Robust 8-color flow cytometry panel reveals enhanced effector function of NKG2C + CD57 + FcεRγ − NK cells in CMV seropositive human blood donors

Increasing evidence suggests that human NK cells may develop memory-like features. Here, we report the establishment of a robust 8-color flow cytometry panel that allows quantification and functional analysis of different memory-like NK cell subsets (NKG2C+/CD57+, FcεRγ-) from relatively small blood samples. We could confirm previous publications reporting an enhanced prevalence of the mentioned memory-like NK cell subsets in CMV seropositive human donors and were able to show a clear congruence between enhanced expression of NKG2C and CD57, the absence of FcεRγ and CMV seropositivity supporting the hypothesis of memory-like NK cell development following viral infections. While we could not detect significant differences in effector functions (i.e. degranulation and production of IFNγ) in regard to age or CMV seropositivity when looking at the overall NK cell population, a significantly enhanced expression of CD107a and IFNγ could be observed in NKG2C+/CD57+ as well as FcεRγ- NK cell subpopulations in CMV+ donors. This enhancement of effector functions was even more pronounced in NKG2C+/CD57+ NK cells that were also negative for FcεRγ; CMV seropositive donors showed a dramatically increased expression of CD107a as well as IFNγ. With only small-sized volumes of blood needed, our proposed 8-color panel and experimental protocol offers easy handling and a reliable and reproducible option for implementation in accompanying clinical research, e.g. for evaluation of immunosuppressed patients suffering from autoimmune diseases or in allograft recipients.