Anti granzyme b

Immunofluorescence staining utilized tyramide signal amplification staining performed using OPAL Reagents (Akoya Biosciences, Inc, Marlborough, MA). Briefly, tumor tissues were formalin fixed and embedded in paraffin blocks and cut into 10 μm sections at the Cedars-Sinai Biobank and Research Pathology Core. Tissue sections were deparaffinized and rehydrated, then antigen retrieval was performed in Tris-EDTA or citrate buffer. Primary antibodies anti-CD8 (ebioscience, 4SM15), anti-CD4 (R&D, GK1.5), anti-CD90 (Sino Biological, Cat #50461-T44), anti-CD73 (Sino Biological, Cat # 50231-T56), anti-GranzymeB (ebioscience, 16G6), anti-CD90.1 (BioLegend, OX-7), anti-CD45.1 (Invitrogen, A20) diluted to 1:200 were incubated overnight at 4°C. Secondary antibodies conjugated to HRP-polymers (Abcam, Cambridge, UK) were incubated for 15 min and then washed before slides were incubated with OPAL fluorophores (OPAL 520, OPAL, 650, OPAL 570) diluted 1:200 for 10 min at RT. Slides were washed in PBST before performing subsequent rounds of antigen retrieval and staining. Tissues were mounted with ProLong Gold Antifade Mountant with DNA Stain DAPI (Invitrogen). Images were acquired on ECHO Revolution upright microscope equipped with sCMOS Mono camera.

pDCs were harvested after activation with IMQ and CpG for 48 hours, and stained with the following mAbs: anti-CD11c, anti-CD11b, anti-B220, anti-CD80, anti-CD86, anti-MHC II (BD Pharmingen, USA), anti-TRAIL, and anti-Granzyme B (eBioscience, USA). Mice were injected i.t. with resting or activated pDCs, and sacrificed on day 2 or 5. Single-cell suspensions were prepared from tumor tissues. After enzymatic digestion for 30 minutes at 37°C with type IA collagenase (1 mg/mL) and DNase (0.1 mg/mL), red blood cells were lysed with Pharmlyse Buffer (BD Biosciences, USA); and stained with the following mAbs: anti-CD45, anti-NK, anti-CD3, anti-CD8, anti-TRAIL, anti-NKG2D, anti-NKG2A, and CD107 (BD Pharmingen, USA). Intracellular staining was performed using a cell permeabilization kit (Fix & Perm; An Der Grub). After incubation with the respective Abs for 20 minutes at 4°C, cells were washed twice and subjected to flow cytometric analysis. FACS plots depict the mean fluorescence intensity (MFI) values of Ab staining after subtraction of the MFI of the respective isotype.

The following antibodies were used in experiments; conjugated antibodies for flow cytometry: anti-CD8α (clone, 53-6.7), anti-CD4 (clone, RM4–5), anti-IFNγ, anti-CD3, anti-Granzyme B (ebioscience). Antibodies for IHC: anti-PD1 and anti-FOXP3 (Cell Signaling Technology), anti-CD8 and anti-CD4 (Abcam)

Cells were stained with fixable viability dye (eBioscience-Thermo Fisher Scientific, Bleiswijk, The Netherlands) and then with the following antibodies for surface staining: anti-TCRVα7.2, anti-CD161, anti-CD3, anti-ST2, anti-CD4 and anti-CD8 (SONY biotechnology, Weybridge, United Kingdom) (eBiosciences-Thermo Fisher Scientific) (Biolegend, Amsterdam, The Netherlands). MAIT cells were identified as TCRVα7.2⁺CD161⁺ or MR1 5-OP-RU tetramer⁺ cells. MR1 6-FP tetramer⁺ was used as negative control (NIH Tetramer Facility, Atlanta, GA, USA) (Figure S1). For intracellular cytokine staining, cells were fixed in 4% paraformaldehyde, washed, and permeabilized with 0.5% saponin (Sigma-Aldrich), and then incubated with anti-IFNγ, anti-TNFα, anti-Granzyme B or isotype controls (eBiosciences-Thermo Fisher Scientific, SONY biotechnology or BioLegend). In some experiments, MAIT cells were stained intracellularly with anti-Tbet (BD Biosciences, Grenoble, France) and anti-PLZF (R&D Systems) antibodies using a transcription factor buffer set (BD Biosciences). Events were acquired on a FACS LSR Fortessa (BD Biosciences) and analyzed using FlowJo software v10.7.1 (Becton, Dickinson and Company, Ashland, OR, USA).

After PET imaging, animals were sacrificed and tissue in the region of LPS/PBS injections were dissected, placed in 4% paraformaldehyde for overnight incubation, and transferred to 30% sucrose solution overnight. Subsequently they were embedded in O.C.T. medium (Fischer Scientific, Waltham, MA, USA) and frozen overnight at −80 degrees. Samples were sectioned on a cryostat at 5–10-micron sections.
Anti-Granzyme B (Cat #4059, Abcam, Cambridge, MA, USA) immunofluorescence was performed according to a standard protocol [21 (link)] using Anti-Granzyme B primary antibody 1:100 and a secondary goat anti-rabbit antibody 1:1000 (Thermo Fisher, Waltham, MA, USA). Slides were imaged on an Echo Revolve 332 microscope (Echo) at 4×, 10×, and 40× magnification.