Expression of cyCD3⁺ in NK cells in vivo. NK cells were enriched by depleting membrane CD3⁺ cells with CD3-biotin and streptavidin Dynalbeads. The NK-cell–enriched cell fraction was analyzed for CD56 and cyCD3 expression after carefully gating out membrane CD3⁺ cells to avoid contamination with peripheral T cells and NKT cells that are cyCD3⁺. (A) Frequencies of NK cells that express cyCD3 in thymus (♦), CB (■), and PB (▴); solid lines represent the means of 6 to 8 independent samples. (B) Dot plot analysis of cyCD3 expression on gated CD56⁺CD3⁻ NK cells. Note the difference in the intensity of cyCD3 expression between PB (low) and CB (high) and thymus (high). Numbers in dot plots show the frequency of cells in the respective top right and bottom right quandrants. (C) Dot plot analysis of CD127 versus CD56 expression on gated CD3⁻ thymus, CB, and PB cells. Note that the CD127⁺ subset is present in the CD56^bright subpopulation, whereas most cyCD3 cells are present in the CD56^dim population.