Large-scale expansion of Vγ9Vδ2 T cells with engineered K562 feeder cells in G-Rex vessels and their use as chimeric antigen receptor-modified effector cells

Cytotherapy. 2018 Mar;20(3):420-435. doi: 10.1016/j.jcyt.2017.12.014. Epub 2018 Feb 3.


Vγ9Vδ2 T cells are a minor subset of lymphocytes in the peripheral blood that has been extensively investigated for their tolerability, safety and anticancer efficacy. A hindrance to the broad application of these cells for adoptive cellular immunotherapy has been attaining clinically appropriate numbers of Vγ9Vδ2 T cells. Furthermore, Vγ9Vδ2 T cells exist at low frequencies among cancer patients. We, therefore, sought to conceive an economical method that allows for a quick and robust large-scale expansion of Vγ9Vδ2 T cells. A two-step protocol was developed, in which peripheral blood mononuclear cells (PBMCs) from healthy donors or cancer patients were activated with Zometa and interleukin (IL)-2, followed by co-culturing with gamma-irradiated, CD64-, CD86- and CD137L-expressing K562 artificial antigen-presenting cells (aAPCs) in the presence of the anti-CD3 antibody OKT3. We optimized the co-culture ratio of K562 aAPCs to immune cells, and migrated this method to a G-Rex cell growth platform to derive clinically relevant cell numbers in a Good Manufacturing Practice (GMP)-compliant manner. We further include a depletion step to selectively remove αβ T lymphocytes. The method exhibited high expansion folds and a specific enrichment of Vγ9Vδ2 T cells. Expanded Vγ9Vδ2 T cells displayed an effector memory phenotype with a concomitant down-regulated expression of inhibitory immune checkpoint receptors. Finally, we ascertained the cytotoxic activity of these expanded cells by using nonmodified and chimeric antigen receptor (CAR)-engrafted Vγ9Vδ2 T cells against a panel of solid tumor cells. Overall, we report an efficient approach to generate highly functional Vγ9Vδ2 T cells in massive numbers suitable for clinical application in an allogeneic setting.

Keywords: antibody-dependent cell-mediated cytotoxicity; artificial antigen-presenting cells; chimeric antigen receptor; gamma delta T cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigen-Presenting Cells / cytology
  • Antigen-Presenting Cells / metabolism
  • CD3 Complex / immunology
  • Cell Culture Techniques / instrumentation*
  • Cell Culture Techniques / methods*
  • Cell Proliferation / drug effects
  • Feeder Cells / metabolism
  • Humans
  • Immunophenotyping
  • Interleukin-2 / pharmacology
  • K562 Cells
  • Leukocytes, Mononuclear / drug effects
  • Leukocytes, Mononuclear / metabolism
  • Receptors, Antigen, T-Cell, gamma-delta / immunology
  • Receptors, Antigen, T-Cell, gamma-delta / metabolism*
  • Receptors, Chimeric Antigen / genetics
  • Receptors, Chimeric Antigen / immunology
  • Receptors, Chimeric Antigen / metabolism*
  • T-Lymphocytes / cytology*
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism


  • CD3 Complex
  • Interleukin-2
  • Receptors, Antigen, T-Cell, gamma-delta
  • Receptors, Chimeric Antigen