lung homeostasis. LR-MSCs exert beneficial effects mainly by local secretion of various growth factors and cytokines that in turn foster pulmonary regeneration including suppression of inflammation. At the same time, MSCs derived from various tissues of origins represent the first choice of cells for cell-based therapeutic applications in clinical medicine. Particularly for various acute as well as chronic lung diseases, the therapeutic applications of exogenous MSCs were shown to mediate beneficial effects, hereby improving lung function and survival. In contrast, endogenous MSCs of normal lungs seem not to be sufficient for lung tissue protection or repair following a pathological trigger; LR-MSCs could even contribute to initiation and/or progression of lung diseases, particularly lung cancer because of their inherent tropism to migrate towards primary tumors and metastatic sites. However, the role of endogenous LR-MSCs to be multipotent tumor-associated (stromal) precursors remains to be unraveled. Here, we summarize the recent knowledge how ‘cancer-educated’ LR-MSCs impact on lung cancer with a focus on mesenchymal stem cell fates.

Understanding the Immune-Stroma Microenvironment in B Cell Malignancies for Effective Immunotherapy.

Cancers, including lymphomas, develop in complex tissue environments where malignant cells actively promote the creation of a pro-tumoral niche that suppresses effective anti-tumor effector T cell responses. Research is revealing that the tumor microenvironment (TME) differs between different types of lymphoma, covering inflamed environments, as exemplified by Hodgkin lymphoma, to non-inflamed TMEs as seen in chronic lymphocytic leukemia (CLL) or diffuse-large B-cell lymphoma (DLBCL). In this review we consider how T cells and interferon-driven inflammatory signaling contribute to the regulation of anti-tumor immune responses, as well as sensitivity to anti-PD-1 immune checkpoint blockade immunotherapy. We discuss tumor intrinsic and extrinsic mechanisms critical to anti-tumor immune responses, as well as sensitivity to immunotherapies, before adding an additional layer of complexity within the TME: the immunoregulatory role of non-hematopoietic stromal cells that co-evolve with tumors. Studying the intricate interactions between the immune-stroma lymphoma TME should help to design next-generation immunotherapies and combination treatment strategies to overcome complex TME-driven immune suppression.

Reconstruction of Intercellular Signaling Network by Cytokine-Receptor Interactions.

Background: The immune system function depends on the coordination activity of the components of system and communications between them which leads to the formation of a complex communication network between immune cells. In this network, cytokines have an important role in the communication between immune cells through the interaction to their specific receptors. These molecules cause to cellular communications and normal function of a tissue. Reconstruction of such a complex network can be a way to provide a better understanding of cytokines’ function.Objective: Our main goal from reconstructing such a network was investigation of expressed cytokines and cytokines receptors in various lineage and tissues of immune cells and identifying the lineage and tissue with the highest expression of cytokines and their receptors.Materials and Methods: In this study, gene expression data related to part of the Immunological Genome Project (ImmGen) and receptor-ligand interactions dataset were used to reconstruct the immune network in mouse. In next step, the topological properties of reconstructed network, expression specificity of cytokines and their receptors and interactions specificity were analyzed.Results: The results of the network analysis were indicated that non- hematopoietic stromal cells have the highest expression of cytokines and cytokine receptors and interactions specificity is very high. Our results show that chemokine receptor of Ccr1 receives the largest number of signals between receptors and only expressed in three hematopoietic lineages.Conclusions: The most of the network communications belonged to non-hematopoietic stromal and macrophage cells. The relationships between stromal cells and macrophages are necessary to create an appropriate environment for differentiation of immune cells. Studying the cellular expression specificity of receptor and ligand genes reveal the high degree of specificity of these genes that indicate non-random transfer of information between cells in multicellular organisms.

