Stromal Cell Subsets Research Articles

Creating a Favorable Microenvironment for Fat Grafting in a Novel Model of Radiation-Induced Mammary Fat Pad Fibrosis.

Radiofibrosis of breast tissue compromises breast reconstruction by interfering with tissue viability and healing. Autologous fat transfer may reduce radiotherapy-related tissue injury, but graft survival is compromised by the fibrotic microenvironment. Elevated expression of receptor for hyaluronan-mediated motility (RHAMM; also known as hyaluronan-mediated motility receptor, or HMMR) in wounds decreases adipogenesis and increases fibrosis. The authors therefore developed RHAMM peptide mimetics to block RHAMM profibrotic signaling following radiation. They propose that this blocking peptide will decrease radiofibrosis and establish a microenvironment favoring adipose-derived stem cell survival using a rat mammary fat pad model. Rat mammary fat pads underwent a one-time radiation dose of 26 Gy. Irradiated (n = 10) and nonirradiated (n = 10) fat pads received a single intramammary injection of a sham injection or peptide NPI-110. Skin changes were examined clinically. Mammary fat pad tissue was processed for fibrotic and adipogenic markers using quantitative polymerase chain reaction and immunohistochemical analysis. Clinical assessments and molecular analysis confirmed radiation-induced acute skin changes and radiation-induced fibrosis in rat mammary fat pads. Peptide treatment reduced fibrosis, as detected by polarized microscopy of picrosirius red staining, increased collagen ratio of 3:1, reduced expression of collagen-1 crosslinking enzymes lysyl-oxidase, transglutaminase 2, and transforming growth factor β1 protein, and increased adiponectin, an antifibrotic adipokine. RHAMM was expressed in stromal cell subsets and was downregulated by the RHAMM peptide mimetic. Results from this study predict that blocking RHAMM function in stromal cell subsets can provide a postradiotherapy microenvironment more suitable for fat grafting and breast reconstruction.

High Proliferative Placenta-Derived Multipotent Cells Express Cytokeratin 7 at Low Level.

The purpose of this study was to investigate the immunophenotypes and gene expression profile of high proliferative placenta-derived multipotent cells (PDMCs) population at different stages of culture. We demonstrated that the colonies resulting from single cells were either positive or negative for CK7, whereas only PDMC clones with weak CK7 expression (CK7low-clones) were highly proliferative. Interestingly, vimentin positive (Vim+) placental stromal mesenchymal cells did not express CK7 in situ, but double CK7+Vim+ cells detection in tissue explants and explants outgrowth indicated CK7 inducible expression in vitro. PCNA presence in CK7+Vim+ cells during placental explants culturing confirmed belonging of these cells to proliferative subpopulation. Transcription factors CDX2 and EOMES were expressed in both CK7low-clones and subset of stromal mesenchymal cells of first-trimester placental tissue in situ. Meanwhile, CK7low -clones and stromal mesenchymal cells of full-term placental tissue in situ expressed ERG heterogeneously. SPP1, COL2A1, and PPARG2 mesodermal-related genes expression by CK7low-clones additionally confirms their mesenchymal origin. Inherent stem cell-related gene expression (IFTM3, POU5F1, and VASA) in CK7low-clones might indicate their enrichment for progenitors. Finally, in CK7low-clones we observed expression of such trophoblast-associated genes as CGB types I and II, fusogenic ERVW-1, GCM1, and GATA3. Thus, our results indicate that PDMCs acquired the representative immunophenotype signature under culture conditions.

High-Throughput Sequence Analysis of Peripheral T-Cell Lymphomas Indicates Subtype-Specific Viral Gene Expression Patterns and Immune Cell Microenvironments.

