Kimberly Haupt, PhD: No relevant disclosure to display
Introduction/Rationale: Granzyme B (GZB), a serine protease secreted by cytotoxic T lymphocytes and natural killer cells, plays a critical role in immune defense by inducing apoptosis in target cells. Monitoring GZB production, e.g., during T cell activation, provides a direct functional readout of cytotoxic potential, distinguishing it from surface markers CD69 and CD25 that indicate activation status. Current methods for measuring GZB protein levels (flow cytometry and ELISA) involve multi-step, lengthy protocols. We developed a bioluminescent GZB activity assay for simplified, rapid GZB analysis to facilitate the incorporation of this functional indicator into experimental workflows.
Methods: The assay reaction consists of a peptide-luciferin prosubstrate and luciferase. Proteolytic cleavage by active GZB releases luciferin, which is oxidized by the luciferase enzyme. The resulting light signal is directly proportional to GZB activity.
Results: We used the assay to monitor GZB production during T cell activation. T cells were isolated from peripheral blood mononuclear cells and stimulated under various media conditions. Samples were collected during the activation phase on Days 1-3; culture medium and cell lysates were analyzed. GZB activity increased over time and varied by activation condition. On day 1, signals from cell lysates were ~2 to10-fold higher than medium background, increasing to ~30 to 550-fold by day 3. Signals from non-activated cell lysates were not above background. GZB detected in culture medium also increased, reaching ~11 to 106-fold above background on day 3. These results were confirmed by measuring intracellular GZB levels by flow cytometry and ELISA. GZB activity also aligned with CD69 and CD25 expression, as well as IFN-γ and TNF-α cytokine secretion.
Conclusion: This bioluminescent GZB activity assay provides a rapid and simple method for monitoring GZB, especially for studies requiring higher-throughput sample analysis.
