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. 2021 Oct 28;12:6230. doi: 10.1038/s41467-021-26446-x

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© The Author(s) 2021

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. 4 — a Schematic illustration of the distribution of thymic DCs in the parenchyma (Par-DC), vessel lumen (Lu-DC), and transendothelial localization (TE-DC) and the expected accessibility of epitopes (red) for IV injected mAbs. b and c C57BL/6 mice were injected IV with anti-CD11c-PE or isotype-PE mAb and euthanized 2 minutes later. b Representative FACS plots showing PE⁺ DCs in ET single-cell suspensions after gating on total CD11c⁺ DCs. c Frequency of thymic PE⁺ DCs from two independent experiments; n = 12 mice/group; n = 5; ****: P < 0.0001 (Student’s two-tailed, unpaired t test). d Mice were injected with anti-CD45-PE mAb and euthanized after 2 mins. The frequency of DCs was determined among total blood and thymic leukocytes and among CD45-PE⁺ thymic leukocytes. n = 5 mice (one-way ANOVA, n.s.: not significant, ****P < 0.0001). e Representative plots of in vivo and ex vivo labeling indices of DCs from blood, thymus, lymph nodes (LN), and spleen that were determined as described in Results and Supplementary Fig. 3. Gates reflecting parenchymal and luminal localization were set based on labeling indices obtained from thymic DCs after injection of isotype-PE and analysis of anti-CD11c-PE stained blood-borne DCs, respectively. For a transendothelial localization, in which only a fraction of CD11c epitopes should be accessible from the blood, a third gate was defined in which cells display greater ex vivo and in vivo labeling indices than luminal and parenchymal DCs, respectively. f Percentage of Lu-DC, Par-DC, and TE-DCs in thymus, spleen, blood, and LN. n = 4 mice/group (two-way ANOVA, ****P < 0.0001). g DC subset composition of thymic Par-DC and TE-DCs as well as blood-borne, splenic, and LN DCs. n = 5 mice/group (two-way ANOVA, ****P < 0.0001). h and i Thymic macrophages (Mϕ), Par-DCs, and PE⁺ DCs were evaluated for Flt3 expression. h Representative FACS histogram; i mean fluorescence intensities (MFI) of each cell population. n = 3 independent experiments with five mice/each (one-way ANOVA, **P = 0.0078, *P = 0.0115). Bars and error bars represent mean ± SEM. See also Supplementary Figs. 2–4.