Certain peripheral T-cell lymphomas (PTCLs) have been associated with viral infection, particularly infection with Epstein-Barr virus (EBV). However, a comprehensive virome analysis across PTCLs has not previously been reported. Here we utilized published whole-transcriptome RNA sequencing (RNA-seq) data sets from seven different PTCL studies and new RNA-seq data from our laboratory to screen for virus association, to analyze viral gene expression, and to assess B- and T-cell receptor diversity paradigms across PTCL subtypes. In addition to identifying EBV in angioimmunoblastic T-cell lymphoma (AITL) and extranodal NK/T-cell lymphoma (ENKTL), two PTCL subtypes with well-established EBV associations, we also detected EBV in several cases of anaplastic large-cell lymphoma (ALCL), and we found evidence of infection by the oncogenic viruses Kaposi's sarcoma-associated herpesvirus and human T-cell leukemia virus type 1 in isolated PTCL cases. In AITLs, EBV gene expression analysis showed expression of immediate early, early, and late lytic genes, suggesting either low-level lytic gene expression or productive infection in a subset of EBV-infected B-lymphocyte stromal cells. Deconvolution of immune cell subpopulations demonstrated a greater B-cell signal in AITLs than in other PTCL subtypes, consistent with a larger role for B-cell support in the pathogenesis of AITL. Reconstructed T-cell receptor (TCR) and B-cell receptor (BCR) repertoires demonstrated increased BCR diversity in AITLs, consistent with a possible EBV-driven polyclonal response. These findings indicate potential alternative roles for EBV in PTCLs, in addition to the canonical oncogenic mechanisms associated with EBV latent infection. Our findings also suggest the involvement of other viruses in PTCL pathogenesis and demonstrate immunological alterations associated with these cancers.IMPORTANCE In this study, we utilized next-generation sequencing data from 7 different studies of peripheral T-cell lymphoma (PTCL) patient samples to globally assess viral associations, provide insights into the contributions of EBV gene expression to the tumor phenotype, and assess the unique roles of EBV in modulating the immune cell tumor microenvironment. These studies revealed potential roles for EBV replication genes in some PTCL subtypes, the possible role of additional human tumor viruses in rare cases of PTCLs, and a role for EBV in providing a unique immune microenvironmental niche in one subtype of PTCLs. Together, these studies provide new insights into the understudied role of tumor viruses in PTCLs.

Intra- and Inter-cellular Rewiring of the Human Colon during Ulcerative Colitis

Genome-wide association studies (GWAS) have revealed risk alleles for ulcerative colitis (UC). To understand their cell type specificities and pathways of action, we generate an atlas of 366,650 cells from the colon mucosa of 18UC patients and 12 healthy individuals, revealing 51 epithelial, stromal, and immune cell subsets, including BEST4+ enterocytes, microfold-like cells, and IL13RA2+IL11+ inflammatory fibroblasts, which we associate with resistance to anti-TNF treatment. Inflammatory fibroblasts, inflammatory monocytes, microfold-like cells, and Tcells that co-express CD8 and IL-17 expand with disease, forming intercellular interaction hubs. Many UC risk genes are cell type specific and co-regulated within relatively few gene modules, suggesting convergence onto limited sets of cell types and pathways. Using this observation, we nominate and infer functions for specific risk genes across GWAS loci. Our work provides a framework for interrogating complex human diseases and mapping risk variants to cell types and pathways.

Role of mesenchymal stem cell-derived soluble factors and folic acid in wound healing.

Background/aimMesenchymal stem cells (MSCs) are a type of adult stem cell consisting of a heterogeneous subset of stromal stem cells that can be isolated from adult tissues. Folic acid is another important contributor to tissue regeneration and repair, which affects the synthesis of some building block molecules used for wound healing. In this study, we examine the effect of folic acid and MSC-derived soluble factors in the wound healing model.Materials and methodsHuman umbilical vein endothelial cells (HUVECs) and bone marrow-derived MSCs (BMSCs) were cultured for this study. Cell proliferation analysis was done with xCELLigence RTCA. After 48 h of cultivation, the cell culture medium was collected as MSC conditional medium containing mesenchymal stem cell-derived soluble factors (MDFs). Different concentrations of MDFs (12%, 25%, 50%, 75%, and 100%) were applied to the HUVEC cell line. Folic acid (25, 30, 50, 60, 75, 90, and 100 µM) was tested by application of three different groups (control, 25 µM folic acid, 625 µM folic acid inhibitors) for proliferation on the HUVEC cell line. The combined effects of folic acid and MDFs were tested on the HUVEC cell line with 25 µM folic acid and 50 µM MDFs. All data were statistically analyzed using SPSS 15.0 for Windows.ResultsSignificant differences were observed between controls and cells treated with folic acid, as well as between controls and both folic acid and MDFs (P < 0.05). Among the treated groups, the fastest wound closure rate was seen in cells treated with both folic acid and MDFs.ConclusionThe results show that both folic acid and MDFs increased the wound healing rate in HUVECs when they were used separately. The strongest benefits were seen in treatment using folic acid and MDFs together.

