Human Lymph Node Stromal Cells Research Articles

Lower Metabolic Potential and Impaired Metabolic Flexibility in Human Lymph Node Stromal Cells from Patients with Rheumatoid Arthritis.

Cellular metabolism is important for determining cell function and shaping immune responses. Studies have shown a crucial role for stromal cells in steering proper immune responses in the lymph node microenvironment. These lymph node stromal cells (LNSCs) tightly regulate immune tolerance. We hypothesize that malfunctioning LNSCs create a microenvironment in which normal immune responses are not properly controlled, possibly leading to the development of autoimmune diseases such as rheumatoid arthritis (RA). Therefore, we set out to determine their metabolic profile during health and systemic autoimmunity. We included autoantibody positive individuals at risk of developing RA (RA-risk individuals), RA patients and healthy volunteers. All study subjects underwent lymph node biopsy sampling. Mitochondrial function in cultured LNSCs was assessed by quantitative PCR, flow cytometry, Seahorse and oleate oxidation assays. Overall, mitochondrial respiration was lower in RA(-risk) LNSCs compared with healthy LNSCs, while metabolic potential was only lower in RA LNSCs. To maintain basal mitochondrial respiration, all LNSCs were mostly dependent on fatty acid oxidation. However, RA(-risk) LNSCs were also dependent on glutamine oxidation. Finally, we showed that RA LNSCs have impaired metabolic flexibility. Our results show that the metabolic landscape of LNSCs is not only altered during established disease, but partly already in individuals at risk of developing RA. Future studies are needed to investigate the impact of restoring metabolic capacity in LNSC-mediated immunomodulation and disease progression.

Abstract 1487: Role of micro-RNA199a-3p in lymph node stromal extracellular vesicles on tumorigenesis in colorectal cancer

Abstract Introduction: MicroRNAs (miRNAs) are short non-coding RNAs implicated in post-transcriptional regulation of gene expression. Abnormal expression of miRNAs in cancer is associated with enhanced tumorigenesis and metastatic potential. Our previous data has shown that extracellular vesicles (EV) secreted by human lymph node stromal cells (LNSC) enhance tumorigenesis in colorectal cancer (CRC). By systemic searching and modifying miRNAs carried by EV through overexpression and inhibition on LNSC, we aim to demonstrate their effect on CRC progression in vitro and orthotopic mouse model. Methods: Total microRNA sequencing was analyzed using in-silico computer prediction models. Four overexpressed miRNAs in the LNSC-EV were selected (miR-155-5p, miR-199a-3p, mi-143-3p, and mi-214-3p). Tumor cell proliferation assay was performed using a CRC SW620 cell line transfected with selected miRNA mimics or inhibitors or controls. In our mouse model, miRNA transfected SW620 cells tagged with luciferase/RFP, were injected into the rectal submucosa of NOD/SCID mice. Tumor growth and metastases were observed by weekly bioluminescent imaging (BLI). At week 7, tumors were weighed and BLI of the tumors and organs were recorded. Immunohistochemistry staining was performed on the paraffin sections of tumors, livers and lung collected from these mice. Results: In vitro proliferation assays, cells transfected with the mimic of miRNA 199a-3p significantly increased CRC SW620 cell proliferation (p<0.01) while cells treated with the inhibitor showed decreased proliferation (p<0.05). In our orthotopic mouse model, mice with SW620 cells transfected with the mimic had increased frequencies of tumorigenesis (94% vs. 14%) and liver and lung metastasis comparing to control group (56% vs 9.5%, 50% vs 0%). Mice injected with SW620 cells transfected with the miRNA inhibitor had decreased tumor growth compared to the negative control (p<0.05). Conclusion: Our study shows that miRNAs carried in LNSC-EV are crucial for CRC progression. Specifically, inhibition of miRNA-199a-3p may serve as a therapeutic target in the treatment of CRC. Understanding the role and mechanisms of this miRNA in CRC could lead to the improved management of patients with CRC. Citation Format: Hallie Baer, Jessica Simon, Lara McKean-Baste, Alicia N. Ray, Heather Green, Grace Maresh, Xin Zhang, David Marjolin, Jennifer Paruch, Li Li. Role of micro-RNA199a-3p in lymph node stromal extracellular vesicles on tumorigenesis in colorectal cancer [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 1487.

