Intracellular profiling of GrM activity by bioluminescent gain-of-function biosensors. (A) Schematic overview of the GrM GloSensor (left) (based on Promega backbone) and the GrM iGLuc sensor (right). The GloSensor circularly permuted firefly luciferase (FLuc) molecule is locked in an inactive conformation by a short peptide linker containing a GrM-specific recognition sequence (AKMPL↓AAEEE). Upon cleavage of the linker by GrM, the FLuc subunits undergo a conformational change, resulting in the formation of active FLuc. The GrM iGLuc sensor is a fusion protein of pro–IL 1β and gaussia luciferase (GLuc). Upon cleavage of the AKMPL↓AAEEE recognition sequence by GrM, the prodomain is separated from the IL-1β and GLuc domains, leading to GLuc monomerization, resulting in sensor activation and secretion. (B) GrM GloSensors and iGLuc sensors were produced in vitro using a cell-free transcription/translation system (Protease-Glo Assay and TNT Sp6 High-Yield Wheat Germ Master Mix; Promega) and were subsequently incubated at 37°C for 30 minutes with increasing concentrations of purified GrM or GrM-SA (an inactive GrM mutant in which the catalytic Ser residue has been mutated to Ala) in 50 mM Tris, 150 mM NaCl, pH 7.4. Resulting relative luminescence units (RLUs) were detected with a Veritas Microplate Luminometer (Promega). (C) The GrM and mock GloSensors and iGLuc sensors were labeled with fluorescent green lysines (FluoroTect Green Lys; Promega) during in vitro transcription/translation and incubated at 37°C for 30 minutes with indicated concentrations of GrM or the maximal dose of GrM-SA. Samples were separated on SDS-PAGE and visualized on a Typhoon 9410 scanner (GE Healthcare). Full-length GloSensor (∼61 kDa) and its cleavage fragments (∼36 and 25 kDa) and full-length iGLuc sensor (∼60 kDa) and its largest cleavage fragment (∼40 kDa) are visible. (D) GrM GloSensors and iGLuc sensors were produced as in panel B and incubated at 37°C for 30 minutes with recombinant GrA, GrB, GrH, GrK, or GrM (50 nM). Gr’s were produced in Pichia pastoris, and Gr activity was verified as described previously.⁶  (E) GrM sensors were cloned into a lentiviral pLV plasmid for transduction of OPM-2 multiple myeloma cells. Sensor expression was verified with immunoblot and flow cytometry (using anti-FLuc and anti-FLAG antibodies). Freeze-thaw lysates (10 µg) of GloSensor- or iGLuc-transduced OPM-2 cells were treated with indicated concentrations of GrM or GrM-SA for 1 hour at 37°C, after which Bright-Glo or Stop&Glo reagent (Promega) was added and luminescence was measured. (F) GloSensor-transduced OPM-2 cells were cocultured with KHYG-1 cells for 2 hours in the presence of 300 µg/mL d-luciferin in increasing effector/target (E:T) ratios. iGLuc sensor-transduced OPM-2 cells were cocultured with KHYG-1 cells in increasing E:T ratios. After 2 hours, 4.4 µM coelenterazine was added, and luminescence was measured. (G) Peripheral blood mononuclear cells were isolated from whole blood using density gradient centrifugation. Sorting by fluorescence-activated cell sorter was then used to isolate CD3⁻ CD56⁺ primary NK cells. NK cells were stimulated with IL-2 for 20 hours and subsequently cocultured with sensor-expressing HeLa target cells for 3 hours. Sensor activation was detected with LARII (Dual Luciferase kit; Promega) or coelenterazine for the GloSensors and iGLuc sensors, respectively. (H) Peripheral blood mononuclear cells were stimulated with IL-2 for 4 days to generate lymphokine-activated killer (LAK) cells. These were then cocultured with sensor-expressing HeLa cells as described in panel G. (I) Target cell death induced by coculture with LAK cells was measured after 16 hours using annexin V/propidium iodide flow cytometry. Annexin V/propidium iodide double-negative cells were considered viable (with viability set at 100% for untreated cells). (J) iGLuc-transduced OPM-2 cells and KHYG-1 cells were pretreated with 100 µM GrM-inhibitor Ac-KVPL-cmk or dimethyl sulfoxide (DMSO) (vehicle control) for 30 minutes, after which they were cocultured in the presence of Ac-KVPL-cmk or DMSO for 2 hours as in panel F. All data in this figure are depicted as mean ± standard deviation and represent at least 3 independent experiments.