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. 2020 Mar 3;9:e52539. doi: 10.7554/eLife.52539

Figure 2. SLAMF6 expressed in trans by a melanoma target inhibits anti-tumor T cell reactivity.

(A) SLAMF6 expression on B16-F10/mhgp100 parental or transfected (SLAMF6 or empty) melanoma cells. (B) Pmel-1 splenocytes were activated for 7 days with gp10025-33 peptide and IL-2 (30 IU/ml), and then incubated overnight with B16-F10/mhgp100/empty or B16-F10/mhgp100/SLAMF6 melanoma cells at the indicated effector-to-target ratios. IFN-γ secretion was measured by ELISA. (C) Pmel-1 splenocytes were activated for 7 days with gp10025-33 peptide and IL-2 (30 IU/ml), and then incubated overnight with B16-F10/mhgp100/empty or B16-F10/mhgp100/SLAMF6 melanoma cells. IFN-γ production was detected by intracellular staining and flow cytometry (gated on CD8+). Three replicates. The gating strategy is illustrated in Figure 2—figure supplement 1. (D, E) Pmel-1 splenocytes were expanded with gp10025-33 peptide (1 µg/ml) and IL-2 (30 IU/ml) for 7 days. On day 7, cells were transferred i.v. into irradiated C57Bl/6 mice bearing palpable (1 week) B16-F10/mhgp100/empty or B16-F10/mhgp100/SLAMF6 tumors. IL-2 (0.25 × 106 IU) was administered i.p. twice a day for 2 days. Tumor growth was measured twice a week. Mice were sacrificed when the tumor reached 15 mm in diameter. (D) Scheme showing experimental layout. (E) Spider plot showing tumor volume [calculated as L (length) x W (width)2 x 0.5]. One-way ANOVA. *, p<0.05, **, p<0.01, ***, p<0.001.

Figure 2.

Figure 2—figure supplement 1. Gating strategy.

Figure 2—figure supplement 1.

Initially, non-single cells were excluded using FSC-A and FSC-H axes. Then, based on the morphology of the cells in the FSC-A SSC-A axes, the live cell population was gated. In this population, only cells that stained positively for CD8+ expression were subjected to further analysis.