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Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids

Nature Methods volume 14, pages 521530 (2017) | Download Citation

Abstract

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCR-αβ+ single-positive CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.

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References

  1. 1.

    & Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 17, 749–756 (2002).

  2. 2.

    , & Human bone marrow CD34+ progenitor cells mature to T cells on OP9-DL1 stromal cell line without thymus microenvironment. Blood Cells Mol. Dis. 33, 227–232 (2004).

  3. 3.

    , & Induction of T cell development from human cord blood hematopoietic stem cells by Delta-like 1 in vitro. Blood 105, 1431–1439 (2005).

  4. 4.

    & T cell potential and development in vitro: the OP9-DL1 approach. Curr. Opin. Immunol. 19, 163–168 (2007).

  5. 5.

    , , & Human CD8 T cells generated in vitro from hematopoietic stem cells are functionally mature. BMC Immunol. 12, 22 (2011).

  6. 6.

    et al. Functionally mature CD4 and CD8 TCR-αβ cells are generated in OP9-DL1 cultures from human CD34+ hematopoietic cells. J. Immunol. 183, 4859–4870 (2009).

  7. 7.

    et al. Human intrathymic lineage commitment is marked by differential CD7 expression: identification of CD7 lympho-myeloid thymic progenitors. Blood 111, 1318–1326 (2008).

  8. 8.

    et al. T lymphoid differentiation potential measured in vitro is higher in CD34+CD38−/lo hematopoietic stem cells from umbilical cord blood than from bone marrow and is an intrinsic property of the cells. Haematologica 96, 646–654 (2011).

  9. 9.

    , , & MHC class II–positive epithelium and mesenchyme cells are both required for T cell development in the thymus. Nature 362, 70–73 (1993).

  10. 10.

    , , , & Human CD34+ fetal liver stem cells differentiate to T cells in a mouse thymic microenvironment. Blood 84, 1587–1593 (1994).

  11. 11.

    et al. Efficient generation of human T cells from a tissue-engineered thymic organoid. Nat. Biotechnol. 18, 729–734 (2000).

  12. 12.

    et al. Engineering the human thymic microenvironment to support thymopoiesis in vivo. Stem Cells 32, 2386–2396 (2014).

  13. 13.

    , & In vitro human T cell development directed by notch–ligand interactions. in Hematopoietic Stem Cell Protocols. (ed. Bunting, K.D.) 135–142 (Humana Press, 2008).

  14. 14.

    , , & A novel method for the generation of reaggregated organotypic cultures that permits juxtaposition of defined cell populations. Genesis 47, 346–351 (2009).

  15. 15.

    et al. Reproducible establishment of hemopoietic supportive stromal cell lines from murine bone marrow. Exp. Hematol. 17, 145–153 (1989).

  16. 16.

    , , & Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J. Neurosci. Res. 35, 567–576 (1993).

  17. 17.

    et al. Technical advance: ascorbic acid induces development of double-positive T cells from human hematopoietic stem cells in the absence of stromal cells. J. Leukoc. Biol. 96, 1165–1175 (2014).

  18. 18.

    et al. Vitamin C promotes maturation of T cells. Antioxid. Redox Signal. 19, 2054–2067 (2013).

  19. 19.

    et al. Long noncoding RNA profiling of human lymphoid progenitor cells reveals transcriptional divergence of B cell and T cell lineages. Nat. Immunol. 16, 1282–1291 (2015).

  20. 20.

    et al. Characterization in vitro and engraftment potential in vivo of human progenitor T cells generated from hematopoietic stem cells. Blood 114, 972–982 (2009).

  21. 21.

    , , & Characterization of distinct stages during the differentiation of human CD69+CD3+ thymocytes and identification of thymic emigrants. J. Immunol. 155, 1862–1872 (1995).

  22. 22.

    , , , & 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. 185, 141–151 (1997).

  23. 23.

    et al. Two subsets of naive T helper cells with distinct T cell receptor excision circle content in human adult peripheral blood. J. Exp. Med. 195, 789–794 (2002).

  24. 24.

    et al. Adoptively transferred effector cells derived from naive rather than central memory CD8+ T cells mediate superior antitumor immunity. Proc. Natl. Acad. Sci. USA 106, 17469–17474 (2009).

  25. 25.

