Figure 1 Gating strategy for the determination of NK and Treg cell subsets. (A) After excluding doublets from the total of acquired events, peripheral blood lymphocytes (PBL) were gated according to (B) FSC/SSC and (C) CD45/SSC dot plot. (D) Then, CD3–CD56+ NK cells, CD3+CD56+ NKT cells and CD3+ T cells were gated in the CD3 APC-Cy7/CD56 PerCPCy5.5 dot plot. (K) CD3–CD56+ NK cells were further analyzed according to the intensity of the CD56 and CD16 expression (CD16 V450/CD56 PerCPCy5.5 dot plot). (E, L, M) Further, dependent on isotype controls, subsets of (F–J) T cells, (N–S) CD56brightCD16dim/− NK cells and (T–Y) CD56dimCD16+ NK cells were analyzed using the depicted gate settings in dot plots of (N, T) NKG2D/NKG2A, (O, U) CD69/CD107, (P, V) perforin/granzymeB, (Q, W) IFNγR/CD25, (R, X) IL4/TGFβ and (S, Y) TGFβRII/IL10R. With respect to the determination of IFNγ+ Treg, CD3+ T lymphocytes were further analyzed and (F) CD4+ lymphocytes were identified using a mixture of CD4 and CD16 monoclonal antibody with the same color (CD4+CD16 V450/CD3 APC-Cy7 dot plot). (G) Further, CD4+CD25+ lymphocytes were gated using CD25 and the mixture of CD4 and CD16 monoclonal antibody (CD25 APC/CD4+CD16 V450 dot plot). (H) Then, CD127–Foxp3+ Treg were determined within the CD4+CD25+ lymphocyte subset (CD127 PE-Cy7/Foxp3 PE dot plot). (I) CD127-Foxp3+ Treg were additionally gated in a CD127/CD25 gate based on all CD3+ T cells (CD127 PE-Cy7/CD25 APC dot plot) and (J) IFNγ+ Treg were determined using a CD56/IFNγ dot plot (CD56 PerCPCy5.5/IFNγ FITC). FSC – forward-scattered light; SSC – side-scattered light.