In contrast to a second dose of the SARS-CoV-2 mRNA vaccine, a third dose elicits potent neutralizing activity against the Omicron variant. To address the underlying mechanism for this differential antibody response, we examined spike receptor-binding domain (RBD)-specific memory B cells in vaccinated individuals. Frequency of Omicron-reactive memory B cells increased ∼9 mo after the second vaccine dose. These memory B cells show an altered distribution of epitopes from pre-second memory B cells, presumably due to an antibody feedback mechanism. This hypothesis was tested using mouse models, showing that an addition or a depletion of RBD-induced serum antibodies results in a concomitant increase or decrease, respectively, of Omicron-reactive germinal center (GC) and memory B cells. Our data suggest that pre-generated antibodies modulate the selection of GC and subsequent memory B cells after the second vaccine dose, accumulating more Omicron-reactive memory B cells over time, which contributes to the generation of Omicron-neutralizing antibodies elicited by the third vaccine dose.

Germinal center (GC) B cells cycle between two states, the light zone (LZ) and the dark zone (DZ), and in the latter they proliferate and hypermutate their immunoglobulin genes. How this functional transition takes place is still controversial. In this study, we demonstrate that ablation of Foxo1 after GC development led to the loss of the DZ GC B cells and disruption of the GC architecture, which is consistent with recent studies. Mechanistically, even upon provision of adequate T cell help, Foxo1-deficient GC B cells showed less proliferative expansion than controls. Moreover, we found that the transcription factor BATF was transiently induced in LZ GC B cells in a Foxo1-dependent manner and that deletion of BATF similarly led to GC disruption. Thus, our results are consistent with a model where the switch from the LZ to the DZ is triggered after receipt of T cell help, and suggest that Foxo1-mediated BATF up-regulation is at least partly involved in this switch.

A still unanswered question is what drives the small fraction of activated germinal center (GC) B cells to become long-lived quiescent memory B cells. We found here that a small population of GC-derived CD38intBcl6hi/intEfnb1+ cells with lower mTORC1 activity favored the memory B cell fate. Constitutively high mTORC1 activity led to defects in formation of the CD38intBcl6hi/intEfnb1+ cells; conversely, decreasing mTORC1 activity resulted in relative enrichment of this memory-prone population over the recycling-prone one. Furthermore, the CD38intBcl6hi/intEfnb1+ cells had higher levels of Bcl2 and surface BCR that, in turn, contributed to their survival and development. We also found that downregulation of Bcl6 resulted in increased expression of both Bcl2 and BCR. Given the positive correlation between the strength of T cell help and mTORC1 activity, our data suggest a model in which weak help from T cells together with provision of an increased survival signal are key for GC B cells to adopt a memory B cell fate.

Positive selection of high-affinity germinal center (GC) B cells is driven by antigen internalization through their B cell receptor (BCR) and presentation to follicular helper T cells. However, the requirements of BCR signaling in GC B cells remain poorly understood. Store-operated Ca2+ entry, mediated by stromal interacting molecule 1 (STIM1) and STIM2, is the main Ca2+ influx pathway triggered by BCR engagement. Here, we showed that STIM-deficient B cells have reduced B cell competitiveness compared with wild-type B cells during GC responses. B cell-specific deletion of STIM proteins decreased the number of high-affinity B cells in the late phase of GC formation. STIM deficiency did not affect GC B cell proliferation and antigen presentation but led to the enhancement of apoptosis due to the impaired upregulation of anti-apoptotic Bcl2a1. STIM-mediated activation of NFAT was required for the expression of Bcl2a1 after BCR stimulation. These findings suggest that STIM-mediated survival signals after antigen capture regulate the optimal selection and maintenance of GC B cells.