Figure 4 from Antigen-Loaded Extracellular Vesicles Induce Responsiveness to Anti–PD-1 and Anti–PD-L1 Treatment in a Checkpoint Refractory Melanoma Model

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Prophylactic administration of EVs and subsequent injection of checkpoint blockade results in a combination effect and improves survival. A, Mice were injected with EVs intravenously before they received OVA-expressing B16 melanoma intraperitoneally, to induce memory T-cell responses as indicated. The mice were sacrificed when tumors reached 1,000 mm3. B, Survival was plotted using the Kaplan–Meier survival curve. C, After inoculation, tumors were measured every 2 to 3 days, and individual tumor growth curves for each group were plotted. Numbers at the bottom right represent surviving mice of the total mice in each group. D, At the endpoint, tumors were collected and analyzed for immune cell infiltration using flow cytometry. Immune cells were identified as CD45+ cells and B16 melanoma cells as CD45–. E, B16 melanoma cells were analyzed for surface MHC class I and PD-L1 expression. F, The frequency of infiltrating of total, CD8+ and antigen-specific T cells in tumors was analyzed by flow cytometry. G, PD-1 expression on the infiltrating CD8+ T cells was determined using flow cytometry. Graphs show the results of two independent experiments. The experiment included 7 to 12 mice per group, of which 4 to 8 mice per group qualified for flow cytometric analysis of the tumor. Dots represent a single mouse, and data are presented as the mean ± SD. Data were analyzed using the Kruskal–Wallis test with Dunn test for multiple comparisons. The Mantel–Cox test was applied for the survival curve. *, P <0.05; **, P < 0.01; and ***, P < 0.001.

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