Detection of tetC⁻ and 2 different subsets of tetC⁺ PCs in the peripheral blood of volunteers, 6 days after tet immunization. (A) PCs were identified as CD19⁺ CD38^HIGH cells in a CD19/CD38 dot plot obtained by FACS (left panel). The presence of tetC^INT and tetC^HIGH cell subsets was established by labeling permeabilized CD19 CD38^HIGH cells with increasing quantities of FITC-tetC. Right panel shows a representative experiment. FITC-tetC concentrations of 500 ng/mL or less only distinguished 2 cell subsets (tetC⁻ and tetC⁺; top left), while the use of higher concentrations revealed the presence of 3 different cell subsets (tetC^NEG, tetC^INT, and tetC^HIGH; bottom left). (B) Representative control experiments showing the expression histograms of FITC-tetC staining (2 μg/mL) when the cells were coincubated with increasing quantities of unlabeled tet (left panels) or with 5% BSA (middle right), and when the cells were not permeabilized (lower right). Pale line denotes background control. (C) Temporal kinetics of tetC^INT and tetC^HIGH cells. The percentages of circulating tetC^INT and tetC^HIGH CD19⁺ CD38^HIGH cells were obtained from day 3 to day 8 after booster. Results represent the mean plus or minus SEM (n = 7).