Molecular signature of human bone marrow-derived mesenchymal stromal cell subsets

In the current study we compared the molecular signature of expanded mesenchymal stromal cells (MSCs) derived from selected CD271+ bone marrow mononuclear cells (CD271-MSCs) and MSCs derived from non-selected bone marrow mononuclear cells by plastic adherence (PA-MSCs). Transcriptome analysis demonstrated for the first time the upregulation of 115 and downregulation of 131 genes in CD271-MSCs. Functional enrichment analysis showed that the upregulated genes in CD271-MSCs are significantly enriched for extracellular matrix (tenascin XB, elastin, ABI family, member 3 (NESH) binding protein, carboxypeptidase Z, laminin alpha 2 and nephroblastoma overexpressed) and cell adhesion (CXCR7, GPNMB, MYBPH, SVEP1, ARHGAP6, TSPEAR, PIK3CG, ABL2 and NCAM1). CD271-MSCs expressed higher gene transcript levels that are involved in early osteogenesis/chondrogenesis/adipogenesis (ZNF145, FKBP5). In addition, increased transcript levels for early and late osteogenesis (DPT, OMD, ID4, CRYAB, SORT1), adipogenesis (CTNNB1, ZEB, LPL, FABP4, PDK4, ACDC), and chondrogenesis (CCN3/NOV, CCN4/WISP1, CCN5/WISP2 and ADAMTS-5) were detected. Interestingly, CD271-MSCs expressed increased levels of hematopoiesis associated genes (CXCL12, FLT3L, IL-3, TPO, KITL). Down-regulated genes in CD271-MSCs were associated with WNT and TGF-beta signaling, and cytokine/chemokine signaling pathways. In addition to their capacity to support hematopoiesis, these results suggest that CD271-MSCs may contain more osteo/chondro progenitors and/or feature a greater differentiation potential.

Assessment of Lymph Node Stromal Cells as an Underlying Factor in Age-Related Immune Impairment.

Aging negatively impacts immunity, resulting in inefficient responses to vaccinations and infections. Fibroblastic reticular cells (FRCs) are the major stromal cell subset in lymph nodes (LNs) and play an intricate role in the orchestration and control of adaptive immune responses. Although stromal cells have a major impact on immune responses, the impact of aging on LN stromal cells remains unclear. Quantitative analysis of LN stromal cells by flow cytometry revealed that there are no significant differences in the number of stromal cells in young and aged LN at steady state but after influenza infection aged FRCs have delayed expansion as a result of reduced proliferation. Aged LNs also produce reduced levels of homeostatic chemokines, which correlates with reduced homing of naive T cells. Image analysis reveals that young and aged T-cell zone FRCs have similar morphology at steady state and after infection. Furthermore, aged FRCs did not appear to be a contributing factor in the reduced proliferation of young T cells transferred into aged LNs after influenza infection. These results demonstrate that aging alters LN stromal cell response to challenge and these age-related changes may be an underlying contributor to impaired immune responses in the elderly people.

Identification of Stromal Cells in Spleen Which Support Myelopoiesis.

Stromal cells in spleen organize tissue into red pulp, white pulp and marginal zone, and also interact with hematopoietic cells to regulate immune responses. This study has used phenotypic information of a previously described spleen stromal cell line called 5G3, which supports restricted hematopoiesis in vitro, to identify an equivalent stromal cell subset in vivo and to test its capacity to support hematopoiesis. Using stromal cell fractionation, phenotypic analysis, as well as cell growth and hematopoietic support assays, the Sca-1+gp38+Thy1.2+CD29+CD51+ fraction of spleen stroma has been identified as an equivalent stromal subset resembling the 5G3 cell counterpart. While heterogeneity may still exist within that subset, it has been shown to have superior hematopoietic support capacity compared with the 5G3 cell line, and all other spleen stromal cell fractions tested.

Molecular profile and proangiogenic activity of the adipose-derived stromal vascular fraction used as an autologous innovative medicinal product in patients with systemic sclerosis

ObjectiveThe autologous stromal vascular fraction (SVF) from adipose tissue is an alternative to cultured adipose-derived stem cells for use in regenerative medicine and represents a promising therapy for vasculopathy and...

Essential Role of Canonical NF-κB Activity in the Development of Stromal Cell Subsets in Secondary Lymphoid Organs.

