Fluorogenic Granzyme A Substrates Enable Real‐Time Imaging of Adaptive Immune Cell Activity

Abstract Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near‐infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity‐based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild‐type and GzmA knock‐out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen‐driven recognition of cancer cells in real time.


S16
Cell co-cultures, flow cytometry and real-time imaging.HaCat cells were purchased from ATCC and grown in complete DMEM.For HaCaT cell imaging, cells were plated at 2,000 cells per well in complete DMEM and incubated with hemicyanine or compound 9 (both at 5 µM) and Hoechst 33342 at 37 ºC.Confocal images were acquired on Leica SP8 and images were analysed using Leica Application Suite X. RAW264.7 cells were purchased from ATCC and were grown in complete RPMI supplemented with 10% foetal calf serum (FCS) and antibiotics (100 U mL -1 penicillin and 100 µg mL -1 streptomycin).For RAW264.7 imaging, cells were plated at 1,000 cells per well in complete RPMI and incubated with hemicyanine or compound 9 (both at 5 µM) and Hoechst 33342 at 37 ºC.For control experiment, the RAW cells were pre-incubated with 1 µM E-64 for 1 h at 37 ºC before incubated with compound 10.
Confocal images were acquired on Leica SP8 and images were analysed using Leica Application Suite X. EL4 cells were purchased from ATCC and grown in complete RMPI supplemented with 10% heat inactivated fetal bovine serum, 1% penicillin/streptomycin, 2 mM L-glutamine and 40 µM β-mercaptoethanol.For coculture experiments, EL4 cells were pulsed with SIINFEKL peptide to be recognized by OT-I T cells.OT-I TCR transgenic T cells were generated in vitro from cell isolates from the spleen and lymph nodes of OT-I mice.Splenocytes were pulsed with SIINFEKL and put in co-culture with cells from the lymph nodes.The TCR based recognition of SIINFEKL resulted in the expansion of antigen specific OT-I T cells.
Cells were expanded for 5 days and used at day 5 for cytotoxicity experiments.EL4 cells were pulsed with SIINFEKL, 2.5 µM compound 10 and 3 µM probe H5 for 1 h at 37 ºC in complete RPMI.After incubation cells were washed three times.EL4 cells were incubated with or without OT-I T cells for 4 h 37 ºC.Samples were stained with S17 CD8-Percp Cy5.5 antibody, DAPI and Annexin V, and acquired on the BD Fortessa flow cytometer.Flow cytometric analysis was performed using Flowjo.
For real-time imaging, EL4 cells were seeded onto fibronectin coated chambers and allowed to settle.OT-I T cells were added to the EL4 and imaged on the Nikon A1R for 4 h.Image analysis was performed using Imaris.
Measurements of mGzmA activity in mouse sera and ex vivo mouse tissues.All animal work was ethically approved and conducted at the University of Zaragoza under the control of the animal welfare committee (project number: 64/17).Blood was collected by cardiac puncture after pentothal administration, and sera was extracted by centrifugation.Spleen and liver tissues were harvested and lysed for ex vivo analysis.Compounds 9 or 10 (20 nM) were incubated with 2.25 µL of mouse serum or spleen/liver lysates in a final volume of 45 µL for 3 h at 37 ºC.Fluorescence measurements were performed in a fluorescence spectrophotometer (λexc: 680 nm).

Chemical synthesis and characterization
Solid-phase peptide synthesis.
Resin loading.Fmoc-AA-OH (1 eq) were attached to the resin with DIPEA (3 eq) in DCM at r.t. for 10 min and then DIPEA (7 eq) for 40 min.The remaining 2-chlorotrityl groups were capped by addition of MeOH (0.8 mL g -1 resin) for 10 min.After that, the resin was filtered off and washed with DCM (5 × 1 min), DMF (5 × 1 min).The loading of the resin was determined by titration of the Fmoc group using UV-Vis measurements.
Amino acid couplings.After the Fmoc groups were removed, resins were washed with DMF (5 × 1 min), DCM (5 × 1 min) and DMF (5 × 1 min).A solution of the appropriate Fmoc-AA-OH (4 eq), COMU (4 eq), Oxyma (4 eq) and DIPEA (8 eq) was stirred for 1 min in DMF before addition to the resin previously swollen in DMF.The mixture was then stirred for 1 h at r.t.The solution was filtered off and the resins were washed with DMF (5 × 1 min) and DCM (5 × 1 min).The completion of the couplings was monitored by the Kaiser test.
Cleavage.Resin-bound peptides were treated with 2% TFA in DCM (5 × 1 min) and washed with DCM.The combined filtered mixtures were poured over DCM and evaporated under reduced pressure.The peptides were isolated by precipitation in ice-cold Et2O generating white solids (HPLC purities > 95%).
Protecting group removal in solution.The peptides were treated with a mixture of 95% TFA, 2.5% TIPS and 2.5% DCM for 1 h and evaporated under reduced pressure.
The peptides were isolated by precipitation in ice-cold Et2O generating white solids.

Figure S2 .Figure S3 .
Figure S2.HPLC-MS traces of compound 9 before and after reaction with hGzmA.Compound 9 (20 µM) was incubated with hGzmA (20 nM) in Tris buffer (pH 8) at 37°C for 90 min.HPLC traces were monitored at 650 nm before and after the

Figure S13 .Figure S14 .
Figure S13.Fluorescence confocal microscopy images of live RAW 264.7 cells upon incubation with compound 10.Live RAW 264.7 cells were incubated with compound 10 (red, 5 µM) for 1 h and Hoechst (blue) was used for nuclear counterstaining.E-64 (1 µM) was used as a cathepsin inhibitor for 1 h prior to labeling.