A Proinflammatory Invariant Natural Killer T Cell Phenotypic State Associates with Human Graft-Versus-Host Disease Onset

Human invariant natural killer T cells (iNKT) are a rare population of lymphocytes that bridges innate and adaptive immune functions. These cells show diverse phenotypes and function, but the current population structure imposed on iNKT cells inadequately differentiates these diverse phenotypes and function. Using single-cell RNA-sequencing and subsequent functional assays and flow cytometric analysis, we present a novel population structure that characterizes human iNKT cell heterogeneity. Markers delineating primary iNKT cells for IL-4 + , IFNg + TNFa + , and cytotoxicity functions were CD4, KLRG1, and CD94, respectively. In addition, iNKT cells follow a temporal evolution of function and associated markers - upon chronic activation, HLA-DR expression associated with increased Th1 skewing. Unsupervised clustering of our iNKT single-cell RNA-sequencing data from a graft-versus-host disease (GvHD) and a GvHD-free post-transplant patient showed a GvHD-enriched subset that is defined by expression of HLA-DR alleles. In a cohort of 38 patients, 19 of whom developed GvHD, flow cytometric analysis of iNKT cells revealed increased HLA-DR and decreased CD161 expression when compared to GvHD-free patients. CD94 expression was overall increased after allogeneic hematopoietic cell transplantation. Functional assessment of HLA-DR hi CD161 − iNKT cells showed that this population produced significantly less IL-4 while maintaining IFNg production. Furthermore, high expression of HLA-DR could be detected before initial GvHD onset compared to in patients who did not develop GvHD. Together, our data suggests HLA-DR as a marker for delineating iNKT cells with a proinflammatory phenotype and is associated with GvHD. In sum, our approach for describing iNKT cell population structure revealed new markers that associate with different iNKT cell phenotypes. This new paradigm for describing iNKT cell heterogeneity can potentially be translated into the clinic as an immune monitoring tool for patients undergoing allogeneic hematopoietic cell transplantation.