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.

  • Original Article
  • Published:

Immunology

In vitro generation of mature, naive antigen-specific CD8+ T cells with a single T-cell receptor by agonist selection

Leukemia volume 28, pages 830–841 (2014)Cite this article

Subjects

Abstract

Peripheral blood T cells transduced with a tumor-specific T-cell receptor (TCR) face problems of auto-reactivity and lack of efficacy caused by cross-pairing of exogenous and endogenous TCR chains, as well as short term in vivo survival due to activation and growth factor-induced differentiation. We here studied an alternative strategy for the efficient generation of naive CD8+ T cells with a single TCR. TCR-transduced human postnatal thymus-derived and adult mobilized blood-derived hematopoietic progenitor cells (HPCs) were differentiated to CD4+CD8+ double-positive T cells using OP9-Delta-like 1 (OP9-DL1) cultures. Addition of the agonist peptide induced double positive cells to cross-present the peptide, leading, in the absence of co-stimulation, to cell cycle arrest and differentiation into mature CD8+ T cells. Comprehensive phenotypic, molecular and functional analysis revealed the generation of naive and resting CD8+ T cells through a process similar to thymic positive selection. These mature T cells show a near complete inhibition of endogenous TCRA and TCRB rearrangements and express high levels of the introduced multimer-reactive TCR. Upon activation, specific cytokine production and efficient killing of tumor cells were induced. Using this strategy, large numbers of high-avidity tumor-specific naive T cells can be generated from readily available HPCs without TCR chain cross-pairing.

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
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Schmitt TM, Ragnarsson GB, Greenberg PD . T cell receptor gene therapy for cancer. Hum Gene Ther 2009; 20: 1240–1248.

    Article  CAS  Google Scholar 

  2. Provasi E, Genovese P, Lombardo A, Magnani Z, Liu PQ, Reik A et al. Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer. Nat Med May 2012; 18: 807–815.

    Article  CAS  Google Scholar 

  3. Bendle GM, Linnemann C, Hooijkaas AI, Bies L, de Witte MA, Jorritsma A et al. Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy. Nat Med 2010; 16: 565–570.

    Article  CAS  Google Scholar 

  4. Ahmadi M, King JW, Xue SA, Voisine C, Holler A, Wright GP et al. CD3 limits the efficacy of TCR gene therapy in vivo. Blood 2011; 118: 3528–3537.

    Article  CAS  Google Scholar 

  5. Cohen CJ, Li YF, El-Gamil M, Robbins PF, Rosenberg SA, Morgan RA . Enhanced antitumor activity of T cells engineered to express T-cell receptors with a second disulfide bond. Cancer Res 2007; 67: 3898–3903.

    Article  CAS  Google Scholar 

  6. Kuball J, Dossett ML, Wolfl M, Ho WY, Voss RH, Fowler C et al. Facilitating matched pairing and expression of TCR chains introduced into human T cells. Blood 2007; 109: 2331–2338.

    Article  CAS  Google Scholar 

  7. Okamoto S, Mineno J, Ikeda H, Fujiwara H, Yasukawa M, Shiku H et al. Improved expression and reactivity of transduced tumor-specific TCRs in human lymphocytes by specific silencing of endogenous TCR. Cancer Res 2009; 69: 9003–9011.

    Article  CAS  Google Scholar 

  8. Porter DL, Levine BL, Kalos M, Bagg A, June CH . Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med 2011; 365: 725–733.

    Article  CAS  Google Scholar 

  9. June CH . Adoptive T cell therapy for cancer in the clinic. J Clin Invest 2007; 117: 1466–1476.

    Article  CAS  Google Scholar 

  10. Schmitt TM, Zuniga-Pflucker JC . Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 2002; 17: 749–756.

    Article  CAS  Google Scholar 

  11. La Motte-Mohs RN, Herer E, Zuniga-Pflucker JC . Induction of T-cell development from human cord blood hematopoietic stem cells by Delta-like 1 in vitro. Blood 2005; 105: 1431–1439.

