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. 2020 Mar 20;10:5095. doi: 10.1038/s41598-020-61736-2

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© The Author(s) 2020

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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Immune cell analyses. (a–f) CD8+ T cells against GP33 (GBM antigen; panel a,c,e) or gB498 (oHSV antigen; panel b,d,f), 3 (left panels) or 7 (middle and right) days after oHSV or PBS injection in GBMs (labeled as CT2Agp33nectin1) implanted in mouse brains. CD8+ T cells were gated from CD45+TCRβ+ population isolated by Percoll gradient-enriched leukocytes from excised brains (Brain TIL) and PBMCs. GP33 or gB498+ CD8+ T-cells were stained with corresponding tetramers. Controls were from tumor-free native mouse brains (Naïve) compared to vehicle or oHSV injected mouse brains. Each dot represents an individual mouse sample. The bars represent the mean and the range represents the SEM. (g) Percent of GBM-infiltrating whole CD8+ T cells and (h) PD-1-negative CD8+ T cells that express IFNγ, 7 days after oHSV injection. *p < 0.05, one-way ANOVA; (i) Unbiased immune marker analysis by t-Distributed Stochastic Neighboring viSNE revealed PD-1 downregulation on the cytotoxic CD8+ T cells in brains from murine GL261nectin1 GBM-bearing C57Bl/6 mice after oHSV treatment (rQNestin34.5 and NG3420) at day 7 compared to naïve control group.