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Optimized retroviral transduction of mouse T cells for in vivo assessment of gene function

Nature Protocols volume 12, pages 19801998 (2017) | Download Citation

Abstract

Retroviral (RV) expression of genes of interest (GOIs) is an invaluable tool and has formed the foundation of cellular engineering for adoptive cell therapy in cancer and other diseases. However, monitoring of transduced T cells long term (weeks to months) in vivo remains challenging because of the low frequency and often poor durability of transduced T cells over time when transferred without enrichment. Traditional methods often require additional overnight in vitro culture after transduction. Moreover, in vitro-generated effector CD8+ T cells enriched by sorting often have reduced viability, making it difficult to monitor the fate of transferred cells in vivo. Here, we describe an optimized mouse CD8+ T-cell RV transduction protocol that uses simple and rapid Percoll density centrifugation to enrich RV-susceptible activated CD8+ T cells. Percoll density centrifugation is simple, can be done on the day of transduction, requires minimal time, has low reagent costs and improves cell recovery (up to 60%), as well as the frequency of RV-transduced cells (sixfold over several weeks in vivo as compared with traditional methods). We have used this protocol to assess the long-term stability of CD8+ T cells after RV transduction by comparing the durability of T cells transduced with retroviruses expressing each of six commonly used RV reporter genes. Thus, we provide an optimized enrichment and transduction approach that allows long-term in vivo assessment of RV-transduced T cells. The overall procedure from T-cell isolation to RV transduction takes 2 d, and enrichment of activated T cells can be done in 1 h.

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Acknowledgements

We thank M.A. Ali for animal care; S. Adamski for technical assistance; K. Lewy for plasmid work; J.E. Wu for protocol verification; T. Yamada and K. Rome for comments on RV transduction of CD4+ T cells; and W.S. Pear (University of Pennsylvania), A. Bhandoola (Center for Cancer Research, National Cancer Institute), S.L. Reiner (Columbia University) and D.P. Beiting (University of Pennsylvania) for plasmids, cell line, template protocols and comments. This work was supported by German Research Foundation fellowship BE5496/1-1 (to B.B.), National Institutes of Health (NIH) grants F30AI129263 (to O.K.) and AI105343, AI112521, AI115712, AI108545, AI117950, AI117718, AI082630, AI095608 (to E.J.W.), U.S. Broad Agency Announcements Grant HHSN272201100018C (to E.J.W.) and funding from the Parker Institute for Cancer Immunotherapy (to E.J.W.).

Author information

Affiliations

  1. Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Makoto Kurachi
    • , Junko Kurachi
    • , Zeyu Chen
    • , Omar Khan
    • , Bertram Bengsch
    • , Erietta Stelekati
    • , John Attanasio
    • , Laura M McLane
    •  & E John Wherry
  2. Institute for Immunology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Makoto Kurachi
    • , Junko Kurachi
    • , Zeyu Chen
    • , John Johnson
    • , Omar Khan
    • , Bertram Bengsch
    • , Erietta Stelekati
    • , John Attanasio
    • , Laura M McLane
    •  & E John Wherry
  3. Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • John Johnson
  4. Laboratory of Immunology, Faculty of Pharmacy, Osaka Ohtani University, Osaka, Japan.

    • Michio Tomura
  5. Department of Molecular Preventive Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

    • Satoshi Ueha

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Contributions

M.K. and E.J.W. designed the protocol. M.K. and J.K. made the RV stock. J.K. prepared mouse T cells and analyzed flow cytometry data. M.K. optimized density centrifugation. Z.C., J.J. and O.K. optimized in vitro T-cell stimulation and RV transduction methods. M.K., J.K., Z.C., J.J., O.K., B.B., E.S., J.A. and L.M.M. performed the in vivo experiments. J.A. and L.M.M. developed and verified a new fixation method for RV-transduced cells. M.T. and S.U. contributed to the development of a new Kusabira Orange 2 retrovirus. J.K. prepared all figures. M.K. and E.J.W. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to E John Wherry.

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https://doi.org/10.1038/nprot.2017.083

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