Expression of CD38 on resting peripheral iNKT cells defines an immature subpopulation with distinct functionality in humans

Human invariant natural killer T cells (iNKT) play an important role in an orchestrated immune response; however, the heterogeneity of iNKT subsets is not yet fully understood. Here, we uncovered CD38 as a marker of iNKT differentiation, decoupling it from its role as a marker of activation by comparing the phenotype, cytokine profile and transcription factor expression of iNKT cell subsets in humans. Expression of CD38 on resting iNKT cells was restricted to cells that were low in well-described maturity markers such as CD161 and CCR5 and co-expressed markers associated with undifferentiated T cells (CD45RA, CCR7, CD62L). High abundance of CD38⁺ iNKT cells in human infant thymus and cord blood supported the immature nature of this subset. Functional analysis revealed that the CD38⁺ phenotype of resting iNKT cells was accompanied by diminished type 1 cytokine release, which was reflected by reduced expression of the transcription factor EOMES. Moreover, in vitro stimulation of sorted CD38⁺ and CD38^- iNKT cells demonstrated the distinct phenotype of cells expressing CD38 in a resting state and activation-induced CD38. These findings suggest a context-dependent role of CD38 expression on iNKT cells, distinguishing activated from resting iNKT cells where CD38 expression marks a subset of undifferentiated cells with altered functionality. Taken together, we describe a population of iNKT cells that extends the remarkable heterogeneity of the iNKT cell compartment beyond the presence of CD4⁺ and CD4^- subsets.