BM-MSCs acquire complete functional characteristics of FRCs under TNF/LT stimulation. (A) Membrane expression of TNFR1 and LTβR on BM-MSCs. Blue lines indicate immunoglobulin control, red lines indicate specific staining. (B) Meshwork induction. BM-MSCs were cultured with or without TNF/LT stimulation for 7 days. Microscopic visualization of a meshwork of extracellular matrix fibers was performed with fibronectin and transglutaminase (TG) staining. Bar represents 20 μm. (C) Adhesion assay. Tonsil leukocytes or monocyte-derived iDCs were incubated for 2 hours with BM-MSCs pretreated or not with TNF/LT for 7 days. Unbound cells were then removed and adherent CD45⁺ cells and stromal CD45⁻ cells were collected using trypsin and quantified by flow cytometry. Results are the mean values of the ratio of CD45⁺ to CD45⁻ cells (n = 4). The error bars indicate the SD of the mean. *Mean value is statistically different from that of coculture with unstimulated BM-MSCs (P<.05). (D) Real-time PCR quantification of chemokine expression. BM-MSCs were cultured for 3 days with TNF/LT and were then analyzed for CXCL9, CXCL10, CCL5, CXCL12, and CCL19 expression. Each sample was normalized to ABL and compared to expression levels in untreated (UT) BM-MSCs. The arbitrary value of 1 was assigned to UT BM-MSCs. CCL19 was not detected in UT Resto cells and the ΔΔC_T was then calculated with an arbitrary C_T value of 40. Results are those of one experiment of 3.