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.

  • Article
  • Published:

Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB

Nature Biotechnology volume 20, pages 143–148 (2002)Cite this article

Abstract

The ex vivo priming and expansion of human cytotoxic T lymphocytes (CTLs) has potential for use in immunotherapy applications for cancer and infectious diseases. To overcome the difficulty in obtaining sufficient numbers of CTLs, we have developed artificial antigen-presenting cells (aAPCs) expressing ligands for the T-cell receptor (TCR) and the CD28 and 4-1BB co-stimulatory surface molecules. These aAPCs reproducibly activate and rapidly expand polyclonal or antigen-specific CD8+ T cells. The starting repertoire of CD8+ T cells was preserved during culture. Furthermore, apoptosis of cultured CD8+ T cells was diminished by this approach. This approach may have important therapeutic implications for adoptive immunotherapy.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Construction of artificial APCs (aAPCs) from the parental K562 cell line.
Figure 2: Long-term growth of primary polyclonal human CD8+ T cells stimulated with aAPCs in the absence of exogenous cytokines.
Figure 3: Propagation of antigen-specific cytotoxic T cells from an HLA A*0201 donor using K32/4-1BBL aAPCs.
Figure 4: Maintenance of the TCR Vβ repertoire in polyclonal CD8+ T cells after expansion with K32/4-1BBL aAPCs.
Figure 5: Expression of genes involved in T-cell growth and survival after stimulation with aAPCs.
Figure 6: Distinct effects on apoptosis in cultures of polyclonal human CD8+ T cells stimulated with various aAPCs.

Similar content being viewed by others

References

  1. Melief, C.J. & Kast, W.M. T-cell immunotherapy of tumors by adoptive transfer of cytotoxic T lymphocytes and by vaccination with minimal essential epitopes. Immunol. Rev. 145, 167–177 (1995).

    Article  CAS  PubMed  Google Scholar 

  2. Riddell, S.R. & Greenberg, P.D. Principles for adoptive T cell therapy of human viral diseases. Annu. Rev. Immunol. 13, 545–586 (1995).

    Article  CAS  PubMed  Google Scholar 

  3. Riddell, S.R. et al. Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones. Science 257, 238–241 (1992).

    Article  CAS  PubMed  Google Scholar 

  4. Yee, C. et al. Melanocyte destruction after antigen-specific immunotherapy of melanoma. Direct evidence of T cell-mediated vitiligo. J. Exp. Med. 192, 1637–1644 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Brodie, S.J. et al. In vivo migration and function of transferred HIV-1-specific cytotoxic T cells. Nat. Med. 5, 34–41 (1999).

    Article  CAS  PubMed  Google Scholar 

  6. Riddell, S.R. et al. Phase I study of cellular adoptive immunotherapy using genetically modified CD8+ HIV-specific T cells for HIV seropositive patients undergoing allogeneic bone marrow transplant. The Fred Hutchinson Cancer Research Center and the University of Washington School of Medicine, Department of Medicine, Division of Oncology. Hum. Gene Ther. 3, 319–338 (1992).

    Article  CAS  PubMed  Google Scholar 

  7. Riddell, S.R. & Greenberg, P.D. The use of anti-CD3 and anti-CD28 monoclonal antibodies to clone and expand human antigen-specific T cells. J. Immunol. Methods 128, 189–201 (1990).

    Article  CAS  PubMed  Google Scholar 

  8. Heslop, H.E. et al. Long-term restoration of immunity against Epstein–Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes. Nat. Med. 2, 551–555 (1996).

    Article  CAS  PubMed  Google Scholar 

  9. Levine, B.L. et al. Effects of CD28 costimulation on long-term proliferation of CD4+ T cells in the absence of exogenous feeder cells. J. Immunol. 159, 5921–5930 (1997).

    CAS  PubMed  Google Scholar 

  10. Deeths, M.J., Kedl, R.M. & Mescher, M.F. CD8+ T cells become nonresponsive (anergic) following activation in the presence of costimulation. J. Immunol. 163, 102–110 (1999).

    CAS  PubMed  Google Scholar 

  11. Laux, I. et al. Response differences between human CD4(+) and CD8(+) T-cells during CD28 costimulation: implications for immune cell-based therapies and studies related to the expansion of double-positive T-cells during aging. Clin. Immunol. 96, 187–197 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. Deeths, M.J. & Mescher, M.F. B7-1-dependent co-stimulation results in qualitatively and quantitatively different responses by CD4+ and CD8+ T cells. Eur. J. Immunol. 27, 598–608 (1997).

