Moreover, memory B cells have been detected early in the immune response, prior to the peak of the GC reaction [21, 23, 34, 36], suggesting that memory B cells emerge early from the GC or, alternatively, independently of GCs. To assess the relative contribution of GC-dependent and GC-independent pathways to memory B cell formation, an antigen-based cell-enrichment strategy was developed [20, 21]. Immunizing mice with the soluble protein phycoerythrin (PE), a fluorescent Td antigen, made

it possible to track PE-binding B cells in order to study memory and GC B cells. In this way, a precursor cell population was identified that could give rise to GC B cells and later differentiate into memory B or plasma cells. Early in the response and independently of the GC reaction, Proteasome assay these precursors could differentiate

directly into memory B cells. The GC-independent memory B cells mainly retained IgM expression and were less mutated compared with the GC-derived memory B cells. In another model [23], conditional ablation GSK458 in vivo of Bcl-6, a transcription factor pivotal for the survival of GC B cells [39], was used to investigate the GC-dependent and GC-independent pathways in response to the Td antigen, NP-CGG, using IgG1+ NP-specific B cells as read-out [23]. Deletion of Bcl-6 in B cells did not affect B cell development per se whereas it did reduce the number of antigen-specific GC B cells after Astemizole immunization. However, antigen-specific memory B cells were still present, indicating that memory B cells develop

independently of GCs. There seems to be a difference although between memory B cells that develop in a GC-independent compared with GC-dependent manner with respect to SHM. Those that developed in a GC-independent manner did not show signs of SHM by contrast to the GC-dependent memory B cells. Early in the primary response, the GC-independent unmutated memory B cells undergo expansion to become long-lived cells that express antibodies with low affinity. As the response progresses, these cells become resting and are later joined by mutated GC progenies. Together these two populations comprise the memory B cell pool at comparable frequencies and mediate secondary antibody responses upon adoptive transfer. Moreover, and consistent with memory B cells expressing CD80, PDL-2 and CD73 [15], these markers were also detected on memory B cells in this study although not analysed in detail. Finally, it was suggested that the memory compartment is generated as two layers of cells: those uniquely tailored to the pathogen and those that are unmutated in order to accommodate cross-reacting specificities of related pathogens. TFH cells have been suggested as an essential cellular component for GC formation [5-8], and Bcl-6 is pivotal also for the differentiation of TFH cells [5]. In the study just discussed [23], Bcl-6 was conditionally deleted selectively in CD4-expressing cells.