*2.4. MHC Class II Expression by Human LNSC In Vitro*

Since direct antigen presentation is very difficult to study in human LNSCs due to MHC restriction and lack of high number of antigen-specific T cells recognizing PTAs, we aimed to study the machinery needed for antigen presentation and lymphocyte modulation in cultured LNSCs. Under homeostatic conditions the human leukocyte antigen-DR (HLA-DR) gene participating in the MHC class II complex, is expressed by cultured human LNSCs (all passage 2) at very low levels in all donor groups tested (Figure 4A). Of interest, a non-significant lower expression of HLA-DR was observed in cultured LNSCs obtained from RA-risk individuals and RA patients. We next used interferon γ (IFNγ) to study regulation of MHC class II as previously reported in mice and humans [26,29]. HLA-DR mRNA was strongly increased after 24 and 48 h of stimulation, when compared to unstimulated (US), with no significant differences between donor groups, though induction was highly variable between donors (Figure 4B, *n* = 5 per donor group). On protein level, HLA-DR was strongly increased after 72 h of IFNγ stimulation (Figure 4C,D). Interestingly, next to FRCs (Podoplanin+, CD31−) also DNs (Podoplanin−, CD31−) clearly upregulated MHC class II. Podoplanin expression was not influenced by IFNγ stimulation (Figure 4C). Furthermore, the number of FRCs and DNs was similar between donor groups (Figure 4D) and within these two subsets HLA-DR expression was similarly increased in all donor groups although a large inter-donor variation in induction was observed (Figure 4E, healthy individuals *n* = 5, RA-risk individuals *n* = 4, and RA patients *n* = 3). These findings did not correlate with clinical parameters such as age, gender, or antibody titers. Overall, these data suggest that human FRCs as well as DNs are equipped to potentially present antigens to CD4+ T cells.

**Figure 4.** Expression of human leukocyte antigen-DR (HLA-DR) (major histocompatibility complex (MHC) class II) in cultured human LNSCs. (**A**) Expression of HLA-DR mRNA in cultured human LNSCs

(all passage 2) from healthy individuals, RA-risk individuals, and RA patients. (**B**) Induction of HLA-DR was assessed by qPCR after stimulation with interferon γ (IFNγ) at different time points (unstimulated (US), 4, 24, and 48 h, all passage 2). Data are represented as relative quantity in each donor (dot) measured over time, where the line indicates the median expression level within one study group over time. (**C**) Flow cytometry gating strategy used to identify CD45− stromal cells according to their Podoplanin (PDPN) and HLA-DR (MHC class II) expression. Gating was based on negative isotype staining. FACS plots display cultured LNSCs from three representative donors (double negative (DN) cells derived from RA-risk individuals passage 5, fibroblastic reticular cells (FRCs) from RA patients passage 7, and LNSCs from healthy individuals passage 5 containing DN and FRCs) out of 13 individuals tested (healthy individuals *n* = 5, RA-risk individuals *n* = 5, and RA patients *n* = 3). Dot blot shows their expression profile under unstimulated condition versus stimulation for 72 h with IFNγ. Numbers adjacent to the outlined areas indicate percentage of cells in the gated population. (**D**) Frequencies of DNs and FRCs per donor under unstimulated conditions and stimulated for 72 h with IFNγ is shown. (**E**) The frequencies of HLA-DR positive cells after stimulation for 72 h with IFNγ in FRCs (PDPN+) and DN cells (PDPN−) are depicted in two separate graphs. Data are represented as median with interquartile range.
