SRP315016 PRJNA722286 GSE172169 Invariant Natural Killer T Cell Subsets Have Diverse Graft-Versus-Host-Disease-Preventing and Anti-Tumor Effects Invariant Natural Killer T (iNKT) cells are a T cell subset with potent immunomodulatory properties. Experimental evidence in mice and observational studies in humans indicate that iNKT cells have antitumor potential as well as the ability to suppress acute and chronic Graft-versus-Host-Disease (GvHD). Murine iNKT cells differentiate during thymic development into iNKT1, iNKT2 and iNKT17 sublineages, which differ transcriptomically and epigenomically, and have subset-specific developmental requirements. Whether distinct iNKT sublineages also differ in their antitumor effect and their ability to suppress GvHD is currently unknown. In this work, we generated highly purified murine iNKTsublineages, characterized their transcriptomic and epigenomic landscape, and assessed specific functions. We demonstrate that iNKT2 and iNKT17, but not iNKT1 cells, efficiently suppress T cell activation in vitro and mitigate murine acute GvHD in vivo. Conversely, we show that iNKT1 cells display the highest antitumor activity against murine B-cell lymphoma cells both in vitro and in vivo. Thus, we demonstrate for the first time that iNKT sublineages have distinct and different functions, with iNKT1 cells having the highest antitumor activity and iNKT2 and iNKT17 cells having immune-regulatory properties. These results have important implications for the translation of i Overall design: Highly purified murine iNKT-sublineages derived from thymi of FVB mice were characterized by bulkRNA and ATAC-sequencing. Total thymic iNKT from single thymi from 2 8 week old FVB/N mice were analyzed by single cell RNAseq. Donor CD4 and CD8 Tcon from FVB mice on day 7 after transplantation in a major mismatch GvHD model (FVB -> Balb/C) and treatment with iNKT1, iNKT2 or iNKT17 cells or no treatment were sorted and analyzed by bulk RNAseq. GSE172169 pubmed 34036317 pubmed 35790103