Transcription factor Tlx1 marks a subset of lymphoid tissue organizer-like mesenchymal progenitor cells in the neonatal spleen

The spleen is comprised of spatially distinct compartments whose functions, such as immune responses and removal of aged red blood cells, are tightly controlled by the non-hematopoietic stromal cells that provide regionally-restricted signals to properly activate hematopoietic cells residing in each area. However, information regarding the ontogeny and relationships of the different stromal cell types remains limited. Here we have used in vivo lineage tracing analysis and in vitro mesenchymal stromal cell assays and found that Tlx1, a transcription factor essential for embryonic spleen organogenesis, marks neonatal stromal cells that are selectively localized in the spleen and retain mesenchymal progenitor potential to differentiate into mature follicular dendritic cells, fibroblastic reticular cells and marginal reticular cells. Furthermore, by establishing a novel three-dimensional cell culture system that enables maintenance of Tlx1-expressing cells in vitro, we discovered that signals from the lymphotoxin β receptor and TNF receptor promote differentiation of these cells to express MAdCAM-1, CCL19 and CXCL13, representative functional molecules expressed by different subsets of mature stromal cells in the spleen. Taken together, these findings indicate that mesenchymal progenitor cells expressing Tlx1 are a subset of lymphoid tissue organizer-like cells selectively found in the neonatal spleen.

From Graft-Versus-Host Disease to Lymphoma Pathogenesis: Emerging Roles for Stromal Notch Ligands in Hematology

Notch signaling is a highly conserved signaling pathway with multiple functions in health and disease. Notch ligands of the Delta-like and Jagged families interact with Notch1-4 on adjacent cells to trigger proteolytic activation of the Notch receptors, followed by context-dependent transcriptional activation of Notch target genes. In the hematopoietic system, Notch was first identified for its oncogenic role in T cell acute lymphoblastic leukemia (T-ALL). More recently, Notch has been recognized as a recurrent oncogenic pathway in multiple B cell lymphoproliferative disorders, including CLL/SLL, mantle cell lymphoma and marginal zone lymphoma. In parallel, essential functions of Notch signaling have been identified in early T cell development and B cell homeostasis, as well as in regulating T and B cell immune responses in secondary lymphoid organs. We have discovered a conserved pathogenic function of Notch signaling in the control of graft-versus-host disease (GVHD), both in mouse and non-human primate models of hematopoietic cell transplantation. Within days after allogeneic hematopoietic cell transplantation, donor-derived T cells interact with Delta-like Notch ligands expressed by specialized subsets of non-hematopoietic fibroblastic stromal cells in spleen and lymph nodes. In turn, Notch signaling programs alloreactive T cells to become pathogenic effector cells that mediate long-term damage in GVHD target tissues, while suppressing the expansion of regulatory T cells. Emerging evidence suggests the existence of an active crosstalk between T cells, B cells and well-defined specialized immunological stromal niches expressing Notch ligands in secondary lymphoid organs, both in GVHD and in other types of immune responses. Moreover, many B cell lymphomas demonstrate oncogenic Notch activation as a result of gain-of-function mutations that increase the persistence of intracellular Notch after proteolytic cleavage, thus requiring the presence of Notch ligands in the microenvironment to trigger oncogenic Notch signals. New evidence suggests that key Notch ligands in human lymphomas are present in the lymph node microenvironment, and that oncogenic Notch activation can happen even in the absence of mutations in a substantial fraction of lymphoma patients, including the majority of patients with CLL/SLL. In these cases, lymphoma cells appear to interact with microenvironmental Notch ligands in secondary lymphoid organs through unmutated Notch receptors, borrowing from the playbook of normal lymphocytes. Furthermore, the expression of Notch ligands by defined subsets of fibroblastic stromal cells illustrates a new paradigm through which microenvironmental signals can profoundly influence the differentiation and function of adjacent lymphoid cells. We speculate that better understanding the regulation of Notch signaling and fibroblastic stromal cells in secondary lymphoid organs will provide valuable new information with translational potential both in immunobiology and in malignant hematology. Disclosures Maillard: Genentech: Consultancy; Regeneron: Consultancy.

