Granzyme b pe

The following Abs were used: CD4‐APC, CD3‐PERCP, CD28‐FITC, CX3CR1‐PE, CD27‐PE, Granzyme‐B‐PE, and isotype‐matched controls (BioLegend). For intracellular staining, cells were fixed, permeabilized with a Fixation/Permeabilization kit (Immunostep, Salamanca, Spain), and stained with anti‐Lck and antiphospho‐Zap‐70 Abs (Cell Signaling Technology, Danvers, MA, USA) and a secondary antibody conjugated to phycoerythrin (Biolegend). The active form of caspase‐1 was determined with the FAM‐FLICA in vitro caspase‐1 detection kit (ImmunoChemistry Technologies, Bloomington, MN, USA). Apoptosis was quantified by staining with CD28‐PE and CD4‐APC and FITC Annexin V/7AAD Apoptosis detection kit, following the manufacturer's instructions. Cells were analyzed on a Gallios Flow Cytometer (Beckman Coulter, Inc. Fullerton, CA, USA)

To obtain single-cell suspensions, the mouse tumor tissues were minced and digested, and then filtered through 70μm strainers. Erythrocytes in the cell suspension were lysed by RBC lysis buffer (Biolegend, USA), and the supernatant was removed by centrifugation. Cells were washed with PBS and stained for flow cytometry with the following antibodies: CD3-APC, CD45-PE-cy7/FITC, CD4-FITC, CD8-PE/pecy7, CD11b-PE, GR1-PC5.5, F4/80-APC, CD206-FITC, His-APC, Ly6G-PC5.5, TREM2-PE, PD-1-FITC, IFN-γ-PE, PE Goat anti-mouse IgG, Granzyme B-PE, and Perforin-PE (BioLegend).

For in vitro assays, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (Lymphoprep) density-gradient centrifugation and incubated on plates coated with anti-CD3 (3 μg/ml) and anti-CD28 (1 μg/ml) mAbs (Biolegend) or with IL-2 (100 U/ml) and IL-15 (10 ng/ml)(PeproTech EC Ltd, London, UK) for activation of CD8⁺ T and NK cells, respectively. CD107a-FITC (clone H4A3) antibody was added to the wells followed by monesin (6 μg/ml, Biolegend) and cultured for 6 h before analysis on a Gallios Flow Cytometer using Kaluza 1.3 analysis software (Beckman Coulter, CA, USA). For IFN-γ production, activated CD8⁺ T lymphocytes were treated for 5 h with brefeldin A (10 μg/ml, Biolegend), and cell surfaces were stained with CD8-PE and CD3-PerCP mAbs, followed by intracellular staining with anti-INFγ-FITC for 30 min at 4°C. Similarly, PBMCs were incubated with CD8-PE, CD3-PerCP and CD16-APC mAbs, followed by intracellular staining with granzyme B-PE and perforin-FITC mAbs (Biolegend). In both cases, a fixation / permeabilization kit (Immunostep, Salamanca, Spain) was used and the 7-Aminoactinomycin D (7AAD) fluoresecent marker (Biolegend) was included to excluded dead cells from the analysis. Cells were further analyzed by flow cytometry.

Tumor cell suspensions were stained, and single-cell analysis was performed by flow cytometry using 8-parameter flow cytometry on a FACS Canto (BD). To identify positive and negative populations, gates were set based on pilot experiments and our experience running similar experiments. Fluorescence minus one, isotype controls, and/or negative samples were used to determine gate boundaries. Additionally, antibody titration was used to optimize antibody panels. Immune phenotyping of samples was purchased from Biolegend (FVD eFluor 660, hCD3-Alexa Fluor 700, hCD8 PerCP-Cy5.5, mCD45 PE-CY7, hCD45 APC-eFluor 780, hCD279 FITC, Granzyme B PE, hCD4 FITC, hCD25 PE-Cy7, Foxp3 PE, hFoxp3 PE, hIFN-γ PerCP-Cy5.5).

Leukocytes from previously immunized (NP, NP-Tag, CpG + Tag, and CpG-NP-Tag) mice were restimulated with tumor cells. A total of 5 × 10⁴ 4T1 tumor cells were seeded in a flat-bottom 24-well plate overnight, and subsequently, 1 × 10⁶ of leukocytes were added and incubated at 37 °C with 5% CO₂. After one hour of incubation, Golgi plug (BD biosciences)—the protein transport inhibitor containing brefeldin A, was added. Unstimulated pneumocytes were used as the negative control. After 5 h of incubation, pneumocytes were harvested and suspended in FACS buffer to stain with the fluorochrome antibody cocktail: CD3-APC (clone 145-2C11; Tonbo Biosciences, San Diego, CA), CD4-BV711 (clone RM 4-5; Biolegend, San Diego, CA, USA), CD8-BV650 (clone 53-6.7; Biolegend, San Diego, CA, USA), CD69-APC-CY7 (clone H1.2F3; Tonbo Biosciences, San Diego, CA, USA), CD103-FITC (clone QA17A24; Biolegend, San Diego, CA, USA). For the staining of intracellular granzyme B (GZMB), cells were fixed and permeabilized with BD cytofix/cytoperm (BD biosciences), according to the manufacturer’s instructions, followed by intracellular staining with granzyme B-PE (clone QA18A28; Biolegend, San Diego, CA, USA) and data acquired as outlined in Section 2.10.

Activated T cells were obtained as described above. Once adhering to the plates, SW480 cells were treated with IFN-γ for 24 h, incubated with ACY-1215 (0, 2.5, 5.0 or 10.0µM) for 24 h, then processed with activated T cells for 24 h. The ratio between tumor cells and activated T cells is 1:8. Before T cells were collected, the tumor cells and T cells were co-cultured for 3 h with the addition of the Golgi transport blocker 25 ng/mL BFA (Selleck). Then T cells were collected and stained with cell membrane antibodies APC/Cvanine7-CD3 (Cat No. 300,317, BioLegend), PE/Cyanine7-CD8a (Cat No. 300,913, BioLegend). After pretreatment with a membrane breaker, the fluorescently labeled cytokine Brilliant Violet 421^TM-IFN-γ (Cat No. 502,531, BioLegend) and PE-Granzyme B (Cat No. 3,722,071, BioLegend) antibody was added to incubate. In addition, to detect the expression of PD-L1 on the membrane of single-cell suspension from human cell lines in culture, the PE-PD-L1 (Cat No. 329,706, BioLegend) antibody is used. Flow cytometry was performed using the BD FACS Calibur flow cytometer. The FlowJo v10 software (Treestar) was used for data analysis.

Antibodies for APC-CD3, PE-CD4, PE/Cy7-CD8, Alexa Fluor647-CD56, PE-CD107a, PE-CCR7, PE/Cy7-CD62L, PE/Cy7-perforin, PE-granzyme B, PE-PD-L1 were purchased from BioLegend. Goat anti-B7-H3 antibody (MAB1027) was purchased from R&D System. Humanized anti-B7-H3 IgG (8H9) was expressed and purified in the laboratory (for details, see Additional file 1). Mouse anti-CD16 antibody (3G8) was purchased from BD Biosciences. PE-conjugated anti-human Fc antibody and HRP anti-human IgG antibody were purchased from Invitrogen. HRP-conjugated rabbit anti-goat IgG antibodies were purchased from Jackson ImmunoResearch. Rabbit antihuman PD-L1 antibody (13,684), HRP-conjugated anti-β-actin and anti-GAPDH antibodies were purchased from Cell Signaling.