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This study compared 6-hour and 18-hour stimulation durations for natural killer cell degranulation using the CD107a assay, finding that the shorter duration was sufficient for assessing NK cell activity. The results showed that longer stimulation resulted in lower CD107a expression. The study's findings have implications for optimizing workflow efficiency in clinical immunology laboratories.

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Abstract

Background: Natural killer (NK) cell degranulation is a key immune defense mechanism where exposure to tumor or virus-infected cells triggers the fusion of cytoplasmic granules containing apoptotic proteins, perforin, and granzyme with the cell membrane. This process transiently expresses CD107a on the NK cell surface, and measuring CD107a is a standard method to assess NK cell activity. Methods: We compared two stimulation protocols differing only in duration (6-hour vs. 18-hour) using K562 target cells to induce NK cell degranulation. Isolated PBMCs without stimulation served as controls to assess spontaneous degranulation. Anti-CD107a-PE antibody was present throughout stimulation in both test and control samples. After stimulation, cells were stained with anti-CD45, anti-CD3, and anti-CD56 and analyzed by flow cytometry. Results: For 6 of 7 healthy controls, results from both methods fell within 2 standard deviations. Notably, longer (18-hour) stimulation resulted in lower CD107a expression than the 6-hour assay. Interlaboratory comparisons of two samples showed no significant difference (p 0.05). In a suspected hemophagocytic lymphohistiocytosis (HLH) case, two labs reported similarly reduced CD107a expression (9% and 7%). Inter-day variability was observed in a donor across both time points. The 6-hour assay showed higher sensitivity and specificity than the 18-hour assay. A resting period before ex vivo PBMC assays was found necessary. Conclusion: Stimulation periods beyond 6 hours are unsuitable for clinical NK degranulation assays. Screening for HLH should include multiple stimulants to improve assay reliability.

Key findings

  • 6-hour stimulation was sufficient for assessing NK cell activity in most healthy controls
  • 18-hour stimulation resulted in lower CD107a expression
  • Interlaboratory comparisons showed no significant difference between the two methods

Keywords

DegranulationStimulationGranzyme BImmunologyGranzymePeripheral blood mononuclear cell

Identifiers

DOI
10.64898/2026.03.02.708872
Journal
bioRxiv (Cold Spring Harbor Laboratory)
Year
2026