To the editor:
With great interest, we read the recent Blood article by Vrazo et al,1 who report on the use of luciferase-based biosensors to detect proteolytic activity of granzymes (Gr’s) A, B, and K. They show that these sensors can be used to profile Gr delivery by natural killer (NK) cells inside tumor cells in real time. However, humans express 5 Gr’s, and a specific sensor for GrM is lacking. GrM is expressed by lymphocytes of both the innate and adaptive immune system and plays a role in the control of cancer, viruses, and inflammation.2 Here, we report the development and comparison of 2 distinct variants of GrM gain-of-function biosensors: the GrM GloSensor, which is similar to the sensors described by Vrazo et al,1 and the GrM pro-interleukin (IL)-1β-gaussia luciferase (iGLuc) sensor3 (Figure 1A).
We previously characterized the extended substrate specificity of human GrM using complementary positional proteomics,4 identifying AKMPL↓AAEEE as an optimal cleavage recognition motif. Based on this sequence, we developed 2 GrM sensors and corresponding mock sensors (P1 Leu replaced by Ala). The GrM GloSensor is based on a circularly permuted FLuc,5 locked in an inactive state by a short peptide linker. Upon GrM-mediated cleavage of the linker, the FLuc becomes active. The GrM iGLuc sensor is a fusion of GLuc and murine pro-IL-1β.3 When GrM cleaves off the IL-1β prodomain, the GLuc monomerizes and becomes active.
We synthesized the GrM and mock sensors in vitro using cell-free transcription/translation and subsequently treated them with purified recombinant GrM or inactive GrM-SA mutant (Figure 1B). Both GrM sensors were activated by GrM in a concentration-dependent manner, but not by GrM-SA. The corresponding mock sensors were not activated, indicating that activation depends on GrM-mediated cleavage after the Leu residue in the peptide recognition sequences. Consistent with these data, visualization of the fluorescently labeled GrM sensors by SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) showed that the full-length Glo (∼61 kDa) and iGluc (∼60 kDa) sensors were cleaved by increasing concentrations of GrM, but not by GrM-SA, resulting in the formation of the expected subunits (∼36/∼25 and ∼40 kDa, respectively) (Figure 1C). To determine the specificity of the GrM sensors, they were incubated with all purified human Gr’s (Figure 1D).6 Of these, only GrM led to robust activation of the GrM sensors, and none of the Gr’s could activate the mock sensors.
We expressed the sensors in OPM-2 multiple myeloma cells and confirmed expression by immunoblot and flow cytometry (data not shown). Recombinant GrM activated the GrM sensors in freeze-thaw lysates of OPM-2 cells in a concentration-dependent manner, whereas GrM-SA did not (Figure 1E). Intracellular delivery of recombinant GrM via pore formation with streptolysin O in tumor cells also led to GrM sensor activation (data not shown).
To determine whether GrM activity can be profiled inside tumor cells that are attacked by cytotoxic lymphocytes, we cocultured GrM sensor-transduced OPM-2 cells with NK cells (KHYG-1), which express high levels of GrM.7 Increasing effector/target ratios resulted in increased activation of both GrM sensors (Figure 1F). Activation of the iGLuc sensor was much stronger, suggesting that the iGLuc sensor may outperform the GloSensor in a more physiological setting. The corresponding mock sensors were not activated. Activation of the sensors occurred within 2 hours, which is compatible with the proposed kinetics of Gr delivery.1,8 Similar results were obtained in HeLa cervix carcinoma cells (supplemental Figure 1, available on the Blood Web site). Primary NK and LAK cells also activated the GrM sensors (Figure 1G-H, respectively). In addition, sensor activation by LAK cells correlated with cell death induction in target cells (Figure 1I). To confirm that GrM sensor activation was attributable to GrM, iGLuc sensor–transduced OPM-2 cells and KHYG-1 NK cells were (pre-)incubated with the cell-permeable GrM-specific inhibitor Ac-KVPL-cmk.9 The tetrapeptide sequence KVPL is specific for GrM and is not recognized by the closely related neutrophil elastase and cathepsin G.10 Treatment with Ac-KVPL-cmk completely inhibited NK cell–mediated activation of the GrM iGLuc sensor (Figure 1J).
Our data nicely complement the recent data of Vrazo and coworkers,1 who developed GrA/K/B biosensors to profile tumor cell death kinetics induced by killer cells. We now report 2 new protease-cleavable biosensors that specifically track GrM proteolytic activity in the course of cytotoxic lymphocyte-induced cell death. This allows further studies to monitor entry and functional activity of all Gr’s in target cells during cancer development, inflammation, and virus infections.
The online version of this article contains a data supplement.
Acknowledgments: The authors thank Dr Veit Hornung (Institute of Molecular Medicine, University Hospital, University of Bonn, Bonn, Germany) for providing the basic iGLuc plasmid. This work was supported by grants from the Dutch Cancer Society (UU-2009-4302) (N.B.) and the Dutch Organization for Scientific Research (916.66.044) (N.B.).
Contribution: S.A.H.d.P. participated in the design of the study, performed experiments, analyzed and interpreted the data, and wrote the manuscript; E.A.v.E., J.M., R.B., M.C.O., and E.M.P.S. performed experiments; R.G. participated in the design of the study and interpreted the data; and N.B. participated in the design of the study, analyzed and interpreted the data, and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Niels Bovenschen, Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, Utrecht 3584 CX, The Netherlands; e-mail: email@example.com.