    , & Adoptive immunotherapy or cancer: harnessing the T cell response. Nat. Rev. Immunol. 12, 269–281 (2012).

  26. 26.

    , , & Expression analysis of surface molecules on human thymic dendritic cells with the 10th HLDA Workshop antibody panel. Clin. Transl. Immunology 4, e47 (2015).

  27. 27.

    et al. A discrete population of IFN-λ-expressing BDCA3hi dendritic cells is present in human thymus. Immunol. Cell Biol. 93, 673–678 (2015).

  28. 28.

    , , , & A functional comparison of CD34+CD38 cells in cord blood and bone marrow. Blood 86, 3745–3753 (1995).

  29. 29.

    et al. Lymphoid priming in human bone marrow begins before expression of CD10 with upregulation of L-selectin. Nat. Immunol. 13, 963–971 (2012).

  30. 30.

    et al. A human postnatal lymphoid progenitor capable of circulating and seeding the thymus. J. Exp. Med. 204, 3085–3093 (2007).

  31. 31.

    , , & Human T, B, natural killer and dendritic cells arise from a common bone marrow progenitor cell subset. Immunity 3, 459–473 (1995).

  32. 32.

    et al. HSV-sr39TK positron emission tomography and suicide gene elimination of human hematopoietic stem cells and their progeny in humanized mice. Cancer Res. 74, 5173–5183 (2014).

  33. 33.

    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).

  34. 34.

    et al. Allelic exclusion and peripheral reconstitution by TCR transgenic T cells arising from transduced human hematopoietic stem and progenitor cells. Mol. Ther. 21, 1044–1054 (2013).

  35. 35.

    et al. Extrathymic generation of tumor-specific T cells from genetically engineered human hematopoietic stem cells via Notch signaling. Cancer Res. 67, 2425–2429 (2007).

  36. 36.

    et al. Functional human antigen-specific T cells produced in vitro using retroviral T cell receptor transfer into hematopoietic progenitors. J. Immunol. 179, 4959–4968 (2007).

  37. 37.

    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).

  38. 38.

    & Exploiting the curative potential of adoptive T cell therapy for cancer. Immunol. Rev. 257, 56–71 (2014).

  39. 39.

    et al. Introduction of exogenous T cell receptors into human hematopoietic progenitors results in exclusion of endogenous T cell receptor expression. Mol. Ther. 21, 1055–1063 (2013).

  40. 40.

    , , & Immunotherapy with TCR-redirected T cells: comparison of TCR-transduced and TCR-engineered hematopoietic stem cell–derived T cells. J. Immunol. 192, 206–213 (2014).

  41. 41.

    et al. A foundation for universal T cell–based immunotherapy: T cells engineered to express a CD19-specific chimeric antigen receptor and eliminate expression of endogenous TCR. Blood 119, 5697–5705 (2012).

  42. 42.

    , , & TALEN-mediated editing of endogenous T cell receptors facilitates efficient reprogramming of T lymphocytes by lentiviral gene transfer. Gene Ther. 21, 539–548 (2014).

  43. 43.

    et al. Multiplex genome-edited T cell manufacturing platform for 'off-the-shelf' adoptive T cell immunotherapies. Cancer Res. 75, 3853–3864 (2015).

  44. 44.

    , & New cell sources for T cell engineering and adoptive immunotherapy. Cell Stem Cell 16, 357–366 (2015).

  45. 45.

    , & Artificial thymic organoid cultures: in vitro human T cell differentiation from hematopoietic stem and progenitor cells. Protoc. Exch. ().

  46. 46.

    , & Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood. Cell Stem Cell 1, 635–645 (2007).

  47. 47.

    et al. Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions. J. Immunol. 180, 6116–6131 (2008).

  48. 48.

    et al. Gene transfer of tumor-reactive TCR confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and tumor-infiltrating lymphocytes. J. Immunol. 177, 6548–6559 (2006).

  49. 49.

    , & IMGT/GENE-DB: a comprehensive database for human and mouse immunoglobulin and T cell receptor genes. Nucleic Acids Res. 33, D256–D261 (2005).

  50. 50.

    BLAT—the BLAST-like alignment tool. Genome Res. 12, 656–664 (2002).

