Murine Leukemia-Derived Extracellular Vesicles Elicit Antitumor Immune Response

Figures & data

Figure 1 C1498 derived EVs induce splenocyte and CD3+ proliferation. Splenocytes (1 x 10⁵ cells) were cultured with PBS, C1498 cells, different concentrations of C1498 derived EVs (C1 = high, C2 = low), or both C1498 cells and C1498 derived EVs. (A) Comparison of tritiated thymidine incorporation (mean ± SD) of combined splenocyte proliferation activity from splenocytes isolated from naïve mice (control) and splenocytes isolated from EV immunized mice (treated) (mean ± SD). (B) CD3+ splenocytes from PBS injected, C1498 derived EV immunized, or C1498 cell immunized mice were labeled with CFSE and co-cultured in vitro with medium, C1498 derived EVs, or C1498 cells. On day 4 the splenocytes were analyzed by flow cytometry and the number of CFSE low cells (proliferating cells) were measured as fold difference from PBS injected naïve mice cultured with medium (mean ± SD). (C) Representative flow cytometry plots of CFSE labeled splenocytes stained with anti-CD3 after four days of culture. CD3+ expression on the vertical axis and CFSE expression on the horizontal axis. Column 1: cells underwent in vivo injection of PBS, column 2 cells underwent in vivo injection of C1498 derived EVs, column 3: cells underwent in vivo injection of C1498 cells. Row 1: cells underwent in vitro culture with medium, row 2: cells underwent in vitro culture with C1498 derived EVs, row 3: cells underwent in vitro culture with C1498 cells. Left upper quadrant represents CD3+ CFSE^lo population. Left bottom quadrant represents CD3- CFSE^lo population. Asterisks indicate significant differences (*p < 0.05, **p<0.01).

Figure 2 C1498 derived EVs preferentially activate CD8+ T cells and C1498 derived EV immunization increases lytic effector function. CFSE labeled splenocytes from in vivo PBS injected, C1498 derived EV immunized, or C1498 cell immunized mice were co-cultured in vitro with medium, C1498 derived EVs, or C1498 cells. On day 4 the cultures were harvested and stained with a panel of antibodies including anti-CD3, anti-CD8, anti-CD4, and underwent immunophenotypic analysis. (A) Table shows the fraction of CD8+ and CD4+ CFSE^lo population that are CD8+ cells. (B) Flow cytometry bar graphs of measured CD3+ CD8+ proliferation of each condition. Experiment repeated at least 3 times. Data is presented as fold difference in CFSE low cells (proliferating cells) from PBS injected naïve mice cultured with medium (mean ± SD). (C) Splenocytes isolated from C57BL/6J mice 7- and 14-days following injection with PBS, C1498 derived EVs, or AML cells were co-cultured in vitro with PBS, C1498 derived EVs, or AML cells for five days and the responding cells were tested for their ability to lyse ⁵¹Cr labeled C1498 cells. Data is presented as LU/10⁶ effector cells and represents replicates from 4 experiments. Asterisks indicate significant differences (*p < 0.05, **p<0.01).

Figure 3 C1498 derived EVs increase cytokine production. Splenocytes obtained from PBS injected, C1498 derived EV immunized, or C1498 cell immunized mice co-cultured in vitro with medium, C1498 derived EVs, or with C1498 cells were incubated for five days at 37°C. Supernatants were then collected and stored at −20°C. These supernatants were tested for cytokine levels using the mouse Th1/Th2/Th17A CBA assay. The cytokines shown are as follows: Panel (A), TNFα; Panel (B), IFNγ; Panel (C) IL6, Panel (D), IL10. Error bars indicate standard deviation of of triplicate samples from a representative of three independent experiments. Asterisks indicate significant differences (*p < 0.05, **p<0.01, ***p<0.001).