Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

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

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. Perfetto, S.P., Chattopadhyay, P.K. & Roederer, M. Seventeen-colour flow cytometry: unravelling the immune system. Nat. Rev. Immunol. 4, 648–655 (2004).

    Article  CAS  Google Scholar 

  2. Lugli, E., Troiano, L. & Cossarizza, A. Investigating T cells by polychromatic flow cytometry. Methods Mol. Biol. 514, 47–63 (2009).

    Article  CAS  Google Scholar 

  3. Sallusto, F., Lenig, D., Forster, R., Lipp, M. & Lanzavecchia, A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401, 708–712 (1999).

    Article  CAS  Google Scholar 

  4. Gattinoni, L. et al. A human memory T cell subset with stem cell-like properties. Nat. Med. 17, 1290–1297 (2011).

    Article  CAS  Google Scholar 

  5. Zhang, Y., Joe, G., Hexner, E., Zhu, J. & Emerson, S.G. Host-reactive CD8+ memory stem cells in graft-versus-host disease. Nat. Med. 11, 1299–1305 (2005).

    Article  CAS  Google Scholar 

  6. Gattinoni, L. et al. Wnt signaling arrests effector T cell differentiation and generates CD8+ memory stem cells. Nat. Med. 15, 808–813 (2009).

    Article  CAS  Google Scholar 

  7. Pitcher, C.J. et al. Development and homeostasis of T cell memory in rhesus macaque. J. Immunol. 168, 29–43 (2002).

    Article  CAS  Google Scholar 

  8. De Rosa, S.C., Herzenberg, L.A. & Roederer, M. 11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity. Nat. Med. 7, 245–248 (2001).

    Article  CAS  Google Scholar 

  9. Mahnke, Y.D. & Roederer, M. Optimizing a multicolor immunophenotyping assay. Clin. Lab. Med. 27, 469–485 (2007).

    Article  Google Scholar 

  10. Schmitz, I. et al. An IL-2-dependent switch between CD95 signaling pathways sensitizes primary human T cells toward CD95-mediated activation-induced cell death. J. Immunol. 171, 2930–2936 (2003).

    Article  CAS  Google Scholar 

  11. Lugli, E. et al. Quercetin inhibits lymphocyte activation and proliferation without inducing apoptosis in peripheral mononuclear cells. Leuk. Res. 33, 140–150 (2009).

    Article  CAS  Google Scholar 

  12. Lugli, E. et al. Transient and persistent effects of IL15 on lymphocyte homeostasis in nonhuman primates. Blood 116, 3238–3248 (2010).

    Article  CAS  Google Scholar 

  13. Geginat, J., Sallusto, F. & Lanzavecchia, A. Cytokine-driven proliferation and differentiation of human naive, central memory, and effector memory CD4+ T cells. J. Exp. Med. 194, 1711–1719 (2001).

    Article  CAS  Google Scholar 

  14. Geginat, J., Lanzavecchia, A. & Sallusto, F. Proliferation and differentiation potential of human CD8+ memory T cell subsets in response to antigen or homeostatic cytokines. Blood 101, 4260–4266 (2003).

    Article  CAS  Google Scholar 

  15. Gattinoni, L. et al. Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells. J. Clin. Invest. 115, 1616–1626 (2005).

    Article  CAS  Google Scholar 

  16. Perfetto, S.P., Ambrozak, D., Nguyen, R., Chattopadhyay, P. & Roederer, M. Quality assurance for polychromatic flow cytometry using a suite of calibration beads. Nat. Protoc. 1, 1522–1530 (2006).

    Article  CAS  Google Scholar 

  17. Roederer, M. Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry 45, 194–205 (2001).

    Article  CAS  Google Scholar 

  18. Kantor, A.B. & Roederer, M. FACS analysis of lymphocytes. in Handbook of Experimental Immunology, Vol. 49 (eds. Herzenberg, L.A., Weir, D.M., Herzenberg, L.A. & Blackwell, C.) 1–13 (Blackwell Science, 1997).

  19. English, D. & Andersen, B.R. Single-step separation of red blood cells. Granulocytes and mononuclear leukocytes on discontinuous density gradients of Ficoll-Hypaque. J. Immunol. Methods 5, 249–252 (1974).

    Article  CAS  Google Scholar 

  20. Altman, J.D. et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94–96 (1996).

    Article  CAS  Google Scholar 

  21. Cavalieri, S. et al. Human T lymphocytes transduced by lentiviral vectors in the absence of TCR activation maintain an intact immune competence. Blood 102, 497–505 (2003).

    Article  CAS  Google Scholar 

  22. Surh, C.D. & Sprent, J. Homeostasis of naive and memory T cells. Immunity 29, 848–862 (2008).

    Article  CAS  Google Scholar 

Download references

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.

Author information

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

Authors
  1. Enrico Lugli
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Luca Gattinoni
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alessandra Roberto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Domenico Mavilio
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. David A Price
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nicholas P Restifo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mario Roederer
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding authors

Correspondence to Enrico Lugli or Mario Roederer.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2012.143

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing