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4--5 h prior to the vaccination, whereas all others received s.c. vaccination on day −3. We assessed the localization of transferred cells using immunofluorescent staining of sections of the axillary lymph nodes and spleen with anti-CD3 antibodies and anti-IgD antibodies or fluorescein isothiocyanate-conjugated anti-IgD antibodies (BD Pharmingen).
Antibody responses were measured by ELISA, as described previously ([@B25]).
Generation of memory B cells. {#s4.5}
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We generated FO B cells from single-cell suspensions of day 84 mLN and spleen of mice in each group, using EasySep kits according to the manufacturer's instructions (STEMCELL Technologies). FO B cells were plated at a density of 1 × 10^6^ cells/well in 96-well round-bottom plates in the presence of 7 µg/ml lipopolysaccharide, 25 ng/ml recombinant murine (rm) IL-4, and 1 µg/ml anti-IL-21 antibody. Cells were incubated for 72 h and then assayed for IgG1 and IgG2c production.
Statistical analyses. {#s4.6}
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GraphPad Prism 7.0 was used for all statistical analyses. The difference between groups was determined using a two-tailed Student's *t* test or a one-way analysis of variance with Tukey's multiple-comparison test. Differences were considered statistically significant at *P* values of .
We are grateful to Susan Watson for assistance with animal care and housing, to Anirban Chakraborty for assistance with flow cytometry, and to Ilse Reim