Organized tissue structure in the secondary lymphoid organs (SLOs) tightly depends on the development of fibroblastic stromal cells (FSCs) of mesenchymal origin; however, the mechanisms of this relationship are poorly understood. In this study, we specifically inactivated the canonical NF-κB pathway in FSCs in vivo by conditionally inducing IκBα mutant in a Ccl19-IκBSR mouse system in which NF-κB activity is likely to be suppressed in fetal FSC progenitors. Given that NF-κB activation in fetal FSCs is essential for SLO development, the animals were expected to lack SLOs. However, all SLOs were preserved in Ccl19-IκBSR mice. Instead, the T cell area was severely disturbed by the lack of CCL21-expressing FSCs, whereas the follicles and associated FSC networks were formed. Fate mapping revealed that IκBSR-expressing cells constituted only a small fraction of stromal compartment outside the follicles. Taken together, our findings indicate an essential role of the canonical NF-κB pathway activity in the development of three FSC subsets common to SLOs and suggest transient or stochastic CCL19 expression in FSC progenitors and a compensatory differentiation program of follicular FSCs.

Donor T Cells Require Notch Signals but Not Alloantigen Presentation from Specialized Secondary Lymphoid Organ Fibroblasts to Drive Graft-Versus-Host Disease

Notch signals drive pathogenic T cell alloreactivity in mouse models of allogeneic bone marrow transplantation (allo-BMT) leading to lethal graft-versus-host disease (GVHD). In a mouse model of MHC-mismatched allo-BMT with high intensity whole body conditioning (11 Gy), residual recipient or donor hematopoietic cells were dispensable sources of Notch signals to drive T cell alloreactivity. Instead, abrogating the expression of Delta-like1 (Dll1) and Delta-like4 (Dll4) Notch ligands in Ccl19-Cre+ secondary lymphoid organ (SLO) fibroblastic stromal cells protected mice from GVHD (Chung et al., JCI 2017). In this study we sought to 1) understand the expression and regulation of Notch ligands in fibroblastic stromal cells during allo-BMT; 2) test whether fibroblastic stromal cells are still critical sources of Notch signals in models of allotransplantation with reduced or no conditioning (when other radiation-sensitive sources of Notch ligands are better preserved); and 3) assess whether SLO fibroblastic stromal also present alloantigens to T cells during GVHD.To characterize stromal sources of Notch ligands early after allo-BMT, we combined a Ccl19-Cre transgene with a ROSA26-YFP Cre-activated allele in Dll4-mCherry BAC reporter mice. We observed combined Ccl19-Cre activity and Dll4-mCherry reporter expression in CD157hi fibroblastic reticular cells, MAdCAM1+ marginal reticular cells, CD21+ follicular dendritic cells, and a subset of MAdCAM1+ lymphatic endothelial cells. 12 hours after high intensity conditioning and allo-BMT, Dll4 mRNA and cell surface expression increased in fibroblastic but not in hematopoietic or endothelial cellular compartments. Dll4 upregulation required transfer of alloreactive T cells and was potentiated by the intensity of myeloablative conditioning, suggesting that fibroblastic stromal cells have increased availability of Notch ligands to drive GVHD after high-intensity conditioning.To assess the functional impact of conditioning on the cellular sources of Notch ligands, we studied a parent-to-F1 allo-BMT model in which recipients are tolerant to donor-derived T cells, and the conditioning intensity can be titrated (C57BL/6 [H-2b] or BALB/c [H-2d] into BALB/c x C57BL/6 F1 [H-2d/b]). Cell-specific genetic loss of function systems combining floxed alleles for Dll1 and Dll4 with Ccl19-Cre+ and Mx-Cre+ transgenes allowed conditional deletion in SLO fibroblastic stromal cells or hematopoietic cells, respectively. Across all conditioning intensities tested, upregulation of core-2 O-glycosylation machinery in alloreactive T cells was critically regulated by Notch signals. Thus, induction of the core-2 glycoform of CD43 could be used as a sensitive surrogate readout of Notch signals received by alloreactive T cells. In models of allotransplantation with high-intensity (11 Gy), low-intensity (3 Gy), and no conditioning, fibroblastic stromal cells remained essential and hematopoietic cells were dispensable as sources of Notch signals driving both T cell core-2 O-glycosylation and lethal alloimmunity.Because previous studies indicated that non-hematopoietic cells can serve as antigen-presenting cells in CD4+ T cell mediated GVHD, we tested if fibroblastic stromal cells could present MHC class II alloantigens in addition to Notch ligands. We used Ccl19-Cre+ crossed to mice with a floxed I-A allele to delete the cell-intrinsic ability of fibroblastic stromal cells to present MHC class II alloantigens. In multiple models of high-intensity conditioned CD4+ T cell mediated GVHD (BALB/c [H-2d] into BALB/c x C57BL/6 F1 [H-2d/b] and TEa TCR Tg into BALB/c x C57BL/6 F1 [H-2d/b]), fibroblastic stromal cells were dispensable as sources of alloantigen to drive T cell alloreactivity and lethal GVHD. Together, these results indicate that across varying conditioning intensities, specialized subsets of SLO fibroblastic stromal cells critically regulate T cell alloreactivity through presentation of Notch ligands independently of antigen presentation. Selective targeting of Delta-like Notch ligand expression on SLO fibroblastic cells may prevent T cell immunopathology while preserving other beneficial antigen-dependent T cell responses after both high-intensity conditioned allo-BMT and allotransplantation with reduced intensity conditioning. DisclosuresBlazar:Kadmon Corporation, LLC: Consultancy, Research Funding. Siebel:Genentech: Employment.

