Abstract 4720: mTOR inhibition is required for conversion of invariant NKT cells into immunosuppressive regulatory cells.
Authors: Huijts Charlotte M. , Schneiders Famke L. , Verheul Henk M. , de Gruijl Tanja D. , van der Vliet Hans J. 
Published: August 2014

Abstract
Invariant Natural Killer T (iNKT) cells recognize glycolipid antigens presented by the CD1d antigen-presenting molecule. They have been shown to play an important role in various types of immune responses, including antitumor immune responses. Upon activation with α-galactosylceramide (α-GalCer) iNKT cells can produce different kinds of cytokines, like IFN-γ, resulting in activation of other T cell subsets. However, production of IL-2 by iNKT cells has been shown to contribute to the expansion of immunosuppressive regulatory T cells (Tregs). Although Tregs are critically dependent on the X-chromosome encoded FoxP3 gene, it is also known that FoxP3 is expressed in conventional T cells upon activation. Recently it was reported that freshly isolated iNKT cells can express FoxP3 upon stimulation with TGF-β and acquire suppressive capacities in the presence of rapamycin. In addition, IL-10 has been reported to stimulate iNKT cells resulting in CD25 upregulation and proliferation.
In order to assess whether it is possible to induce FoxP3 expression and suppressive capacities in iNKT cell lines, iNKT cell lines were cultured with IL-10, TGF-β and/ or rapamycin. Phenotypic analysis was performed and upregulation of FoxP3 was seen in all conditions cultured with IL-10 and rapamycin while, in contrast to a previous report, TGF-β was found to inhibit iNKT cell FoxP3 expression. To investigate whether these FoxP3+ iNKT cells acquired suppressive abilities, they were co-cultured with CFSE labeled responder cells. These assays showed that while IL10 resulted in a moderate increase in FoxP3 expressing iNKT cells, only iNKT cultured in the presence of rapamycin were able to suppress responder cells. Therefore, while IL-10 can enhance FoxP3 expression in proliferating iNKT cells, rapamycin is required and responsible for the induction of suppressive function of iNKT cells. As recent evidence indicates that FoxP3 is differentially localized in subcellular compartments in suppressive T cells versus activated T cells, we are currently performing FoxP3 localization experiments to identify different localization patterns in suppressive vs. non-suppressive FoxP3+ iNKT cells.
With these experiments we show that rapamycin is required for the induction of suppressive capacities in iNKT cells. Further studies are required to evaluate the clinical relevance of this modulating effect of mTOR inhibitors on iNKT cells in the field of transplantation medicine and anticancer therapies in renal cell carcinoma, breast cancer, and pancreatic neuroendocrine carcinomas.
Citation Format: Charlotte M. Huijts, Famke L. Schneiders, Henk M. Verheul, Tanja D. de Gruijl, Hans J. van der Vliet. mTOR inhibition is required for conversion of invariant NKT cells into immunosuppressive regulatory cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4720. doi:10.1158/1538-7445.AM2013-4720