Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iVα14-Jα18 transgenic mice (iVα14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iVα14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.