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.