    Article  CAS  Google Scholar 

  12. De Smedt M, Hoebeke I, Plum J . Human bone marrow CD34+ progenitor cells mature to T cells on OP9-DL1 stromal cell line without thymus microenvironment. Blood Cells Mol Dis 2004; 33: 227–232.

    Article  CAS  Google Scholar 

  13. Van Coppernolle S, Verstichel G, Timmermans F, Velghe I, Vermijlen D, De Smedt M et al. Functionally mature CD4 and CD8 TCRalphabeta cells are generated in OP9-DL1 cultures from human CD34+ hematopoietic cells. J Immunol 2009; 183: 4859–4870.

    Article  CAS  Google Scholar 

  14. Timmermans F, Velghe I, Vanwalleghem L, De Smedt M, Van Coppernolle S, Taghon T et al. Generation of T cells from human embryonic stem cell-derived hematopoietic zones. J Immunol 2009; 182: 6879–6888.

    Article  CAS  Google Scholar 

  15. van Lent AU, Nagasawa M, van Loenen MM, Schotte R, Schumacher TN, Heemskerk MH et al. Functional human antigen-specific T cells produced in vitro using retroviral T cell receptor transfer into hematopoietic progenitors. J Immunol 2007; 179: 4959–4968.

    Article  CAS  Google Scholar 

  16. Zhao Y, Parkhurst MR, Zheng Z, Cohen CJ, Riley JP, Gattinoni L et al. Extrathymic generation of tumor-specific T cells from genetically engineered human hematopoietic stem cells via Notch signaling. Cancer Res 2007; 67: 2425–2429.

    Article  CAS  Google Scholar 

  17. Bluthmann H, Kisielow P, Uematsu Y, Malissen M, Krimpenfort P, Berns A et al. T-cell-specific deletion of T-cell receptor transgenes allows functional rearrangement of endogenous alpha- and beta-genes. Nature 1988; 334: 156–159.

    Article  CAS  Google Scholar 

  18. von Boehmer H, Aifantis I, Azogui O, Feinberg J, Saint-Ruf C, Zober C et al. Crucial function of the pre-T-cell receptor (TCR) in TCR beta selection, TCR beta allelic exclusion and alpha beta versus gamma delta lineage commitment. Immunol Rev 1998; 165: 111–119.

    Article  CAS  Google Scholar 

  19. Nishimura T, Kaneko S, Kawana-Tachikawa A, Tajima Y, Goto H, Zhu D et al. Generation of rejuvenated antigen-specific T cells by reprogramming to pluripotency and redifferentiation. Cell Stem Cell 2013; 12: 114–126.

    Article  CAS  Google Scholar 

  20. Dervovic DD, Liang HC, Cannons JL, Elford AR, Mohtashami M, Ohashi PS et al. Cellular and molecular requirements for the selection of in vitro-generated CD8 T cells reveal a role for notch. J Immunol 2013; 191: 1704–1715.

    Article  CAS  Google Scholar 

  21. Heemskerk MH, Hoogeboom M, Hagedoorn R, Kester MG, Willemze R, Falkenburg JH . Reprogramming of virus-specific T cells into leukemia-reactive T cells using T cell receptor gene transfer. J Exp Med 2004; 199: 885–894.

    Article  CAS  Google Scholar 

  22. Xue SA, Gao LQ, Thomas S, Hart DP, Xue JZ, Gillmore R et al. Development of a Wilms' tumor antigen-specific T-cell receptor for clinical trials: engineered patient's T cells can eliminate autologous leukemia blasts in NOD/SCID mice. Haematol-Hematol J 2010; 95: 126–134.

    Article  Google Scholar 

  23. Jorritsma A, Gomez-Eerland R, Dokter M, van de Kasteele W, Zoet YM, Doxiadis II et al. Selecting highly affine and well-expressed TCRs for gene therapy of melanoma. Blood 2007; 110: 3564–3572.

