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. 2022 Apr 18;79(5):247. doi: 10.1007/s00018-022-04279-1

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© The Author(s) 2022

Open AccessThis 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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Fig. 3 — Impacts of platelet-derived TGFβ1 on naïve CD4⁺ T cell responses. A, C Naïve CD4⁺ T cells were stimulated with αCD3/αCD28 antibodies in the absence (blue squares) or presence (red triangles and green circles) of platelets (Tn:plt = 1:250), and cultured for 5 days with () or without () a TGF-β neutralizing antibody (20 µg/ml). Data plotted are flow cytometric phenotyping of T helper cells (A) and the total/active TGFβ1 levels in the supernatants as measured by ELISA (C), n = 7. Panels B: Naïve CD4⁺ T cells were stimulated with αCD3/αCD28 antibodies in the absence () or presence () of platelets (Tn:plt = 1:250), and cultured with () or without () a TGFBRII-blocking antibody (15 µg/ml) for 5 days. Flow cytometric phenotyping of T helper cells were plotted (n = 5). D–F Platelet-specific TGFβ1 knockout mice or control mice were adoptively transferred with CD4⁺ T cells from CD45.1 OT-II mice, followed by OVA challenge. Plasma levels of the total and active TGFβ were monitored by ELISA during 7 days (D; n = 5). Mononuclear cells were isolated from the spleen of recipient mice and assayed for flow cytometric phenotyping of Treg, Th1, Th2, and Th17 cells (E; n = 5), as well as phosphorylation levels of Smad2/3 of OT-II T cells (F; n = 5). Data were presented as mean ± SEM. For data presented in A–C, the comparisons among the treatments were performed using RM ANOVA followed by Tukey’s multiple comparison test. For data in D, comparisons between the groups were performed using two-way ANOVA followed by Sidak’s multiple comparison test. For data presented in E–F, Mann–Whitney test was used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Inline graphic — Impacts of platelet-derived TGFβ1 on naïve CD4⁺ T cell responses. A, C Naïve CD4⁺ T cells were stimulated with αCD3/αCD28 antibodies in the absence (blue squares) or presence (red triangles and green circles) of platelets (Tn:plt = 1:250), and cultured for 5 days with () or without () a TGF-β neutralizing antibody (20 µg/ml). Data plotted are flow cytometric phenotyping of T helper cells (A) and the total/active TGFβ1 levels in the supernatants as measured by ELISA (C), n = 7. Panels B: Naïve CD4⁺ T cells were stimulated with αCD3/αCD28 antibodies in the absence () or presence () of platelets (Tn:plt = 1:250), and cultured with () or without () a TGFBRII-blocking antibody (15 µg/ml) for 5 days. Flow cytometric phenotyping of T helper cells were plotted (n = 5). D–F Platelet-specific TGFβ1 knockout mice or control mice were adoptively transferred with CD4⁺ T cells from CD45.1 OT-II mice, followed by OVA challenge. Plasma levels of the total and active TGFβ were monitored by ELISA during 7 days (D; n = 5). Mononuclear cells were isolated from the spleen of recipient mice and assayed for flow cytometric phenotyping of Treg, Th1, Th2, and Th17 cells (E; n = 5), as well as phosphorylation levels of Smad2/3 of OT-II T cells (F; n = 5). Data were presented as mean ± SEM. For data presented in A–C, the comparisons among the treatments were performed using RM ANOVA followed by Tukey’s multiple comparison test. For data in D, comparisons between the groups were performed using two-way ANOVA followed by Sidak’s multiple comparison test. For data presented in E–F, Mann–Whitney test was used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001