Distinct Immune Regulatory Potential of Invariant Natural Killer T (iNKT) Cell Subsets: iNKT2 and iNKT17, but Not iNKT1, Protect from Graft-Versus-Host-Disease

Invariant natural killer T (iNKT) cells can potently inhibit graft-versus-host-disease (GVHD) in animal models through the production of Interleukin-4. Once considered a homogeneous population, it is now well established that murine iNKT cells differentiate during thymic development into three distinct sublineages, distinguished based on the expression of transcription factors and effector molecules: Th1-like iNKT (iNKT1) cells, Th2-like iNKT (iNKT2) cells, and Th17-like iNKT (iNKT17) cells. In this study, we investigated the immune regulatory potential of iNKT1, iNKT2 and iNKT17 cell subsets. In order to identify surface molecules whose expression would allow us to isolate viable iNKT1, iNKT2 and iNKT17 cells, we performed single cell RNA sequencing analysis on murine thymic iNKT cells isolated from FVB/N mice by flow cytometry based on PBS-57-CD1d-Tetramer staining. The results of this analysis together with previous knowledge allowed us to develop a sorting strategy to isolate iNKT1 (ICOS- PD1- CD27+ CD24-), iNKT2 (ICOS+ PD1+ CD27+ CD4+ CD24-), iNKT17 (ICOS+ PD1+ CD4- CD27- CD24-) cells, whose purity was confirmed by intra-nuclear staining for the transcription factors PLZF and RORgT. To assess the immune regulatory potential of the three iNKT sublineages, we employed a murine major histocompatibility complex (MHC)–mismatched bone marrow transplantation model. Lethally irradiated BALB/c (H-2Kd) mice were injected intravenously with T cell depleted-bone marrow (TCD-BM) cells and conventional CD4 and CD8 T cells (Tcon, 10e6 cells/mouse) from FVB/N (H-2kq) mice. Additionally, 5 × 10e4 sorted iNKT1, iNKT2 or iNKT17 cells from FVB/N donors were injected the same day. A significant survival benefit was observed when iNKT2 (p=0.0166) and iNKT17 (p=0.033) cells were adoptively transferred compared to mice that only received TCD-BM and Tcon, whereas there was no survival benefit in the group that received iNKT1 cells. In addition, body weight was improved in mice that received iNKT2 (day +41: p=0.009, day +49: p=0.005 and day +59: p= 0.005) or iNKT17 (day +59: p= 0.006) compared to mice that received iNKT1 cells. Clinical GVHD scores were also improved in mice that received iNKT2 (day +41: p= 0.012, day +51: p= 0.005) or iNKT17 (day +28: p=0.05, day +51: p=0.007) compared to mice that received iNKT1 cells. Interestingly, we found that even 1 × 10e4 iNKT2 (p= 0.008) and iNKT17 (p= 0.04) significantly suppressed GVHD. In summary, we demonstrate here that only iNKT2 and iNKT17 cells mitigated GVHD, whereas iNKT1 had no detectable immune regulatory potential. To our knowledge, this is the first study to show functional differences between iNKT sublineages, indicating that iNKT1, iNKT2 and iNKT17 cells have diverse functions and providing new biological insights that will be useful for developing iNKT cell-based cell therapies.