Tonic 4-1BB Costimulation in Chimeric Antigen Receptors Impedes T Cell Survival and Is Vector-Dependent

Cell Rep. 2017 Oct 3;21(1):17-26. doi: 10.1016/j.celrep.2017.09.015.


Antigen-independent tonic signaling by chimeric antigen receptors (CARs) can increase differentiation and exhaustion of T cells, limiting their potency. Incorporating 4-1BB costimulation in CARs may enable T cells to resist this functional exhaustion; however, the potential ramifications of tonic 4-1BB signaling in CAR T cells remain unclear. Here, we found that tonic CAR-derived 4-1BB signaling can produce toxicity in T cells via continuous TRAF2-dependent activation of the nuclear factor κB (NF-κB) pathway and augmented FAS-dependent cell death. This mechanism was amplified in a non-self-inactivating gammaretroviral vector through positive feedback on the long terminal repeat (LTR) promoter, further enhancing CAR expression and tonic signaling. Attenuating CAR expression by substitution with a self-inactivating lentiviral vector minimized tonic signaling and improved T cell expansion and anti-tumor function. These studies illuminate the interaction between tonic CAR signaling and the chosen expression platform and identify inhibitory properties of the 4-1BB costimulatory domain that have direct implications for rational CAR design.

Keywords: 4-1BB; T cells; adoptive T cell therapy; chimeric antigen receptor; costimulation.

MeSH terms

  • 4-1BB Ligand / genetics*
  • 4-1BB Ligand / immunology
  • Animals
  • Antigens, Neoplasm / genetics*
  • Antigens, Neoplasm / immunology
  • Cell Death
  • Cell Survival
  • Gammaretrovirus / genetics
  • Gammaretrovirus / metabolism
  • Gene Expression Regulation, Leukemic*
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Humans
  • Lentivirus / genetics
  • Lentivirus / metabolism
  • Leukemia-Lymphoma, Adult T-Cell / genetics*
  • Leukemia-Lymphoma, Adult T-Cell / immunology
  • Leukemia-Lymphoma, Adult T-Cell / pathology
  • Mice
  • Mice, Inbred NOD
  • Mutant Chimeric Proteins / genetics*
  • Mutant Chimeric Proteins / immunology
  • NF-kappa B / genetics
  • NF-kappa B / immunology
  • Neoplasm Transplantation
  • Receptors, Antigen, T-Cell / genetics
  • Receptors, Antigen, T-Cell / immunology
  • Signal Transduction
  • T-Lymphocytes / immunology*
  • T-Lymphocytes / pathology
  • T-Lymphocytes / transplantation
  • Tumor Necrosis Factor Receptor Superfamily, Member 9 / genetics*
  • Tumor Necrosis Factor Receptor Superfamily, Member 9 / immunology
  • fas Receptor / genetics
  • fas Receptor / immunology


  • 4-1BB Ligand
  • Antigens, Neoplasm
  • FAS protein, human
  • Mutant Chimeric Proteins
  • NF-kappa B
  • Receptors, Antigen, T-Cell
  • TNFRSF9 protein, human
  • Tumor Necrosis Factor Receptor Superfamily, Member 9
  • fas Receptor