Figure 1: Identification of CD1d–α-GalCer reactive atypical NKT cells.

(a) Flow cytometry of CD1d–α-GalCer reactive cells enriched and expanded from PBMCs from three healthy human donors. TRDV1⁻ γδTCR⁻ cells were analysed for the expression of TRBV25-1 versus CD1d-endogenous tetramer or CD1d–α-GalCer tetramer (left-hand density plots). TRDV1⁻ γδTCR⁻ CD1d–α-GalCer tetramer⁺ cells were analysed for the expression of TRAV10 (middle density plots). CD1d–α-GalCer tetramer⁺ TRBV25-1⁺ type I cells and CD1d–α-GalCer tetramer⁺ TRBV25-1⁻ cells were analysed for the expression of CD4 and CD8α (right-side density plots). (b) The mean percentage of double negative (DN), CD4⁺ and CD8⁺ cells among CD1d–α-GalCer tetramer⁺ TRBV25⁺ Type I cells (dark grey) and CD1d–α-GalCer tetramer⁺ TRBV25⁻ cells (light grey). Each symbol represents cells from a different donor (n=6). (c) The mean percentage of CD1d–α-GalCer tetramer⁺ TRBV25⁻ cells of total CD1d–α-GalCer reactive NKT cells, from 19 individual donors. Donors that showed no clear population of atypical NKT cells were given an arbitrary value of 0.01%.