S1P1 deficiency does not affect T-cell motility within the popliteal LN. (A-D) Differentially fluorescently labeled S1P1+/ and S1P1–/– CD4+ SP cells were adoptively transferred into WT mice and their migratory behavior was analyzed in the same LN after 24 hours and 48 hours using MP-IVM (pooled data, n = 2 mice/time-point). Parameters analyzed are (A) the median instantaneous 3D velocity (S1P1+/: 12.0 ± 0.7 μm minute–1 versus S1P1–/–: 12.4 ± 0.4 μm minute–1), (B) the cumulative instantaneous 3D velocity, (C) motility coefficients (S1P1+/: 73.5 ± 5.1 μm2 minute–1 versus S1P1–/–: 74.8 ± 4.0 μm2 minute–1), and (D) turning angles. (E-G) Differentially fluorescently labeled untreated (control) or CD4+ T cells from FTY720-treated WT donors were adoptively transferred into WT recipients (n = 2 mice, 2-3 recordings/mouse). After 3 hours, cell motility was analyzed in the popliteal LN. (E) Median instantaneous 3D velocity (control: 11.3 ± 0.5 μm minute–1 versus FTY720: 13.2 ± 0.9μm minute–1), (F) cumulative instantaneous 3D velocity, (G) motility coefficients (control: 82.3 ± 8.2 μm2 minute–1 versus FTY720: 95.6 ± 8.5 μm2 minute–1). Horizontal bars in panels A, C, E, G represent the mean of the measurements. Dashed horizontal lines in panels B and F indicate a frequency of 0.5, corresponding to the median. Vertical dashed lines connect these data points to the x-axis, indicating the median values for instantaneous 3D velocity.
