Recombinant human il 21r fc chimera protein

Purified B cells were pre-activated with a BCR agonist affinity purified F(ab′)2 fragment of goat anti-human IgG + IgM (H + L) (2.5 μg/mL; Jackson ImmunoResearch, Baltimore, PA, USA) in the presence of recombinant human IL-2 and IL-10 proteins (both 50 ng/mL; PeproTech, Cranbury, NJ, USA) for 24 h at 37 °C in 5% CO₂ milieu in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% heat-inactivated fetal bovine serum in 96-well flat-bottom plates. Activated B cells (5 × 10⁴) were co-cultured with equal numbers of autologous CD4⁺CXCR5⁺ cT_FH cells in 200 μL of complete RPMI medium in 96-well U-bottom plates and stimulated with SEB (1 μg/mL; Sigma-Aldrich; Steinheim, Germany). To modulate T/B cell interaction, recombinant human IL-21R Fc chimera protein (2.5 μg/mL, R&D Systems, MN, USA), as the isotype control, and recombinant human IgG1 Fc chimera protein (2.5 μg/mL, R&D Systems) was added to the culture. After 7 days, cells were harvested and stained for plasmablast phenotype using FITC-conjugated anti-CD38, PE-conjugated anti-CD27, APC-conjugated anti-CD4 monoclonal antibodies and 7-AAD viability solution. CD38^hiCD27⁺ plasmablasts were analyzed and data acquired on a FACS Calibur flow cytometer equipped with CellQuest v3.3 software. Data were further analyzed using FlowJo v10.0.7 software. Supernatants were collected and stored at −20 °C until further measurements.

For IL-13 blocking, IL-13 neutralizing antibody (1 µg/ml, BD Pharmingen™, USA) and IgG control antibody (1 µg/ml, BD Pharmingen™, USA) were used. For IL-21 blocking, recombinant human IL-21R Fc chimera protein (1 µg/ml, R&D systems) and recombinant human IgG1 Fc (1 µg/ml, R&D systems) were used. After 6 days’ culture with cells in different groups, the differentiation of CD4⁺ Tfh cells and PCs were determined by FACS LSRII.