Identification of an Fgd5-positive subset of iNKT1 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 infections and are involved in diseases such as allergic asthma and cancer. Despite 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 hematopoietic stem cells (HSCs) can dually serve to identify 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. RNA sequencing of CD5+EPCR-Fgd5ZsGreen+ bone marrow cells showed greatest similarity to 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 coexpressed 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. Together, these data implicate Fgd5 expression as a powerful new tool for phenotyping and tracking cytotoxic iNKT1 cell subsets in vivo.