Dll1 and Dll4 Notch Ligands Prime T Cell Alloimmunity and Are Expressed in Non-Overlapping Populations of Fibroblastic Stromal Cells in Spleen and Lymph Nodes at the Onset of Gvhd

Notch signaling drives graft-versus-host-disease (GVHD) pathogenesis in preclinical models of allogeneic hematopoietic cell transplantation (allo-HCT). Short-term systemic blockade of the Notch ligands Delta-like1 (Dll1) and Delta-like4 (Dll4) within two days of allo-HCT conferred long-term protection from acute GVHD lethality in mice, with a dominant impact of Dll4. Surprisingly, critical Notch ligands in GVHD were provided by radioresistant non-hematopoietic fibroblastic stromal cells lineage traced by a Ccl19-Cre transgene in secondary lymphoid organs (SLO) (Chung, JCI 2017). Our discovery revises prevailing GVHD models and identifies a pathogenic role for a stromal cell niche expressing Notch ligands in SLOs. However, little is known about the distribution and regulation of Dll1/Dll4-expressing cells among SLO stromal cells. To address this question, we deployed genetic and biochemical tools to map expression of Notch ligands in SLO stromal cells, characterize the transcriptional landscape of these cells, and identify putative regulators of Notch ligand expression. To map Dll1 and Dll4 expression, we combined a Ccl19-Cre transgene with a ROSA26-YFP Cre-activated allele and Dll1-mCherry or Dll4-mCherry BAC reporters. Using this model, we fluorescently traced all cells derived from Ccl19-Cre+ cells which contain the essential Dll1/Dll4 source in GVHD, while detecting Dll1 vs. Dll4 expression with high sensitivity. Within the lymph node (LN) Ccl19-Cre+ROSA26-YFP+ compartment, Dll4-mCherry was present predominantly among CD157hi CD45-Ter119-CD31-gp38+ fibroblastic stromal cells, including fibroblastic reticular cells (FRCs), marginal reticular cells (MRCs) and follicular dendritic cells (FDCs). In the spleen, Dll4-mCherry was detected among both CD157+ cells (predominant in the T-zone) and CD157- cells. In contrast, Dll1-mCherry was abundant among spleen CD157+ and CD157- cells, but not detected in the corresponding LN compartments. Thus, Dll1 and Dll4 are expressed with a non-overlapping distribution pattern in different SLOs and stromal cell subsets. Next, we studied expression of the dominant Dll4 ligand after MHC-mismatched allo-HCT (BALB/c into C57BL/6). Although Dll4-mCherry fluorescence did not increase, we detected a rise in surface Dll4 protein, peaking 12 hrs after allo-HCT in LN Ccl19-Cre+ CD157hi FRCs, MRCs and FDCs. Increased Dll4 abundance coincided with the critical timing of pathogenic Notch signals within days after allo-HCT and required irradiation conditioning as well as infusion of allogeneic T cells, suggesting a crosstalk between alloreactive T cells and Dll4+ stromal cells. The mechanisms underlying increased surface Dll4 abundance are under investigation, but could involve post-transcriptional effects as the Dll4-mCherry transcriptional reporter did not change. We next characterized transcriptomic features of Dll4+ fibroblastic stromal cells. We performed RNA-Seq on rare SLO stromal cells sort-purified from LNs at baseline and 12 hrs after allo-HCT. We compared CD157hi FRCs that do or do not express Dll4-mCherry, while including CD157lo FRCs that do not express Dll4-mCherry and lymphatic endothelial cells that express Dll4 at high levels as controls. These populations were sort-purified for RNA-Seq analysis, using the data to produce a comprehensive picture of gene expression and regulatory machinery in the SLO niche in alloimmunity, as well as identify new putative regulators of Notch ligand expression. Interestingly, we found only 13 differentially expressed genes between Ccl19-Cre-expressing CD157hi fibroblastic stromal cells that do and do not express the Dll4-mCherry reporter 12 hrs after transplant (log-fold change ≥ 1, corrected p ≤ 0.01). The list of up-regulated transcripts included Cxcl13, encoding a chemokine produced by FDCs, and Rankl, encoding a TNF-family member important in LN development and homeostasis. In summary, our data uncover key new features of the specialized fibroblastic stromal cells expressing Delta-like Notch ligands in SLOs. Ongoing work is testing how qualitative and quantitative differences in Dll1 and Dll4's effects during GVHD may relate to their distribution, regulated expression, or biochemical properties as they interact with Notch receptors in T cells. Our ultimate goal is to design strategies that target Notch ligand expression in SLOs as new prophylactic approaches in GVHD. Disclosures Blazar: Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; Tmunity: Other: Co-Founder; KidsFirst Fund: Research Funding; Childrens' Cancer Research Fund: Research Funding; Leukemia and Lymphoma Society: Research Funding; Abbvie Inc: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees. Maillard:Genentech: Consultancy; Regeneron: Consultancy.

