Highlights d Unbiased scRNA-seq on thymic iNKT reveals discrete subsets in iNKT1 and iNKT2 d Transcriptomically defined iNKT2 cells comprise precursors to iNKT1 and iNKT17 d Most of thymic iNKT subsets seem to have the potential to egress the thymus d The transcription cofactor FHL2 controls specification of the iNKT1 effector fate

IL-27 is a cytokine of the IL-12 family that plays a key role in the regulation of inflammatory and T cell responses. Its receptor is composed of IL-27Rα and gp130 and activates the STAT pathway. We show in this study, using an ELISA that we developed, that a naturally occurring soluble form of IL-27Rα (sIL-27Rα) is produced by human activated CD4+ and CD8+ T cells, B cells, myeloid cells, and various cell lines. sIL-27Rα is present at a mean concentration of 10,344 ± 1,274 pg/ml in the sera from healthy individuals. Biochemical studies showed that sIL-27Rα is released as two N-glycosylated variants of ∼90 and ∼70 kDa. In IL-27Rα–transfected COS7 cells, primary cells, and cell lines, production of sIL-27Rα is inhibited by the metalloprotease inhibitors GM6001 and TAPI-0. Importantly, natural sIL-27Rα binds rIL-27, inhibits IL-27 binding to its cell surface receptor, and is a potent inhibitor of IL-27 signaling, as shown by its ability to specifically block IL-27–mediated STAT activation, at low molar excess over IL-27. Also, we found that serum levels of sIL-27Rα were elevated in patients with Crohn’s disease, a Th1-mediated disease. These findings suggest that sIL-27Rα may play important immunoregulatory functions under normal and pathological conditions.