    Article  CAS  Google Scholar 

  24. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 2005; 102: 15545–15550.

    Article  CAS  Google Scholar 

  25. van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, Hummel M, Lavender FL et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 2003; 17: 2257–2317.

    Article  CAS  Google Scholar 

  26. Van Coppernolle S, Vanhee S, Verstichel G, Snauwaert S, van der Spek A, Velghe I et al. Notch induces human T-cell receptor gammadelta+ thymocytes to differentiate along a parallel, highly proliferative and bipotent CD4 CD8 double-positive pathway. Leukemia 2012; 26: 127–138.

    Article  CAS  Google Scholar 

  27. Hu Y, Smyth GK . ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods 2009; 347: 70–78.

    Article  CAS  Google Scholar 

  28. Menssen HD, Siehl JM, Thiel E . Wilms tumor gene (WT1) expression as a panleukemic marker. Int J Hematol 2002; 76: 103–109.

    Article  CAS  Google Scholar 

  29. Rosenfeld C, Cheever MA, Gaiger A . WT1 in acute leukemia, chronic myelogenous leukemia and myelodysplastic syndrome: therapeutic potential of WT1 targeted therapies. Leukemia 2003; 17: 1301–1312.

    Article  CAS  Google Scholar 

  30. Pobezinsky LA, Angelov GS, Tai XG, Jeurling S, Van Laethem F, Feigenbaum L et al. Clonal deletion and the fate of autoreactive thymocytes that survive negative selection. Nat Immunol 2012; 13: 569–578.

    Article  CAS  Google Scholar 

  31. Res P, Blom B, Hori T, Weijer K, Spits H . Downregulation of CD1 marks acquisition of functional maturation of human thymocytes and defines a control point in late stages of human T cell development. J Exp Med 1997; 185: 141–151.

    Article  CAS  Google Scholar 

  32. Vanhecke D, Leclercq G, Plum J, Vandekerckhove B . Characterization of distinct stages during the differentiation of human CD69+CD3+ thymocytes and identification of thymic emigrants. J Immunol 1995; 155: 1862–1872.

    CAS  PubMed  Google Scholar 

  33. Vanhecke D, Verhasselt B, De Smedt M, Leclercq G, Plum J, Vandekerckhove B . Human thymocytes become lineage committed at an early postselection CD69+ stage, before the onset of functional maturation. J Immunol 1997; 159: 5973–5983.

    CAS  PubMed  Google Scholar 

  34. Gangadharan D, Lambolez F, Attinger A, Wang-Zhu Y, Sullivan BA, Cheroutre H . Identification of pre- and postselection TCRalphabeta+ intraepithelial lymphocyte precursors in the thymus. Immunity 2006; 25: 631–641.

    Article  CAS  Google Scholar 

  35. Annunziato F, Cosmi L, Liotta F, Lazzeri E, Romagnani P, Angeli R et al. CXCR3 and alphaEbeta7 integrin identify a subset of CD8+ mature thymocytes that share phenotypic and functional properties with CD8+ gut intraepithelial lymphocytes. Gut 2006; 55: 961–968.

    Article  CAS  Google Scholar 

  36. Dik WA, Pike-Overzet K, Weerkamp F, de Ridder D, de Haas EF, Baert MR et al. New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling. J Exp Med 2005; 201: 1715–1723.

    Article  CAS  Google Scholar 

  37. Ghisi M, Corradin A, Basso K, Frasson C, Serafin V, Mukherjee S et al. Modulation of microRNA expression in human T-cell development: targeting of NOTCH3 by miR-150. Blood 2011; 117: 7053–7062.

    Article  CAS  Google Scholar 

  38. Moran AE, Holzapfel KL, Xing Y, Cunningham NR, Maltzman JS, Punt J et al. T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse. J Exp Med 2011; 208: 1279–1289.

