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. 2019 Dec 5;10:5546. doi: 10.1038/s41467-019-13471-0

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© The Author(s) 2019

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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Fig. 3 — a C57Bl/6 mice were implanted with 1 × 10⁶ of 4MOSC1 cells into the tongue. After tumors reached ~30 mm³, mice were treated IP with 10 mg/kg of isotype control or anti-PD-1 (n = 10 per group). Individual growth curves of 4MOSC1 tumor-bearing mice are shown. b A Kaplan–Meier curve showing the survival of mice from a. The death of animals occurred either naturally, when tumor compromised the animal welfare, or when tumor volume reached 100 mm³ (n = 10 mice per group; Log-Rank/Mantel–Cox test.). c Absolute number of live CD45⁺CD3⁺CD8⁺ T cells infiltrating 4MOSC1 tumors with or without anti-PD-1 treatment. Shown is the average of the number of live CD8 T cells infiltrating per mm³ of tumor (n = 4 mice per group; two-sided Student’s t test; data are represented as mean ± SEM). d Immunofluorescent staining of CD8 highlights an increase in CD8 T-cell recruitment with anti-PD-1 treatment. Shown is the average CD8 positivity was by three regions of interest (ROI) per mouse (n = 3 mice per group; two-sided Student’s t test; data are represented as mean ± SEM). e Dependency of anti-PD-1 on CD8 T cells. C57Bl/6 mice were treated with CD8 T cell depleting antibody daily for 3 days before tumor implantation and then once a week after. Mice were then implanted with 1 × 10⁶ of 4MOSC1 cells into the tongue. After tumors reached ~30 mm³, mice were treated IP with 10 mg/kg isotype control or 10 mg/kg anti-PD-1 (n = 5 per group). Individual growth curves of 4MOSC1 tumor-bearing mice are shown. f Immunofluorescence staining of CK5 and Ki-67 in cervical lymph nodes of control or CD8-depleted 4MOSC1-bearing mice. Metastatic lesions in the lymph nodes showed abundant Ki-67⁺ proliferating tumor cells (n = 5 mice per group). g C57Bl/6 mice were implanted with 1 × 10⁶ of 4MOSC2 cells into the tongue. After tumors reached ~30 mm³, mice were treated IP with 10 mg/kg isotype control or 10 mg/kg anti-PD-1 (n = 5 per group). Individual growth curves of 4MOSC2 tumor-bearing mice are shown.