Type-I NKT cells (NKT cells) express semi-invariant TCRs and recognise self and non-self glycolipids presented in complex with MHC-I-like CD1d molecules. Once activated NKT cells function in an innate-like manner and play important roles in anti-microbial and anti-cancer immunity, but also have immune regulatory roles and are associated with allergy and autoimmune disease. Despite the importance of NKT cells in health and disease only a few in situ studies have investigated NKT cell location and behavior. Most of these studies have utilized exogenous cell transfer and/or surrogate markers such as CXCR6 expression and as such may not have fully represented the NKT cell population or their location.

With the use of CD1d tetramers loaded with the prototypical NKT cell antigen a-GalCer we have aimed to specifically detect endogenous NKT cells in situ. Within spleen and thymus sections of wt BALB/c mice tetramer staining was observed that was co-located with TCR staining. Within the spleen the tetramer positive cells were particularly evident within the T cell zone. While some of the tetramer positive cells were observed within the red pulp, none occurred within the B cell zone. Furthermore, and consistent with previous data on the development of NKT cells, staining of wt thymus sections revealed a prominent a population of CD1d/a-GalCer tetramer positive cells within the thymic medulla. Conversely, staining was not observed within the spleen or thymus sections of CD1d^-/- mice, which are deficient in NKT cells, or within wt sections stained with negative control CD1d tetramer.

This technique not only resulted in novel insights into the location of NKT cells and their subsets including CD4+ and CD4-, within spleen, thymus, and other tissues, but also provides a means for future investigations into the behavior of NKT cells during development and disease.