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Abstract
Targeting cancer through the use of effector T cells bearing chimeric Ag receptors (CARs) leads to elimination of tumors in animals and patients, but recognition of normal cells or excessive activation can result in significant toxicity and even death. CAR T cells based on modified NKG2D receptors are effective against many types of tumors, and their efficacy is mediated through direct cytotoxicity and cytokine production. Under certain conditions, their ligands can be expressed on nontumor cells, so a better understanding of the potential off-tumor activity of these NKG2D CAR T cells is needed. Injection of very high numbers of activated T cells expressing CARs based on murine NKG2D or DNAM1 resulted in increased serum cytokines (IFN-γ, IL-6, and MCP-1) and acute toxicity similar to cytokine release syndrome. Acute toxicity required two key effector molecules in CAR T cells-perforin and GM-CSF. Host immune cells also contributed to this toxicity, and mice with severe immune cell defects remained healthy at the highest CAR T cell dose. These data demonstrate that specific CAR T cell effector mechanisms and the host immune system are required for this cytokine release-like syndrome in murine models.
Copyright © 2016 by The American Association of Immunologists, Inc.
Figures
(a) overview of approach to determine MTD for single and multi-dose treatment with murine NKG2D CAR T cells. (b) The health score for mice injected with 2 x 107 wild type NKG2D T cells (WT) or NKG2D CAR T cells (CH) in non-tumor bearing (upper) and tumor-bearing (lower) mice is shown. Each symbol represents an individual mouse at the time of euthanasia. (c–j) The percent change in initial weight in the days following injection of NKG2D CAR T cells, wild type NKG2D T cells, or HBSS is plotted for 3 to 28 d after T cell injection. Average values for each group are shown (n=10/group). Mice were treated with a single dose of 5 x106 T cells (c, d), 107 T cells (e, f), or 2 x 107 T cells (g, h). Mice were RMA tumor bearing (c, e, g) or non-tumor bearing (d, f, h). (i) RMA tumor-bearing mice (RMA cells i.v. day -5) were injected with 107 T cells or HBSS on days 0 and 7. (j) Non-tumor bearing mice were injected with 107 T cells or HBSS on days 0, 7 and 14. Data are from two independent experiments (n=10/group). A Student’s t-test was used to compare differences between groups using a two-tailed test assuming unequal variance. An * indicates significant differences compared to WT; * p < 0.05, ** p <0.01, *** p <0.001.
HBSS, NKG2D CAR T cells (CH) or wtNKG2D T cells (WT) were injected i.v. into mice at 5 x 106, 107, or 2 x 107 cells. (a) Overall health score on day 1 after transfer. (b) Serum samples were analyzed for G-CSF, IFNγ, IL-1β, IL-6, IL-10, MCP1, MIG, and MIP-1β by multiplex cytokine analysis. Each symbol represents an individual mouse (n=6–14/group). ANOVA was performed using a nonparametric Kruskal-Wallis test with Dunn’s test for multiple comparisons. An * indicates significant differences compared to WT; * p < 0.05, ** p <0.01, *** p <0.001.
A single dose of 2 x 107 NKG2D CAR T cells or wild type NKG2D T cells were injected into mice. Changes in health status, body weight (a), and serum cytokines (b–d) were measured after 20 h. The amounts (pg/mL) of G-CSF, IFNγ, IL-1β, IL-6, IL-10, MCP1, MIG, and MIP-1β were measured by multiplex analysis. Serum samples from mice given B6-derived T cells that expressed wild type NKG2D (WT) or the NKG2D CAR (CH) were compared to samples from B6 mice given NKG2D CAR T cells derived from (b) IFNγ-(c) GM-CSF-, or (d) perforin-deficient mice. Each symbol represents an individual mouse. ANOVA was performed using a nonparametric Kruskal-Wallis test with Dunn’s test for multiple comparisons. An * indicates significant differences compared to WT; * p < 0.05, ** p <0.01, *** p <0.001.
A single dose of 2 x 107 B6-derived NKG2D CAR T cells (CH) or wild type NKG2D T cells (WT) was injected i.v. into mice and health status and body weight were evaluated after 20 h. (a) Recipients were B6, NSG, NOD/SCID, and NOD mice (n=8/group). (b) At the time of euthanasia, serum samples were isolated and analyzed for G-CSF, IFNγ, IL-1β, IL-6, IL-10, MCP1, MIG, and MIP-1β by multiplex analysis. In other experiments, recipients were (c) Rag1-deficient or IL-2Rγ-deficient mice, (d) B6 mice pretreated with anti-NK1.1 mAbs (PK136), IgG controls, CD1-deficient mice pretreated with anti-NK1.1 mAbs, or anti-Ly6G (1A8). Each symbol represents an individual mouse. ANOVA was performed using a nonparametric Kruskal-Wallis test with Dunn’s test for multiple comparisons. An * indicates significant differences compared to B6 WT; * p < 0.05, ** p <0.01, *** p <0.001. A + indicates significant differences compared to the similar deficient or depleted WT; + p < 0.05, ++ p <0.01, +++ p <0.001.
A single dose of 2 x 107 B6-derived NKG2D CAR T cells (CH) or wild type NKG2D T cells (WT) was injected i.v. into mice and health status and body weight were evaluated after 20 h. Recipients were (a) IL-6-deficient, IL-1R-deficient or GM-CSF-deficient mice, (b) IFNγ-deficient or IFNγR-deficient mice, (c) MyD88-deficient or DR5-deficient mice, (d) NKG2D-deficient mice, (e) BALB/c, A/J, DBA/1, or DBA/2 mice, (f) B6 mice injected with CAR or control T cells derived from B6, 129 or (B6x129) F1 mice. Each symbol represents an individual mouse. ANOVA was performed using a nonparametric Kruskal-Wallis test with Dunn’s test for multiple comparisons. An * indicates significant differences compared to B6 WT; * p < 0.05, ** p <0.01, *** p <0.001. A + indicates significant differences compared to the similar deficient or depleted WT; + p < 0.05, ++ p <0.01, +++ p <0.001.
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.
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