Direct observations of the kinetics of migrating T-cells suggest active retention by endothelial cells with continual bidirectional migration

monolayer within 15 minutes, as in the other models. This proportion increased to about 50%
after 1 hour, but <5% were found in the gel at this time. By 3 hours, about 10% were in the gel,
but these had not penetrated far compared to the neutrophils. Even at 24 hours only 10% were in
gel, and most of these were still in the first 100μm.

We also recorded the behaviour of the lymphocytes at the endothelial surface after 15min,
to allow comparison to the observations on solid substrate. Again, we observed cells undergoing
forward and backward migration, with some making multiple transits through the endothelial
monolayer, at frequencies comparable to those seen on the clear plastic (Table 1D). The phase-
dark cells were highly motile and had velocity averaging 5-6μm∕min (Table 1D).

Effects of lymphocyte activation or the presence of a subendothelial chemokine

We considered whether activated T-cells would migrate more efficiently into gels.

However, while PHA activation significantly increased lymphocyte migration through the
endothelial monolayer (64.6 ± 3.5% vs. 45.2 ± 6.4% of adherent cells migrated at 3h following
PHA treatment vs. freshly isolated PBL respectively; mean ± SEM, n=3; p<0.05 by paired t-test),
migration into the gel was not significantly altered (9.1 ± 3.9% vs. 5.1 ± 4.9% migrated at 24h
with or without PHA respectively; mean ± SEM, n=3). Furthermore, we still observed multiple
transits back and forth across EC by PHA-activated lymphocytes. In fact, the proportions of cells
undergoing at least one transit (~14%), or undergoing multiple transits (~5%) in a six-minute
period were similar to freshly isolated lymphocytes. Thus, activation was insufficient to induce
migration away from the subendothelial space, suggesting the need for a second signal
presumably from within the tissue.

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