Diffuse Large B-Cell Lymphoma Remodels the Fibroblastic Reticular Network That Acquires Aberrant Immunosuppressive Capabilities; Implications for the Regulation of Anti-Tumor Immunity in the Immuno-Oncology Era

Immunotherapy has demonstrated potential to reactivate or transfer T cell immunity and regress tumors, offering hope to relapsed or refractory diffuse large B-cell lymphoma (DLBCL) patients. However, many DLBCL patients do not experience therapeutic benefit, likely owing to a lack of pre-existing anti-tumor immunity and/or poorly understood immunosuppressive mechanisms in the tumor microenvironment (TME). Understanding the different obstacles that cytotoxic T cells face in the DLBCL TME will help the development of novel therapeutic approaches to overcome them and optimize immunotherapy. Stroma-associated gene signatures reflecting fibroblast and immune cell activity as well as angiogenesis have been associated with outcome in DLBCL but the biology underlying these signatures has been understudied. Here we have examined beyond tumor ‘effacement’ and hypothesized that, rather than being sparse bystanders, lymph node stromal cells may be important players in driving immune suppression in lymphoma.Multiplex immunofluorescence (IF) confocal microscopy analysis of the major stromal cell subsets revealed a marked expansion and remodeling of podoplanin, PDPN+ fibroblastic reticular cells (FRCs) in DLBCL lymph node tissue TME biopsies across GCB and ABC subtypes (n=40) compared to reactive control tissues (n=10). FRC myofibroblasts were similarly remodeled in tumors from the transgenic mouse model of DLBCL (Iμ-HABCL6, n=10) compared to wild type littermates (n=5). These altered PDPNhigh, αSMAhigh FRC networks were interspersed within effaced lymph node tissues in close proximity to DLBCL tumor cells.To model the interactions between tumor cells and FRCs, we established 2D and 3D co-culture platforms that combined DLBCL cells (or non-malignant control B-cells) and PDPN+ FRCs derived from human or murine lymph node tissues. These quantitative assays have shown that tumor cells activate FRCs promoting their proliferation, increased expression of PDPN, marked elongation/stretching and subsequent reduced ability to contract 3D collagen matrix (non-contractile) (P<.01). Exposure to tumor cells caused uncoupling of PDPN from RHOA signaling and redistribution of PDPN into lipid rafts, permitting FRC stretching. FRCs purified from both human and murine tumor lymph nodes showed the same activated morphology and phenotype, demonstrating that our co-culture systems recapitulate in vivo findings. Screening experiments have shown that lymphoma-expressed membrane and soluble lymphotoxins (LTα1β2, LTα3) as well as TNFα significantly contribute to the remodeling of FRCs (P<.01). Co-culture assays have revealed evidence for mutualistic interaction as FRCs, that express BAFF, promote the survival of DLBCL cells in 3D matrix gels (P<.01). Expanded lymphoma PDPNhigh FRCs in situ co-expressed BAFF compared to a more restricted expression profile (marginal zone FRCs) in reactive lymph node tissues. Flow cytometry revealed that lymphoma FRCs exhibit a cancer-associated fibroblast (CAF)-like immunophenotype including upregulation of fibroblast activation protein (FAP) and αSMA, as well as immunomodulatory MHC class I, PD-L1 and PD-L2 molecules compared to control FRCs (P<.01).An important function of FRCs in regulating immunity is attracting and maintaining T cells by secreting chemokines and promoting their migration along the network. Functional assays revealed that T cells show significantly reduced chemotaxis as well motility (quantitative time-lapse movies) across 2D and 3D lymphoma FRC networks compared to control FRCs (P<.01). Multiplex IF analysis revealed reduced CCL19 and CCL21-expressing FRCs in DLBCL compared to the reactive control FRC network as well as low numbers of tumor-infiltrated CD8+ T cells, which localized closely with remodeled FRCs. We next determined whether lymphoma FRCs (CAFs) could negatively regulate T cell function. Triple culture autologous assays (murine and human) have shown that prior exposure of tumor-infiltrated CD8+ T cells to FRCs significantly decreased their cytolytic killing activity against tumor cells (P<.01).In conclusion our data indicate that DLBCL tumor cells convert FRCs into immunosuppressive CAFs, which exhibit altered immumomodulatory activities at different levels that we believe has important implications for the regulation of anti-tumor immunity as well as response to immunotherapy. DisclosuresVardi:Gilead: Research Funding; Janssen: Honoraria. Ramsay:Roche Glycart AG: Research Funding; MedImmune: Research Funding; Celgene Corporation: Research Funding.

