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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

Abstract

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Introduction

Chimeric antigen receptors (CARs) provide a promising approach to redirect T cells against distinct tumor antigens. CARs are synthetic receptors that bind an antigen target. While their precise composition is variable, CARs generally contain 3 distinct domains. The extracellular domain directs binding to a target antigen and is typically comprised of a single chain antibody fragment linked to the CAR via an extracellular hinge. The second domain, commonly derived from the CD3ζ chain of the T cell receptor (TCR) complex, promotes T cell activation following CAR engagement. A third costimulatory domain is included to enhance T cell function, engraftment, metabolism....

Protocol

All animal studies are approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

1. T Cell Activation, Transduction, and Expansion

  1. Activate fresh or cryopreserved primary human T cells by mixing with anti CD3/CD28 magnetic beads (e.g., dynabeads) at a ratio of 3 beads per T cell in 6-well cell culture dishes. Culture T cells in X-VIVO 15 medium supplemented with 5% normal human AB serum, 2 mM L-glutamine, 20 mM HEPES, and IL2 (100 units/mL). Mai.......

Representative Results

Using the methods described above, we stimulated and expanded T cells for either 3 or 9 days (Figure 1A,B). We also analyzed their differentiation profile, as indicated by the gating strategy outlined in Figure 1C, by measuring the abundance of distinct glycoproteins expressed on the cell surface. We show a progressive shift towards effector differentiation over time during ex vivo culture (Figure 1.......

Discussion

Here we describe approaches to measure the function and efficacy of CAR T cells harvested at varying intervals throughout ex vivo culture. Our methods provide comprehensive insight into assays designed to assess proliferative capacity as well as effector function in vitro. We describe how to measure CAR T cell activity following stimulation through the CAR and detail xenograft models of leukemia using CAR T cells harvested at day 3 vs day 9 of their logarithmic expansion phase.

There are inher.......

Acknowledgements

This work was supported in part through funding provided by Novartis Pharmaceuticals through a research alliance with the University of Pennsylvania (Michael C. Milone) as well as St. Baldrick's Foundation Scholar Award (Saba Ghassemi).

....

Materials

NameCompanyCatalog NumberComments
Anti CD3/CD28 dynabeadsThermo Fisher40203D
APC Mouse Anti-Human CD8BD Biosciences555369RRID:AB_398595
APC-H7 Mouse anti-Human CD8 AntibodyBD Biosciences560179RRID:AB_1645481
BD FACS Lysing Solution 10X ConcentrateBD Biosciences349202
BD Trucount Absolute Counting TubesBD Biosciences340334
Brilliant Violet 510 anti-human CD4 AntibodyBioLegend317444RRID:AB_2561866
Brilliant Violet 605 anti-human CD3 AntibodyBioLegend317322RRID:AB_2561911
CellTrace CFSE Cell Proliferation KitLife TechnolohgiesC34554
CountBright Absolute Counting Beads,InvitrogenC36950
FITC anti-Human CD197 (CCR7) AntibodyBD Pharmingen561271RRID:AB_10561679
FITC Mouse Anti-Human CD4BD Biosciences555346RRID:AB_395751
HEPESGibco15630-080
Human AB serumValley BiomedicalHP1022
Human IL-2 IS, premium gradeMiltenyi130-097-744
L-glutamineGibco28030-081
Liquid scintillation counter, MicroBeta triluxPerkin Elmer
LIVE/DEAD Fixable VioletMolecular ProbesL34964
Multisizer Coulter CounterBeckman Coulter
Na251CrO4Perkin ElmerNEZ030S001MC
Pacific Blue anti-human CD14 AntibodyBioLegend325616RRID:AB_830689
Pacific Blue anti-human CD19 AntibodyBioLegend302223
PE anti-human CD45RO AntibodyBD Biosciences555493RRID:AB_395884
PE/Cy5 anti-human CD95 (Fas) AntibodyBioLegend305610RRID:AB_493652
PE/Cy7 anti-human CD27 AntibodyBeckman CoulterA54823
Phenol red-free mediumGibco10373-017
UltraPure SDS Solution, 10%Invitrogen15553027
Via-ProbeBD Biosciences555815
X-VIVO 15Gibco04-418Q
XenoLight D-Luciferin - K+ SaltPerkin Elmer122799

References

  1. Brentjens, R. J., et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Science Translational Medicine. 5 (177), 177ra138 (2013).
  2. Grupp, S. A., et al.

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