POS0391 ASSESSING SENESCENT HUMAN LYMPH NODE STROMAL CELLS DURING RHEUMATOID ARTHRITIS DEVELOPMENT

Background:Cellular senescence is a state of proliferation arrest of cells. The persistence and accumulation of senescent cells has been implicated in the pathogenesis of age-related diseases like rheumatoid arthritis (RA). Clinical disease is preceded by loss of immune tolerance and autoimmunity. Lymph node stromal cells (LNSC) are important regulators of this tolerance. Therefore, senescent LNSC may affect tolerance and the development of systemic autoimmunity.Objectives:To determine the extent of cellular senescence of LNSC during early phases of systemic autoimmunity.Methods:We included individuals with arthralgia without any evidence of arthritis who were positive for IgM rheumatoid factor (IgM-RF) and/or anti-citrullinated protein antibodies (ACPA; RA-risk group), early arthritis patients (ACR/EULAR 2010 criteria; disease duration < 1 year) and seronegative healthy controls. All study subjects underwent ultrasound-guided inguinal lymph node biopsy. LNSC were isolated and cultured from freshly collected lymph node needle biopsies and passages 0-9 were used for experiments. Cellular senescence was assessed by measuring cell size, granularity, autofluorescence, senescence-associated gene expression levels, DNA damage and senescence-associated β-galactosidase (SA β-gal) activity.Results:Preliminary flow cytometry data shows that the cell size and autofluorescence of LNSC from RA patients (n=11) and RA-risk individuals (n=7) is increased compared with healthy LNSC (n=7), while granularity was specifically increased in LNSC from RA patients (n=9). Initial stainings indicate higher SA β-gal activity and more DNA damage in LNSC from RA-risk (n=3) and RA patients (n=4) compared with healthy controls (n=3). Expression levels of senescence-associated genes such as p21 and p53 were significantly higher in LNSC from RA patients compared with healthy controls (n=6 per group).Conclusion:These preliminary findings suggest senescence already in early RA and provide a rationale for investigating the consequence of senescent LNSC on immune cell responsesDisclosure of Interests:None declared

Lymph Node Stromal Cells From Different Draining Areas Distinctly Regulate the Development of Chronic Intestinal Inflammation

The balance between the responsiveness of the intestinal immune system and the gut environment is fundamental for the maintenance of intestinal homeostasis, which is required for an adequate recognition of entering antigens. The disruption of this homeostasis by exaggerated immune response to harmless antigens can lead to the development of intestinal disorders such as inflammatory bowel disease. Stromal cells are sessile non-hematopoietic cells that build the backbone of the lymph node, an important site for the immune response induction, but also contribute to immune response and tolerance induction. However, the knowledge about the role of stromal cells in the regulation of inflammatory responses is still limited. Therefore, in this study we analyzed the influence of stromal cells on the development of chronic intestinal inflammation. Here, we show that intestinal inflammation alters the immune activation of the mesenteric lymph node-derived stromal cells. Podoplanin+ and CD21/35+ stromal cells showed increased expression of MHC class II molecules, but CD106 expression on CD21/35+ cells was reduced. Stromal cells secreted cytokines and chemokines such as CCL7 and CXCL16 influenced the gut-homing phenotype and proliferation of CD4+ and CD8+ T cells. Furthermore, stromal cells of peripheral lymph nodes transplanted into the mesentery attenuated colitis severity in B6-Il10-/- mice. The reduced colitis severity in these mice was associated with increased expression of IL4 and distinct activation pattern of stromal cells derived from transplanted peripheral lymph nodes. Altogether, our results demonstrate that lymph node stromal cells impact development of chronic colitis via T cell induction. Moreover, lymph node stromal cells from different draining area due to neonatally imprinted processes distinctly regulate the induction of immune responses.

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.