Fig 2: Antibodies in sera after intradermal DNA vaccination.(a-g) Mice were immunized once i.d. with 25 µg DNA/EP as indicated (n = 6/group), and assayed for total IgG (a), IgG1 (b), IgG2a (c), IgG2b (d) and IgG3 (e) against PR8 in ELISA (mean+/-SEM). f) Hemagglutination-inhibition (HI) titers (mean+/-SEM) in sera. g) Sera from day 14 were assayed in a micro neutralization assay (PR8 virus). Dotted line indicates threshold for positive neutralization (50%). (h,i) Mice were immunized twice i.d. at days 0 and 50 as indicated by arrows (↑), and sera assayed in ELISA against PR8 for induced IgG1 (h) and IgG2a (i) (mean +/- SEM).

Fig 3: CSR and SHM are impaired in ROD1exon3-/- mice. a The absence of ROD1 protein in ROD1exon3-/- B cells. Diagram of gRNAs and the major AUG start codon in the ROD1 loci (upper panel). ROD1 and AID expression were examined by Western blotting. ß-actin served as the loading control. b RT-qPCR examination of post-switch transcripts containing Iµ and each of the CH exons. The RNA levels were normalized to GAPDH (n = 3). c Flow cytometric analysis of IgG1 and IgG3 with ex vivo cultured splenic B cells activated either by LPS or LPS plus IL4. Splenic B cells were isolated from wild-type and ROD1exon3-/- mice. Exogenous ROD1 was transduced into ROD1exon3-/- B cells before stimulation to rescue CSR defects (n = 3). d Quantification of IgG1 and IgG3 CSR efficiency by normalizing to WT levels as shown in (c). *P < 0.05, **P < 0.01 (two-tailed Student’s t test). e Flow cytometric analysis of Peyer’s patch germinal center B cells in wild-type (WT) and ROD1 knockout (KO) mice. Percentage of GL-7+/Fas+ germinal center B cells amongst all B220+ cells is indicated. f SHM analysis of Peyer’s patch germinal center B cells at AID hypermutation substrates located at the JH4 intron. The mean values were determined by Sanger sequencing from 73 (WT) and 88 (-/-) sequenced clones