*4.3. Lymph Node Stromal Cell Culture and Stimulation*

LNSC culture was performed as previously described [63,64]. In short, after depletion of lymphocytes through a 70 μm cell strainer (BD Falcon, San Jose, CA, USA) the remaining stromal tissue of a freshly collected LN needle core biopsy was plated on a 6-well culture dish (Greiner CELLSTAR®, Sigma Aldrich, Zwijndrecht, The Netherlands) (passage 0; P0). Complete cell culture medium was added which consists of Dulbecco's Modified Eagle Medium (DMEM) low glucose (Gibco, Bleiswijk, The Netherlands) supplemented with 0.1% penicillin (Astellas Pharma Inc, Leiden, The Netherlands), 0.1% streptomycin, 0.05 mg/mL gentamicin, 10 mM HEPES buffer, 2 mM L-glutamine (all Gibco, Bleiswijk, The Netherlands), and 10% fetal calf serum (FCS) (GE Healthcare, Zeist, The Netherlands). To expand cell numbers and for passaging, cultured monolayers of human LNSCs were treated with

trypsin (0.05% trypsin/5 mM ethylenediaminetetraacetic acid (Thermo Fisher Scientific, Landsmeer, The Netherlands)) in phosphate buffer saline (PBS, Fresenius Kabi Nederland BV, Zeist, The Netherlands) for 7 min at 37 ◦C. For harvesting, cells were washed with sterile PBS, trypsinized and the cell suspension was collected and centrifuged for 10 min, 1000 rpm (212 g) at 4 ◦C. Cells were resuspended in cold complete medium and counted using trypan blue (Sigma Aldrich) in a Bürker-Türk chamber (LO Labor Optik, Lancing, UK). Subsequently, human LNSCs were seeded for different experiments. For flow cytometry LNSCs were plated in a 6-well plate (100,000–200,000/well) and stimulated with 50 ng/mL IFNγ (Affymetrix eBioscience, Landsmeer, The Netherlands). For real-time PCR analysis LNSCs were seeded in a 24-well plate (30,000/well) and stimulated with 10 ng/mL IFNγ. For chamber slides (Thermo Fisher Scientific, Landsmeer, The Netherlands) 5000 LNSCs/well were used. As described previously, this ex vivo LNSC culture model contains a mixture of FRCs and DNs [64].

#### *4.4. Immunohistochemical Analysis and (Confocal) Microscopy*

LN needle biopsies were snap frozen in Tissue-Tek OCT (Thermo Fisher Scientific, Landsmeer, The Netherlands), cryostat sectioned (7 μm), and stored at −80 ◦C till further use. Before staining, sections were fixed for 20 min using 2% formaldehyde (Sigma-Aldrich, Zwijndrecht, The Netherlands). Cultured LNSCs were plated out (5000/well) in chamber slides (Thermo Fisher Scientific, Landsmeer, The Netherlands), rested for 24 h in complete medium, washed, fixed with cold methanol (Sigma Aldrich, Zwijndrecht, The Netherlands) for 10 min, and stored at −80 ◦C till further use. LNSCs on chamber slides were blocked with 1% H2O2 (Sigma Aldrich, Zwijndrecht, The Netherlands) and 20% human serum (Akademiska pharmacy, Stockholm, Sweden). The following antibodies were generated at the Karolinska Institutet as previously described [35]: human biotinylated monoclonal ACPAs (1325:04C03 = C03, 1325:01B09 = B09) are second generation validated ACPAs as described in Steen et al. [35]. A human biotinylated concentration-matched antibody (E02, reactive against tetanus) was used as a non-ACPA control antibody. Furthermore, in this study rabbit polyclonal anti-PADI2 (Cosmo Bio, Tokyo, Japan), mouse monoclonal anti-PADI4 (Abcam, Cambridge, UK) and corresponding isotype controls were used. Slides were incubated overnight in a moist chamber at 4 ◦C with the primary/detection antibodies. The next day, slides were first blocked with 1% normal goat serum (Dako, Stockholm, Sweden) and then incubated for 30 min with either biotin-conjugated goat anti-mouse secondary antibody (Invitrogen, Stockholm, Sweden) or a biotin anti-rabbit IgG (H + L) (Vector Laboratories, Stockholm, Sweden). Staining was performed using the VECTASTAIN Elite ABC kit (Vector Laboratories, Stockholm, Sweden) and visualized with 3,3-diaminobenzidine (DAB, Vector Laboratories, Stockholm, Sweden). Slides were counterstained with Mayer's hematoxylin (Sigma Aldrich Zwijndrecht, The Netherlands), permanently mounted and viewed by a light microscope (Reichert Polyvar 2 type 302001, Leica Microsystems, Wetzlar, Germany). Sections of LN tissue were scored by two independent researchers with a scale ranging from 0 to 4 with 0 = no signal and 4 = very strong signal. For AIRE staining, LNSCs on chamber slides were blocked and permeabilized with PBS buffer containing 3% bovine serum albumin (BSA) and 0.3% Triton (both Sigma Aldrich, Zwijndrecht, The Netherlands) for 30 min and then incubated overnight at 4 ◦C with monoclonal mouse IgG1 anti-human AIRE antibody (Santa Cruz, Huissen, The Netherlands) or control mouse IgG1 antibody (Dako, Amstelveen, The Netherlands). Subsequently, secondary anti-mouse IgG1-AlexaFluor488 (Invitrogen, Landsmeer, The Netherlands) was used for labelling and chamber slides were mounted using Vectashield Hardset (Vector Laboratories, Stockholm, Sweden). After hardening overnight staining was analyzed by confocal microscope (TCS SP8, Leica Microsystems, Wetzlar, Germany).