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Acknowledgements

We thank J. Scholes and F. Codrea at the UCLA Broad Stem Cell Research Center (BSCRC) Flow Cytometry Core for assistance with FACS sorting, R. Chan for assistance with specimen processing, C. Parekh (Children's Hospital Los Angeles) for generous assistance with thymus samples, M. Sehl (UCLA) for assistance with MPB collection, and A. Cooper (UCLA) for helpful advice and discussion. We thank I. Antoshechkin (Millard and Muriel Jacobs Genetics and Genomics Laboratory, Caltech), who developed the method for, and who assisted with, TCR sequencing analysis, A. Ribas (UCLA) for the NY-ESO-1 and MART-1 TCR constructs, J. Zuniger-Pflucker (University of Toronto) for OP9-DL1 cells, L. Coulombel for MS-5 cells and J. Chute (UCLA) for U266 cells. This work was supported by the NIH (grants R01 AG049753 (G.M.C.), 1R21AI119927 (G.M.C. and A.M.-H.), P01 HL073104 (G.M.C. and D.B.K.) and T32HL066992 (C.S.S.)), the Tower Cancer Research Foundation (C.S.S.), a UCLA BSCRC Innovation award (G.M.C. and D.B.K.) and a BSCRC Clinical Fellowship (C.S.S.). M.T.B. and D.B. are supported by Prostate Cancer Foundation Challenge Award 15CHAL02, and M.T.B. is the recipient of a Jane Coffin Childs Postdoctoral Fellowship. Core services were supported by the UCLA Jonsson Comprehensive Cancer Center shared facility (TPCL, grant 5P30CA016042), the UCLA Immunogenetics Center, the UCLA Center for AIDS Research Virology Core Lab and the UCLA AIDS Institute (grant 5P30 AI028697), and the Millard and Muriel Jacobs Genetics and Genomics Laboratory at Caltech.

Author information

Author notes

    • Gay M Crooks
    •  & Amélie Montel-Hagen

    These authors contributed equally to this work.

Affiliations

  1. Division of Hematology–Oncology, Department of Medicine, David Geffen School of Medicine (DGSOM), University of California Los Angeles (UCLA), Los Angeles, California, USA.

    • Christopher S Seet
  2. Department of Pathology and Laboratory Medicine, DGSOM, UCLA, Los Angeles, California, USA.

    • Chongbin He
    • , Suwen Li
    • , Brent Chick
    • , Yuhua Zhu
    • , Kenneth Kim
    • , Gay M Crooks
    •  & Amélie Montel-Hagen
  3. Division of Biology and Biological Engineering, California Institute of Technology (Caltech), Pasadena, California, USA.

    • Michael T Bethune
    •  & David Baltimore
  4. Department of Microbiology, Immunology and Molecular Genetics, DGSOM, UCLA, Los Angeles, California, USA.

    • Eric H Gschweng
    •  & Donald B Kohn
  5. Division of Pediatric Hematology–Oncology, Department of Pediatrics, DGSOM, UCLA, Los Angeles, California, USA.

    • Donald B Kohn
    •  & Gay M Crooks
  6. Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, California, USA.

    • Donald B Kohn
    •  & Gay M Crooks
  7. Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, California, USA.

    • Donald B Kohn
    •  & Gay M Crooks

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Contributions

C.S.S. and A.M.-H. designed and performed experiments, analyzed data, prepared figures and co-wrote the manuscript; C.H. performed histological experiments and, with B.C., assisted with in vivo experiments; S.L. assisted with ATO analysis and T cell functional assays; K.K. assisted with ATO cultures; Y.Z. performed human specimen processing and cultured the cell lines; E.H.G. and D.B.K. provided critical reagents and conceptual advice and edited the manuscript; M.T.B. and D.B. devised the approach for, and performed, TCR repertoire sequencing analysis and provided critical reagents; and G.M.C. and A.M.-H. co-directed the project and co-wrote the manuscript.

Competing interests

Kite Pharma, Inc. is supporting the preclinical research of the ATO system at UCLA with G.M.C. as principal investigator. Kite Pharma, Inc. also holds an exclusive license to certain intellectual property that relates to the ATO system.

Corresponding author

Correspondence to Gay M Crooks.

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    Supplementary Text and Figures

    Supplementary Figures 1–9

  2. 2.

    Supplementary Protocol

    DETAILED ARTIFICIAL THYMIC ORGANOID (ATO) METHOD

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DOI

https://doi.org/10.1038/nmeth.4237

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