Increased cell expansion on CCL21+ICAM1-coated surfaces depends on ICAM1-LFA1 signaling. (A-C) Representative images of OT-II T cells activated for 24 hours with antigen-loaded DCs and reseeded for an additional 48 hours on different surface coatings, with or without an immobilized blocking antibody against LFA1. The degree of cell spreading was similar when CCL21 (A) was combined with either ICAM1 (B) or blocking antibody against LFA1 (C). Scale bars, 100 μm. (D) Bar graph illustrating the number of live cells, measured using a metabolic cell viability assay (data are representative of at least 3 independent experiments with 3 to 4 replicates; error bars represent standard error of the mean). Number of T cells seeded per well: 150 × 103. Despite similar degrees of cell spreading and attachment to the surface through the LFA1 T-cell receptor, there were 4.5 times more live cells when CCL21 coating was combined with ICAM1 than when combined with the anti-LFA1 blocking antibody, indicating that the effect on expansion required actual ICAM1-LFA1 signaling and could not be replaced by the immobilized anti-LFA antibody. Calculated P values (using t test) are as indicated in the figure.
