Figure 4

CD57⁺ GC-Th cells have the capacities to induce AID expression and to support CSR in B cells. IgD⁺CD38^- naïve B cells were cultured with CD57⁺ GC-Th cells for indicated time periods followed by RT-PCR analysis for (A) AID expression and (B) CSR. The sizes of specific PCR products are 152 bp (IgM); 416 bp (IgG1, G2, G3), 904 bp (IgG4); 904 bp (IgA1); 891 bp (IgA2); and 179 bp (IgE). Shown are productive recombination products. (C) The expression kinetics of AID and productive IgG3 transcripts over an 8 day period are shown together in a graph. In this panel, normalized expression levels calculated after dividing the levels of AID amplification by β-actin levels are shown. The time gap to reach the peak levels of the expression between AID and productive IgG3 transcripts is shown by an arrow. Representative data from at least three independent experiments are shown (panels A and B). (D) Identification of extrachromosomal reciprocal DNA recombination products. Naïve B cells were cultured with CD57⁺ GC-Th cells for indicated time periods and were processed to isolate genomic DNA. Fresh GC-B cells were examined for positive controls. The switch circles were detected by a nested PCR method. Representative data out of three independent experiments are shown. (E) Detection of switch circles by a DC-PCR technique. Naive B cells, CD38⁺ GC-B cells and naïve B cells cultured with GC-Th cells for 5 days were examined for the presence of γ3 and α1/2 switch circles.