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. 2019 Apr 16;9:6136. doi: 10.1038/s41598-019-42237-3

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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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Pre-treatment with NextA further improves anti-tumor immune response when using anti-PD-1 immune checkpoint blockade. (A) C57BL/6 mice were subcutaneously injected with 1 × 10⁶ SM1 murine melanoma tumor cells. Mice were treated with a vehicle control, 15 mg/kg anti-PD-1, 25 mg/kg NextA, or a combination of both agents for 30 days. The NextA pre-treatment noted in the figure started 10 days before other conditions. (B) Kaplan-Meier survival plot of the previous study. (C) Individual group plots representation for the previous study. (D) Tumors were collected at the end point and the presence of M1 and M2 surface markers were evaluated by flow cytometry. M1/M2 ratios were calculated from three independent populations from M1 and M2. Similarly, tumors were screened by flow cytometry to evaluate the infiltration of CD8+, CD4+, NKT, NK, MDSC, pDC and Tregs (E–L), and specific markers of memory formation were evaluated as noted in the figure (F,G). Representative panels for all conditions are shown in Supplementary Figs 8 and 9.