Abstract
The recent Natural Killer (NK) cell maturation model postulates that CD34(+) hematopoietic stem cells (HSC) first develop into CD56(bright) NK cells, then into CD56(dim)CD57(-) and finally into terminally maturated CD56(dim)CD57(+). The molecular mechanisms of human NK cell differentiation and maturation however are incompletely characterized. Here we present a proteome analysis of distinct developmental stages of human primary NK cells, isolated from healthy human blood donors. Peptide sequencing was used to comparatively analyze CD56(bright) NK cells versus CD56(dim) NK cells and CD56(dim)CD57(-) NK cells versus CD56(dim)CD57(+) NK cells and revealed distinct protein signatures for all of these subsets. Quantitative data for about 3400 proteins were obtained and support the current differentiation model. Furthermore, 11 donor-independently, but developmental stage specifically regulated proteins so far undescribed in NK cells were revealed, which may contribute to NK cell development and may elucidate a molecular source for NK cell effector functions. Among those proteins, S100A4 (Calvasculin) and S100A6 (Calcyclin) were selected to study their dynamic subcellular localization. Upon activation of human primary NK cells, both proteins are recruited into the immune synapse (NKIS), where they colocalize with myosin IIa.
Highlights
Among those proteins, S100A4 (Calvasculin) and S100A6 (Calcyclin) were selected to study their dynamic subcellular localization
The challenge here was to overcome the scarcity of CD56bright Natural killer (NK) cells, which account for only 5–10% of all NK cells
6 ϫ 108 Peripheral blood mononuclear cells (PBMCs) were sorted for CD56ϩ NK cell subsets to obtain about 1 ϫ 106 CD56bright NK cells from each donor, sufficient material to study the first developmental step from CD56bright to CD56dim
Summary
Monoclonal Antibodies and Reagents—For fluorescence-activated cell sorting (FACS), anti-CD56 (clone AF12–7H3, mouse IgG1, Miltenyi Biotec, Auburn, CA), anti-CD3 (clone HIT3a, mouse IgG1 , BD Bioscience), and anti-CD57 (clone TB03, mouse IgM, Miltenyi Biotec) mouse monoclonal antibodies (mAbs) were used. In our statistical model we assume that the log2-regulation factors (RF) of each protein follow a normal distribution (5 ϫ log RFs per protein because of five analyzed donors per NK cell subset comparison) with different expected values, but with the same standard deviation 0. The MAD was corrected by the factor of 1.4826 (determined by Monte Carlo simulation) according to the number of k ϭ 5 data sets (donors) to obtain an unbiased and robust estimator for the standard deviation 0. In this way we obtain many estimates for the MAD with a large variance.
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