Transduction and expansion of primary T cells in nine days with maintenance of central memory phenotype

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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.

Original languageEnglish (US)
Article numbere60400
JournalJournal of Visualized Experiments
Issue number157
StatePublished - Mar 2020

Bibliographical note

Funding Information:
This study was supported by NIH grants 5R01AI096966-06S1 (PS, EC, and EB), 1UM1AI26617 (PS, EC and EB), P51OD011106/P51RR000167 (ER), MN REACH grant 5U01HL127479-03 (PS), 1R01A143380-01 (PS and EB), 1UM14126617 (PS and EC) as well as funds provided by the NIAID Division of Intramural Research and the NIH Intramural AIDS Targeted Antiviral Program. Anti-CD3 and anti-CD28 used in these studies was provided by the NIH Nonhuman Primate Reagent Resource (R24 OD010976, U24 AI126683). IL-2 used in these studies was provided by The NCI Preclinical Repository. We thank our collaborators in this CD4-MBL CAR/CXCR5 project, Dr. Elizabeth Connick at the University of Arizona, Dr. Edward A Berger at NIAID, NIH, Dr. Eva G Rakasz at the Wisconsin National Primate Research Center and Dr. Geoff Hart and Ms. Preethi Haran at the University of Minnesota, Dr. Leslie Kean at Harvard Medical School and Dr. Catherine Bollard at the Children's Research Institute. We also thank Dr. Scott McIvor at the University of Minnesota, Dr. Christopher Peterson at the Fred Hutchinson Cancer Center, Dr. Matthew Trivett at the NIH, Dr. Agne Taraseviciute at Seattle Children's Hospital, and Dr. Conrad Russell Cruz at The Children's Research Institute for their very helpful assistance in optimizing this protocol. We also gratefully acknowledge Ms. Chi Phan and Ms. Jhomary Alegria-Berrocal at the University of Minnesota for gammaretroviral production, and Ms. Kim Weisgrau at the University of Wisconsin-Madison for isolation of rhesus macaque PBMC.

Publisher Copyright:
© 2020 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.


  • Bioengineering
  • CAR-T cells
  • Central memory
  • Expansion
  • Issue 157
  • PBMC
  • Retrovirus
  • Transduction


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