Non-Hematopoietic Lymphoid Stromal Cells Prime Alloreactive CD4+ T Cells in Acute Graft-Versus-Host Disease

Allogeneic T cell priming is considered as an essential event determining the outcome of allogeneic hematopoietic stem cell transplantation (allo-HCT), ideally triggering anti-leukemic responses (GvL effect) or, at worst, causing life-threatening acute graft-versus-host disease (aGvHD). During acute GvHD initiation, alloreactive T cells are activated by host antigen presenting cells (APCs), rapidly expand and subsequently exert tissue damage. Recently, it was discovered that absence of host hematopoietic APCs does not prevent acute GvHD, suggesting a crucial role of non-hematopoietic APCs for priming alloreactive T cells (Toubai et al., Blood 2012, Li et al., J Immunol. 2012). Furthermore, it was even suggested that in the absence of professional APCs allogeneic CD4+ T cells can be activated in the lamina propria by MHC class II expressing myofibroblasts (Koyama et al., Nat Med 2012). As exact location and identity of host non-hematopoietic APCs triggering alloreactive T cell responses are essential to dissect the priming of GvHD-inducing vs. GvL-mediating allogeneic T cells, we investigated the role of lymph node stromal cells (LNSCs) in the initiation phase of aGvHD and their potential role as non-hematopoietic APCs. Employing allo-HCT mouse models in combination with flow cytometry and advanced microscopy techniques, we explored early alloreactive T cells activation first in a myeloablatively conditioned MHC major mismatch allo-HCT setting (FVB→B6). Under these conditions, CD4+ and CD8+ T cells activation and proliferation occurred exclusively in secondary lymphoid organs (SLOs) and not in the intestinal lamina propria early after allo-HCT within the first three days after allo-HCT. To study non-hematopoietic antigen presentation early after allo-HCT, we generated bone marrow B6.MHCIIΔ/Δ→B6.WT chimeras that lacked MHC II expression in the host hematopoietic compartment. Subsequent allo-HCT of these chimeras revealed activation and expansion of allogenic donor CD4+ T-cells exclusively in SLOs and not the lamina propria in the first three days after transplantation. Next, we generated recipient mice that selectively lacked MHCII expression either in CD11c expressing cells or under the control of the Vav1-promoter in all hematopoietic cells. In both type of recipients, allogenic donor CD4+ T-cells were activated within 3 days after allo-HCT in SLOs and no other tissues. After irradiation of B6.WT mice we observed LNSCs upregulate co-stimulatory receptors early after irradiation (24 and 72 hours), in vitro and in vivo suggesting that they may act as active non-hematopoietic APCs. Next, we performed mixed lymphocyte reactions (MLRs) of irradiated APCs derived from TgVav1-Cre+MHCIIΔ/Δmice with alloreactive CD4+ T cells from FVB mice. Here, we observed in the total absence of hematopoietic MHCII antigen presentation reduced but still pronounced activation of alloreactive CD4+ T cells. Therefore, in these transgenic allo-HCT models, hematopoietic cells in the SLOs were dispensable for alloreactive CD4+ T cell activation in the initiation phase of aGvHD. To ascertain that SLOs are the exclusive priming sites of alloreactive CD4+ T cells, we transplanted mesenteric lymph nodes (mLNs) from a B6.CD11c.DTR mice, depleted of CD11c+ cells into B6.MHCIIΔ/Δmice. After successful engraftment of donor mLNs in these mice, allo-HCT revealed these as unique priming sites in MHCII deficient hosts for alloreactive CD4+ T cells, which differentiated into CD44hiCD62Llow effector T cells that were detectable in the transplanted mLNs. Conclusively, these results indicate that specialized non-hematopoietic lymph node stromal cells prime alloreactive CD4+ T cell within the SLOs early after allo-HCT. Pinpointing the molecular pathways in these non-hematopoietic APCs within SLOs that trigger alloreactive T cell responses may prove fruitful for selective therapeutic intervention after allo-HCT. Disclosures No relevant conflicts of interest to declare.

