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. 2019 May 17;10:2220. doi: 10.1038/s41467-019-09727-4

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — Both AIRE⁺ mTECs and DCs engage polyclonal SP thymocytes undergoing TCR signaling. a, b Maximum intensity projections of 2PM imaging volumes collected at ×20 magnification showing a WT CD4SP thymocytes (Indo1 ratio) migrating in Aire^EGFP (green) or b WT CD8SP thymocytes (Indo1 ratio) migrating in CD11c-EYFP (yellow) slices. Thymocyte tracks are color-encoded for time; scale bar is 30 µm. c Frequency of polyclonal CD8SP (red) and CD4SP (blue) thymocytes undergoing activation in WT thymic slices. Data points represent the average of all cells in an experiment, and bars show mean ± SEM. d Mean cell velocities of unactivated and activated WT CD8SPs and CD4SPs in WT thymic slices. Data points represent individual cells in all experiments, and the red bar and numbers show means. e Average [Ca²⁺]_i of WT CD8SP (red; N = 3) and CD4SP thymocytes (blue; N = 19) undergoing initial activation on WT thymic slices. Single-cell calcium traces were aligned such that the lowest ratio value before the increase in [Ca²⁺]_i was set at time point 0. f Average [Ca²⁺]_i of all activated WT CD8SPs and CD4SPs on WT thymic slices. Data points represent the average of all cells in an experiment, and bars show mean ± SEM. g Frequency of activated WT CD8SP and CD4SP thymocytes contacting AIRE⁺ mTECs or DCs on Aire^EGFP (green) or CD11c-EYFP (yellow) thymic slices. Data points represent the average of all cells in an experiment, and bars show mean ± SEM. h Frequency of activated WT CD8SPs and CD4SPs contacting DCs, that interacted with cDC2 (yellow) or cDC1 (gray) on CD11c-EYFP thymic slices. Graphs show mean + SEM. Data compiled from 7 to 8 separate imaging experiments per condition, number of cells: CD8SP (N_unactivated = 399, N_activated= 46), CD4SP (N_unactivated = 419, N_activated= 60). Analyzed by unpaired t tests, except for h which was analyzed by t tests with Sidak–Bonferroni correction for multiple comparisons, p-values: * < 0.05, ** < 0.01, **** < 0.0001, ns: not significant. Source data are provided as a Source Data file. See also Supplementary Movies 9, 10