br Results br Discussion The process by
Discussion The process by which MBCs differentiate from GC B cell precursors remains mostly uncharacterized, in large part because of the uncertainty surrounding the nature of the precursor aminolevulinic acid within the GC. The identification here of a distinct subset of GC B cells, CCR6+ LZ B cells, as the direct precursors of GC-dependent MBCs therefore provides an important step forward in the understanding of both GC function and the molding of long-term immunity. In particular, our finding that predominantly low-affinity cells but also significant numbers of high-affinity CCR6+ LZ B cells exit the cell cycle and transition into MBCs indicates that this process is not tightly linked to antigen affinity but allows a blend of antigen affinities to contribute to the MBC pool. Because of the well-established fragility of GC B cells ex vivo, it was not possible to show the direct transition of CCR6+ LZ B cells into MBCs either in vitro or upon in vivo transfer. However, in view of the strong links in phenotype, somatic mutation pattern, gene expression, and function between these cells, there seems to be no likely alternative explanation of the data presented here. The possibility that CCR6+ LZ B cells might represent a “dead-end” population is not supported by their failure to show increased levels of apoptosis or by their unique capability among LZ B cells to sustain antibody production. In addition, although phenotypic and functional similarities between CCR6+ LZ B cells and MBCs might be explained by the re-entrance of MBCs into the GC, this possibility is not consistent with our BrdU kinetic analysis, which showed that CCR6+ LZ B cells become quiescent rather than undergo rapid re-activation. Similarly, BrdU kinetics indicate that CCR6+ LZ B cells are also not recently activated naive B cells entering the GC, as does the fact that >80% of these cells carry somatic mutations in the heavy-chain variable region and so are derived from, rather than become, GC B cells. A recent step forward in characterizing the mechanisms that drive MBC differentiation within the GC was provided by studies showing that expression of the transcription factor Bach2 is required for the production of GC-derived MBCs (Shinnakasu et al., 2016). Given that signals from T cells reduce Bach2, the authors suggested that GC B cells that receive reduced inputs from Tfh cells are prone to increase Bach2 and thus undergo MBC differentiation. The prevalence of low-affinity GC B cells entering the MBC pool in both our study and that of Shinnakasu et al. is consistent with this model if, as has been suggested (Victora et al., 2010), LZlo GC B cells receive less Tfh cell help than do LZhi cells. In this case, the smaller but significant contribution of CCR6+ LZhi cells to the MBC pool could be due to the limiting nature of Tfh cell help. Thus, despite having a high affinity for antigen, some LZhi cells might simply fail to encounter cognate Tfh cells and therefore undergo MBC differentiation. Because we showed that both LZlo and LZhi CCR6+ MBC precursors exit the cell cycle, we propose that a failure to receive strong stimulatory signals from Tfh cells and/or a direct encounter with antigen is a key step in MBC differentiation and explains the predominance of low-affinity specificities entering the MBC pool. The model proposed above indicates a fundamental distinction between the mechanisms driving the differentiation of MBCs and PCs from GC precursors. We have previously shown that PCs differentiate exclusively from high-affinity precursors in the GC (Phan et al., 2006) and recently demonstrated that this process initiates among a subset of LZhi cells (Kräutler et al., 2017). Thus, there appears to be a symmetry to the differentiation of the key effector populations that emerge from the GC, such that PCs derive from LZhi precursors and MBCs primarily from the LZlo compartment. This dichotomy stems in part from the fact that the initiation of PC differentiation is tightly linked to antigen affinity through its requirement for a positive signal delivered upon direct contact with antigen (Kräutler et al., 2017). As discussed above, MBC differentiation appears instead to be associated with a paucity of extracellular inputs and so is more prevalent for (but not exclusive to) LZlo B cells. Why CCR6+ LZ B cells differentiate into MBCs under such circumstances instead of undergoing apoptosis and ingestion by tingible body macrophages will be an important question to answer in future studies.