High-resolution phenotyping identifies NK cell subsets that distinguish healthy children from adults

<div><p>Natural killer (NK) cells are critical in immune defense against infected, stressed or transformed cells. Their function is regulated by the heterogeneous expression of a wide array of surface receptors that shape its phenotypic diversity. Although NK cells develop in the bone marrow and secondary lymphoid tissues, substantive differentiation is apparent in the peripheral blood including known age-related variation. In order to gain greater insight into phenotypic and functional variation within peripheral blood NK cells across age groups, we used multi-parametric, polyfunctional flow cytometry to interrogate the NK cell variability in 20 healthy adults and 15 5–10, 11–15 and 16–20 year-old children. We found that the normative ranges in both adults and children displayed great inter-individual variation for most markers. While the expression of several receptors did not differ, among those that did, the majority of the differences existed between adults and the three pediatric groups, rather than among children of different ages. Interestingly, we also identified variation in the individual expression of some markers by sex and ethnicity. Combinatorial analysis of NK cell receptors revealed intermediate subsets between the CD56^bright and CD56^dim NK cells. Furthermore, on examining the NK cell diversity by age, adults were discovered to have the lowest developmental diversity. Thus, our findings identify previously unappreciated NK cell subsets potentially distinguishing children from adults and suggest functional correlates that may have relevance in age-specific host defense.</p></div

Most relevant markers and combinatorial subsets in unstimulated NK cells.

<p>Most relevant markers and combinatorial subsets in unstimulated NK cells.</p

NK cell phenotyping panels.

<p>NK cell phenotyping panels.</p

Short-term NK cell stimulation leads to significant receptor modulation.

<p>NK cells were stimulated for 4 hours using PMA/ ionomycin and evaluated using flow cytometry using panels shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181134#pone.0181134.t001" target="_blank">Table 1</a>. (A-B) Percentage difference from baseline of NK cells expressing markers after stimulation among 4 age groups. (C-D) Median fluorescent intensity (MFI) difference from baseline of NK cells with positive expression of markers after stimulation among four age groups. 5–10 y.o. (n = 15) (red); 11–15 y.o. (n = 15) (green); 16–20 y.o. (n = 15) (blue) and adults (n = 20) (black). All bars for Panels 1–4 are indicative of surface expression. Panel 5 markers except CD3, CD45, CD56 and CD107a were detected intracellularly as described in the Materials and methods section. All data shown is mean ± S.D. *p<0.05 among four age groups by Kruskal-Wallis and post-hoc comparison by Dunn test with Bonferroni adjustment.</p

Significant inter-individual variation of NK cell markers between age groups of healthy adults and children.

<p>NK cells were defined using flow cytometry as CD3^-CD45^highCD56⁺ and gated above fluorescence minus one (FMO) controls for all NK cell markers (panels shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181134#pone.0181134.t001" target="_blank">Table 1</a>). (A-E) Percentage of NK cells positively expressing markers without stimulation among four age groups. (F-J) Median fluorescent intensity (MFI) of NK cells with positive expression of markers without stimulation among 4 age groups. 5–10 y.o. (n = 15) (red); 11–15 y.o. (n = 15) (green); 16–20 y.o. (n = 15) (blue) and adults (n = 20) (black). All bars for Panels 1–4 are indicative of surface expression. Panel 5 markers except CD3, CD45, CD56 and CD107a were detected intracellularly as described in the Materials and methods section. All data shown is mean ± S.D. *p<0.05 among four age groups by Kruskal-Wallis and post-hoc comparison by Dunn test with Bonferroni adjustment.</p

Highly significant phenotypic differences in combinatorial NK subsets between stimulated and unstimulated cells.

<p>Highly significant phenotypic differences in combinatorial NK subsets between stimulated and unstimulated cells.</p

Significantly different markers by frequency within CD56^bright NK cells of healthy adults and children.

<p>Significantly different markers by frequency within CD56^bright NK cells of healthy adults and children.</p

Highly significant phenotypic differences in combinatorial NK subsets between adults and children.

<p>Highly significant phenotypic differences in combinatorial NK subsets between adults and children.</p

Most relevant markers and combinatorial subsets in stimulated NK cells.

<p>Most relevant markers and combinatorial subsets in stimulated NK cells.</p

Significantly different markers by MFI within CD56^dim NK cells of healthy adults and children.

<p>Significantly different markers by MFI within CD56^dim NK cells of healthy adults and children.</p