Granzyme b

The levels of IFN-γ (R&D Systems, Minneapolis, MN, USA), granzyme B (R&D Systems, Minneapolis, MN, USA), IL-12p70 (R&D Systems, Minneapolis, Minnesota, USA), IL-12p40 (Invitrogen, Waltham, MA, USA), IL-15 (Invitrogen, Waltham, MA, USA), and IL-18 (R&D Systems, Minneapolis, MN, USA) in the BALF, lung extract, and cell supernatants were measured using ELISA kits, following the manufacturer’s instructions.

Supernatants from experiments were analyzed using ELISA according to the manufacturer’s instructions (IFNγ, Thermo Fisher Scientific, 15501107; IFN-α, PBL Assay Science, 42120-2; IL-2, Thermo Fisher Scientific, 15133787; IL-12p70, Thermo Fisher Scientific, 12384003; granzyme B, R&D Systems, DY1865).

4T1_P and 4T1_M tumor tissues were placed in a 1.6 mL tube containing RIPA buffer (5M NaCl (Fisher Chemical, Cat# 231-598-3), 0.5M EDTA (Sigma-Aldrich, Cat# EDS) pH=8, 1M Tris (Alfa Aesar, Cat# A12274) pH=8, 1% NP-40, 10% sodium deoxycholate (Sigma-Aldrich, Cat#D6750), 10% SDS (Fisher Scientific, Cat# BP2436-1), protease inhibitor cocktail (1:100, Roche, Cat# 11697498001) and phosphatase inhibitor (1:20 PhosSTOP, Roche, Cat# 4906845001). Stainless steel beads (Cat# SSB14B, Next Advance, New York, USA) were added and tumor tissue was homogenized using the Bullet Blender Tissue Homogenizer (Next Advance) according to the manufacturer’s protocol. The homogenate was centrifuged and supernatant was collected. The protein concentration of the tumor lysates was determined using Protein Assay Dye Reagent Concentrate (Bio-Rad, California, USA, Cat# 500-0006). The quantification of IFNγ and TNFα was carried out by using the LEGENDplex Mouse Th1/Th2 Panel (BioLegend, San Diego, CA, USA, Cat# 741053), in accordance with the manufacturer’s instructions. In addition, IFNα (Invitrogen, Cat# BMS6027) and Granzyme B (R&D Systems, Minneapolis, MN, USA, Cat# DY1865-05) were quantified by specific ELISA according to the manufacturer's instructions. All experiments were performed using at least three biological repeats.

Cloned 100 k CD8⁺ T cells were co-cultured with HLA-matched 50 k T2 cells line pulsed with 1 μM of the peptide in 200 μL of CTL media. After 16 hours of incubation, each 50 μL of the supernatant was extracted for analysis by standard ELISA protocols for TNF-α (R&D Systems, DY210–05), IFN-γ (R&DSystems, DY285B-05), and Granzyme B (R&D Systems, DY2906–05) and by Non-Radioactive Cytotoxicity Assay (Promega, G1780) for LDH measurement. Every experiment was duplicated.

Cytotoxic T cell activity was determined as described before [69 (link)]. Briefly, five days post infection splenocytes from naive syngeneic donor mice were prepared, split into equal populations, loaded with or without 1μM OVA peptide S8L (SIINFEKL) and with CFSE (1 μM or 0.1 μM respectively). Both populations were mixed at equal numbers, every mouse received 1x10⁷ of total cells. 18h later, mice were sacrificed and spleens were isolated. CFSE positive cells were acquired by flow cytometry and both cell populations were determined. The specific lysis was determined as follows: 100-(ratio sample/ratio average of naive) x 100. Splenocytes and brain leukocytes were stained and analyzed for expression of CD4, CD8, CD11b, CD11c, CD45, CD69, CCR5 and Granzyme B in a FACS Canto II flow cytometer (BD, San Jose, USA).
Data were analysed with FlowJo Software (Treestar Inc., Ashland, USA). ELISA IFNγ (e-Bioscience, San Diego, USA), TNF, CCL5 and Granzyme B (R&D, Minneapolis, USA) ELISA-kits were used according to the manufacturers protocol. The TNF antibody has not been tested by the company for crossreactivity with lymphotoxin (LT).