Bispecific T-cells expressing polyclonal repertoire of endogenous γδ T-cell receptors and introduced CD19-specific chimeric antigen receptor

Mol Ther. 2013 Mar;21(3):638-47. doi: 10.1038/mt.2012.267. Epub 2013 Jan 8.


Even though other γδ T-cell subsets exhibit antitumor activity, adoptive transfer of γδ Tcells is currently limited to one subset (expressing Vγ9Vδ2 T-cell receptor (TCR)) due to dependence on aminobisphosphonates as the only clinically appealing reagent for propagating γδ T cells. Therefore, we developed an approach to propagate polyclonal γδ T cells and rendered them bispecific through expression of a CD19-specific chimeric antigen receptor (CAR). Peripheral blood mononuclear cells (PBMC) were electroporated with Sleeping Beauty (SB) transposon and transposase to enforce expression of CAR in multiple γδ T-cell subsets. CAR(+)γδ T cells were expanded on CD19(+) artificial antigen-presenting cells (aAPC), which resulted in >10(9) CAR(+)γδ T cells from <10(6) total cells. Digital multiplex assay detected TCR mRNA coding for Vδ1, Vδ2, and Vδ3 with Vγ2, Vγ7, Vγ8, Vγ9, and Vγ10 alleles. Polyclonal CAR(+)γδ T cells were functional when TCRγδ and CAR were stimulated and displayed enhanced killing of CD19(+) tumor cell lines compared with CAR(neg)γδ T cells. CD19(+) leukemia xenografts in mice were reduced with CAR(+)γδ T cells compared with control mice. Since CAR, SB, and aAPC have been adapted for human application, clinical trials can now focus on the therapeutic potential of polyclonal γδ T cells.

Publication types

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

MeSH terms

  • Animals
  • Antigen-Presenting Cells / immunology
  • Antigen-Presenting Cells / metabolism
  • Antigens, CD19 / immunology*
  • Antigens, CD19 / metabolism
  • Cell Line, Tumor
  • Electroporation
  • Humans
  • Immunotherapy, Adoptive / methods*
  • Leukemia / therapy
  • Leukocytes, Mononuclear / immunology
  • Leukocytes, Mononuclear / metabolism
  • Lymphocyte Activation / immunology
  • Mice
  • Mice, Knockout
  • Receptors, Antigen, T-Cell, gamma-delta / immunology*
  • T-Lymphocytes / immunology*
  • Transposases / genetics
  • Transposases / metabolism


  • Antigens, CD19
  • Receptors, Antigen, T-Cell, gamma-delta
  • Transposases
  • sleeping beauty transposase, human