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. 2021 Jan 21;11:1949. doi: 10.1038/s41598-020-80102-w

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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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Differential adherence of allogeneic PBMC to cytokine-activated endothelial cells. Endothelial monolayers were stimulated with TNFα (20 ng/mL) or IL-1β (20 ng/mL) for 4 h or 18 h. Stimulation medium was removed, and whole PBMC fractions were added at a ratio of 3 PBMC to 1 endothelial cell and allowed to adhere for 45 min. Nonadherent cells were removed by two washes with HBSS with Ca²⁺ and Mg²⁺, and adherent cells were detached by a third wash with PBS without Ca²⁺ and Mg²⁺ followed by treatment Accutase. Adherent cells were stained with an immunophenotyping panel and acquired by flow cytometry. (a) Endothelial cells and total PBMC as a percent of live cells in the adherent fraction is shown in the spaghetti plots, comparing untreated endothelial cells with (a) TNFα 4 h-treated EC and (b) TNFα 18 h-treated EC. Each pair represents a unique experiment (n = 21). ****p < 0.0001. (c) Adherence of T cells (gated on CD105^neg CD3⁺) to endothelial cells (gated on CD11a^neg CD105⁺) from different vascular beds after pre-activation with TNFα at 4 h (black bars) or IL-1β at 4 h (grey bars). #p < 0.1, *p < 0.05 comparing HAEC or HCAEC to HLSEC. (d) Adherence of B cells (gated on CD105^neg CD19⁺) to endothelial cells (gated on CD11a^neg CD105⁺) from different vascular beds after pre-activation with TNFα at 4 h (black bars) or IL-1β at 4 h (grey bars). #p < 0.1, *p < 0.05 comparing HAEC or HCAEC to HLSEC. (e) Adherence of NK cells (gated on CD105^neg CD3^neg CD19^neg CD56⁺) to endothelial cells (gated on CD11a^neg CD105⁺) from different vascular beds after pre-activation with TNFα at 4 h (black bars) or IL-1β at 4 h (grey bars). #p < 0.1, *p < 0.05 comparing HAEC or HCAEC to HLSEC. (f) Adherence of monocytes (gated on CD105^neg CD3^neg CD19^neg CD14⁺) to endothelial cells (gated on CD11a^neg CD105⁺) from different vascular beds after pre-activation with TNFα at 4 h (black bars) or IL-1β at 4 h (grey bars). #p < 0.1, *p < 0.05 comparing HAEC or HCAEC to HLSEC. Results are presented as the fold increase in the ratio of PBMC to endothelial cells as mean ± SEM (n = 3 unique EC-PBMC donor combinations).