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. 2020 Mar 13;11:1395. doi: 10.1038/s41467-020-15229-5

Fig. 6. Enhanced T cell responses against dominant and subdominant tumor neoantigen epitopes.

Fig. 6

a Enhanced T cell responses against a pool of neoantigen peptides. MC38 tumor-bearing mice were intratumorally injected with various VVs at days 0, 3, and 7. One group of C57BL/6 mice were i.v. injected with 200 μg of anti-PD-L1 antibody. Ten days later, splenocytes were cultured in the presence of a mixture of 11 neoepitope peptides (10 μg/mL/each). After 80 h of incubation, supernatants were collected for IFN-γ ELISA (right). [3H] thymidine incorporation was measured (left). The graph shows the results from three mice of each group. Data presented as the means ± SD. *P < 0.05 by two-tailed Student’s t-test. b Enhanced T cell responses against individual neoantigens. The splenocytes from VV-treated mice were cocultured with each of the 11 neoepitope peptides (100 μg/ml) as above described above. [3H] thymidine incorporation (left) and ELISA IFN-γ concentrations (right) are shown; n = 3 mice. One bar or one dot represents one mouse. Data presented as the means ± SD. *P < 0.05 by two-tailed Student’s t-test. c Enhanced T cell responses against the neoantigenic peptide 11. The splenocytes isolated from VV-treated mice were cocultured with various concentrations of the neoepitope peptide 11 as above described above. [3H] thymidine incorporation was used to analyze T cell proliferation; n = 3 mice. Data presented as the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t-test. d, e Enhanced tumor infiltration of neopeptide 4-specific T cells. Tumor cell suspensions from various VV-treated mice using the same treatment schedule as Fig. 5a were stained with the neopeptide 4 (Pep4, ASMTNMEL)-loaded, H-2Db-labeled pentamers, anti-CD45, and anti-CD8. Data are representative of five independent experiments. d Dot plots of flow cytometry; e quantification of peptide 4-pentamer+ CD8+ T cells. Data presented as the means ± SD. *P < 0.05 by two-tailed Student’s t-test. f Enhanced generation of neopeptide-specific memory T cells. Forty days after the virus injection, splenocytes were restimulated with neopeptide 4-loaded DCs in the presence of Golgi-plug followed by surface staining with anti-CD8 and intracellular staining with anti-107a, anti-IFN-γ, anti-IL-2, and anti-TNF-α.