Defining a novel subset of CD1d‐dependent type II natural killer T cells using natural killer cell‐associated markers

Natural killer T (NKT) cells are αβ T cell receptor (TCR) expressing innate‐like T cells that display natural killer (NK) cell markers. Based on TCR characteristics, they are divided into two groups restricted to the MHC class I‐like molecule CD1d. Type I NKT cells, most extensively studied, are identified by a semi‐invariant Vα14‐Jα18 (mouse, Vα24‐Jα18 in humans) TCR reactive to the prototypic ligand α‐galactosylceramide presented on CD1d. In contrast, type II NKT cells display diverse TCR reacting to different CD1d‐presented ligands. There are no reagents that identify all type II NKT cells, limiting their exploration. Here, we searched for novel type II NKT cells by comparing Jα18(−/−)MHCII(−/−) mice that harbour type II but not type I NKT cells, and CD1d(−/−)MHCII(−/−) mice, lacking all NKT cells. We identified significantly larger populations of CD4(+) and CD4(−)CD8(−) (double negative, DN) TCRβ(+) cells expressing NKG2D or NKG2A/C/E in Jα18(−/−)MHCII(−/−) mice compared with CD1d(−/−)MHCII(−/−) mice, suggesting that 30%‐50% of these cells were type II NKT cells. They expressed CD122, NK1.1, CXCR3 and intermediate/low levels of CD45RB. Further, the CD4(+) subset was CD69(+), while the DN cells were CD49b(+) and CD62L(+). Both subsets expressed the NKT cell‐associated promyelocytic leukaemia zinc finger (PLZF) transcription factor and Tbet, while fewer cells expressed RORγt. NKG2D(+) CD4(+) and DN populations were producers of IFN‐γ, but rarely IL‐4 and IL‐17. Taken together, we identify a novel subset of primary CD4(+) and DN type II NKT cells that expresses NKG2 receptors have typical NKT cell phenotypes and a TH1‐like cytokine production.