Brefeldin a

Ascites cell pellets were incubated with 5 ml of red cell lysis buffer for 5 min and then equal numbers of cells (2.0 x 10⁶) were resuspended in Iscove's Modified Dulbecco's culture media (Gibco) supplemented with 10% heat inactivated fetal calf serum, 1% Glutamax (Gibco), 0.5% gentamycin and 50 μM 2-mercaptoethanol. Cells were treated for 4 hours at 37°C with ionomycin (500 ng/ml) and phorbol myristate acetate (PMA) (50 ng/ml) to stimulate activated T cells to produce IFN-γ in the presence of Brefeldin A (eBioscience) to block cytokine secretion. Cells were surface stained with PE-Cy7-conjugated anti-CD8α and APC-Cy7-conjugated anti-CD45 in the presence of Brefeldin A. Cells were fixed, permeabilized, and intracellularly stained with APC-conjugated anti-IFN-γ (eBioscience). Flow-cytometric data were acquired on a BD FACSCanto II and analyzed using FACSDiva software (BD Biosciences). Viable cells were gated based on forward and side scatter profiles.

For detection of SEB or C. albicans-reactive Th cells from healthy pigs, isolated PBMCs were rested overnight in complete IMDM (PAN-Biotech) supplemented with 10% FCS (PAN-Biotech), 100 U/ml Penicillin, and 100 µg/ml Streptomycin (PAN-Biotech) and stimulated on the following day with superantigen (SEB, 1 µg/ml, Sigma-Aldrich) or PMA (20 ng/ml, Sigma-Aldrich) plus ionomycin, (1 µg/ml, Sigma-Aldrich) or C. albicans lysate (40 µg/ml, GREER^®) for 6 h and in presence of BrefeldinA (3 µg/ml, eBioscience) during the last 4 h of restimulation. C. albicans lysate was produced by dissolving statically grown cellular antigen, defatted, powdered, and dried by GREER^® in PBS. For detection of A. suum-reactive Th cells, splenocytes and lung parenchymal cells were rested overnight in complete IMDM (PAN-Biotech) supplemented with 10% FCS, 100 U/ml Penicillin, and 100 µg/ml Streptomycin and stimulated on the following day with 20 µg/ml of Ascaris L3 worm lysate (Asc Lys) or L3 excretory–secretory products (Asc ES) for 6 h and in presence of BrefeldinA (3 µg/ml, eBioscience) during the last 4 h of restimulation.

THP1 cells (ECACC, UK) were incubated overnight with paraformaldehyde (PFA)-fixed Escherichia coli (E. coli; DH5α, Invitrogen) at a ratio of 25 bacteria per cell, or a sterility control. THP1 cells were washed extensively, and PBMCs, intrahepatic lymphocytes, enriched CD8+ T cells, or sorted CD161++ Vα7.2+ T cells were added to the THP1 cells for a 5-h stimulation. Brefeldin A (eBioscience) was added for the final 4 h of the stimulation. Alternatively, for the assessment of degranulation, anti-CD107a-PE-Cy7 (BioLegend) was added from the start of the stimulation. For the assessment of GrB and perforin upregulation, PBMCs were added to E. coli-treated THP1 cells for 24 h. MAIT cell apoptosis was detected by Annexin V surface staining (Miltenyi Biotec), in Annexin V binding buffer (Miltenyi Biotec) for 15 min. Staurosporine (Sigma Aldrich) was added as a positive control. Alternatively, MAIT cells were stimulated with IL-12+ IL-18 (both Miltenyi Biotech) at 50 ng/ml for 20 h, and Brefeldin A (eBioscience) was added at 3 µg/ml for the final 4 h of the incubation. For blocking experiments, anti-MR1 antibody (gift from Professor Ted Hansen or from Biolegend), anti-IL-12p40/70, anti-IL-18 antibody (both Miltenyi Biotech), or the appropriate isotype controls were added at 10 µg/ml. Anti-CD8α antibody (clone LT8; Novus Biologicals) was added at the indicated concentrations.

Single cell suspensions were prepared from mouse spleen or colonic LP or human LPMC or PBMC cells as described above. For intracellular cytokine staining mouse or human were directly cultivated for 3 or 4 hr in the presence of phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) (100ng/ml), ionomycin (Sigma-Aldrich) (1μg/ml), monensin (BD Biosciences) and brefeldin A (eBioscience). Where indicated, mouse and human cLP cells were stimulated overnight with 10 ng/ml of recombinant IL-12 (Peprotech, Rocky Hill, NJ) or IL-23 (mouse - R&D Systems (Minneapolis, MN), human - Peprotech), and then for the last 4 hr PMA, ionomycin and brefeldin A were added.
Cells were incubated with CD16/32 (eBioscience) to block Fcγ receptors, prior to incubation with fixable viability dye (eFluor780, eBioscience), and mAb conjugates in 100 μl PBS containing 2% BSA and 1 mM EDTA (PBS-E) for 30 min. Cells were subsequently washed twice in PBS-E. For cytokine staining, cells were stained with antibodies for 1 hr using the Cytofix/Cytoperm kit (BD Biosciences). For transcription factor staining, cells were stained with transcription factor antibodies for 1 hr using the Foxp3 staining kit (eBioscience). Cells were washed twice in PBS-E. All labelled cells were then acquired on a LSRII SORP (BD Biosciences) and analysed using Flowjo software (Tree star, Ashland, OR).