*2.1. Citrullinated Antigens Targeted by ACPAs Are Present in Human LN Tissue and in Cultured LNSCs*

First we investigated by immunohistochemistry the presence of PADI enzymes required for citrullination in LN tissue and cultured LNSCs of a small cohort of individuals (healthy individuals, RA-risk ACPA− individuals, RA-risk ACPA+ individuals, RA ACPA− patients, and RA ACPA+ patients; for each subgroup *n* = 3, total *n* = 15). Both PADI2 and PADI4 enzymes were abundantly present in LN tissue. In cultured LNSCs, PADI2 and PADI4 enzymes were detected intracellularly with PADI4 showing a nuclear expression pattern as reported before [33] (Figure 1A,B, upper panels). Using unique monoclonal ACPAs isolated from RA patients' synovial fluid, which are cross-reactive to one or more citrullinated antigens [34,35], we next analyzed the presence of RA-associated citrullinated proteins in LN tissues and cultured LNSCs. Staining with monoclonal ACPAs C03 and B09 revealed the presence of ACPA-targets in LN tissue as well as in cultured LNSCs derived from all study groups (Figure 1A,B, lower panels). Overall, each antibody showed a distinct staining pattern with variable intensity between donors tested. C03-binding was only detected in a few donors, while B09 was highly abundant in most donors tested, with expression mainly restricted to nuclei. These data show that citrullinated proteins, targeted by ACPAs, are present in LN tissue and cultured LNSCs, and at similar levels in healthy individuals, RA-risk individuals, and RA patients.
