Lack of in situ collagen degradation by T blasts. (A) Dose-dependent inhibition of MMP-2, MMP-9, and recombinant MT1-MMP activity by BB-2615 (marimastat) in gelatin zymography. (B) Lack of FITC release from FITC-labeled collagen matrices by T cells, but not by HT-1080/MT1 cells (positive control). ConA-stimulated CD4⁺ T cells or HT-1080/MT1 cells were cultured in 3D collagen lattices containing 2% FITC-collagen in the absence or presence of protease inhibitor cocktail for 40 hours. Data show the means + SDs for 3 independent experiments. (C) The percentage of viable CD4⁺ cells remained unaffected after 40 hours of culture in collagen. (D) CD4⁺ T cell and (E) HT-1080/MT1 cell incorporated in a 3D collagen lattice containing 5% quenched FITC-collagen. The false-color encoded confocal FITC channel (color code inset: fluorescence channel for lowest (–) and highest (+) intensity, respectively) of the 3D reconstructed in focus sections (left) was superimposed on the shape of the cell body (right; red false color) obtained from the transmission image. In CD4⁺ T cell, physical contact with collagen fibers (arrowheads) did not result in increased cleavage-related fluorescence (blue false color; arrowheads), whereas HT-1080/MT1 cell (positive control) generated focal FITC-fluorescence at fiber binding sites (yellow false color; arrowheads). Fiber cleavage by HT-1080 cells in situ was confirmed by staining with cleavage-site specific antibody (not shown). Images are representative for 10 to 15 cells. Bars, 5 μm. Black arrows, direction of migration.