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. 2017 Aug 21;7:8952. doi: 10.1038/s41598-017-08357-4

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© The Author(s) 2017

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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Reduced surface TCR levels but not impaired immune synapse formation accounts for compromised peripheral T cell homeostasis in Arpc2-TKO mice. (a) Flow cytometry analysis of surface and total TCR levels in CD8⁺ T cells of OT-1 TCR Tg mice and non TCR Tg mice. (b) The ratio of surface to total TCR levels from OT-1 TCR Tg mice and non TCR Tg mice. (n = 4). (c) Flow cytometry analysis of surface TCR levels of CD8⁺ T cells in lymph node from Ctrl OT-1 TCR Tg and Arpc2-TKO OT-1 TCR Tg mice. (d) Histogram shows the MFI of surface TCRβ level of CD8⁺ T cells in lymph node from Ctrl OT-1 TCR Tg and Arpc2-TKO OT-1 TCR Tg mice. (n = 6). (e–h) Flow cytometry analysis in CD4 and CD8 expression of T cells in (e) lymph nodes, (g) spleen obtained from Ctrl OT-1 TCR Tg and Arpc2-TKO OT-1 TCR Tg mice. Total numbers of CD8⁺ T cells in (f) lymph nodes (n = 9) and (h) spleen (n = 7). (i) Expression of CD44 and CD62L on T cells from Ctrl OT-1 Tg and Arpc2-TKO OT-1 Tg mice. (J) Total number of CD8⁺ naive T cell in lymph nodes (n = 8) and spleen (n = 6) were quantified from Ctrl OT-1 Tg and Arpc2-TKO OT-1 Tg mice. (k) Naïve CD8⁺ T cells isolated from Ctrl or Arpc2-TKO OT-1 TCR Tg mice were labeled with CFSE and transferred into Rag1 ^−/− mice. After 7 days, the mice were sacrificed, and the lymphocytes were analyzed using FACS. (l) Isolated CD8⁺ T cells from the lymph nodes of Ctrl and Arpc2-TKO OT-1 TCR Tg mice were incubated with anti-CD3 and anti-CD28 activator beads for 15 min and then co-stained antibodies against CD3ζ, phalloidin and Arpc2. They were then imaged using microscopy. Bar is 5 μm. Representative images are shown. (m) Histogram shows the percentage of conjugates that harbored CD3ζ and F-actin at the immune synapse. At least 30 cells were analyzed for each experiment. (experiment, n = 3). The data are means ± S.D., for all panels: **P < 0.01; ***P < 0.001 by Student’s t-test, N.S.: no significance. All results are representative from at least three independent experiments.