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

We did not observe marked differences in migration behaviour in the presence or absence of
flow. Flow is required to model the capture processes operating in the vasculature, and here, as
expected, cytokine-treated EC supported much greater adhesion than unstimulated EC in
presence of flow. However, we found that the proportion of adherent cells transmigrating, and
the frequency with which cells migrated in and out of the monolayer, were similar with or
without flow, for EC treated with TNF, IFN or both. Interferon tended to induce the highest level
of migration and to induce lymphocytes to migrate more rapidly, but the trends were similar with
or without flow. Throughout the study, treatment of EC with cytokines increased the efficiency of
transmigration, as well as capture in flow-based assays, but migration did not require addition of
an exogenous chemokine. Previously, presence of flow and binding of SDF to the surface of
TNF-treated HUVEC were found necessary to obtain efficient trans-endothelial migration of T-
cells [6]. However, the definition of transmigration was different from that used here (only cells
which were observed to change once from phase-bright to phase-dark were counted) and the T-
cells had been cultured for 15-18h before analysis.

Lymphocytes were not purposefully activated in most of our studies, on the basis that PBL
are recruited directly from the circulation during inflammation in vivo. While this recruitment
may be more efficient for memory cells, naive cells are also recruited to non-lymphoid tissue
[17]. Here, migration behaviour was broadly similar for PBL, T-cells, and CD4⁺ and CD8⁺
subsets. We have found preferential migration of CD45RA-negative (i.e., memory phenotype),
CD4⁺ or CD8⁺ cells through Transwell filters (assessed using flow cytometry; unpublished
observations) in line with reports by others [15, 16, 28]. Thus, it is likely that PBL migrating
through endothelial monolayers were enriched in memory cells, but pre-separation of naive and
memory populations would be required to define their relative migration efficiencies e.g., in flow

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