Granzyme B activity in target cells detects attack by cytotoxic lymphocytes

Area of Science:

• Immunology
• Cell Biology

Background:

• Lymphocyte-mediated cytotoxicity is crucial for immune surveillance.
• Granzyme B (GzB) is a key effector protease delivered by cytotoxic lymphocytes.
• Understanding GzB's fate within target cells is vital for studying cell death.

Purpose of the Study:

• To develop and utilize novel fluorogenic substrates to measure protease activity in living cells.
• To investigate the spatiotemporal dynamics of Granzyme B activity and caspase 3 activation in target cells following cytotoxic lymphocyte attack.
• To assess the impact of Bcl-2 overexpression on these cytotoxic pathways.

Main Methods:

• Development of a cell-permeable fluorogenic substrate for Granzyme B.
• Use of a second fluorogenic substrate specific for caspase 3.
• Application of flow cytometry and fluorescence confocal microscopy.
• Experiments involving cytotoxic T lymphocytes (CTLs), NK92 cells, and target cells (with and without Bcl-2 overexpression).

Main Results:

• Granzyme B activity was detected in the cytoplasm of target cells after effector-target engagement, but not in effector cells.
• Caspase 3 activation was observed subsequent to Granzyme B activity in all tested target cells.
• Overexpression of Bcl-2 had minimal impact on target cell lysis, GzB delivery, or caspase 3 activation.
• The sequential detection of GzB and caspase 3 activity provides a novel readout for cytotoxic lymphocyte-mediated cell death.

Conclusions:

• The study demonstrates a novel method for tracking lymphocyte-mediated cytotoxicity in real-time.
• Granzyme B activation precedes caspase 3 activation in the cytotoxic pathway.
• This sequential protease activation is a key indicator of lethal immune cell attack.