Intracellular cytokine staining kit

CD8 T cells were harvested and incubated with HLA-multimers for 15 min at 4 °C and washed three times with cold PBS/BSA prior to staining with antibodies. Cells were incubated with anti-CD3 (clone SK-7, BD), anti-CD4 (clone SK-3, BD), anti-CD8 (clone SK-1, BD) antibodies for 30 min at 4 °C and washed three times with cold PBS/BSA. T cell activation was measured by intracellular IFNγ staining (XMF1.2, BioLegend) using an intracellular cytokine staining kit (BioLegend) according to manufactures protocol. moDC’s were stained with anti-CD1a (clone HI149, BD), anti-CD14 (clone M5E2, BD), anti-CD80 (clone L307.4, BD), anti-CD83 (clone HB15e, BD), anti-CD86 (clone IT2.2, BioLegend), and anti-HLA-DR (clone G46-6, BD) antibodies for 30 min at 4 °C and washed three times with cold PBS/BSA. Samples were acquired using a BD LSRFortessa™ flow cytometry machine and analyzed using FlowJo software (Tree Star).

CD4⁺ T cells were harvested from the lymph nodes and spleens of naive NR1 mice and enriched with a mouse naïve CD4-negative isolation kit (BioLegend) following the manufacturer’s protocol. CD4⁺ T cells were cultured in media consisting of RPMI 1640 (Invitrogen), 10 % FCS, l-glutamine, HEPES, 50 μM 2-ME, 50 U/ml penicillin, and 50 mg/ml streptomycin. NR1 cells were activated by coculture with mitomycin-treated splenocytes pulsed with 5 μM Cta1_133–152 peptide at a stimulator/T cell ratio of 4:1. Th1 polarization was achieved by supplying cultures with 10 ng/ml IL-12 (PeproTech, Rocky Hill, NJ) and 10 μg/ml anti–IL-4 (Biolegend) One week after initial activation resting NR1 cells were co-incubated with untreated or IFNγ-treated macrophages of different genotypes, that were or were not pulsed with Cta1 peptide. Six hours following co-incubation NR1 cells were harvested and stained for intracellular IFNγ (BioLegend) using an intracellular cytokine staining kit (BioLegend) as done previously. Analyzed T cells were identified as live, CD90.1⁺ CD4⁺ cells.

Cytokine release assays were performed upon stimulation with Cell Stimulation Cocktail (BioLegend) containing phorbol 12-myristate 13-acetate (PMA), ionomycin, and brefeldin A for 4 hours at 37°C. Cells were labeled using surface antibodies followed by fixation, permeabilization, and intracellular cytokine staining using the Intracellular Cytokine Staining kit (BioLegend) according to the manufacturer’s protocol.