Figure 2

sMUC16 predominantly binds to Siglec-9^pos/CD16^pos/CD56^dim NK cells. Peripheral blood mononuclear cells (PBMC) from healthy donors (HD) and patients (PT) and peritoneal fluid mononuclear cells (PFMC) of PT were labeled with VK8 followed by a fluorophore conjugated secondary reporter antibody. The cells were subsequently labeled with fluorescently tagged anti-CD3, -CD16, -CD56, -Siglec-9, and -NKp46 antibodies as described earlier [20]. NK cells identified as CD3^neg/NKp46^pos cells were classified into CD16^pos/CD56^dim and CD16^neg/CD56^bright cells by flow cytometry. A, Siglec-9 expression on the two NK cell subpopulations, CD16^pos/CD56^dim and CD16^neg/CD56^bright, was analyzed. Each bar represents a mean and standard deviation of three independent experiments. B and C, sMUC16 binding and Siglec-9 expression on CD16^pos/CD56^dim NK isolated from PBMC of healthy donors and PBMC and PFMC of cancer patients was determined. B, shows dot plots of representative healthy donor (HD) peripheral blood NK and patient (PT) NK cells from peripheral blood (PB) and peritoneal fluid (PF). Majority of events in the lower left quadrant are located on the axes and therefore not visible in the dot plots. C, Each value is mean of PBMC from 6 healthy donor and PBMC and PFMC from five cancer patients showing percent of the cells positive for Siglec-9 and sMUC16 and those double positive for both Siglec-9 and sMUC16 are shown.