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

**Janine S. Hähnlein 1,2,**†**, Reza Nadafi 3,4,**†**, Tineke A. de Jong 1,2, Johanna F. Semmelink 1,2, Ester B. M. Remmerswaal 5, Mary Safy 1, Krijn P. van Lienden 6, Mario Maas 6, Danielle M. Gerlag 1, Paul P. Tak 1,7,8,9, Reina E. Mebius 3, Heidi Wähämaa 10, Anca I. Catrina <sup>10</sup> and Lisa G. M. van Baarsen 1,2,\***


Received: 15 July 2020; Accepted: 5 August 2020; Published: 9 August 2020

**Abstract:** 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.

**Keywords:** lymph node stromal cells; rheumatoid arthritis; tolerance; autoimmunity

#### **1. Introduction**

Rheumatoid arthritis (RA) is a debilitating inflammatory autoimmune disease hallmarked by disease-specific autoantibody production against citrullinated proteins, but the underlying etiopathogenesis remains largely unknown [1,2]. Citrullination is a post-translational modification changing arginine side chain residues to citrulline, thereby altering structure and charge of the protein. This process occurs regularly under homeostatic conditions like apoptosis of cells where high levels of calcium activate the peptidylarginine deiminase (PADI) enzymes catalyzing citrullination. PADI activity is also detected in a wide range of inflammatory tissues [3] including RA synovial tissue where high expression levels of PADI2 and PADI4 enzymes have been reported [4]. However, *anti-citrullinated protein antibodies* (ACPAs) can be present years before the actual onset of clinical disease [5], while synovial inflammation seems absent [6,7] during this pre-clinical RA-risk phase [8]. Therefore, breaking of tolerance against citrullinated proteins is probably generated at an extra-articular site like lymphoid organs.

Tolerance by negative selection, anergy, or by generation of regulatory T cells (Tregs) is induced during lymphocyte maturation in thymus and maintained in the periphery. Through presentation of peripheral tissue antigens (PTAs) by medullary thymic epithelial cells (mTECs) in the thymus, self-reactive thymocytes are deleted or become unresponsive [9]. Unsurprisingly, loss of expression of these PTAs, which is driven by the transcription factors autoimmune regulator (AIRE), deformed epidermal autoregulatory factor 1 (DEAF1), and FEZ family zinc finger 2 (Fezf2) [10–13], leads to autoimmunity [10,12,14]. In humans, where AIRE expression is observed in the thymus and in dendritic cells (DCs) [15,16], AIRE mutations cause a multi-systemic autoimmune syndrome, known as autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) [17].

Some self-reactive lymphocytes escape the thymic negative selection and are present in healthy individuals [18]. Safeguarding tolerance in the periphery is therefore crucial and studies in mice show that lymph node (LN) stromal cells (LNSCs) have therein a dominant role. LNSCs possess an impressive arsenal to shape T and B cell responses for maintenance of the delicate balance between tolerance and appropriate immune response [19,20]. Several subsets of LNSCs have been described, and although the number of subsets is expanding, six subsets are well defined according to their function, location within the LN, and the expression of surface markers podoplanin (PDPN, gp38) and CD31 (PECAM-1): fibroblastic reticular cells (FRCs: CD31− gp38+), follicular dendritic cells (FDCs: CD31− gp38+/−), marginal reticular cells (MRCs: CD31− gp38+/−), the rather poorly studied double negative cells (DNs: CD31− gp38−), lymphatic endothelial cells (LECs: CD31+ gp38+), and blood endothelial cells (BECs: CD31+ gp38−) [21,22]. Among others, FDCs and LECs serve as antigen libraries since they catch, preserve, and present antigens over longer periods, thereby enhancing T cell memory [23,24]. FRCs and LECs have the ability to limit T cell proliferation during ongoing inflammation by secretion of nitric oxide (NO) and expression of other negative regulators such as indoleamine 2,3-dioxygenase (IDO) to protect LN integrity and to contract immune responses for return to steady state [25,26]. Furthermore, studies have convincingly demonstrated that several LNSC subsets present PTAs on major histocompatibility complex (MHC) class I and induce clonal deletion [10,11,27,28]. Additionally, CD4+ T cells can be tolerized via PTA presentation on MHC class II or by presentation of MHC-II-peptide complexes acquired from DCs [29,30]. Moreover, expression and subsequent presentation of PTAs by LNSC in the context of MHC class II to CD4+ T cells can also lead to maintenance of Tregs [31]. Moreover, recently we demonstrated that LNSCs convert naïve

autoreactive CD4+ T cells into antigen-specific Tregs cells and suppress autoreactive T follicular helper (Tfh) and B cells responses [32].

Taking into account the tremendous influence of LNSCs on peripheral tolerance and lymphocyte regulation we hypothesize that malfunctioning of LNSCs might lead to a microenvironment causing loss of tolerance and autoantibody production. In this study we investigated for the first time in humans whether the LN is a potential place where citrullination of RA-related PTAs occurs and whether human LNSCs, like murine LNSCs, exhibit the tools for tolerance induction. Finally, we compared the expression of citrullinated proteins, PTAs, and immunomodulatory molecules on human LNSCs of healthy individuals to LNSCs from RA patients and autoantibody positive individuals at risk of developing RA (RA-risk individuals). Our data reveal that human LNSCs express citrullinated proteins targeted by ACPAs and are well equipped to regulate (RA-related) tolerance.
