Figure 6. Mechanisms of DNAM1 CAR mediated toxicity.
A single dose of 107 DNAM1 based CAR T cells were injected into B6 mice and health status and body weight were evaluated. (a) The CARs used were constructed using the entire murine DNAM1 protein combined with cytoplasmic portions of CD3z (DZ), CD28 and CD3z (D28Z), or OX40 and CD3z (D40Z). (b) Health status and body weight of B6 mice after a single injection of HBSS or 107 D28Z CAR T cells derived from B6, (B6x129)F1, or GM-CSF-, perforin-, or IFNγ-deficient mice. All groups had 8 mice, except the (B6x129)F1 that had 4 mice. All mice in groups treated with HBSS, or GM-CSF- or perforin-deficient T cells had a health score of 1. (c) At the time of euthanasia, serum samples were isolated and analyzed for G-CSF, IFNγ, IL-6, and MCP1 by multiplex analysis. (d) Health status at different times after CAR T cells derived from B6 mice or HBSS injections from two independent experiments (experiment 1, experiment 2). (e) Specific lysis of MC38 tumor cells at various effector:target (E:T) ratios by T cells expressing a DNAM1 CAR – DZ, D28Z, D40Z, DBBZ (DNAM1 protein with cytoplasmic domains from 4-1BB and CD3z), or mock T cells. Error bars are SD. ANOVA was performed using a nonparametric Kruskal-Wallis test with Dunn’s test for multiple comparisons. For analysis of cell cytotoxicity in vitro (6e), a one way ANOVA with Dunnett’s method for multiple comparisons was used. An * indicates significant differences compared to WT; * p < 0.05, ** p <0.01, *** p <0.001.