Five day B cell ELISpot assays were performed as previously described (50 (link)). Briefly, 1 × 106 PBMC were cultured in complete media in 24 well plates alone or with stimulation consisting of 2.5 µg CpG oligodeoxynucleotide ODN-2006 (Operon Technologies, CA), 1/10,000 dilution of protein A from Staphylococcus aureus Cowan (SAC; Sigma-Aldrich, MO), 1/100,000 dilution of pokeweed mitogen (Sigma-Aldrich, MO) and 25 ng/mL IL-10 (R&D Systems, MN)/mL in a volume of 200 µL for 5 days. Cells were then washed in complete media and plated on prepared ELISpot plates (Millipore S2EM004M99, MA) previously coated with either PBS, 10 µg polyclonal goat anti-human IgG (Caltag, CA) to detect all antibody secreting cells (ASC), 2.5 µg TT, or 4.0 µg DT/mL. Stimulated PBMC were incubated with antigen for six hours unless otherwise specified. Plates were developed, scanned, and spots counted by Immunospot satellite analyzer (Cellular Technology, OH). For the ex vivo B cell ELISpot assay, cryopreserved PBMC were thawed, rested, and plated onto plates that were coated with either human anti-IgG or 4 µg DT/mL for six hours. No stimulation was used.