Tnfα clone mp6 xt22

The induction of collagen induced arthritis (CIA) in the C57BL/6 strain was performed as previously published [17 ]. Male WT and Ptpn22^−/− mice of 10–14 weeks of age were injected intradermally at the base of the tail with 100 μg chicken type II collagen (Sigma) emulsified in complete Freund's adjuvant. Clinical signs of arthritis were assessed visually in the wrist and ankle joints 3 times weekly, using a previously described severity scale: 0 = no arthritis; 1 = 1 inflamed digit; 2 = 2 inflamed digits; 3 = more than 2 digits and footpad inflamed; 4 = all digits and footpad inflamed [17 ]. Scoring was conducted under blinded conditions for up to 96 days. At day 96 single cell suspensions from lymph nodes (LN) and spleens were restimulated for 6 h with PMA (Sigma; 50 ng/ml) ionomycin (Sigma; 10 ng/ml) and monensin (Biolegend; 1 in 1000) and expression of IFNγ (clone XMG1.2; Biolegend), IL-17 (clone TC11-18H10.1; Biolegend), TNFα (clone MP6-XT22; Biolegend), IL-4 (clone 11B11; Biolegend) and IL-10 (clone JES5-16E3; Biolegend) determined by intracellular flow cytometry.

Poxvirus-specific T cell responses in murine splenocytes were measured as previously described [20] (link). Briefly, splenocytes isolated from VACV-infected mice (2×10⁶ PFU/mouse) at 8 days p.i. were stimulated with A20 cells infected with CPXV or VACV (MOI = 5, 16 h) in the presence of Brefeldin A for 6 h. Cells were stained overnight at 4°C with αCD3ε and αCD4 Ab's (clones 145-2C11 and RM 4–5, respectively, BD Biosciences), αCD8 Ab (clone 5H10, Life Technologies), Fc Block (BD Biosciences) and mouse IgG (Sigma). The next day, the cells were washed, fixed, and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) followed by intracellular staining with Ab to IFNγ (clone XMG1.2, BD Biosciences PharMingen) and TNFα (clone MP6-XT22, BioLegend, San Diego, CA). The samples were analyzed by flow cytometry as described above. Non-viable cells were excluded using a live cell gate based on Aqua staining, gated for lymphocytes based on forward and side scatter characteristics followed by gating for CD3ε+. Next, CD3ε+ T cells were gated on either CD4+ or CD8+ and IFNγ+TNFα+ T cells were quantified. Background IFNγ+TNFα+ events from uninfected samples were subtracted. T cell responses to CPXV and CPXV deletion mutants were normalized to VACV.

Mice were perfused with PBS before the brains were harvested. Brain tissues were pretreated with 2 μg/ml collagenase D and 1 μg/ml DNAse I (both Roche Diagnostics), and total cells were isolated by cell straining (100-μm mesh). Brain homogenates were separated into neuronal and leukocyte populations by discontinuous density gradient centrifugation using isotonic Percoll (GE HealthCare, Uppsala, Sweden) as described by Pino and Cardona [40 ]. After isolation, cells were stimulated in vitro with PMA and Ionomycin plus BD GolgiStop™ (BD Biosciences, San Jose, CA, USA) for at least 4 h at 37 °C. After stimulation, the cells were fixed with BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit and intracellular staining was carried out following the manufacturer’s instructions (BD Biosciences). Flow cytometry was performed using a FACS Verse (BD Biosciences) with the specific antibodies listed: anti-CD45 clone A20, anti-CD4 clone GK1.5, CD8α clone 5H10-1, IFN-γ clone XMG1.2, TNF-α clone MP6-XT22, CD3 clone 17A2, IL-10 clone JES5-16E3, F4/80 clone BM8 (all from BioLegend Inc., San Diego, CA, USA), IL-33 clone 396118 (R&D Systems, Minneapolis, MN, USA), and anti-iNOS (NOS2) clone N-20 (Santa Cruz Biotechnology, San Diego, CA, USA).

Single-cell suspensions from tumors were plated in a 12-well plate (2,500,000 cells per sample per well) and stimulated with 25 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma Aldrich), 485 ng/mL ionomycin (Sigma Aldrich), 5 µg/mL BFA (Biolegend), 10% FBS, 1× Antibiotic–Antimycotic (Gibco), and 100 μM β-mercaptoethanol in RPMI media for 4 h at 37 °C. Stimulated cells were removed from the plate, washed, and stained with the following stains and surface antibodies: Live/Dead Aqua (Invitrogen), CD8 (clone 53-6.7, BioLegend cat. 100747, 1:250), CD45 (clone 30-F11, BD Bioscience cat. 564279, 1:350), and CD4 (clone RM4-5, BioLegend cat. 100563, 1:250). After surface staining, cells were fixed and permeabilized with the BD Fixation/Permeabilization kit and stained with intracellular antibodies: IFN-γ (clone XMG1.2, eBioscience cat. 12-7311-41, 1:100), TNFα (clone MP6-XT22, BioLegend cat. 506307, 1:100), and granzyme B (clone QA16A02, BioLegend cat. 372203, 1:100). Cells were analyzed using a BD LSRFortessa X-20 cytometer and data were analyzed by FlowJo software.

Single cell suspensions were washed and resuspended in FB. For intracellular cytokine analysis, cells were resuspended in 500 μL of eBioscience^TM Cell Stimulation Cocktail (plus protein transport inhibitors) in RPMI medium supplemented with 10% FCS, 1% l‐glutamine, and 0.01% β‐mercaptoethanol and the technique was carried out as previously outlined [53 (link)]. These cells remained at 37°C, 5% CO₂ for 4 h before being washed in FB, centrifuged at 400 × g for 5 min at 4°C and subjected to surface staining as above. Following overnight incubation in Fix/Perm buffer, cells were washed twice with 2 mL Perm Buffer (both Foxp3/Transcription Factor staining buffer set, eBioscience). Cells were then resuspended in 200 μL of Perm Buffer supplemented with intracellular antibodies (1:200 for each) for 1 h in the dark. Antibodies used were against: Ki67 (clone 16A8, Biolegend), IFN‐γ (clone 554413, BD Biosciences), IL‐6 (clone MP520F3, BD Biosciences), TNF‐α (clone MP6‐XT22, Biolegend), IL‐12p23 (clone C15.6, Biolegend), pro‐IL‐1β (clone NJTEN‐3, eBioscience). Appropriate isotype controls for intracellular stains were included in all experiments.