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Single-Fluorescent Protein Reporters Allow Parallel Quantification of Natural Killer Cell-Mediated Granzyme and

Clarissa Liesche1, Patricia Sauer1, Isabel Prager2

  • 1Division of Theoretical Bioinformatics at German Cancer Research Center (DKFZ), Department for Bioinformatics and Functional Genomics, Institute for Pharmacy and Molecular Biotechnology, BioQuant Center, Heidelberg University, Heidelberg, Germany.

Frontiers in Immunology
|August 24, 2018
PubMed

Insights

This study introduces novel reporters to simultaneously measure protease activity in single cells, revealing caspase-3 activation follows granzyme B activity in cytotoxic lymphocyte responses.

Area of Science:

  • Immunology
  • Cell Biology
  • Biochemistry

Background:

  • Natural killer (NK) cells induce target cell death via granzymes or death receptors.
  • Quantifying single-cell responses is crucial for understanding NK cell cytotoxic mechanisms.
  • Existing reporters limit the simultaneous measurement of multiple protease activities.

Purpose of the Study:

  • To develop a novel strategy for comparing two protease activities within individual target cells.
  • To enable the simultaneous measurement of granzyme and caspase activities.
  • To investigate the temporal relationship between granzyme B and caspase activation.

Main Methods:

  • Development of single-fluorescent protein reporters with specific cleavage sites (RIEAD, VGPD) for granzyme B.
  • Application of reporters in combination with caspase-8 and caspase-3 reporters.
  • Simultaneous measurement of protease activities in single target cells upon NK cell engagement.

Main Results:

  • Demonstrated the ability to compare two different protease activities within individual target cells.
  • Showed that caspase-3 activity follows granzyme B activity with a delay of approximately 6 minutes.
  • Did not find evidence of granzyme B activating caspase-8.

Conclusions:

  • The developed reporter system allows for the simultaneous measurement of multiple protease activities in single cells.
  • Provides insights into the temporal dynamics of cell death pathways mediated by cytotoxic lymphocytes.
  • Facilitates comparative analysis of various granzyme activities (A, M, K, H) in conjunction with caspases.
Keywords:
apoptosis and cell deathcytotoxic lymphocytesgranzyme and caspase activitynatural killer cellssingle-fluorescent protein reporters

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