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. 2018 Dec 3;215(12):2984–2993. doi: 10.1084/jem.20181345

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© 2018 James et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure 3. — LTβR expression on the endothelium regulates thymocyte egress via the perivascular route. (A) Analysis of LTβR on TER119⁻CD45⁻EpCAM-1⁻PDPN⁻CD31⁺ thymic endothelium in Flk-1^cre (blue, n = 6) and LTβR^ENDO (red, n = 5), where the gray filled histogram indicates staining in an Ltbr^−/− mouse. Data are pooled from two independent experiments. (B) CD69⁺CD62L⁻ immature and CD69⁻CD62L⁺ mature CD4⁺CD8⁻TCRβ^HICD25⁻Foxp3⁻ SP4 thymocytes in Flk-1^cre (black, n = 9) and LTβR^ENDO (white, n = 7). Data are pooled from three independent experiments. (C) Thymus sections stained with anti-CD8 (green) and ERTR5 (red) in Flk-1^cre (left) and LTβR^ENDO (right). Scale bars: 500 µm. Images represent n ≥ 4 mice. (D) Thymus sections from Flk-1^cre (top) and LTβR^ENDO (bottom) stained for ERTR7 (red), CD31 (green), and CD4 (blue) to identify thymocytes around and in the PVS. Scale bars: 20 µm. Images represent n = 3 mice. (E) Percentage and number of i.v. anti-CD4 PE-labeled thymocytes in Flk-1^cre (black, n = 6) and LTβR^ENDO mice (white, n = 6). Data are pooled from two independent experiments. (F) Quantitation of SP4 thymocytes in medullary regions of Flk-1^cre (black bars) and LTβR^ENDO mice (white bars). Data represent mean number of cells counted per medullary area (n = 3 mice per strain). (G) Number of CD31⁺ thymic endothelial cells in Flk-1^cre (black, n = 7) and LTβR^ENDO (white, n = 6). Data are pooled from two independent experiments. (H) Confocal quantitation of medullary ERTR7⁺ perivascular-associated CD31⁺ vessels located within 100 µm of the CMJ in Flk-1^cre (black, n = 3) and LTβR^ENDO mice (white, n = 3). (I) qPCR of indicated genes in purified CD31⁺ WT and Ltbr⁻^/⁻ thymic endothelium. Data are from three independent repeats. (J) CD62p and Ly6C expression on CD31⁺ thymic endothelium and percentage of CD62p⁺Ly6C⁻, CD62p⁺Ly6C⁺, and CD62p⁻Ly6C⁺ CD31⁺ thymic endothelium in Flk-1^cre (black, n = 7) and LTβR^ENDO mice (n = 6). Data are pooled from two independent experiments. An unpaired Student’s t test was used for statistical analysis. All bar charts and error bars represent means ± SEM. *, P < 0.05; **, P < 0.001; ****, P < 0.0001.