040 Single-cell RNA sequencing identifies a disease-dominant CD8+ T cell population co-expressing both activating and inhibitory receptors of the NKG2 family

Alopecia areata (AA) is a cell-mediated, autoimmune form of hair loss characterized by lymphocytic infiltration of the bulb region of the hair follicles (HFs), inflammation, and destruction of the HF. We previously showed that CD8+ NKG2D+ T cells are necessary and sufficient to induce AA in the graft-induced C3H/HeJ mouse model. Here, we used single-cell RNA sequencing to comprehensively profile the T cell component of the inflammatory infiltrate in AA. We first isolated CD45+ cells from the skin of affected and unaffected control mice and focused our analysis on CD8+ T cells. We observed a marked expansion of all CD8+ T cells in AA mice (41% versus 4% of all CD45+ T cells in control mice). CD8+ T cells in both AA and control mice were clustered into 5 distinct populations, each with an associated set of marker genes. Shared CD8+ populations included antigen-experienced effector cells with high expression of IFNG, GZMA, and a memory T cell population with high CD69 and CD40LG, both of which were expanded in AA mice. Interestingly, we discovered a population of CD8+ T cells that is largely predominant in AA mice, marked by increased expression of not only NKG2D, an activating receptor, but also NKG2A, a known inhibitory receptor of the NKG2 family. These cells were also characterized by increased expression of T cell exhaustion markers (PD1, TIM3, CTLA4) as well as the co-stimulatory markers CD137 and ICOS. To probe the NKG2A receptor pharmacologically, we found that treatment of mouse T cells in vitro with an NKG2A agonist antibody resulted decreased IFNG production, and treatment of C3H/HeJ mice with a blocking antibody against Qa-1 (an NKG2A ligand) prior to disease induction via engraftment resulted in earlier AA onset and accelerated disease progression in vivo. Therapeutic manipulation of the NKG2 family of activating and inhibitory receptors represents a novel treatment approach in AA.