Human and mouse iNKT cell expansion and engineering with viral vectors

Invariant natural killer T (iNKT) cells are a non-conventional T-cell population characterized by the expression of a conserved semi-invariant T-cell receptor (TCR) with specificity towards self or microbial lipid antigens, presented by the non-polymorphic MHC class I-related molecule CD1d. iNKT cells play a pivotal role in tumor immunosurveillance and serve as a potent tool for anti-cancer therapies. Notably, iNKT cells can be effectively redirected against both hematological and solid malignancies through genetic engineering using either Chimeric Antigen Receptors (CARs) or TCRs targeted to tumor antigens. However, due to their low frequency, iNKT cell expansion in vitro is an essential step to obtain suitable cell number for adoptive cell therapy (ACT). Here we describe two robust methods for efficiently isolating primary mouse and human iNKT cells that can be easily genetically modified and expanded. iNKT cells are isolated from the spleens of iVα14-Jα18 transgenic mice or from human buffy coats resulting in highly enriched populations. Both mouse and human iNKT cells are activated with anti-CD3/CD28 beads, IL-2 and IL-7, and subsequently transduced with tumor-specific receptors, yielding millions of ready-to-use, highly pure, and stably transduced tumor-redirected iNKT cells. The final cell product is suitable for in vitro investigation of iNKT cell activation and function mechanisms, as well as for pre-clinical ACT studies.