(A) Two-photon microscopy of migrating human T cells, showing eGFP-E106A transfected cells in green and CMTMR-labeled mock transfected cells in red, within intact mouse lymph node 18 hr after adoptive co-transfer of 5 × 106 of each cell type. (B) Average cell velocities of eGFP-E106Ahi (n = 50) versus CMTMR-labeled control (n = 71) T cells; bars represent mean ± SEM, data from independent experiments using four different donors (12.8 ± 0.5 µm/min vs. 11.1 ± 0.5 µm/min for E106Ahi vs CMTMR cells, p=0.0268). (C) Maximum and minimum cellular instantaneous velocities of eGFP-E106Ahi (green) versus CMTMR-labeled (red) control T cells. (Hodges-Lehmann median difference of −0.21 µm/min, −2.82 to 2.16 µm/min 95% confidence interval for maximum velocity and −0.36 µm/min, −1.02 to 0.36 µm/min 95% confidence interval for minimum velocity) (D) Superimposed tracks with their origins normalized to the starting point. Cells were tracked for more than 20 min. n = 111 (CMTMR), n = 58 (eGFP-E106Ahi) (E) Directionality ratio (displacement/distance) over elapsed time. For Orai1-blocked cells in green, tau = 397 s; vs CMTMR controls in red, tau = 238 s, n = 49 time points. (F) Histogram of turn angles in eGFP-E106Ahi (green) and CMTMR controls (red). Mean ± SEM, 74.5 ± 1.0 degrees for Orai1 blocked cells vs 86.5 ± 1.5 degrees for CMTMR controls, p=0.0001, two-tailed T test. (G) Arrest coefficients of eGFP-E106Ahi compared with CMTMR-labeled control T cells, defined as fraction of time with instantaneous velocity <2 µm/min. (For Orai1-blocked cells in green, 0.02 ± 0.01; vs. CMTMR controls in red, 0.12 ± 0.03, p=0.0406) (H) Frequency distribution of average cell velocities for eGFP-E106Ahi (top) and CMTMR-labeled control T cells (bottom), cells with average velocity <7 µm/min are highlighted in gray; tick marks denote the center of every other bin. (I,J) Average cell velocities (I) and arrest coefficients (J) of eGFP-E106Ahi (green, n = 102) vs CMTMR-labeled control (red, n = 278) human T cells in NK-cell-depleted immunodeficient mouse lymph nodes. Average cell velocities: 11.0 ± 0.5 µm/min vs. 8.8 ± 0.3 µm/min, p=0.0004; Arrest coefficients: 0.10 ± 0.02 vs. 0.16 ± 0.01, p=0.0516 for E106Ahi vs CMTMT cells; bars represent mean ± SEM, data from independent experiments using eight different donors, ***p<0.005.