*2.3. Variable Expression of Disease-Related PTAs in Human LNSCs*

Given that we found the presence of the transcription factors potentially driving PTA expression in LNSCs, we next investigated the expression levels of several disease-related PTAs. We selected PTAs previously reported to be expressed by mouse LNSCs [27,28] and involved in human diseases: Ras Related Glycolysis Inhibitor and Calcium Channel Regulator (RRAD), Glutamate Decarboxylase 1 (GAD1), Proteolipid Protein 1 (PLP1), and Alpha Fetoprotein (AFP). RRAD is implicated in several

cancers [36] and plays an important role in type II diabetes [37]. GAD1 is a major autoantigen in type I diabetes and highly expressed in brain and pancreas [38]. PLP1 encodes the most abundant myelin protein which is a main target of autoreactive T cells in multiple sclerosis (MS) and is detected in cervical LNs of MS patients [39]. AFP levels serve as diagnostic marker of liver injury such as hepatocellular carcinoma or Hepatitis C infection [40]. With the exception of AFP, all these disease-related PTAs were expressed by human LNSCs (passages 3 and 4) (Figure 3A). While we did not observe a significant difference in PLP1 expression by LNSCs from different donor groups, we interestingly found that GAD1 and RRAD has a variable expression pattern in LNSCs (all passage 2) derived from healthy individuals, RA-risk individuals and RA patients (healthy individuals *n* = 14, RA-risk individuals *n* = 23, and RA patients *n* = 24). RRAD was significantly expressed at a lower level in LNSCs of RA patients (*p* = 0.037), whereas GAD1 was highly expressed in LNSCs of RA-risk individuals (*p* = 0.0129) when compared to healthy individuals (Figure 3B). Furthermore, after stratification according to ACPA status, the difference in PTA expression was especially clear in ACPA negative RA patients with almost no RRAD expression in LNSCs from ACPA negative RA patients (Figure S2). The expression of these PTAs did not correlate with any other clinical parameters such as age, gender, or antibody titers.

**Figure 3.** Expression of disease-related PTAs in human LNSCs. (**A**) mRNA expression of Ras Related Glycolysis Inhibitor and Calcium Channel Regulator (RRAD), Glutamate Decarboxylase 1 (GAD1), Proteolipid Protein 1 (PLP1), and Alpha Fetoprotein (AFP) in five LNSC individuals (1: healthy individuals passage 4; 2: RA-risk individuals passage 4; 3: RA patients passage 4; 4: RA patients passage

4; 5: RA patients passage 3) analyzed by PCR and visualized on agarose gel. cDNA prepared from an arbitrary in house-made RNA sample pool containing all human tissues (RefRNA) as well as a human thymus sample served as positive control and H2O in exchange for cDNA as negative control. (**B**) Expression of PLP1, RRAD, and GAD1 and was assessed by qPCR in different donor groups (healthy individuals *n* = 14, RA-risk individuals *n* = 23, and RA patients *n* = 24, all passage 2). Relative quantity is displayed as median and interquartile range. Differences between donor groups were assessed by Kruskal–Wallis followed by a post Dunn's test. \* *p* < 0.050.
