Varying expression of NKp46, CD8α, CD16 and CD27 on NK-cell subsets in blood and spleen. (A) Following five-colour staining, PBMC and splenocytes were gated on CD3- cells and further subgated according to their NKp46/CD8α expression pattern in NKp46- and NKp46+ NK cells in blood (upper graph) and NKp46-, NKp46+ and NKp46high NK cells in spleen (lower graph). (B) 14 healthy 6–7 month old pigs were investigated for NKp46 and CD8α expression levels in the NKp46-defined NK-cell subsets. Box-plots show the mean fluorescence intensity of the two markers. (C) NK-cell subsets defined in (A) were further analysed for their expression of the surface markers CD16 and CD27. Histograms show the expression of the two markers within the respective NKp46-defined subsets (CD8α+NKp46-: blue histograms, CD8α+NKp46+: green histograms, CD8αdim/-NKp46high: red histograms) in blood (upper graphs) and spleen (lower graphs) according to the corresponding isotype control (grey histrograms with dotted lines). Box-plots show the mean fluorescence intensity of CD16 and CD27 of the NKp46-defined NK-cell subsets in blood and spleen of 14 healthy 6–7 month old pigs. (B + C) Significant differences between the subsets in blood or spleen are indicated (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).