Many immune activities are attributed to NKT cells, although they are associated most often with providing effective immunity against cancer, infections and autoimmune diseases [2-4]. Given these varied roles [5, 6], it is surprising (and an issue of conjecture [7, 8]) that usually only the CD4+ and CD4− subsets of mature human NKT cells are assayed when clinically assessing the human NKT cell pool [9]. CD4+ NKT cells produce cytokines associated with T helper PI3K inhibitor type 0 (Th0) responses,

and CD4− NKT cells are associated with Th1 responses [10, 11]. The extent to which additional functionally distinct human NKT cell subsets exist is not known, but others have been defined in mice, and human NKT cells express differentially several cell surface antigens used to define conventional T cell subsets [8, 10-13]. A recent study showed MAPK inhibitor that both the CD4+

and CD4− NKT cell subsets were highly heterogeneous in their expression of cell surface antigens and cytokine production, which suggested that unidentified functionally distinct subsets may exist within both these subsets [14]. This was an important finding, however, similar to earlier reports that examined the significance of CD8 expression by human NKT cells [15, 16], the study used expanded NKT cell lines to obtain sufficient cell numbers and it is uncertain whether or not the phenotype of the expanded cells accurately reflected the in situ (i.e. non-expanded) human NKT cell pool. Like many other NKT cell studies, the analysis was conducted using only NKT cells sourced from peripheral blood. This is an important issue to consider because, although analysis of blood is the dominant source of cells for assessing patient immunity, NKT cell tissue location is an important determinant of their function in mice [17]. Mouse studies have also shown that the profile of blood NKT cells often does not reflect NKT cells from other tissue

sites [18]. It is not known whether this also applies to human NKT cells, although NKT cells from human thymus are functionally unresponsive compared to blood-derived NKT cells Clomifene [19] and liver NKT cells are distinct from blood NKT cells in their expression of cell surface proteins [20]. In this study, we characterize the heterogeneity of the human NKT cell pool by analysing cell surface antigen and cytokine expression of the overall NKT cell pool and of the CD4+ and CD4− subsets from different tissues, with an emphasis on testing freshly isolated, rather than in-vitro-expanded, NKT cells. We detail significant heterogeneity within the established CD4+ and CD4− NKT cell subsets from peripheral blood, thymus, spleen and cord blood and identify several candidate antigens where differential expression correlates with distinct patterns of cytokine production by blood-derived NKT cells. Our findings provide a platform for an improved understanding of the complex organization of the normal human NKT cell pool.