Despite the key role that Ab memory plays in protection from a variety of infectious diseases (1
), little is known about the responses of human MBCs and naïve B cells to Ag. High-resolution live cell imaging is providing the tools necessary to define and order the early events in BCR signaling that ultimately lead to B cell proliferation and differentiation. Here, we used these tools to compare the responses of human PB IgM+
B cells and IgG+
B cells as representative naïve B cells and MBCs respectively. Our results show that human MBCs are more robust at each step in the initiation of BCR signaling, from interrogation of the lipid bilayer, to the formation of sub-microscopic BCR oligomers, to the recruitment and activation of kinases in the BCR signaling cascade. We recently provided evidence in mouse primary B cells that these early events in the initiation of BCR signaling were highly sensitive to the affinity of the BCR for Ag (40
). As human MBCs have undergone somatic hypermutation and express high- affinity BCRs, we predict that in response to specific Ags, the differences in the response of human MBC and naïve B cells would be even more dramatic than that shown here using anti-Ig as a surrogate Ag. The robust responses of MBCs to anti-Ig and anti-κ that we observed in vitro
could provide an advantage in responding to Ag in the competitive environments of the lymphoid tissues in vivo
, explaining in part the rapid, high-titered Ab responses characteristic of B cell memory. In addition, a number of studies have provided evidence for cell-intrinsic differences between human naïve and MBCs in gene expression and responses to a variety of stimuli, including T cell help, cytokines, TLR ligands and anti-Ig, that are consistent with the ability of MBCs to respond quickly and robustly during an immune response, giving MBCs an intrinsic advantage over naïve B cells (4
). Such differences would serve to further amplify the intrinsic advantage conferred on MBCs through their efficient Ag-driven initiation of BCR signaling.
We observed in human B cells a new phenomenon that we first described in mouse IgG-expressing B cells (27
), namely that IgG BCRs, unlike IgM BCRs, show an Ag-ligation-independent phase in the recruitment of BCRs to BCR-microclusters. From 30 s to 150 s following anti-Ig encounter, the FI of IgG BCRs in the interface between the B cells and the bilayer increased more quickly than did the FI of the underlying anti-Ig, indicating an accumulation of IgG BCRs that were not bound to anti-Ig. In contrast, the FIs of IgM BCRs and underlying anti-Ig accumulated at the same rate. We speculate that this may be a novel mechanism to rapidly amplify early IgG BCR signaling in MBCs.
We also found differences between naïve B cells and MBCs that were independent of BCR ligation. We observed that in the absence of anti-Ig, MBCs were more signaling active, having more spontaneously oligomerized BCRs that recruit more pPI3K, pERK and pp38 to the membranes. Rajewsky and colleagues recently demonstrated a key role of PI3K in BCR tonic signaling (46
). These authors suggested that modulation of PI3K signaling may be the key element in the control of mature B cell survival mediated by the BCR. While studies have suggested that Ag binding is not required for the maintenance of IgG MBCs (47
), the possibility that a constitutive or tonic BCR signal is needed has not been ruled out. In fact, Hikida et al
. proposed that the BCR uses PLC-γ2 for MBC generation and maintenance (48
), supporting the involvement of BCR signaling in maintaining MBC longevity. Other recent work suggested that tonic BCR signals are propagated via ERK showing that treatment with a MEK inhibitor blocked immature B cell differentiation and that immature B cells with low BCR expression were rescued by expression of a constitutively active N-Ras (49
). It is possible that the higher constitutive level of pPI3K and pERK that we observed in MBCs plays a role in the survival of MBCs, allowing them to achieve their remarkable characteristic longevity. Adachi and Davis (50
) recently provided evidence that the phosphorylation of ERK and p38 are dramatically different in naïve and Ag-experienced T cells following T cell receptor stimulation and suggested that these differences could account for differences in responses to Ag. It is possible that the difference in naïve and MBC recruitment of pERK and pp38 in resting cells may subsequently impact Ag-driven signaling in an analogous fashion.
We also show that the robust early responses of MBCs to anti-Ig were completely inhibited by coligating the BCR and FcγRIIB. Thus, differentiation to MBCs does not alter the B cell’s sensitivity to FcγRIIB- mediated inhibition. We also observed a difference in the spatial relationship between the BCR and FcγRIIB following BCR crosslinking alone in naïve and MBCs. Only in naïve B cells did FcγRIIB show significant microscopic colocalization with the BCR. It is possible that the spatial proximity of FcγRIIB and the BCR serve to limit BCR signaling in naïve B cells.
It is becoming increasingly clear that MBCs are not a homogeneous subpopulation of cells, but rather a phenotypically and functionally heterogeneous population of Ag-experienced B cells (42
). CD27 expression was considered to be a reliable marker for human MBCs, but it has more recently become apparent that up to 30% of IgG+
B cells are CD27−
subpopulations may have distinct origins and functions in memory responses. IgM+
human B cells have also been described, but their origins and functions remain less well understood. We found that B cells that expressed CD27 were more robust in the early events in B cell activation, but that IgG+
B cells demonstrated stronger responses to anti-Ig than did IgM+
Collectively our results suggest that differences in the early events following anti-Ig binding between IgM+ naïve B cells and IgG+ MBCs may explain in part the heightened recall Ab responses observed in vivo. A better understanding of the molecular mechanisms that determine the responses to Ag at the early stages of B cell activation in normal human naïve and MBCs may provide new strategies to attenuate this process in systemic autoimmune diseases and to design more effective vaccines.