Fig. 3.

FRCs from swollen LNs exhibit an activated phenotype. (A) (Upper) Representative histograms and mean fluorescence intensity (MFI) for the surface expression of gp38 and PDGFRα on FRCs in the LNs of PBS-injected (black line) or OVA/Mont-injected (red line) mice at 5.5 d after immunization. Surface protein expression on CD45⁺ cells served as a negative control (gray area). (Lower) Kinetics of surface gp38 expression on FRCs at indicated time points after OVA/Mont immunization (closed circles) relative to PBS control (open circles). (B) Representative images showing expression of the myofibroblast marker α-SMA in LN sections obtained at the indicated time points after OVA/Mont immunization, as assessed by immunofluorescence labeling. Asterisks indicate HEVs surrounded by smooth muscle cells, and open arrows indicate reticular FRCs that costained for PDGFRβ. Labeling, exposure time, and image processing were identical for all time points shown. (C) mRNA expression of Il7, Ccl19, and Ccl21 were measured in stroma-enriched (white bars) and lymphocyte-enriched (gray bars) fractions from draining LNs at 0, 3.5, and 8.5 d after immunization and then normalized to two housekeeping genes, as described in Materials and Methods. (D) Representative images of in situ hybridization analysis showing Ccl19 and Ccl21 transcripts (green) in LN sections on days 0, 5.5, and 8.5 after OVA/Mont immunization, along with B220 antibody staining (red). All slides were treated similarly (ISH development, exposure time for photos, and processing of images). Data in A, B, and D are mean ± SD, representative of two or three independent experiments, with n ≥3 mice per group. n.s., statistically not significant. (Scale bars: 100 μm.)