    Article  CAS  Google Scholar 

  39. Williams JA, Lumsden JM, Yu X, Feigenbaum L, Zhang J, Steinberg SM et al. Regulation of thymic NKT cell development by the B7-CD28 costimulatory pathway. J Immunol 2008; 181: 907–917.

    Article  CAS  Google Scholar 

  40. Gattinoni L, Klebanoff CA, Palmer DC, Wrzesinski C, Kerstann K, Yu Z 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 2005; 115: 1616–1626.

    Article  CAS  Google Scholar 

  41. Gattinoni L, Powell DJ Jr., Rosenberg SA, Restifo NP . Adoptive immunotherapy for cancer: building on success. Nat Rev Immunol 2006; 6: 383–393.

    Article  CAS  Google Scholar 

  42. Cieri N, Camisa B, Cocchiarella F, Forcato M, Oliveira G, Provasi E et al. IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors. Blood 2013; 121: 573–584.

    Article  CAS  Google Scholar 

  43. Vizcardo R, Masuda K, Yamada D, Ikawa T, Shimizu K, Fujii S et al. Regeneration of human tumor antigen-specific T cells from iPSCs derived from mature CD8(+) T cells. Cell Stem Cell 2013; 12: 31–36.

    Article  CAS  Google Scholar 

  44. Themeli M, Kloss CC, Ciriello G, Fedorov VD, Perna F, Gonen M et al. Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy. Nat Biotechnol 2013; 31: 928–933.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors would like to thank the staff of the Cardiac Surgery and Hematology department and the Cell Processing Lab of Ghent University Hospital for providing thymus and MB samples. We are indebted to Katleen De Preter and Frank Speleman of the Center for Medical Genetics (Ghent University) for providing mRNA expression data of thymic subsets. Finally, we would like to thank Dr T Boterberg for the irradiation of the feeder cells, Karin Weening for help with the retroviral constructs, Sophie Vermaut for help with flow cytometry and cell sorting and Christian De Boever for the art work.

Disclosures

This work was supported by the Research Foundation - Flanders (Fonds voor Wetenschappelijk Onderzoek Vlaanderen, FWO), Stichting tegen Kanker, the geconcerteerde onderzoeksactiviteiten of Ghent University, and the Interuniversity Attraction Poles Program supported by the Belgian Science Policy. YVC, SV and GV are supported by the Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen. SS, TT and TK are supported by the Research Foundation-Flanders (FWO).

Author information

Author notes

  1. T Kerre and B Vandekerckhove: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent, Belgium

    S Snauwaert, G Verstichel, S Bonte, G Goetgeluk, S Vanhee, Y Van Caeneghem, K De Mulder, T Taghon, G Leclercq, J Plum, T Kerre & B Vandekerckhove

  2. Laboratory of Molecular & Cellular Therapy of the Department of Physiology and Immunology, Free University Brussels (VUB), Brussels, Belgium

    C Heirman & K Thielemans

  3. Department of Immunology, University College London, London, UK

    H Stauss

  4. Laboratory of Experimental Hematology of the Department of Hematology, Leids Universitair Medisch Centrum, Leiden, The Netherlands

    M H M Heemskerk

  5. Department of Immunology, Erasmus Medical Center, Rotterdam, The Netherlands

    A W Langerak

Authors
  1. S Snauwaert
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. G Verstichel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S Bonte
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. G Goetgeluk
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. S Vanhee
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Y Van Caeneghem
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. K De Mulder
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. C Heirman
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. H Stauss
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. M H M Heemskerk
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. T Taghon
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. G Leclercq
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. J Plum
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. A W Langerak
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. K Thielemans
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. T Kerre
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. B Vandekerckhove
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to B Vandekerckhove.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Leukemia website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Snauwaert, S., Verstichel, G., Bonte, S. et al. In vitro generation of mature, naive antigen-specific CD8+ T cells with a single T-cell receptor by agonist selection. Leukemia 28, 830–841 (2014). https://doi.org/10.1038/leu.2013.285

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/leu.2013.285

Keywords

This article is cited by

Search

Advanced search

Quick links