Human il 2

A maximum of 100 CD154⁺CD45RA⁻CD4⁺ T cells were sorted per well (U-bottom 96-well plate) after the CD154 expression assay and expended in the presence of 1.5 × 10⁵ autologous irradiated PBMCs, 1 µg/mL PHA (Sigma), human IL-2 (10 U/mL, Roche), and T cell growth media. After 10–14 days, cells were transferred to a flat bottom 48-well plate and restimulated with irradiated PBMCs, 1 µg/mL PHA (Sigma), human IL-2 (10 U/mL, Roche), and T cell media. Cells were split and fed as appropriate. Once the cells were successful expanded, epitope mapping experiments were performed. For mapping, 10⁵ expanded T cells were stimulated for 3 h at 37°C with 5 µg/mL of synthesized Ara h 2 peptide pools (Ara h 2 peptides were divided into four pools with five peptides per pool) in 96-well plate in the presence of 1 µg/mL anti-CD40 (HB14, Miltenyi Biotec) and 10⁵ autologous PBMCs, in 10% human serum RPMI medium. After 3 h, cells were stained with PE-CD154 and APC-CD4 antibodies. Pool giving a positive response was retested with 40 µg/mL blocking antibodies anti-HLA-DR (L243) or anti-HLA-DQ (SPVL3) to examine DR or DQ restriction. Peptides from pool giving a positive response were then tested with individual peptides from the positive pool. Individual identified peptides (epitope) were loaded into the biotinylated HLA-DR or HLA-DQ proteins to generate tetramers for staining as described (22 (link)).

Abs used were CD19 PE (LT19; Miltenyi Biotec), CD138 allophycocyanin (B-B4; Miltenyi Biotec), CD38 PE-Cy7 (HB7; BD Biosciences), CD38 AF700 (HIT2; BioLegend), CD20 eFluor V450 (2H7; eBioscience); CD27 AF647 (LT27; AbD Serotec), CD27 FITC (M-T271), CD19 PerCP-Cy5.5 (SJ225C1; BD Biosciences), CD19 PE-Cy7 (SJ225C1; BD Biosciences), CD24 FITC (ML5; BD Biosciences), CD84 PE (CD84.1.21; BioLegend), CD38 PerCP-Cy5.5 (HIT2; BD Biosciences), CD95 BV421 (DX2; BioLegend), CD20 allophycocyanin-H7 (L27; BD Biosciences), CD27 BV605 (O323; BioLegend), CD3 VioGreen (BW264/56; Miltenyi Biotec), Ki67 FITC (B56; BD Biosciences), unconjugated goat anti-IRF4 (M-17; Santa Cruz Biotechnology), and donkey anti-goat IgG AF488 (polyclonal; Invitrogen). Controls were isotype-matched mouse mAbs. Annexin V FITC was from eBioscience, and 7-AAD was from BD Biosciences.
Reagents included human IL-2 (Roche), IL-6 (PeproTech), IFN-α (Sigma), IL-21 (PeproTech), goat anti-human F(ab′)₂ fragments (anti-IgM and anti-IgG; Jackson ImmunoResearch), HybridoMax hybridoma growth supplement (Gentaur), Lipid Mixture 1, Chemically Defined (200×), and MEM Amino Acids Solution (50×, Sigma).

Peripheral blood was obtained from healthy donors and the affected patient after informed consent. Mononuclear cells were obtained following lymphoprep density centrifugation. B cell selection was carried out with a Miltenyi memory B-cell isolation kit, selecting for untouched total B cells using the first step of the manufacturer’s protocol which depletes non-B cells. Alternatively, naïve B cells were enriched by depletion of memory cells using anti-CD27 MACs beads (Mitenyi). B-cells were cultured as previously described [30 (link), 31 (link)] in 24-well plates at 1×10⁵ cells/ml in IMDM + 10% FBS with MEM amino acid solution (1:50) and Lipid Mixture 1, chemically defined (1:200) (all Thermo Fisher Scientific) and the addition of 20 U/ml human IL-2 (Roche), 50 ng/ml human IL-21 (Peprotech) and 10 μg/ml F(ab′)₂ goat anti-human IgM/IgG (Jackson Immunoresearch) on previously irradiated CD40L-expressing fibroblasts. After 3 days, B cells were removed from the CD40L-cells by gentle pipette mixing. B cells were reseeded at in media containing 20 U/ml human IL-2 and 50 ng/ml human IL-21. On day 6, cells were seeded in media containing human IL-6 (10 ng/ml), human IL-21 (50 ng/ml), 100 U/ml multimeric APRIL (Adipogen), and 100nM gamma secretase inhibitor (GSI) inhibitor (L-685458; Sigma) and re-fed at day 10.