    Article  CAS  PubMed  Google Scholar 

  13. Pollok, K.E. et al. Inducible T cell antigen 4-1BB. Analysis of expression and function. J. Immunol. 150, 771–781 (1993).

    CAS  PubMed  Google Scholar 

  14. Goodwin, R.G. et al. Molecular cloning of a ligand for the inducible T cell gene 4-1BB: a member of an emerging family of cytokines with homology to tumor necrosis factor. Eur. J. Immunol. 23, 2631–2641 (1993).

    Article  CAS  PubMed  Google Scholar 

  15. Hurtado, J.C., Kim, Y.J. & Kwon, B.S. Signals through 4-1BB are costimulatory to previously activated splenic T cells and inhibit activation-induced cell death. J. Immunol. 158, 2600–2609 (1997).

    CAS  PubMed  Google Scholar 

  16. Hurtado, J.C., Kim, S.H., Pollok, K.E., Lee, Z.H. & Kwon, B.S. Potential role of 4-1BB in T cell activation. Comparison with the costimulatory molecule CD28. J. Immunol. 155, 3360–3367 (1995).

    CAS  PubMed  Google Scholar 

  17. Saoulli, K. et al. CD28-independent, TRAF2-dependent costimulation of resting T cells by 4-1BB ligand. J. Exp. Med. 187, 1849–1862 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Shuford, W.W. et al. 4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses. J. Exp. Med. 186, 47–55 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Takahashi, C., Mittler, R.S. & Vella, A.T. Cutting edge: 4-1BB is a bona fide CD8 T cell survival signal. J. Immunol. 162, 5037–5040 (1999).

    CAS  PubMed  Google Scholar 

  20. Tan, J.T. et al. 4-1BB costimulation is required for protective anti-viral immunity after peptide vaccination. J. Immunol. 164, 2320–2325 (2000).

    Article  CAS  PubMed  Google Scholar 

  21. Melero, I. et al. Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors. Nat. Med. 3, 682–685 (1997).

    Article  CAS  PubMed  Google Scholar 

  22. Melero, I. et al. Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB ligand: synergy with the CD28 co-stimulatory pathway. Eur. J. Immunol. 28, 1116–1121 (1998).

    Article  CAS  PubMed  Google Scholar 

  23. DeBenedette, M.A., Shahinian, A., Mak, T.W. & Watts, T.H. Costimulation of CD28− T lymphocytes by 4–1BB ligand. J. Immunol. 158, 551–559 (1997).

    CAS  PubMed  Google Scholar 

  24. Guinn, B.A., DeBenedette, M.A., Watts, T.H. & Berinstein, N.L. 4-1BBL cooperates with B7-1 and B7-2 in converting a B cell lymphoma cell line into a long-lasting antitumor vaccine. J. Immunol. 162, 5003–5010 (1999).

    CAS  PubMed  Google Scholar 

  25. Latouche, J.B. & Sadelain, M. Induction of human cytotoxic T lymphocytes by artificial antigen-presenting cells. Nat. Biotechnol. 18, 405–409 (2000).

    Article  CAS  PubMed  Google Scholar 

  26. Prakken, B. et al. Artificial antigen-presenting cells as a tool to exploit the immune 'synapse'. Nat. Med. 6, 1406–1410 (2000).

    Article  CAS  PubMed  Google Scholar 

  27. Almand, B. et al. Clinical significance of defective dendritic cell differentiation in cancer. Clin. Cancer Res. 6, 1755–1766 (2000).

    CAS  PubMed  Google Scholar 

  28. Rosenberg, S.A. et al. Gene transfer into humans—immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N. Engl. J. Med. 323, 570–578 (1990).

    Article  CAS  PubMed  Google Scholar 

  29. Dudley, M.E. et al. Adoptive transfer of cloned melanoma-reactive T lymphocytes for the treatment of patients with metastatic melanoma. J. Immunother. 24, 363–373 (2001).

    Article  CAS  PubMed  Google Scholar 

  30. Yee, C., Riddell, S.R. & Greenberg, P.D. In vivo tracking of tumor-specific T cells. Curr. Opin. Immunol. 13, 141–146 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Rooney, C.M. et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein–Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 92, 1549–1555 (1998).

    CAS  PubMed  Google Scholar 

  32. Lord, J.D., McIntosh, B.C., Greenberg, P.D. & Nelson, B.H. The IL-2 receptor promotes proliferation, bcl-2 and bcl-x induction, but not cell viability through the adapter molecule Shc. J. Immunol. 161, 4627–4633 (1998).

