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. 2018 Jul 13;9:2724. doi: 10.1038/s41467-018-05072-0

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© The Author(s) 2018

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. 6 — DP CD8 TILs recognize and kill autologous tumor cells. DN, SP, and DP CD8 TILs were sorted from tumor digest and expanded in vitro. a Expanded CD8 TILs were tested for tumor reactivity by cultivating them for 20 h with increasing numbers of autologous tumor cells, and tumor recognition was assessed by measuring the frequency of 4-1BB expression (filled symbols) and IFN-γ secretion (open symbols). Results are shown for four melanoma patients. b Reactivity of DP CD8 TILs was confirmed by culture with autologous tumor cells with and without MHC-I blocking antibody, allogeneic tumor cells, and plate-bound anti-CD3. The upregulation of 4-1BB after 20 h is shown for four melanoma patients. c The reactivity of expanded DN, SP, and DP CD8 TILs against a pool of 32 peptides derived from EBV, CMV, and influenza virus was assessed by measuring IFN-γ secretion by ELISPOT. Experiments were performed in triplicate and results are shown for three melanoma patients. All TIL sub-populations showed similar levels of IFN-γ when stimulated with anti-CD3. d Expanded DN, SP, and DP CD8 TILs were cultured with and without autologous tumor cells, in presence and absence of MHC-I blocking antibody. Caspase 3/7+ events (green fluorescence) were monitored every hour with the Incucyte live-cell imaging system over a 20 h period and analyzed as described in the Methods section. Small horizontal lines indicate the mean ± SEM. e Representative images for DN and DP CD8 T cells taken at the beginning (T = 0) and at the end of the co-culture with autologous tumor cells (T = 20 h). Green fluorescence indicates caspase 3/7 activity. Results shown in d and e are representative of two patients