Invariant Natural Killer T Cell Subsets Have Diverse Functions: iNKT2 and iNKT17 Protect from Graft-Versus-Host-Disease, Whereas iNKT1 Have Antitumor Potential

Invariant natural killer T (iNKT) cells are an interesting subpopulation of T cells that can potently inhibit graft-versus-host-disease (GVHD) through the production of Interleukin 4 (IL-4), while also carrying anti-tumor potential (Leveson-Gower et al. Blood. 2011;117:3220-9; Schneidawind et al. Blood. 2014;124:3320-8). Murine iNKT cells differentiate during thymic development into three distinct sublineages, named according to the classification of conventional T cells: Th1-like iNKT (iNKT1) cells, Th2-like iNKT (iNKT2) cells, and Th-17 like iNKT (iNKT17) cells (Brennan et al. Nat Rev Immunol. 2013; 13:101-1). In this study we investigated the immune regulatory and anti-tumor potential of iNKT1, iNKT2 and iNKT17 cell subsets.

Thymic iNKT1 cells, iNKT2 cells and iNKT17 cells were isolated from 8-10 week-old FVB/NJ mice by flow cytometry based on PBS-57-CD1d-Tetramer and a combination of cell surface molecules (iNKT1: ICOS^- PD1^- CD27⁺ CD24^-; iNKT2: ICOS⁺ PD1⁺ CD27⁺ CD4⁺ CD24^-; iNKT17: ICOS⁺ PD1⁺ CD4^- CD27^- CD24^-) and purity was confirmed by intra-nuclear staining for the transcription factors PLZF and RORγT. RNA sequencing analysis determined that iNKT1 cells were the main subset expressing proinflammatory and cytotoxic genes, such as Interferon gamma (IFN-γ), Fas Ligand (FasL), Perforin, and Granzyme B (Gzmb), whereas IL-4 was expressed by iNKT2 cells and, at a lesser extent, by iNKT17 cells. To assess the immuno-regulatory potential of the three iNKT sublineages, we employed a murine major histocompatibility complex (MHC)-mismatched bone marrow transplantation model. BALB/c (H-2K^d) recipients were lethally irradiated with 8.8 Gy; on the same day, 4 x 10⁶ TCD-BM cells and 1 x 10⁶ conventional CD4 and CD8 T cells (Tcon) from FVB/NJ (H-2k^q) mice were injected intravenously. Additionally, 5 x 10⁴ purified iNKT1, iNKT2 or iNKT17 cell subsets from FVB donors were injected. A significant survival benefit was observed when iNKT2 (p=0.017) 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 x10⁴ iNKT2 (p= 0.008) and iNKT17 (p= 0.04) significantly suppressed GVHD. As iNKT1, iNKT2 and iNKT17 have a very different gene expression profile, we tested the ability of the sublineages to kill a B-cell lymphoma cell line transduced to express high levels of CD1d (A20-CD1d) in vitro and found that iNKT1 cells killed A20-CD1d cells significantly better than iNKT2 (p=0.006) or iNKT17 (p=0.0001) cells. These findings are in line with the sequencing data mentioned above, showing that iNKT1 cells express a more inflammatory phenotype.

In summary, we demonstrate here that only iNKT2 and iNKT17 cells protect from GVHD, whereas iNKT1 cells have cytotoxic function. To our knowledge, this is the first study to show functional differences between the iNKT sublineages, suggesting that iNKT1, iNKT2 and iNKT17 cells have diverse functions. Therefore, these data provide new biological insights, which will be useful for developing iNKT cell-based cell therapy.