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. 2017 Jan 11;7:40623.
doi: 10.1038/srep40623.

Multi-cellular Natural Killer (NK) Cell Clusters Enhance NK Cell Activation Through Localizing IL-2 Within the Cluster

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Free PMC article

Multi-cellular Natural Killer (NK) Cell Clusters Enhance NK Cell Activation Through Localizing IL-2 Within the Cluster

Miju Kim et al. Sci Rep. .
Free PMC article

Abstract

Multi-cellular cluster formation of natural killer (NK) cells occurs during in vivo priming and potentiates their activation to IL-2. However, the precise mechanism underlying this synergy within NK cell clusters remains unclear. We employed lymphocyte-laden microwell technologies to modulate contact-mediated multi-cellular interactions among activating NK cells and to quantitatively assess the molecular events occurring in multi-cellular clusters of NK cells. NK cells in social microwells, which allow cell-to-cell contact, exhibited significantly higher levels of IL-2 receptor (IL-2R) signaling compared with those in lonesome microwells, which prevent intercellular contact. Further, CD25, an IL-2R α chain, and lytic granules of NK cells in social microwells were polarized toward MTOC. Live cell imaging of lytic granules revealed their dynamic and prolonged polarization toward neighboring NK cells without degranulation. These results suggest that IL-2 bound on CD25 of one NK cells triggered IL-2 signaling of neighboring NK cells. These results were further corroborated by findings that CD25-KO NK cells exhibited lower proliferation than WT NK cells, and when mixed with WT NK cells, underwent significantly higher level of proliferation. These data highlights the existence of IL-2 trans-presentation between NK cells in the local microenvironment where the availability of IL-2 is limited.

Figures

Figure 1
Figure 1. Schematic illustration of experimental settings.
(A) Dimensions of social and lonesome microwells. (B) Experimental scheme for NK cell-laden microwell-based IL-2 stimulation assays.
Figure 2
Figure 2. Single cell-based quantitative analysis of NK cells in microwells revealed that contact-mediated interactions significantly enhanced IL-2 signaling in NK cells.
(A,B) Representative fluorescence images of NK cells in lonesome (A) and social (B) microwells. NK cells in microwells were stimulated with IL-2 for 18 h, fixed, stained, and imaged. Scale bar: 10 μm. (C,D) CD25 vs. pSTAT5 fluorescence intensity (arbitrary unit, a.u.) of NK cells in lonesome (C) and social (D) microwells. Individual dots represent the fluorescence intensity of individual cells. Red box: CD25lowpSTAT5high population; blue box: CD25high population. N = 86 (social) and 75 (lonesome). (E) pSTAT5 and CD25 fluorescence intensity comparison between NK cells in lonesome vs. social microwells. (NK cells were stimulated with IL-2 for 18 h.) N = 86 (social), and N = 86 (lonesome). (F) CD25 and Ki-67 fluorescence intensity comparison between NK cells in lonesome vs. social microwells. (NK cells were stimulated with IL-2 for 36 h.) N = 83 (social), and N = 83 (lonesome). Data are representative of three independent experiments. Error bars: standard deviation. The Mann-Whitney test was performed. NS: not significant, * p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3
Figure 3. NK cells in social microwells polarized CD25, MTOC, and lytic granules toward neighboring NK cells.
(A) Representative images showing the distributions of CD25, CD132, and the MTOCs of NK cells in social microwells. (B) Representative images showing the distributions of lytic granules and the MTOCs of NK cells in social microwells. (C) Quantification of lytic granule convergence to the MTOC. N = 248 (social), and N = 60 (lonesome) (D) Percentage of NK cells exhibiting lytic granules pointing toward neighboring NK cells (Face NK; black bar) or pointing away from neighboring NK cells (Face away; white bar). N = 258. (E) Distribution of lytic granule polarization durations. N = 200. (A,B) Scale bar: 10 μm. (C,D) Data are representative of three independent experiments. Error bars: standard deviation. The Mann-Whitney test was performed. **p < 0.01, ***p < 0.001.
Figure 4
Figure 4. Proliferation of CD25KO NK cells was significantly enhanced when they were in contact with WT NK cells indicating that IL-2 trans-presentation occurred within multi-cellular clusters of NK cells.
Microwell experiments using WT NK cells alone (WT-alone), CD25KO NK cells alone (CD25KO-alone), and a 50:50 mixture of WT (WT-mix) and CD25KO NK cells (CD25KO-mix) were performed, and the percentage of Ki-67 + NK cells were measured using immunofluorescence microscopy. For each case, N > 300 for social microwells, and N > 150 for lonesome microwells. (A) Representative images showing fluorescence staining of WT NK cells and Ki-67 in MIX experiments. White arrows: Ki-67 + WT NK cells. Yellow arrows: Ki-67 + CD25KO NK cells. Scale bar: 10 μm. (B,C) Percentage of Ki-67 + NK cells in each experimental condition. Statistical analysis is conducted for the same type of cells in different types of microwells (B) or different types of cells in the same type of microwell (C). Data are representative of three independent experiments. Error bars: standard deviation. The Mann-Whitney test was performed for pair-wise comparison, and one-way ANOVA test was performed for group comparison. NS: not significant, *p < 0.05, ***p < 0.001.

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