Novel Subsets of Human Invariant Natural Killer T Cells, Including a Temra-like CD4−CD8− Population, Revealed through Single Cell RNA-Sequencing

Introduction Invariant natural killer T (iNKT) cells, with their inherent lack of allorecognition, are a promising platform for universal donor cellular therapies. However, murine studies have shown that the iNKT cell population is comprised of subsets with different functions (ie. iNKT2 and iNKT17 suppress graft-versus-host disease, while iNKT1 are cytotoxic towards malignant cells). This suggests that iNKT-based cellular therapies could be optimized by using different subsets in different disease settings. Objectives Because the heterogeneity of human iNKT cells remains poorly understood, we sought to reveal distinct subsets through single cell RNA-sequencing (scRNA-seq). Methods Human iNKT cells were purified by magnetic bead-based enrichment and fluorescence activated cell sorting. Whole transcriptome scRNA-seq libraries were generated using the Rhapsody platform (Becton Dickenson). Data was processed with BD Rhapsody pipeline (Seven Bridges) followed by analysis in R (Seurat v4.1.0). Results ScRNA-seq of human iNKT cells from peripheral blood (PB), cord blood, thymus, and bone marrow revealed 9 transcriptionally distinct clusters derived from cells from each tissue (Fig 1A). While iNKT cells from some tissues dominated certain clusters, PB-derived iNKT cells were relatively equally distributed. We found that, in contrast to murine iNKT cells, clusters of human iNKT cells expressing TBX21 also express RORC (Clusters 2 & 6, Fig 1A-B). These iNKT cells also upregulated genes important for cytotoxicity and natural killer cell function, therefore we designated these cells as a Th1/17/NK-like population. Oligomer-conjugated antibody use revealed that Cluster 6 was CD45RA+ with absent CCR7 transcript, similar to T effector memory CD45RA+ (TEMRA) cells. Other clusters with low/absent TBX21 upregulated ZBTB16 and may represent Th2-like iNKT cells (Clusters 1, 4, & 7). These cells also expressed IL4R and other genes important for T cell function. Surprisingly, we found several clusters with relatively minimal expression of any of these canonical transcription factors (Clusters 0, 3, 5, & 8). These clusters also upregulated SELL, CCR7, and CD45RA, suggesting a naïve or undifferentiated phenotype, and were especially abundant in thymus, though found in PB as well. We further demonstrated that CD4+ iNKT cells were comprised of naïve and Th2-like clusters as well as a Th1/17/NK-like cluster (Cluster 2), a population previously described in mice, suggesting that CD4 expression may not clearly distinguish functionally distinct subsets. Conclusion These data reveal novel insights into the heterogeneity of human iNKT cells from multiple immunologic tissues, which provide a critical foundation for optimal use of human iNKT cells in the development of cellular therapies. Studies to correlate transcriptome and function in these novel human iNKT cell populations are underway.