Isolation of B-lymphocytes

For enzyme-linked immunospot (ELISPOT) assays CD27⁺ memory B cells were isolated from leukocyte filters obtained from tetanus-vaccinated blood donors. The local ethics committee approved this procedure. The donors gave written informed consent and were asked for their status of vaccination. First, total B cells were isolated by negative selection using the RosetteSep™ Human B Cell Enrichment Cocktail (STEMCELL Technologies SARL, Grenoble, France) and subsequent density gradient centrifugation with Ficoll-Paque PLUS (GE Healthcare, Munich, Germany). After two washing steps with PBS containing 0.5 % BSA (Sigma-Aldrich, Taufkirchen, Germany) the B cells were immediately labeled with CD27 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany; Catalogue number: 130-051-601) and subsequently isolated using LS columns and a MidiMACS Separator according to the manufacturer’s instructions (Miltenyi Biotec). Thereafter, the isolated CD27⁺ memory B cells were incubated at a concentration of 10⁵ cells per ml in RPMI 1640 medium supplemented with 10 % v/v FCS, 100 U/ml penicillin and 100 mg/ml streptomycin (all from Life Technologies GmbH, Darmstadt, Germany) on a 96-well round-bottomed plate (Greiner Bio-One, Frickenhausen, Germany) with 2.5 μg/ml CpG oligodeoxynucleotide 2006 (5’-TCG TCGTTTTGTCGTTTTGTCGTT-3’, TIB MolBiol, Berlin, Germany) and 50 ng/ml interleukin (IL)-21 (Life Technologies GmbH, Darmstadt, Germany). After polyclonal activation with the TLR-9 agonist CpG and IL-21, 10 nM TTC or TTC-ETA was added to the culture medium on day 2. On day 4, activated cells were harvested and ELISPOT assays were carried out to determine the frequencies of IgG, anti-tetanus toxoid (TT) IgG as well as anti-TTC specific IgG antibody secreting cells (ASCs). For flow cytometric analyses CD19 MicroBeads (Miltenyi Biotec; Catalogue number: 130-097-055) instead of CD27 MicroBeads were used for positive selection and peripheral blood mononuclear cells (PBMCs) were isolated from a donor 6 and 14 days after booster vaccination with tetanus toxoid to determine the binding of the TTC fusion protein to ex vivo plasma cells and memory B cells, respectively. Briefly, PBMCs were isolated by density gradient centrifugation with Ficoll-Paque PLUS (GE Healthcare) and stained for TTC-specific CD27⁺⁺CD38⁺⁺ plasma cells or CD27⁺ memory B cells.