Multiplex Analysis of Secreted Biomarkers from Stimulated Natural Killer Cells

R. Fuerstenberg, S. Anderson, K. Brumbaugh, J. Schmidt

ABSTRACT

NK (natural killer) cells form a branch of the innate immune system that destroys compromised host cells, such as tumor cells or virus infected cells, by recognizing such cells through a “missing self” mechanism. Over the past decade, NK cells have come under greater scrutiny as major regulators of the host’s immune response via the secretion of cytokines, chemokines, and other factors. In addition, studies of the mechanisms of NK cells as antitumor effector cells have advanced our understanding of potential cancer therapies. NK cells have traditionally been identified phenotypically by the detection of cell surface markers using flow cytometric methods. The known ability of NK cells to secrete cytokines and other factors provides an opportunity to identify the secretory profile of NK cells under specific conditions as an alternative to flow cytometry. In this study, human NK cells were enriched from peripheral blood using a negative selection method and cultured overnight either unstimulated or stimulated with IL-12. The cell culture supernatants from both conditions were analyzed for 158 secreted biomarkers in large multiplex immunoassays using Luminex^® Screening Assays. The biomarkers that were produced at increased levels following stimulation were analyzed further to establish a secretory profile for IL-12 activated NK cells.

INTRODUCTION

Human Natural Killer (NK) cells were first recognized as a lymphocyte subset in 1975. Mature NK cells represent approximately 15% of the circulating lymphocytes.¹ In humans, mature NK cells are usually defined as CD3⁻CD56⁺ cells and can be further subdivided based on CD56 expression. In general, CD56^dim NK cells include the majority (90%) of peripheral blood NK cells, whereas CD56^bright NK cells are more abundant in secondary lymphoid tissues.²

NK cells differ from T and B lymphocytes in that they do not show germ-line receptor rearrangement. Their effector functions are ruled by a mixture of activating and inhibitory receptors. These receptors allow the recognition of altered-self at a cellular level, rather than at the molecular level as with T-cell receptors and immunoglobulins.¹

NK cells recognize and destroy malignant and virus infected cells by responding to signals generated by activating and inhibitory receptors on their surfaces.² Upon contact with an appropriate target, NK cells release the membrane-disrupting protein perforin and granzyme proteases from secretory granules.²

A second essential function of NK cells, especially in viral infections, is to release antiviral cytokines such as IFN-gamma and TNF-alpha, which act as immunodefensive agents that serve to activate and recruit resident inflammatory and other cells.² These NK cell-drived cytokines regulate dendritic cells, T cells, and B cells and regulate TNF-mediated apoptosis of the NK cells and neighboring cells.² Thus, cytokine production by NK cells influences both innate and adaptive immune responses.²

Luminex Screening Assays are large multiplex panels that are valuable as tools for discovery. Luminex Screening Assay panels provide the highest plex sizes in the industry and offer an ever increasing menu of biomarkers available for screening. They are excellent tools for a wide array of research topics and require only small volumes of sample. Luminex Screening Assays maximize efficiency, sample use, and data generation by allowing the quantitative analysis of up to 100 biomarkers in a single assay.

In this study, we address the measurement of several protein biomarkers by using Human Luminex Screening Assays (Catalog # LXSAH). Human Luminex Screening Assays are designed for the simultaneous measurement of multiple human biomarkers in cell culture supernatants, serum, and plasma. Using the available markers, we evaluated levels in stimulated versus unstimulated NK cells, thereby establishing a secretory biomarker profile.

METHODS

Cells

NK cells were prepared from PBMCs using the MagCellect Human NK Cell Isolation Kit (Catalog # MAGH109). NK cells were either left unstimulated or were stimulated for 24 hours with 10 ng/mL recombinant human IL-12 (Catalog # 219-IL/CF). Cell culture supernatants were collected and evaluated in Human Luminex Screening Assays.

Biomarker Secretion Measurements

NK cell supernatant samples were diluted 1:2 in Diluent RD6-52 prior to assaying. Luminex Screening Assays (Catalog # LXSAH) were configured using the Luminex Assay Customization Tool to allow analysis of 158 biomarkers in the fewest possible assays. Four assays were configured to test all 158 analytes in the current menu. The concentration of each biomarker in each cell culture supernatant was quantified by comparison to standard curves.

Principle of the Assay

Analytes Tested

Figure 1

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Figure 1. A) Heat map showing the tested biomarkers. Colors correspond to high, mid, and low abundance. B) Quantitative comparison of biomarker concentrations between stimulated and unstimulated NK cells.

Figure 2

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Figure 2. A) Heat map showing the tested biomarkers. Colors correspond to high, mid, and low abundance. B) Quantitative comparison of biomarker concentrations between stimulated and unstimulated NK cells. *The increased IL-12 levels in stimulated cells was due to addition of IL-12 during the stimulation procedure.

Figure 3

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Figure 3. A) Heat map showing the tested biomarkers. Colors correspond to high, mid, and low abundance. B) Quantitative comparison of biomarker concentrations between stimulated and unstimulated NK cells.

DISCUSSION and CONCLUSION

These results illustrate the broad utility of R&D Systems Luminex Screening Assays by quantitatively comparing 158 different protein biomarkers simultaneously in a small volume of cell culture supernatant. Some of our observations are as follows:

• Increased levels of secreted biomarkers commonly associated with IL-12 stimulation of NK cells were observed. These included the cytokines TNF-alpha and IFN-gamma.
• An increase in MPO seems counterintuitive as MPO is often identified as an inhibitor of NK cell cytotoxicity, reducing its antitumor functionality.³ MPO is been shown in neutrophils to have antimicrobial activity suggesting a similar role for NK cells.^4,5
• Additionally, higher levels of chemokines such as CXCL10/IP10, CXCL4/PF4, CCL8/MCP-2, and CCL5/RANTES were also observed in stimulated NK cells when compared to the unstimulated control. Consistant with reports, secretion of these chemo-attractants indicates a role of NK cells in the recruitment of adaptive immune cells.⁶
• FGF-21, SPARC, MIF, PCSK9, and many others were also elevated in stimulated versus unstimulated NK cell supernatants.

These and other examples, when taken together, provide a comprehensive secretory profile for NK cells and a basis for further inquiry into the function of NK cells as an integral part of the immune system.

REFERENCES

1. Cheent, K. et al. (2009) J. Immunol. 126:449.
2. Reefman, E. et al. (2010) J. Immunol. 184:4852.
3. Betten, A. et al. (2001) J. Leukoc. Biol. 70:65.
4. Klebanoff, S. et al. (2013) J. Leukoc. Biol. 93:185.
5. Kumar,V. et al. (2010) Int. Immunopharmacol. 10:1325.
6. Roda, J. et al. (2006) Cancer Res. 66:517.

For research use only. Not for use in diagnostic procedures.

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