Localization of Splenic B Cells Activated for Switch Recombination by In Situ Hybridization with Iγ1 Switch Transcript and Rad51 Probes

Abstract

AbstractB cells are activated for switch recombination by signals from Th cells, but the site at which this first occurs in vivo has yet to be identified. By in situ hybridization of splenic sections using riboprobes specific for the Iγ1 switch transcript and Rad51 mRNA, we have visualized B cells that are newly activated for switch recombination and characterized the spatial and temporal patterns of Iγ1 and Rad51 mRNA expression. Within 2 days after immunization with (4-hydroxy-3-nitrophenyl)acetyl-chicken gamma-globulin, expression of Iγ1 switch transcripts and Rad51 mRNA was evident and was localized to B220+ B cells clustered within the T cell-rich periarteriolar lymphoid sheath (PALS) and surrounding follicles. By Ab staining, we have shown previously that cells switching from IgM to IgG expression can be visualized at 3 to 5 days postimmunization and colocalize to clusters of Rad51+ cells. Hybridization of adjacent sections with probes for Cμ and Cγ1 mRNA now shows that switching from μ to γ expression occurs within Rad51+Iγ1+ regions of the PALS and peaks between days 3 and 5. Colocalized expression of Iγ1 and Rad51 transcripts was observed from days 2 through 12 of the immune response. Iγ1 and Rad51 transcripts were down-regulated but still detectable at 12 days postimmunization, when they were evident in peanut agglutinin-positive germinal center B cells. Taken together, these observations show that B cells are first activated for switch recombination in the T cell-rich PALS.