Identification of an Fgd5-positive subset of iNKT type 1 cells with enhanced cytotoxicity

Invariant natural killer T (iNKT) cells are unconventional ɑβ T cells that respond to lipid-based antigens. They play a vital role in response to bacterial and viral infections and are also involved in a wide range of diseases such as allergic asthma and cancer. Despite these clear immunological roles across multiple diseases, remarkably few tools exist to study iNKTs in vivo. In this study, we report that the Fgd5ZsGreen/+ reporter mouse widely used to study haematopoietic stem cells (HSCs) can dually serve to identify other rare immune cell populations, including a subset of iNKTs. Specifically, we show that a ‘non-HSC’ population of CD45+Fgd5ZsGreen+ cells reside in multiple organs including the lungs, liver, spleen and thymus. The majority of these Fgd5ZsGreen+ cells do not express canonical HSC markers, but instead express CD5, a marker of mature lymphocytes. RNA-sequencing of bone marrow resident CD5+EPCR-Fgd5ZsGreen+ cells showed greatest similarity to invariant natural killer T (iNKT) cells and these findings were corroborated by additional cell surface marker analysis of TCRβint CD1d-PBS57+ iNKT cells in reporter mouse tissues. We found that roughly 20% of total thymic iNKT cells were Fgd5-ZsGreen+ and that these cells predominantly co-expressed NK1.1 and CD122, consistent with an iNKT1 cell phenotype. Transcriptomic and proteomic profiling of sorted Fgd5-ZsGreen- and Fgd5-ZsGreen+ iNKT cells revealed a number of key genes involved in cytotoxic responses as distinct between the two iNKT cell populations. These different cytotoxic profiles were further supported by cytokine expression following stimulation, indicating the potential existence of disparate iNKT1 subsets with distinct cytotoxic functions. Together, these data implicate Fgd5 expression as a powerful new tool for phenotyping and tracking cytotoxic iNKT1 cell subsets in vivo.