Single-cell reconstruction of the early maternal-fetal interface in humans.

During early human pregnancy the uterine mucosa transforms into the decidua, into which the fetal placenta implants and where placental trophoblast cells intermingle and communicate with maternal cells. Trophoblast-decidual interactions underlie common diseases of pregnancy, including pre-eclampsia and stillbirth. Here we profile the transcriptomes of about 70,000 single cells from first-trimester placentas with matched maternal blood and decidual cells. The cellular composition of human decidua reveals subsets of perivascular and stromal cells that are located in distinct decidual layers. There are three major subsets of decidual natural killer cells that have distinctive immunomodulatory and chemokine profiles. We develop a repository of ligand-receptor complexes and a statistical tool to predict the cell-type specificity of cell-cell communication via these molecular interactions. Our data identify many regulatory interactions that prevent harmful innate or adaptive immune responses in this environment. Our single-cell atlas of the maternal-fetal interface reveals the cellular organization of the decidua and placenta, and the interactions that are critical for placentation and reproductive success.

A Distinct Subset of Fibroblastic Stromal Cells Constitutes the Cortex-Medulla Boundary Subcompartment of the Lymph Node

The spatiotemporal regulation of immune responses in the lymph node (LN) depends on its sophisticated tissue architecture, consisting of several subcompartments supported by distinct fibroblastic stromal cells (FSCs). However, the intricate details of stromal structures and associated FSC subsets are not fully understood. Using several gene reporter mice, we sought to discover unrecognized stromal structures and FSCs in the LN. The four previously identified FSC subsets in the cortex are clearly distinguished by the expression pattern of reporters including PDGFRβ, CCL21-ser, and CXCL12. Herein, we identified a unique FSC subset expressing both CCL21-ser and CXCL12 in the deep cortex periphery (DCP) that is characterized by preferential B cell localization. This subset was clearly different from CXCL12highLepRhigh FSCs in the medullary cord, which harbors plasma cells. B cell localization in the DCP was controlled chiefly by CCL21-ser and, to a lesser extent, CXCL12. Moreover, the optimal development of the DCP as well as medulla requires B cells. Together, our findings suggest the presence of a unique microenvironment in the cortex-medulla boundary and offer an advanced view of the multi-layered stromal framework constructed by distinct FSC subsets in the LN.

Neonatally imprinted stromal cell subsets induce tolerogenic dendritic cells in mesenteric lymph nodes

Gut-draining mesenteric lymph nodes (mLNs) are important for inducing peripheral tolerance towards food and commensal antigens by providing an optimal microenvironment for de novo generation of Foxp3+ regulatory T cells (Tregs). We previously identified microbiota-imprinted mLN stromal cells as a critical component in tolerance induction. Here we show that this imprinting process already takes place in the neonatal phase, and renders the mLN stromal cell compartment resistant to inflammatory perturbations later in life. LN transplantation and single-cell RNA-seq uncover stably imprinted expression signatures in mLN fibroblastic stromal cells. Subsetting common stromal cells across gut-draining mLNs and skin-draining LNs further refine their location-specific immunomodulatory functions, such as subset-specific expression of Aldh1a2/3. Finally, we demonstrate that mLN stromal cells shape resident dendritic cells to attain high Treg-inducing capacity in a Bmp2-dependent manner. Thus, crosstalk between mLN stromal and resident dendritic cells provides a robust regulatory mechanism for the maintenance of intestinal tolerance.

