Tim-3 expression on human NK cells. (A) Plots depict Tim-3 expression on NK cells for 3 representative healthy donors. PBMCs were gated on forward angle light scatter (FSC-height and FSC-area) to eliminate doublets, followed by side light scatter (SSC) and FSC gating to define lymphocytes. Dead cells were excluded using an Amine Aqua reactive dye, and CD14 and CD19 staining was used to exclude monocytes and B cells, respectively. CD56 and CD16 were used to identify NK cells within the CD14⁻CD19⁻CD3⁻ population. (B) The plot shows expression of Tim-3 on NK cells in comparison to Tim-3 expression on CD4⁺ and CD8⁺ T cells in a representative donor. (C) Graph depicts the frequency (percentage) of Tim-3 on NK cells (solid circles) and CD4⁺ and CD8⁺ T cell (open circles) from multiple donors (n = 27). (D) Plot shows NK cells from a healthy donor, gated into CD56^brightCD16⁻ and CD56^dimCD16⁺ NK-cell subsets with the expression of Tim-3 on these subsets presented as histograms. Graphs show the frequency (E) and MFI (F) of Tim-3⁺ CD56^brightCD16^– (solid circles) and CD56^dimCD16⁺ NK cells (open circles) for 27 healthy individuals. P values were calculated using the Mann-Whitney test. (G) Representative plot shows Tim-3 expression on NK-cell subsets (CD56^brightCD16^– [gray shade] and CD56^dimCD16⁺ NK cells [black line]) from cord blood obtained at the time of birth. (H) Graph shows Tim-3 expression on NK-cell subsets (CD56^brightCD16^– [solid circles] and CD56^dimCD16⁺ NK cells [open circles]) from cord blood of 3 healthy newborn infants. (I) Representative plots of Tim-3 coexpression with CD94 on CD56^brightCD16^– (left) and CD56^dimCD16⁺ (right) NK cells.