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Testing the Specificity of Compounds Designed to Inhibit CPT1A in T Cells

  • Roddy S. O’Connor
  • Michael C. MiloneEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2097)

Abstract

In response to antigen and costimulation, T cells undergo a series of metabolic transitions that fulfill the biosynthetic demands of clonal expansion, differentiation, and effector function. Following antigen clearance, the oxidation of long-chain fatty acids (LCFAO) has been implicated in the transition from effector to central memory T cells. However, studies demonstrating a role for LCFAO in memory T-cell development have largely relied on the use of etomoxir (ETO), a small molecule inhibitor of the long-chain fatty acid transporter CPT1A. Understanding how the depletion of nutrients including LCFA that might occur in tumor microenvironments affects T-cell proliferation, differentiation, and function has important implications for tumor immunotherapy. Here, we combine the analysis of posttranscriptional gene silencing with extracellular flux assays to determine if etomoxir exerts nonspecific effects on oxidative metabolism. The off-target effects of ETO that we describe highlight the challenges of using pharmacologic inhibitors in loss-of-function approaches in T cells.

Keywords

Etomoxir CPT1A shRNA 

Notes

Acknowledgments

This work was supported by grants from the University of Pennsylvania-Novartis Alliance.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Center for Cellular ImmunotherapiesPerelman School of Medicine at the University of PennsylvaniaPhiladelphiaUSA
  2. 2.Department of Pathology and Laboratory MedicinePerelman School of Medicine at the University of PennsylvaniaPhiladelphiaUSA

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