Characterization of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific germinal center B cells and antigen-binding B220^- cells after primary NP challenge in mice

Previous studies examining the primary germinal center (GC) response to SRBC in mice demonstrated a steady ratio of IgM⁺ to isotype-switched GC B cells and a persistent population of GC B cells with a founder phenotype. These characteristics held true at the inductive, plateau, and dissociative phases of the GC response, suggesting a steady-state environment. To test whether these characteristics apply to the primary response of other T cell-dependent Ags, the present study examined the GC response after challenge with (4-hydroxy-3-nitrophenyl)acetyl (NP) in C57BL/6 mice. Multiparameter flow cytometric analysis was used to assess the phenotype of splenic NP-reactive cells at multiple time points after immunization. Results of these studies demonstrated the characteristics of the SRBC-induced GC reaction to be fully maintained in the NP response. In particular, there was a steady ratio of nonswitched to switched B cells, with the majority of NP-reactive GC B cells displaying IgM. In addition, a substantial frequency of B220^- NP-binding cells was observed in the spleen at later time points after NP challenge. Although these cells were IgE⁺, they were found to express both κ and λ L chains and display the high-affinity IgE Fc (FcεRI) receptor, suggesting that this population is not of B cell origin. Adoptive transfer studies further demonstrated the B220^- NP-binding subset to be derived from the myeloid lineage.