Functionality of lung type II NKT cells developing in CD1d−/− animals

Abstract

Abstract NKT cells are lymphocytes that recognize lipid antigens presented by CD1d. Type I NKT cells with semi-invariant TCR recognize α-galactosylceramide while many type II NKT cells with a more diverse TCR repertoire recognize sulfatide. We developed a stable sulfatide-loaded CD1d tetramer that allows the specific identification of type II NKT cells and found that they are enriched in the lung, a major site of tumor metastasis. An in-depth phenotypical analysis revealed that lung type II NKT cells were CD4 or CD8 single positive cells, like conventional T cells, whereas type I NKT cells were either CD4+CD8- or CD4-CD8-. We showed that type II NKT cells expressed markers of myeloid cells, c-Kit, CD11b and Ly6C, even though morphological analysis revealed lymphocytic morphology. In contrast to type I NKT cells, type II NKT existed in PLZF−/− and CD1d−/− mice. Thus, we asked the importance of CD1d in the in vivo and in vitro activation of type II NKT cells. In the steady state, lung type II NKT cells expressed granzyme A but not granzyme B or perforin. By injecting sulfatide in vivo, we observed that WT but not CD1d−/− lung type II NKT cells upregulated granzyme A and Ki67 expression. Using syngeneic WT bone marrow-derived dendritic cells (BMDC) as APCs, ex-vivo stimulation of WT but not CD1d−/− lung type II NKT cells with sulfatide induced the secretion of IFN-γ, IL-13 and IL-17. This was consistent with the expression of specific transcription factors, T-bet and RORγt, by lung type II NKT cells. In conclusion type II NKT cells still develop in CD1d−/− mice but are not functional. The ability to study responses of type II NKT cells in vitro can now help elucidate the mechanism by which these regulatory cells interact with other cells and modulate tumor immunity in the lung.