    CAS  PubMed  Google Scholar 

  33. Dahl, A.M. et al. Expression of bcl-X(L) restores cell survival, but not proliferation of effector differentiation, in CD28-deficient T lymphocytes. J. Exp. Med. 191, 2031–2038 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Dunbar, P.R. et al. Direct isolation, phenotyping and cloning of low-frequency antigen-specific cytotoxic T lymphocytes from peripheral blood. Curr. Biol. 8, 413–416 (1998).

    Article  CAS  PubMed  Google Scholar 

  35. Yee, C., Savage, P.A., Lee, P.P., Davis, M.M. & Greenberg, P.D. Isolation of high avidity melanoma-reactive CTL from heterogeneous populations using peptide–MHC tetramers. J. Immunol. 162, 2227–2234 (1999).

    CAS  PubMed  Google Scholar 

  36. Curtsinger, J.M., Lins, D.C. & Mescher, M.F. CD8+ memory T cells (CD44high, Ly-6C+) are more sensitive than naive cells to (CD44low, Ly-6C−) to TCR/CD8 signaling in response to antigen. J. Immunol. 160, 3236–3243 (1998).

    CAS  PubMed  Google Scholar 

  37. Sagerstrom, C.G., Kerr, E.M., Allison, J.P. & Davis, M.M. Activation and differentiation requirements of primary T cells in vitro. Proc. Natl. Acad. Sci. USA 90, 8987–8991 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Wang, B., Maile, R., Greenwood, R., Collins, E.J. & Frelinger, J.A. Naive CD8+ T cells do not require costimulation for proliferation and differentiation into cytotoxic effector cells. J. Immunol. 164, 1216–1222 (2000).

    Article  CAS  PubMed  Google Scholar 

  39. Voltz, R. et al. A serologic marker of paraneoplastic limbic and brain-stem encephalitis in patients with testicular cancer. N. Engl. J. Med. 340, 1788–1795 (1999).

    Article  CAS  PubMed  Google Scholar 

  40. Tan, E.M. Autoantibodies as reporters identifying aberrant cellular mechanisms in tumorigenesis. J. Clin. Invest 108, 1411–1415 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. June, C.H., Ledbetter, J.A., Gillespie, M.M., Lindsten, T. & Thompson, C.B. T-cell proliferation involving the CD28 pathway is associated with cyclosporine-resistant interleukin 2 gene expression. Mol. Cell Biol. 7, 4472–4481 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Riley, J.L. et al. ICOS costimulation requires IL-2 and can be prevented by CTLA-4 engagement. J. Immunol. 166, 4943–4948 (2001).

    Article  CAS  PubMed  Google Scholar 

  43. Claret, E.J. et al. Characterization of T cell repertoire in patients with graft-versus-leukemia after donor lymphocyte infusion. J. Clin. Invest 100, 855–866 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank William DeMuth for flow sorting, Patricia Rivers and Alanna Toll for technical help, and Coral Hass for administrative excellence. MHC tetramers were supplied by the NIAID Tetramer Facility and the NIH AIDS Research and Reference Reagent Program. Peptide synthesis was provided by the Protein Chemistry Laboratory of the Medical School of the University of Pennsylvania supported by core grants of the Diabetes and Cancer Centers (DK-19525 and CA-16520). M.V.M. was supported in part by training grant DK07748 from the NIH; A.K.T. was supported in part by the Dr. Mildred-Scheel-Stiftung Deutsche Krebschilfe Foundation Grant.

Author information

Authors and Affiliations

  1. Abramson Family Cancer Research Institute at the University of Pennsylvania Cancer Center, Philadelphia, 19104, PA

    Marcela V. Maus, Anna K. Thomas, David Allman, Katia Schlienger, James L. Riley & Carl H. June

  2. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, 19104, PA

    Debra G.B. Leonard, David Allman, James L. Riley & Carl H. June

  3. Molecular Diagnostic Core, University of Pennsylvania, Philadelphia, 19104, PA

    Debra G.B. Leonard & Kathakali Addya

Authors
  1. Marcela V. Maus
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anna K. Thomas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Debra G.B. Leonard
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David Allman
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kathakali Addya
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Katia Schlienger
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. James L. Riley
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Carl H. June
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Carl H. June.

Ethics declarations

Competing interests

A patent application on the work has been submitted on behalf of the University of Pennsylvania.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Maus, M., Thomas, A., Leonard, D. et al. Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB. Nat Biotechnol 20, 143–148 (2002). https://doi.org/10.1038/nbt0202-143

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0202-143

This article is cited by

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