Human Lymph Node Stromal Cells Have the Machinery to Regulate Peripheral Tolerance during Health and Rheumatoid Arthritis.

Background: In rheumatoid arthritis (RA) the cause for loss of tolerance and anti-citrullinated protein antibody (ACPA) production remains unidentified. Mouse studies showed that lymph node stromal cells (LNSCs) maintain peripheral tolerance through presentation of peripheral tissue antigens (PTAs). We hypothesize that dysregulation of peripheral tolerance mechanisms in human LNSCs might underlie pathogenesis of RA. Method: Lymph node (LN) needle biopsies were obtained from 24 RA patients, 23 individuals positive for RA-associated autoantibodies but without clinical disease (RA-risk individuals), and 14 seronegative healthy individuals. Ex vivo human LNs from non-RA individuals were used to directly analyze stromal cells. Molecules involved in antigen presentation and immune modulation were measured in LNSCs upon interferon γ (IFNγ) stimulation (n = 15). Results: Citrullinated targets of ACPAs were detected in human LN tissue and in cultured LNSCs. Human LNSCs express several PTAs, transcription factors autoimmune regulator (AIRE) and deformed epidermal autoregulatory factor 1 (DEAF1), and molecules involved in citrullination, antigen presentation, and immunomodulation. Overall, no clear differences between donor groups were observed with exception of a slightly lower induction of human leukocyte antigen-DR (HLA-DR) and programmed cell death 1 ligand (PD-L1) molecules in LNSCs from RA patients. Conclusion: Human LNSCs have the machinery to regulate peripheral tolerance making them an attractive target to exploit in tolerance induction and maintenance.

Molecular Characterization of Human Lymph Node Stromal Cells During the Earliest Phases of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a progressive, destructive autoimmune arthritis. Break of tolerance and formation of autoantibodies occur years before arthritis. Adaptive immunity is initiated in lymphoid tissue where lymph node stromal cells (LNSCs) play a crucial role in shaping the immune response and maintaining peripheral tolerance. Here we performed the first epigenomic characterization of LNSCs during health and early RA, by analyzing their transcriptome and DNA methylome in LNSCs isolated from lymph node needle biopsies obtained from healthy controls (HC), autoantibody positive RA-risk individuals and patients with established RA. Of interest, LNSCs from RA-risk individuals and RA patients revealed a common significantly differential expressed gene signature compared with HC LNSCs. Pathway analysis of this common signature showed, among others, significant enrichment of pathways affecting the extracellular matrix (ECM), cholesterol biosynthesis and immune system. In a gel contraction assay LNSCs from RA-risk individuals and RA patients showed impaired collagen contraction compared to healthy LNSCs. In RA LNSCs a significant enrichment was observed for genes involved in cytokine signaling, hemostasis and packaging of telomere ends. In contrast, in RA-risk LNSCs pathways in cancer (cell cycle related genes) were differentially expressed compared with HC, which could be validated in vitro using a proliferation assay, which indicated a slower proliferation rate. DNA methylation analyses revealed common and specific differentially methylated CpG sites (DMS) in LNSC from RA patients and RA-risk individuals compared with HC. Intriguingly, shared DMS were all associated with antigen processing and presentation. This data point toward alterations in cytoskeleton and antigen-processing and presentation in LNSC from RA-risk individuals and RA patients. Further studies are required to investigate the consequence of this LNSC abnormality on LNSC-mediated immunomodulation.

MP47-18 LYMPH NODE STROMAL CELLS ENHANCE RENAL CELL CARCINOMA GROWTH, TRANSMIGRATION, AND METASTASIS IN AN ORTHOTOPIC XENOGRAFT MODEL

You have accessJournal of UrologyKidney Cancer: Basic Research II1 Apr 2015MP47-18 LYMPH NODE STROMAL CELLS ENHANCE RENAL CELL CARCINOMA GROWTH, TRANSMIGRATION, AND METASTASIS IN AN ORTHOTOPIC XENOGRAFT MODEL John Nelson, Jessie Gills, Ravan Moret, Xin Zhang, Grace Maresh, Ashley Richman, M'Liss Hudson, Marc Matrana, Ryan Hedgepeth, Shams Halat, Stephen Bardot, Li Li, Christudas Morais, Glenda Gobe, and David Johnson John NelsonJohn Nelson More articles by this author , Jessie GillsJessie Gills More articles by this author , Ravan MoretRavan Moret More articles by this author , Xin ZhangXin Zhang More articles by this author , Grace MareshGrace Maresh More articles by this author , Ashley RichmanAshley Richman More articles by this author , M'Liss HudsonM'Liss Hudson More articles by this author , Marc MatranaMarc Matrana More articles by this author , Ryan HedgepethRyan Hedgepeth More articles by this author , Shams HalatShams Halat More articles by this author , Stephen BardotStephen Bardot More articles by this author , Li LiLi Li More articles by this author , Christudas MoraisChristudas Morais More articles by this author , Glenda GobeGlenda Gobe More articles by this author , and David JohnsonDavid Johnson More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1538AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The incidence of renal cell carcinomas (RCC) is on the rise with an estimated 63,920 new cases in 2014. Despite increased incidental detection of lower clinically staged tumors, metastatic RCC still affects up to 25% of patients at the time of diagnosis. Lymph node (LN) involvement is a strong negative prognostic indicator. LN stromal cells have been shown to enhance tumor cell growth, tumorgenicity and chemotherapy resistance in breast and colon cancer models. However, there are currently no described RCC xenograft models that explore the role of RCC/LN interactions in RCC metastasis. Our objective is the identification of molecular signals that play key roles in human RCC tumor formation and metastasis, and the characterization their activity using a unique orthotopic patient-derived xenograft (PDX) intra-renal sub-capsular (IK) injection NOD/SCID mouse model that mimics metastatic RCC under the influence of LN stromal cells. METHODS Freshly resected human RCC specimen (KiCa-Pt58) was obtained via radical nephrectomy. Six human RCC cell lines and KiCa-Pt58 cancer cells were tagged with luciferase (firefly) to enable bioluminescent imaging (BLI). RCC cells were cultured with or without human LN stromal cells (HK) for proliferation using transmigration assays. A unique PDX IK model was used to monitor tumor growth and metastasis weekly by BLI for up to 16 weeks. H&E, immunohistochemistry (IHC) staining and RT-PCR were performed on primary tumor and mouse lung specimens. RESULTS The presence of HK cells significantly enhanced the proliferation and transmigration of RCC cells. In our PDX IK model, co-injection of HK cells enhanced RCC tumor formation (in 3 out of 6 cell lines) and spontaneous distant metastasis to lung (in 4 out of 6 cell lines). Figure 1 CONCLUSIONS Our PDX model provides a platform to study the determinant factors of tumor formation and metastasis with regard to LN stromal/RCC interaction. It can lead to the development of realistic, durable, and individualized treatments for RCC patients and establish co-clinical trials. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e558 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information John Nelson More articles by this author Jessie Gills More articles by this author Ravan Moret More articles by this author Xin Zhang More articles by this author Grace Maresh More articles by this author Ashley Richman More articles by this author M'Liss Hudson More articles by this author Marc Matrana More articles by this author Ryan Hedgepeth More articles by this author Shams Halat More articles by this author Stephen Bardot More articles by this author Li Li More articles by this author Christudas Morais More articles by this author Glenda Gobe More articles by this author David Johnson More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

FRI0353 Human Lymph Node Stromal Cells Obtained from Rheumatoid Arthritis Patients as Well as from Healthy Individuals Express the Autoimmune Regulator Aire

BackgroundRheumatoid arthritis (RA) is an autoimmune disease with unknown etiology. Immune activation in peripheral lymphoid tissues seems to precede synovial tissue inflammation. The stromal compartment of lymph nodes is crucial...