*2.2. Cultured Human LNSCs Express the Transcription Factors AIRE and DEAF1*

The expression of an abundant number of PTAs is regulated by transcription factor AIRE [17] and DEAF1 [10,11]. In culture expanded LNSCs (all passage 2) DEAF1 mRNA was strongly expressed at similar levels in all donor groups (Figure 2A, healthy individuals *n* = 5, RA-risk individuals *n* = 12, and RA patients *n* = 14). In contrast, AIRE mRNA was very low but detected by qPCR while AIRE protein expression was easily measured by flow cytometry (Figure 2B,C), immunohistochemistry (Figure 2D), and Western blot (Figure 2E). Mean fluorescence intensity (MFI) of AIRE expression (intracellular) in cultured LNSCs (passages 3–5) was comparable between healthy individuals, RA-risk individuals, and RA patients (Figure 2C, healthy individuals *n* = 5, RA-risk individuals *n* = 9 and RA patients *n* = 4). Moreover, comparing intracellular versus intranuclear staining revealed most AIRE protein within the nucleus, which was confirmed by immunohistochemistry (Figure 2D and Figure S1). Additionally, also by Western blot, we observed that AIRE protein was ubiquitously expressed in cultured LNSCs from healthy individuals, RA-risk individuals, and RA patients (Figure 2E) and that the protein band in LNSCs corresponded to the AIRE expression observed in thymus tissue. Overall, these data show that both AIRE and DEAF1 are present in cultured human LNSCs.

**Figure 1.** Expression of peptidylarginine deiminases (PADIs) and citrullinated proteins targeted by anti-citrullinated protein antibodies (ACPAs) in lymph node (LN) tissue and cultured lymph node stromal cells (LNSCs). (**A**) LN tissue was stained for PADI2 and PADI4 proteins and citrullinated proteins targeted by ACPAs using human monoclonal antibodies C03 and B09. Staining with isotype control (iso) (healthy LN tissues for PADI2 and PADI4 are represented) or control antibody E02 (rheumatoid arthritis (RA) ACPA+ patients LN tissue is represented) was negative. A representative picture of each donor group of the immunohistochemical analysis in LN tissue is displayed (*n* = 3 per donor group; healthy individuals, RA-risk ACPA− individuals, RA-risk APCA+ individuals, RA ACPA− patients, and RA ACPA+ patients). (**B**) Cultured LNSCs were stained against PADI2 and PADI4 protein and citrullinated proteins targeted by ACPAs using human monoclonal antibodies C03 and B09. Staining with isotype controls (iso; RA-risk ACPA+ individuals LNSCs for PADI2 and healthy individuals LNSCs for PADI4 are represented) and control antibody E02 (RA ACPA+ patients LNSCs is represented) was negative. A representative picture of each donor per group of the immunohistochemical analysis in cultured LNSCs is displayed (*n* = 3 per donor group; healthy individuals, RA-risk ACPA− individuals, RA-risk APCA+ individuals, RA ACPA− patients and RA ACPA+ patients, passages 3–11). The larger bar in the left corner of images represents 200 μm and the smaller bar inside the magnified images in (**A**) represents 20 μm.

**Figure 2.** Expression of the peripheral tissue antigen (PTA) driving transcription factors autoimmune regulator (AIRE) and deformed epidermal autoregulatory factor 1 (DEAF1) in human LNSCs. (**A**) Expression of DEAF1 in cultured LNSCs of passage 2 was assessed by qPCR and compared between different donor groups (healthy individuals *n* = 5, RA-risk individuals *n* = 12 and RA patients *n* = 14). Relative quantity is displayed as median and interquartile range. (**B**) Intracellular expression of AIRE protein in cultured LNSCs was measured by flow cytometry. Histograms presenting % of positive cells in comparison to isotype staining. (**C**) The scatter plot represents the mean fluorescence intensity (MFI) of intracellular AIRE expression in cultured human LNSCs (passages 3–5) from individuals in different donor groups (healthy individuals *n* = 8, RA-risk individuals *n* = 9 and RA patients *n* = 4). Relative quantity is presented as median and interquartile range. (**D**) Representative pictures of immunofluorescence staining combined with confocal microscopy displaying AIRE (green) and nucleus (blue) in LNSCs (RA patient; passage 3) cultured on chambers slides. Isotype controls were negative. (**E**) Western blot analysis of AIRE protein expression in cultured LNSCs of 12 donors (passages 4–8) is shown. Actin was used as loading control and thymic tissue was used as positive control for AIRE expression.