Differential cellular composition of human palatine and pharyngeal tonsils

ObjectiveThe goal of this study was to gain a better understanding of the potential functional specialization of palatine and pharyngeal tonsils, by comparing their cellular composition in paired specimens from a large cohort of adenotonsillectomy patients. DesignResident B cell, T cell, dendritic cell, and stromal cell subsets were characterized using multicolor flow cytometry in palatine and pharyngeal tonsil specimens from 27 patients, age 2–34 years. ResultsPaired comparisons showed highly significant intra-individual differences in resident cell subsets of palatine and pharyngeal tonsils. Palatine tonsils harbored higher fractions of germinal center B cells/plasmablasts and IgD- CD27- double-negative B cells, and conversely lower fractions of IgD + CD38- resting naïve B cells compared to pharyngeal tonsils. Palatine tonsils also showed lower fractions of plasmacytoid dendritic cells, and higher percentages of two subsets of stromal cells - fibroblastic reticular cells and lymphatic endothelial cells - compared to pharyngeal tonsils from the same individual. ConclusionsDespite their physical proximity and histological similarities, palatine and pharyngeal tonsils display marked intra-individual differences in their cellular composition with regard to functionally important immune and stromal subsets. These differences are likely to have immunologic, pathologic, and physiologic significance.

Bone Research Society Abstracts

Bone Research Society Abstracts

Reversibly immortalized human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are responsive to BMP9-induced osteogenic and adipogenic differentiation.

Human mesenchymal stem cells (MSCs) are a heterogeneous subset of nonhematopoietic multipotent stromal stem cells and can differentiate into mesodermal lineage, such as adipocytes, osteocytes, and chondrocytes, as well as ectodermal and endodermal lineages. Human umbilical cord (UC) is one of the most promising sources of MSCs. However, the molecular and cellular characteristics of UC-derived MSCs (UC-MSCs) require extensive investigations, which are hampered by the limited lifespan and the diminished potency over passages. Here, we used the piggyBac transposon-based simian virus 40 T antigen (SV40T) immortalization system and effectively immortalized UC-MSCs, yielding the iUC-MSCs. A vast majority of the immortalized lines are positive for MSC markers but not for hematopoietic markers. The immortalization phenotype of the iUC-MSCs can be effectively reversed by flippase recombinase-induced the removal of SV40T antigen. While possessing long-term proliferation capability, the iUC-MSCs are not tumorigenic in vivo. Upon bone morphogenetic protein 9 (BMP9) stimulation, the iUC-MSC cells effectively differentiate into osteogenic, chondrogenic, and adipogenic lineages both in vitro and in vivo, which is indistinguishable from that of primary UC-MSCs, indicating that the immortalized UC-MSCs possess the characteristics similar to that of their primary counterparts and retain trilineage differentiation potential upon BMP9 stimulation. Therefore, the engineered iUC-MSCs should be a valuable alternative cell source for studying UC-MSC biology and their potential utilities in immunotherapies and regenerative medicine.

Abstract B59: Single-cell sequencing as a prognostic and predictive tool for ovarian cancer therapy

