Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype

J Vis Exp. 2020 Mar 18;(157). doi: 10.3791/60400.


Emerging immunotherapies to treat infectious diseases and cancers often involve transduction of cellular populations with genes encoding disease-targeting proteins. For example, chimeric antigen receptor (CAR)-T cells to treat cancers and viral infections involve the transduction of T cells with synthetic genes encoding CAR molecules. The CAR molecules make the T cells specifically recognize and kill cancer or virally infected cells. Cells can also be co-transduced with other genes of interest. For example, cells can be co-transduced with genes encoding proteins that target cells to specific locations. Here, we present a protocol to transduce primary peripheral blood mononuclear cells (PBMCs) with genes encoding a virus-specific CAR and the B cell follicle homing molecule chemokine receptor type 5 (CXCR5). This procedure takes nine days and results in transduced T cell populations that maintain a central memory phenotype. Maintenance of a central memory or less differentiated phenotype has been shown to associate with persistence of cells post-infusion. Furthermore, cells produced with this method show high levels of viability, high levels of co-expression of the two transduced genes, and large enough quantities of cells for immunotherapeutic infusion. This nine-day protocol may be broadly used for CAR-T cell and other T cell immunotherapy approaches. The methods described here are based on studies presented in our previous publications.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Video-Audio Media

MeSH terms

  • Cell Culture Techniques
  • Cell Survival
  • Immunotherapy, Adoptive
  • Leukocytes, Mononuclear / immunology
  • Leukocytes, Mononuclear / metabolism
  • Phenotype
  • Receptors, Antigen, T-Cell / immunology*
  • Receptors, Antigen, T-Cell / metabolism
  • Receptors, CXCR5 / genetics
  • Receptors, CXCR5 / metabolism
  • Receptors, Chimeric Antigen / genetics
  • Receptors, Chimeric Antigen / immunology*
  • Receptors, Chimeric Antigen / metabolism
  • T-Lymphocytes / immunology*
  • T-Lymphocytes / metabolism
  • Transduction, Genetic


  • Receptors, Antigen, T-Cell
  • Receptors, CXCR5
  • Receptors, Chimeric Antigen