Fig. 1: A proportion of OT-I thymocytes survive negative selection, and exhibit confined migration.

a, b Total OT-I thymocytes were overlaid onto the indicated thymic slices with reference thymocytes, and slices were harvested after 16 h for flow cytometric analysis. a Negative selection displayed as the ratio of live OT-I thymocytes relative to live reference thymocytes, normalized to no OVA controls. b CD69 expression on gated OT-I thymocytes. Data are representative of b or pooled from a 4 independent experiments, with mean and SEM of n = 19–20 thymic slices, where each dot represents an individual slice. ****p < 0.0001 (one-way ANOVA with Bonferroni’s correction). c–f OT-I thymocytes were depleted of mature CD8 single positives (Supplementary Fig. S1) and labeled with the fluorescent ratiometric calcium indicator dye Indo-1 LR prior to overlay on WT or RIPmOVA thymic slices. Thymocytes were allowed to migrate into slices for 2 h, then imaged by two-photon microscopy 0–7 h later. Data are pooled from 4 (RIPmOVA) or 2 (WT) imaging runs. c Average speed versus relative calcium (calculated as described in Materials and Methods) where each dot represents an individual thymocyte track (RIPmOVA, n = 757; WT, n = 188). Numbers indicate the % of tracked thymocytes with low speed and high calcium tracks (gray box: average speed <9 μm/min and average corrected calcium >0.08). Solid rectangle represents cutoff for fast tracks (average speed >9 μm/min) used for the plots shown in d–f. (d–e) Average corrected calcium ratio (d) and displacement index (e) of fast (>9 microns/min) tracks, where n = 154 (RIPmOVA) or 120 (WT) tracks. f Average displacement versus the square root of time for fast (>9 microns/min) tracks on WT or RIPmOVA thymic slices. Black dotted lines indicate estimated plateau of confined migration, and error bars indicate SEM.