Comparison of CAR T-cell cytokine expression by CAR T cells cocultured with different feeder cell lines.

<p>G1 (1x10⁶), G3 (1x10⁶), or del<i>ζ</i> (8x10⁴)-T-cells were stimulated in 7-day cycles with irradiated CD32-80-137L (A. left panel), CD32-80-137L-EGFRVIIIΔ654 (B. middle panel) (both preloaded with anti-CD3, and anti-CD28), or EGFRVIIIΔ654aAPCs (C. right panel) at a 2:1 ratio. CAR T-cells were then exposed to U87-EGFRvIII tumor cells and supernatants were collected 24 hours thereafter. IFN-<i>γ</i> (solid black bar), and IL-2 (grey bar) expression was measured by ELISA. Significant difference in IFN-<i>γ</i> expression was observed in G3 CAR T-cells when cocultured with CD32-80-137L aAPC compared to delζ CAR T-cells (F_2,2 = 8717, <i>p</i> = .0002), as well as in G1 CAR-T cells compared to del<i>ζ</i> CAR T-cells [(<i>F</i>_2,2 = 8157, <i>p</i> = .0002), A]. When cocultured with EGFRVIII654 aAPC, G3 CAR T-cells demonstrated significantly robust IFN-<i>γ</i> expression compared to G1 (<i>F</i>_2,2 = 91.47, <i>p</i> = .0216) and del<i>ζ</i> CAR T-cells [(<i>F</i>_2,2 = 98.59, <i>p</i> = .0201), B]. Significant IFN-<i>γ</i> production was observed when G3 CAR-T cells were cocultured with highly modified CD32-80-137L-EGFRVIII654 aAPC compared to G1 CAR-T cells [(<i>F</i>_2,2 = 1194, <i>p</i> = .0017), <b>C</b>]. No exogenous IL-2 was utilized during the preceding restimulation cycles or during the co-incubation with tumor cells. Mean cytokine expression values ±SEM is shown for n = 3 for all groups. Asterisks indicate significant differences (***, <i>P</i> ≤ 0.0001; **, <i>P</i> ≤ 0.001, *, <i>P</i> ≤ 0.01, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199414#sec011" target="_blank">Results</a>).