Abstract Technological advances allow genomic and transcriptomic analyses to be conducted at the single-cell level. Analysis of gene expression and DNA sequence at this detailed level could lead to prognostic and predictive biomarkers as well as an enhanced understanding of cancer cell and stromal subpopulations including stem cells and chemotherapy-resistant populations. This knowledge will improve our ability to implement a “precision medicine” approach to the treatment of women with ovarian cancer. We have initiated a project to prospectively study the transcriptome and DNA exome of freshly isolated, high-grade serous ovarian cancer solid tumor samples at the single-cell level. To date we have enrolled 8 patients and have initiated single-cell whole-exome DNA sequencing, using the Fluidigm C1 chip, and single-cell whole-transcriptome RNA sequencing, using the 10X Genomics platform. For comparison of RNA sequencing technologies, we previously performed RNA sequencing of a patient’s sample using the Fluidigm C1 chip. In addition to sequencing the primary tumor samples, we are concurrently attempting to establish patient-derived xenografts (PDXs) and cell lines for each patient. We have successfully generated PDX mice from the first patient and are currently treating a subset of the mice with carboplatin/paclitaxel. Patients will be followed prospectively and information on their clinical response will be incorporated into the analysis. Analysis of the RNA sequencing results from the first patient using the Fluidigm C1 platform identified several distinct populations of cells characterized by gene expression patterns that correlate with specific signaling pathways and disease states. We could clearly differentiate stromal cells from cancer epithelial cells based on gene expression patterns. Furthermore, using known functional markers, we could define subsets within each population, including three subgroups of cancer cells and four subgroups of stromal cells. Based on their gene expression patterns, we could determine the frequency of cancer epithelial cells compared to cancer cells undergoing epithelial-to-mesenchymal transition, as well as activated and nonactivated fibroblasts and myofibroblasts. We were also able to identify rare cells expressing stem cell markers. Using the 10X Genomics platform, we have completed RNA sequencing of five patients (~370 million reads/patient). We quantified gene expression on an average of 8,707 cells/patient and 1,723 genes/cell. Using graph-based clustering combined with the t-Distributed Stochastic Neighbor Embedding technique for high dimensionality reduction, we have identified approximately 9 to 15 subsets of cells within these cancer samples based on an unbiased analysis of their gene expression patterns. Using bioinformatic tools, including CellRanger, Seurat, Ingenuity Pathway Analysis, and Gene Set Enrichment Analysis, we can define several of these subsets based on known functional markers, including subsets of immune cells, stromal cells, and cancer epithelial cells. We can estimate the frequency of each subset and can also subdivide the groups using cell-type specific markers, for example, by distinguishing macrophages from dendritic cells within the immune subset. We will present unpublished data, including RNA sequencing and DNA exome sequencing of single cells on the first 8 enrolled patients. In addition to sequencing the primary tumor samples, we plan on performing single-cell sequencing of matched platinum-resistant tumors generated by treating the PDX mice with carboplatin and paclitaxel. We will also correlate presence and percentage of cell subpopulations with clinical outcomes of the patients. Our long-term goal is to use single-cell data as a prognostic biomarker for chemotherapy resistance as well as a tool for predicting effective therapeutic options, including targeted therapy and immune therapy. Citation Format: Boris J. Winterhoff, Christopher R. Clark, Sidharth Ramesh, Mihir Shetty, Locke Uppendahl, Amit Kumra Mitra, Attila Sebe, Martina Bazzaro, Melissa A. Geller, Juan E. Abrahante, Klein Molly, Raffaele Hellweg, Sally Mullany, Kenneth Beckman, Jerry Daniel, Timothy K. Starr. Single-cell sequencing as a prognostic and predictive tool for ovarian cancer therapy. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B59.

Allergic inflammatory memory in human respiratory epithelial progenitor cells.

Barrier tissue dysfunction is a fundamental feature of chronic human inflammatory diseases1. Specialized subsets of epithelial cells-including secretory and ciliated cells-differentiate from basal stem cells to collectively protect the upper airway2-4. Allergic inflammation can develop from persistent activation5 of type 2 immunity6 in the upper airway, resulting in chronic rhinosinusitis, which ranges in severity from rhinitis to severe nasal polyps7. Basal cell hyperplasia is a hallmark of severe disease7-9, but it is not known how these progenitor cells2,10,11 contribute to clinical presentation and barrier tissue dysfunction in humans. Here we profile primary human surgical chronic rhinosinusitis samples (18,036 cells, n = 12) that span the disease spectrum using Seq-Well for massivelyparallel single-cell RNAsequencing12, report transcriptomes for human respiratory epithelial, immune and stromal cell types and subsets from a type 2 inflammatory disease, and map key mediators. By comparison with nasal scrapings (18,704 cells, n = 9), we define signatures of core, healthy, inflamed and polyp secretory cells. We reveal marked differences between the epithelial compartments of the non-polyp and polyp cellular ecosystems, identifying and validating a global reduction in cellular diversity of polyps characterized by basal cell hyperplasia, concomitant decreases in glandular cells, and phenotypic shifts in secretory cell antimicrobial expression. We detect an aberrant basal progenitor differentiation trajectory in polyps, and propose cell-intrinsic13, epigenetic14,15 and extrinsic factors11,16,17 that lock polyp basal cells into this uncommitted state. Finally, we functionally demonstrate that ex vivo cultured basal cells retain intrinsic memory of IL-4/IL-13 exposure, and test the potential for clinical blockade of the IL-4 receptor α-subunit to modify basal and secretory cell states in vivo. Overall, we find that reduced epithelial diversity stemming from functional shifts in basal cells is a key characteristic of type 2 immune-mediated barrier tissue dysfunction. Our results demonstrate that epithelial stem cells may contribute to the persistence of human disease by serving as repositories for allergic memories.