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Identification, isolation and in vitro expansion of human and nonhuman primate T stem cell memory cells

Nature Protocols volume 8, pages 33–42 (2013)Cite this article

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Abstract

The T cell compartment is phenotypically and functionally heterogeneous; subsets of naive and memory cells have different functional properties, and also differ with respect to homeostatic potential and the ability to persist in vivo. Human stem cell memory T (TSCM) cells, which possess superior immune reconstitution and antitumor response capabilities, can be identified by polychromatic flow cytometry on the basis of the simultaneous expression of several naive markers together with the memory marker CD95. We describe here a protocol based on the minimum set of markers required for optimal identification of human and nonhuman primate (NHP) TSCM cells with commonly available flow cytometers. By using flow sorters, TSCM cells can thereby be isolated efficiently at high yield and purity. With the use of the 5.5-h isolation procedure, depending on the number of cells needed, the sorting procedure can last for 2–15 h. We also indicate multiple strategies for their efficient expansion in vitro at consistent numbers for functional characterization or adoptive transfer experiments.

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Figure 1: Gating strategy for the identification of human and rhesus TSCM cells.
Figure 2: Reproducibility and robustness of three versus seven marker-based TN cell definitions for TSCM identification.
Figure 3: Co-staining strategies to improve the identification and isolation of human TSCM cells.
Figure 4: Flow cytometric sorting of TSCM cells.
Figure 5: Differential response of T cell subsets to different stimuli in vitro.

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Acknowledgements

We thank J. Yu and M. Beddall (ImmunoTechnology Section, VRC) for antibody conjugation; S.P. Perfetto, D. Ambrozak and R. Nguyen (Flow Cytometry Core, VRC) for assistance with cell sorting; M. F. Quigley (Immunology Laboratory, VRC) for providing the HLA-A*0201+ donor; and the other members of the ImmunoTechnology section for continuous discussion. This work was supported by the NIH Intramural Research Program and by the Associazione Italiana per la Ricerca sul Cancro to E.L.

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Author notes

  1. Luca Gattinoni

    Present address: Present address: Experimental Transplantation and Immunology, National Cancer Institute, NIH, Bethesda, Maryland, USA.,

Authors and Affiliations

  1. ImmunoTechnology Section, Vaccine Research Center (VRC), National Institute of Allergy and Infectious Diseases, US National Institutes of Health (NIH), Bethesda, Maryland, USA

    Enrico Lugli & Mario Roederer

  2. Unit of Clinical and Experimental Immunology, Humanitas Clinical and Research Center, Rozzano, Italy

    Enrico Lugli, Alessandra Roberto & Domenico Mavilio

  3. Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA

    Luca Gattinoni & Nicholas P Restifo

  4. Department of Medical Biotechnologies and Translational Medicine, University of Milan, Milan, Italy

    Domenico Mavilio

  5. Institute of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK

    David A Price

  6. NIH Center for Regenerative Medicine, NIH, Bethesda, Maryland, USA

    Nicholas P Restifo

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  1. Enrico Lugli
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Contributions

E.L. conceived the protocol; E.L., L.G. and A.R. performed the experiments; D.A.P. and D.M. provided reagents and critical support; N.P.R. and M.R. supervised the research; L.G., D.M., D.A.P. and N.P.R. edited the manuscript; E.L. and M.R. wrote the manuscript.

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Correspondence to Enrico Lugli or Mario Roederer.

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Lugli, E., Gattinoni, L., Roberto, A. et al. Identification, isolation and in vitro expansion of human and nonhuman primate T stem cell memory cells. Nat Protoc 8, 33–42 (2013). https://doi.org/10.1038/nprot.2012.143

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