Siinfekl

WT, Il1r^−/− or Il18r^−/− OT-1 mice were used for T-cell isolation. Mice were sacrificed by cervical dislocation. Spleen and lymph nodes were meshed through a 30 μm strainer (Miltenyi, Gladbach, Germany). After centrifugation and erythrocyte lysis, the cells were adjusted to 4 × 10⁷ cells/mL. Then, 5 μM of the OVA peptide SIINFEKL (Invitrogen, Waltham, MA, USA) was added to the naïve T cells and incubated at 37 °C for 1 hour. T cells were seeded with T-cell media (RPMI-1640 medium supplemented with 10% FCS, 1% Pen-Strep, 0.1% β-mercaptoethanol, and 1% L-glutamine) and 10 ng/mL IL 12 (Peprotech, Cranbury, NJ, USA) in T75 flasks. After 48 h, the medium was changed, and 20 ng/mL IL-2 (Peprotech, Cranbury, NJ, USA) was added. This was followed by another 48 h of incubation with a subsequent medium change and IL-2 administration. After 5 days of incubation, the effector cells were used for further experiments.

For ELISpot assay, 10⁵ blood leukocytes were added to each well in triplicate in pre-coated PVDF-96 well plates and cultured in complete RPMI in the presence of 10⁴ irradiated (20Gy) tumor cells, OVA_257–264 peptide (SIINFEKL, 10 µg/mL, Invitrogen) or Concanavalin-A (Con-A, 5 μg/mL) as previously described [18 ]. The spot number was counted by using an ELISpot reader (Aelvis) and expressed as the number of spot-forming cells (SFC)/10⁵ cells.

Collagenase D (Cat#1108888200) and DNase I (Cat#1010415900) were purchased from Roche. Collagenase IV (Cat#C4-BIOC), Ovalbumin (Cat#S7951) and anti-chicken egg albumin (Ovalbumin) (Cat#C6534) were from Sigma. Ovalbumin-Alexa Fluor^TM 488 (Cat#O34781), Ovalbumin-Alexa Fluor 647^TM (Cat#O34784), Alexa Flour^TM 647 NHS Ester (Cat#A37573), CFSE (Cat#C34554), and SIINFEKL were from Invitrogen. Diphenyleneiodonium chloride (HY-100965) was from MEC. The expression plasmid for mCherry:Galectin-3 (Cat#85662) was purchased from Addgene. PE Annexin V Apoptosis Detection Kit I was purchased from BD bioscience (Cat#559763). Mouse IL-10 ELISA Kit was from ABclonal (Cat#RK00016). TNF-α was purchased from eBioscience. Z-VAD-FMK was from Calbiochem and Smac mimetic was from APExBIO (Cat#SM164).

For cell sorting and analysis, monoclonal antibodies to CD41 (MWReg30, eBioscience), CXCR4 (2B11, eBioscience), CD11b (M1/70, eBioscience), F4/80 (BM8, eBioscience), Gr-1 (RB6-8C5, Biolegend), Ly6C (HK1.4, Biolegend), CD11c (N418, eBioscience), CD45.1 (A20, eBioscience), CD45.2 (104, Biolegend), CD4 (GK1.5, eBioscience), CD8 (53–6.7, Biolegend), INF-γ (XMG1.2, Biolegend), IL4 (11B11, Biolegend), CD34 (RAM34, eBioscience), Sca-1 (D7, Biolegend), c-kit (2B8, Biolegend), CD135 (A2F10, Biolegend), CD3ε (145–2 C11, Biolegend), CD45R (RA3-6B2, Biolegend), TER-119 (Ter-119, Biolegend), IgM (II/41, eBioscience), FγRII (93, Biolegend), IL-7R (A7R34, Biolegend), TNFα (MP6-XT22, Invitrogen), IL-6 (MP5-20F3, Biolegend), OVA257-264 (SIINFEKL) peptide bound to H-2K^b (eBio25-D1.16 (25-D1.16), Invitrogen) and IL-2 (JES6-5H4, eBioscience) were used where indicated.

Blood leukocytes (10⁵ cells/well) were seeded in triplicate in pre-coated PVDF-96 well plates and incubated with OVA_257–264 peptide (SIINFEKL, 10 µg/mL, Invitrogen) or Concanavalin-A (Con-A, 5 μg/mL, Sigma) as previously described [43 (link)]. Unstimulated wells served as negative control. Spots were counted by using an ELISpot reader (Aelvis).