Rapid and reliable generation of invariant natural killer T-cell lines in vitro

Authors

  • Asako Chiba,

    1. Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA
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  • Nadia Cohen,

    1. Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA
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  • Manfred Brigl,

    1. Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA
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  • Patrick J. Brennan,

    1. Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA
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  • Gurdal S. Besra,

    1. School of Biosciences, The University of Birmingham, Edgbaston, Birmingham, UK
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  • Michael B. Brenner

    1. Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA
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Michael B. Brenner, Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Smith Building, Room 552, One Jimmy Fund Way, Boston, MA 02115, USA.
Email: mbrenner@rics.bwh.harvard.edu
Senior author: Michael B. Brenner

Summary

Several tools have proved useful in the study of invariant natural killer T (iNKT) cells, including CD1d-deficient mice, Jα281-deficient mice, synthetic lipid antigens and antigen-loaded CD1d tetramers. However, the generation and examination of long-term primary murine iNKT cell lines in vitro has been challenging. Here, we show the rapid generation of iNKT cell lines from splenic iNKT cells of Vα14 T-cell receptor (TCR) transgenic (Tg) mice. These purified iNKT cells were stimulated by bone marrow-derived dendritic cells (BMDCs) loaded with α-galactosylceramide (αGalCer) and cultured with interleukin (IL)-2 and IL-7. iNKT cells proliferated dramatically, and the cell number exhibited a 100-fold increase within 2 weeks and a 105-fold increase in 8 weeks after repeated stimulation with αGalCer. The iNKT cell lines consisted of iNKT cells expressing Vβ chains including Vβ8.1/8.2, Vβ14, Vβ10, Vβ6 and Vβ7, and responded to stimulation with αGalCer presented both by BMDCs and by plate-bound CD1d. In addition, the iNKT cell lines produced interferon (IFN)-γ when activated by lipopolysaccharide (LPS) or CpG oligodeoxynucleotide (ODN)-stimulated BMDCs. Further, we show that iNKT cell lines produced cytokines in response to microbial antigens. In summary, high-yield iNKT cell lines were generated very rapidly and robustly expanded, and these iNKT cells responded to both TCR and cytokine stimulation in vitro. Given the desire to study primary iNKT cells for many purposes, these iNKT cell lines should provide an important tool for the study of iNKT cell subsets, antigen and TCR specificity, activation, inactivation and effector functions.

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