Human ab serum

Monocytes were isolated using the Classical Monocyte Isolation Kit, human 130-117-337 from Miltenyi Biotec (Bergisch Gladbach, Germany) from PBMCs obtained from buffy coats of healthy, anonymous donors. Buffy coats were purchased from the Regional Center for Blood Donation and Blood Treatment, Łódź, Poland, as waste material. The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (PAN Biotech, Aidenbach, Germany) and 10% human AB serum (PAN Biotech).

Monocytes were cultured at a density of 0.5 to 1.0 × 10⁶/mL in RPMI-1640 (Gibco, Karlsruhe, Germany) supplemented with 2% human AB serum (PAN Biotech, Aidenbach, Germany), l-glutamine (2 mmol/L), penicillin (50 U/mL), and streptomycin (50 mg/mL; all from Life Technologies, Karlsruhe, Germany). For differentiation into DC, 0.5 to 1.0 × 10⁶ monocytes/mL were cultured for six days in medium as described above, supplemented with 10% fetal calf serum (PAN Biotech), IL-4 (144 U/mL), and granulocyte macrophage colony-stimulating factor (GM-CSF, 225 U/mL; both from PeproTech, Hamburg, Germany). Monocytes and DC were incubated for 4 h, 24 h, and 48 h, respectively, with or without LPS (from Salmonella abortus equi S-form, Enzo Life Sciences, Lörrach, Germany), ATG (Fresenius, Bad Homburg, Germany) (now named Grafalon^®, distributed by Neovii Biotech, Gräfelfing, Germany) (100 µg/mL), IgG isotype control (polyclonal, rabbit, Molecular Innovations, Novi, MI, USA) (100 µg/mL) or Alemtuzumab (monoclonal, human, anti-CD52, Genzyme, Cambridge, MA, USA) (100 µg/mL).

The generation of Ag-specific T cell lines was performed as described by Bacher et al.^{25 (link)}. In brief, the stimulated and isolated CD154⁺Ascaris ES antigen F-specific T cells were cultured 1:100 with autologous, mitomycin C (Sigma-Aldrich) treated feeder cells in X-VIVO™ 15 (Lonza, Basel, Switzlerland) supplemented with 5% human AB serum (PAN-Biotech, Aidenbach, Germany), 100 U/mL penicillin, 100 μg/mL streptomycin (PAN-Biotech, Aidenbach, Germany), and 50 ng/mL IL-2 (PeproTech, NJ, US). Cells were expanded for 14 days and culture medium was replenished with IL-2 containing media when needed. For restimulation after 14 days, autologous PBMC were CD3-depleted using BD FACSAria™ III cell sorter or CD3-bead MACS and co-cultured 1:1 with expanded T cell lines in the presence of the indicated antigens. For assessing the frequency of total Ascaris ES antigen F and/or M reactive T cells after expansion, co-cultured cells were restimulated with 40 µg/mL ES antigen F for 6 h. For addressing peptide specificities, restimulation was performed with single, synthetic peptides (25 µg/mL) or a pool of all peptides (25 µg/mL of each peptide). Brefeldin A (3 µg/mL) was added after the first 2 h of stimulation.

Purified recombinantly expressed HLA molecules were treated with Thrombin and subsequently subjected to size exclusion chromatography (Sephadex S200). The placeholder peptide CLIP was exchanged by the indicated peptides incubating HLA molecules with 50 molar excess of the desired peptide for 72 h in the presence of molecular loading enhancers. In brief, the FR dipeptide (150 μM) and AdaEtOH (100 μM) were used to promote CLIP exchange for the corresponding peptides. After gel filtration the peptide loading of HLA-II complexes was verified by MS. The generated peptide HLA class II complexes were biotinylated in a BirA sequence (DRB chain) using a BirA ligase (Avidity). The Biot-peptide-HLA class II complexes were then used to generate tetramers using Streptavidin-PE. Tetrameric complexes were finally separated by gel filtration and stored in PBS + NaAz (0.02%).
For peptide-specific tetramer staining, expanded cells were incubated in sodium azide (5 mM) containing X-VIVO™ 15 (Lonza) supplemented with 5% human AB serum (PAN-Biotech, Aidenbach, Germany), 100 U/mL penicillin, 100 μg/mL streptomycin (PAN-Biotech, Aidenbach, Germany) for 2 h at 37 °C and 5% CO₂ in the presence of the PE-SAv-Tetramers specific for CLIP, Ov17, or RtBp at final concentrations of 20 µg/mL. Following incubation, cells were washed and counterstained for CD8, CD4, CD20, CD14, and viability.

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by centrifugation on a Ficoll density gradient. Blood was purchased from the Regional Center for Blood Donation and Blood Treatment, Łódź, Poland, and had been obtained from anonymous healthy donors. The plasma samples corresponding to the buffy coats were tested for the presence of anti-SARS-CoV2 antibodies using the Accu-Tell COVID-19 in vitro diagnostic IgG/IgM test (AccuBio Tech, Beijing, China) and the EDI^TM Novel Coronavirus COVID-19 IgG ELISA Kit (Epitope Diagnostics Inc., San Diego, CA, USA). All the serum samples were negative; thus, the isolated cells were treated as having been obtained from unexposed individuals. Monocytes were isolated using the Classical Monocyte Isolation Kit, human 130-117-337 from Miltenyi Biotec (Bergisch Gladbach, Germany). The cells were cultured in RPMI 1640 medium containing 10% FBS (fetal bovine serum) and 10% human AB serum (PAN Biotech, Aidenbach, Germany) for 3 days. For macrophage differentiation, monocytes were isolated as described above and then cultured in RPMI 1640 medium containing 10% FBS and 10% human AB serum (PAN Biotech) supplemented with 10 ng/mL GM-CSF (R&D Systems, Bio-Techne, Minneapolis, MN, USA) for 5 days.