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. 2019 Sep 9;9:12901. doi: 10.1038/s41598-019-49579-y

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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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The accumulation of CD8+ lymphocytes and decreased tumor growth in melanoma-transplanted mice after vaccination with bivalent BC-PIV/gp100 & WT1. (a) The protocol for transplantation of mouse melanoma B16 cells and subsequent vaccination of syngeneic mice. Vac, vaccination; SC, subcutaneous injection. (b) Effects of the vaccination. BC-PIV/gp100 & WT1 harbors both a melanoma gp100-specific CTL epitope KVPRNQDWL fused with hPIV2 HN (the peptide is located outside the viral membrane, see Fig. 1b,d) and a WT1-specific CTL epitope RMFPNAPYL fused with hPIV2 F (the peptide is located inside the viral membrane). The anticancer drug dacarbazine was intraperitoneally injected (100 mg/kg) at the same timing as each vaccination in order to compare the effects. BC-PIV/gp100 & WT1 was treated with 0.01% β-propiolactone (BPL) to inactivate the viral genome before injection³. The 0.01% BPL treatment was confirmed to be sufficient to abolish the expression of the transgene in BC-PIV (data not shown). Five mice per group were used, and the means ± SD are shown. PBS-injected mice were euthanized at day 12. (c) Hematoxylin and Eosin staining of the transplanted tumors. Left, PBS-injected group at day 12; middle, dacarbazine-administered group at day 21. Tumor cells were diffusely dying; right, vaccinated group with BC-PIV/gp100 & WT1 at day 21. The center was necrotic, and massive infiltration of lymphocytes was found at the tumor boundary. (d) The accumulation of CD8+ T-lymphocytes towards the tumor cells in the mice vaccinated with BC-PIV/gp100 & WT1. CD8 was stained with Cy3. Blue regions correspond to nuclei. IDs are the same as in c. CD8+ cells were not found in the PBS-injected group, and a few CD8+ cells were scattered in the dacarbazine-administered group. However, CD8+ cells accumulated at the tumor boundary in the vaccinated group.