Figure 3. Metalloproteinases are expressed by sinus-lining cells and contribute to rapid antigen degradation in LNs.

(A and B) Resting LNs from C57BL/6 mice (n =3 animals/group) were flash frozen and cryosectioned. (A) Anti-CD35 (green) and anti-metalloproteinase or isotype control mAbs (magenta) staining of LN sections and magnified views of the red dashed regions of interest. Scale bar, 200 μm. (B) Magnified images of the SCS stained for CD169⁺ macrophages (left column) or LYVE1⁺ LECs (right column) and the indicated proteases. Scale bar, 20 μm. (C) C57BL/6 mice (n =3 animals) were immunized with 5 μg of saponin adjuvant and 10 μg of eOD-60mer₄₀. After 24 hours, LNs were harvested, sectioned, and collectively stained for ADAM10, ADAM17, and MMP14 before FRET imaging. Two representative SCS regions showing protease expression, eOD-60mer localization, and false color overlay of proteases (white), total degraded antigen (green), and degraded antigen colocalized with proteases (red) are shown. Scale bar, 20 μm. Bottom graph quantifies degraded eOD-60mer colocalized with metalloproteinases. Each point represents one region and data were collected from at least six tissue sections from six LNs. LN boundaries are indicated by white (A), green (B), or yellow (C) dashed lines. (D) C57BL/6 mice (n =3 animals/group) were immunized as in (C) and LNs were collected after 2 hours before vibratome slicing. Live LN slices were left untreated or incubated in marimastat (Mari) with or without broad-spectrum protease inhibitors for 6 hours ex vivo before FRET imaging. Each point represents one region and data were collected from at least ten tissue sections from six LNs. P values were determined by one-way ANOVA with Tukey’s post test. (E) C57BL/6 mice were treated with Mari for 5 days and then immunized as in (C)., LNs were analyzed 24 hours after by pull-down assay (n=3 pools/group, each pool containing four LNs from two mice) or FRET imaging (n =3 animals/group). Shown are Mari or vehicle control (VC) dosing and immunization schedule (top panel), intact extracellular antigen recovered by pull-down assay (bottom left panel) and FRET imaging analysis for intact antigen (bottom right panel). Each point represents one region for FRET imaging and data collected from at least six tissue sections from six LNs. P values were determined by Student’s t test. All plots show mean ± s.d.