U bottom 96 well plate

Total leukocyte populations from individual fish were dispensed into 96-well U bottom plates (Greiner bio-one, Germany) at a density of 1 × 10⁶ cells/ml. Following two washing steps with ice-cold bovine serum albumin (BSA) 0.5% in phosphate-buffered saline (PBS), cells were incubated with 50 μl of FITC labeled mouse anti-IgT mAb (1.1 mg/ml, 1:10 dilution) for 1 h at 37 °C. After two washing steps with BSA-PBS 0.5%, cells were resuspended in 1% PBS. Two replicates per each fish were set. Just before the reading, 10 μl of propidium iodide (Sigma, working stock at 50 μg/ml) were added to each sample for the detection of viable cells from forward scatter (FSC) vs PI graphics. Positive IgT B cells were quantified by flow cytometry in a CyFlow Space Cytometer (Partec) equipped with FloMax® Software. Leukocytes without the FITC- mouse anti-IgT mAb conjugate were used as non-stained controls (NS) to optimize and standardize the system. Further analysis was performed with FlowJo v.10 (FlowJo LLC, Tree Star).

A second experiment was done to evaluate specific IgT⁺ B cell immune response. Six tilapias per group (50.0 ± 6.3 g) were used for the study of the specific B cell response against the synthetic P0 peptide. Two experimental groups were settled. One group of tilapia was immunized by intraperitoneal (i.p.) injection on days 0 and 15 with the TT-P0 Ls antigen at the dose of 100 μg per fish. The second group was the control non-immunized. Samples of blood from the caudal vein, head kidney and spleen were collected of all fish at 7 days after booster (day 22) and were used for lymphocyte isolation as described before. Total leukocyte populations from individual fish were dispensed into 96-well U bottom plates (Greiner bio-one, Germany), at a density of 2.5 × 10⁵ cells/ml in 100 μl of l-15 containing 5% FBS and P/S. The cells were incubated with 100 μl of supplemented l-15 medium containing 10 μg/ml of synthetic P0 peptide or with supplemented medium only (negative controls), respectively. After 72 h of incubation at 25 °C, the leukocytes harvested and were analyzed based on side and forward light scatter parameters for the quantification of P0-specific IgT⁺ cells as described before.

PBMCs were freshly isolated from leukaphereses or buffy coats using Ficoll (Eurobio) density gradient centrifugation. Isolated PBMCs were cryopreserved in freezing medium containing 90% heat-inactivated FCS (Eurobio) and 10% dimethyl sulfoxide (DMSO; Applichem, Darmstadt, Germany) and stored in liquid nitrogen.
CD4⁺ T cells in the CSF of HLA-DR15⁺ RRMS patients were purified by positive selection using CD4 microbeads, human (Miltenyi, Bergisch Gladbach, Germany). Isolated bulk CD4⁺ T cells were seeded at 1.5 × 10³ cells/well in 96-well U-bottom plates (Greiner Bio-One, Kremsmunster, Austria) together with 1.5 × 10⁵ irradiated allogeneic PBMC (45Gy), 1 μg/ml PHA (REMEL, Thermo Fisher Scientific), and hIL-2 in RPMI-1640 medium containing 5% human serum (Blood Bank Basel), 2 mM glutamine (Thermo Fisher Scientific), 1% (vol/vol) nonessential amino acids (GIBCO, Thermo Fisher Scientific), 1% (vol/vol) sodium pyruvate (GIBCO), 5μM β-Mercaptoethanol (GIBCO), 100 U/ml penicillin (Corning), and 100 μg/ml streptomycin (Corning). The medium was changed by aspirating half of the old medium and adding the same amount of fresh medium containing hIL-2 every 3-4 days for up to 25 days until cells were fully rested.