Reversible Transgene Expression Reduces Fratricide and Permits 4-1BB Costimulation of CAR T Cells Directed to T-cell Malignancies

Cancer Immunol Res. 2018 Jan;6(1):47-58. doi: 10.1158/2326-6066.CIR-17-0126. Epub 2017 Oct 27.


T cells expressing second-generation chimeric antigen receptors (CARs) specific for CD5, a T-cell surface marker present on normal and malignant T cells, can selectively kill tumor cells. We aimed to improve this killing by substituting the CD28 costimulatory endodomain (28.z) with 4-1BB (BB.z), as 28.z CD5 CAR T cells rapidly differentiated into short-lived effector cells. In contrast, 4-1BB costimulation is known to promote development of the central memory subpopulation. Here, we found BB.z CD5 CAR T cells had impaired growth compared with 28.z CD5.CAR T cells, due to increased T-cell-T-cell fratricide. We demonstrate that TRAF signaling from the 4-1BB endodomain upregulated the intercellular adhesion molecule 1, which stabilized the fratricidal immunologic synapse between CD5 CAR T cells. As the surviving BB.z CD5 CAR T cells retained the desired central memory phenotype, we aimed to circumvent the 4-1BB-mediated toxicity using a regulated expression system that reversibly inhibits CAR expression. This system minimized CAR signaling and T-cell fratricide during in vitro expansion in the presence of a small-molecule inhibitor, and restored CAR expression and antitumor function of transduced T cells in vivo These studies reveal a mechanism by which 4-1BB costimulation impairs expansion of CD5 CAR T cells and offer a solution to mitigate this toxicity. Cancer Immunol Res; 6(1); 47-58. ©2017 AACR.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Neoplasm / immunology
  • Apoptosis / immunology
  • CD5 Antigens / metabolism
  • Cell Line, Tumor
  • Cytotoxicity, Immunologic
  • Disease Models, Animal
  • Female
  • Gene Expression*
  • Genetic Vectors
  • Immunological Synapses / immunology
  • Immunological Synapses / metabolism
  • Immunotherapy, Adoptive* / adverse effects
  • Immunotherapy, Adoptive* / methods
  • Lymphocyte Activation / genetics
  • Lymphocyte Activation / immunology
  • Male
  • Mice
  • Receptors, Antigen, T-Cell / genetics*
  • Receptors, Antigen, T-Cell / metabolism
  • Receptors, Chimeric Antigen / genetics*
  • Receptors, Chimeric Antigen / metabolism
  • Signal Transduction
  • T-Lymphocytes / immunology*
  • T-Lymphocytes / metabolism*
  • TNF Receptor-Associated Factor 2 / metabolism
  • Transgenes
  • Tumor Necrosis Factor Receptor Superfamily, Member 9 / metabolism*
  • Xenograft Model Antitumor Assays


  • Antigens, Neoplasm
  • CD5 Antigens
  • Receptors, Antigen, T-Cell
  • Receptors, Chimeric Antigen
  • TNF Receptor-Associated Factor 2
  • TRAF2 protein, mouse
  • Tumor Necrosis Factor Receptor Superfamily, Member 9