Multiple Interactions Between Cancer Cells and the Tumor Microenvironment Modulate TRAIL Signaling: Implications for TRAIL Receptor Targeted Therapy.

Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) signaling is far more complex than initially anticipated and can lead to either anti- or protumorigenic effects, hampering the successful clinical use of therapeutic TRAIL receptor agonists. Cell autonomous resistance mechanisms have been identified in addition to paracrine factors that can modulate apoptosis sensitivity. The tumor microenvironment (TME), consisting of cellular and non-cellular components, is a source for multiple signals that are able to modulate TRAIL signaling in tumor and stromal cells. Particularly immune effector cells, also part of the TME, employ the TRAIL/TRAIL-R system whereby cell surface expressed TRAIL can activate apoptosis via TRAIL receptors on tumor cells, which is part of tumor immune surveillance. In this review we aim to dissect the impact of the TME on signaling induced by endogenous and exogenous/therapeutic TRAIL, thereby distinguishing different components of the TME such as immune effector cells, neutrophils, macrophages, and non-hematopoietic stromal cells. In addition, also non-cellular biochemical and biophysical properties of the TME are considered including mechanical stress, acidity, hypoxia, and glucose deprivation. Available literature thus far indicates that tumor-TME interactions are complex and often bidirectional leading to tumor-enhancing or tumor-reducing effects in a tumor model- and tumor type-dependent fashion. Multiple signals originating from different components of the TME simultaneously affect TRAIL receptor signaling. We conclude that in order to unleash the full clinical potential of TRAIL receptor agonists it will be necessary to increase our understanding of the contribution of different TME components on outcome of therapeutic TRAIL receptor activation in order to identify the most critical mechanism responsible for resistance, allowing the design of effective combination treatments.

SIRPα+ dendritic cells promote the development of fibroblastic reticular cells in murine peripheral lymph nodes.

Nonhematopoietic stromal cells contribute to the organization and homeostasis of secondary lymphoid organs by producing cytokines and chemokines. The development and maintenance of these stromal cells are thought to be regulated by innate immune cells. Indeed, we recently showed that signal regulatory protein α (SIRPα)-positive dendritic cells (DCs) are essential for the proliferation and survival of podoplanin (Pdpn)-positive fibroblastic reticular cells (FRCs) in mouse spleen. We have now established an in vitro culture system for lymph node stromal cells (LNSCs) isolated from mouse peripheral LNs. Activated DCs and TNF-α each promoted the proliferation of cultured LNSCs, most of which were found to be Pdpn+ FRCs. Furthermore, ablation of SIRPα in CD11c+ cells attenuated this effect of DCs on LNSC proliferation. Transplantation of activated DCs together with cultured LNSCs into the renal subcapsular space markedly increased the number of ER-TR7+ stromal cells as well as induced the accumulation of T cells and increased the expression of Ccl19 in the transplants. Ablation of SIRPα in CD11c+ cells greatly impaired the development of LN-like structure in the transplants. Our findings thus suggest that SIRPα+ DCs are important for the proliferation and differentiation of Pdpn+ FRCs in peripheral LNs.