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. 2017 Oct 25;7:14049. doi: 10.1038/s41598-017-14417-6

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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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Spleen cells from mice cured by combination therapy with anti-PD-1 and anti-CD4 mAb display CTL responses in vitro.(a) Spleen cells from mice treated by combined anti-PD-1/anti-CD4 mAb therapy show higher frequency of CD8⁺CD107a⁺ CTLs relative to naïve mice. CD8⁺CD107a⁺ slightly increased after in vitro re-stimulation with Neuro2apc cells. Percentages of double positive cells are given. (left panel: a representative mouse is shown, right panel: M ± SD values of CD8⁺CD107a⁺ T cells from 5 mice per group are given. (b) Cytolytic activity of spleen cells from naïve (open boxes) and cured mice (full boxes) against Neuro2a-luc cells, at different Effector:Target (E:T) ratios (upper panel). Cytolytic activity of spleen cells from naïve (open boxes) and cured mice (full boxes), after 5-day in vitro MLTC stimulation with Neuro2apc cells, against Neuro2a-luc cells, at different E:T ratios (lower panel). Percentages are evaluated as indicated in the material and method section. P values are indicated (T-test). (c) IFN-γ production of MLTC stimulated spleen cells from naïve (open histogram) and mice cured by combined anti-PD-1/anti-CD4 mAb therapy (black histogram) after further 72 hrs stimulation with Neuro2a-luc cells, at different E:T ratios. P values are indicated (T-test).