Figure 3. Mst1 is dispensable for integrin-dependent T cell polarization by required for CCL19-induced migration.

A) Wt and Mst1^h/h CD4 T cells were seeded into slide chambers pre-coated with 100 ng/mL ICAM-1-Fc prior to stimulation with CCL19. Polarization of CD44 to the uropod in comparison to LFA-1 expression was visualized by confocal microscopy. B) Computational scoring of CD44 and LFA-1 clustering during live imaging of wt and Mst1^h/h CD4 T cells on ICAM-1 coated chamberslides stimulated for 30 minutes with 100 ng/mL CCL19 in presence of 0.08 ng/mL Alexa647-anti-CD11a/LFA-1 (M17/4) and Alexa488-anti-CD44. For each time point, 99–166 individual cells were analyzed for receptor clustering. Student's t-test was performed to compare clustering efficiency for Mst1^wt and Mst1^h/h T cells. C) Transmigration of purified wt and Mst1^h/h CD4 T cells in response to 100 ng/mL CCL19 through 3 μm or 5 μm pores pre-coated with BSA or ICAM-1 Fc. Data is displayed as mean ± SEM of triplicate samples in a single experiment representative of 3–5 independent experiments. Student's t-test was performed to compare migration efficiency for Mst1^wt and Mst1^h/h T cells, * p<0.002, ** p<0.0001. D) CCR7 expression was determined by flow cytometry.