In Vivo Cytotoxicity by α-GalCer-transactivated NK Cells.

Invariant natural killer T (iNKT) cells are innate-like, lipid-reactive T lymphocytes known for their potent immunomodulatory properties. In addition to expressing and utilizing cytolytic effector molecules of their own against certain target cells, iNKT cells can be stimulated with α-galactosylceramide (α-GalCer) to augment the cytotoxic capacity of natural killer (NK) cells. Herein, we describe a flow cytometry-based in vivo killing assay that enables examination of α-GalCer-promoted cytotoxicity against β2 microglobulin knockout (β2M-/-) target cells, which mimic tumor and virus-infected cells displaying little to no MHC class I molecules on their surface. Using an anti-asialo GM1 antibody, which depletes NK cells but not iNKT cells, we confirmed that the increased clearance of β2M-/- cells in α-GalCer-primed recipients was mediated by NK cells. The protocol detailed here can be leveraged to assess the functional fitness of iNKT cells and their crosstalk with NK cells and to further our understanding of α-GalCer-promoted cytotoxicity in preclinical immunotherapeutic applications.