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. 2018 Jul 13;6(1):71.
doi: 10.1186/s40425-018-0385-z.

CART cells are prone to Fas- and DR5-mediated cell death

Benjamin O Tschumi  1 , Nina Dumauthioz  1 , Bastien Marti  1 , Lianjun Zhang  1 , Evripidis Lanitis, Melita Irving, Pascal Schneider  2 , Jean-Pierre Mach  2 , George Coukos, Pedro Romero  1 , Alena Donda  3
Affiliations

CART cells are prone to Fas- and DR5-mediated cell death

Benjamin O Tschumi et al. J Immunother Cancer. .

Erratum in

Abstract

Adoptive transfer of T cells transduced with Chimeric Antigen Receptors (CAR) are now FDA-approved for the treatment of B-cell malignancies. Yet, the functionality of the endogenous TCR in CART cells has not been fully assessed. Here, we demonstrate that CART cells progressively upregulate Fas, FasL, DR5 and TRAIL, which result in their programmed cell death, independently of antigen-mediated TCR or CAR activation. CART cell apoptosis occurs even when the CAR contains a single (co-)activatory domain such as CD3ζ, CD28 or 4-1BB. Importantly, the dominant role of the Fas and DR5 pathways in CART cell apoptosis is demonstrated by the significant rescue of CART cells upon in vivo blockade by combined Fas-Fc and DR5-Fc recombinant proteins. These observations are of crucial importance for the long-term persistence of CART cells and for the development of new applications including the combined TCR and CAR activation against solid tumors.

Keywords: 4-1BB; Annexin V; CAR T cells; CD28; CD3ζ; CD8 T lymphocytes; Fas; FasL; HER2; Listeria monocytogenes.

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All co-authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
After their initial expansion, HER2-specific CART cells undergo apoptosis. a Schemes of tested HER2-CAR configurations. b Schematic representation of the experimental protocol c Frequencies of BFP or HER2-CART cells bearing various CAR co-stimulatory domains out of total live cells in the spleen at day 7 post rLm-OVA infection. d Representative FACS plots of CART cell frequencies of CART cells at day 5, 6 and 7 in the transferred OT-1 population. e Fas, FasL, DR5, TRAIL and Annexin V expression and viability staining in BFP or CAR-positive OT-1 T cells at day 6 and 7 post infection. f Scheme of the in vivo rescue of CART cells with recombinant Fas-Fc and DR5-Fc recombinant proteins. g Percentage of HER2- positive OT-1 T cells at day 7 post infection with rLm-OVA in the spleen and liver of mice that received either the control hIgG1 or both Fas-Fc and DR5-Fc. Statistical analysis by One-way ANOVA (c and e) and by T-test (g), n = 4 (c-e) or 5 (g) mice per group. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001
Fig. 2
Fig. 2
The programmed cell death of CD8 CART cells occurs independently of TCR engagement. a Scheme of the experiment involving either BFP or HER2-BBz CAR transduced OT-1 T cells in mice that were conditioned with cyclophosphamide. b Frequencies of BFP or HER2-CART cells out of total live cells in the spleen at day 14 post transfer. c Fas, FasL, DR5, TRAIL and Annexin V expression in BFP or HER2-CAR OT-1 T cells at day 14 post transfer. Statistical analysis by T-test, n = 4 mice per group. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001
Fig. 3
Fig. 3
The susceptibility of CART cells to programmed cell death progressively decreases their anti-tumoral capacity. a Scheme of the experiment in which mice were engrafted with B16-OVA tumors, conditioned with cyclophosphamide and treated with BFP or HER2-BBz OT-1 CART cells. b Evolution of tumor volumes over time. c Expression of Fas, FasL, DR5 and TRAIL in BFP or CART cells in the spleen at day 7 post transfer. d Tumor volume and percentage of BFP or CART cells in total live cells in the tumors at day 21 post transfer. e Scheme of the experiment in which mice were engrafted with B16-OVA-HER2 tumors, conditioned with cyclophosphamide and treated with BFP or HER2-BBz OT-1 or polyclonal CART cells. f Evolution of tumor volumes over time. g Expression of Fas, FasL, DR5 and TRAIL in BFP or CART cells in the spleen at day 9 post transfer. h Tumor volume and percentage of BFP or CART cells in total live cells in the tumors at day 22 post transfer. Statistical analysis by T-test (c, d, h) and by One-way ANOVA (g, h). Each sample were compared to OT1⋅BFP (g), n = 10 mice per group. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001
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