Central to the ability of B cells to discriminate between foreign and self antigens is the difference in the responses to antigen of immature vs. mature B cells. Engagement of the BCR of immature B cells in the bone marrow induces expression of the RAG1 and RAG2 genes and subsequent Ig light chain rearrangements with the goal of changing specificity away from self-reactivity, a process called receptor editing(16
). If this loss of self-reactivity is not achieved or if self-antigen triggers BCR signaling of T1 B cells in the spleen, then the cell undergoes apoptosis, resulting in clonal deletion. In contrast, strong BCR engagement of mature B cells induces proliferation and in combination with cytokines or other signals can facilitate a productive immune response. A variety of studies have indicated that immature B cells are more sensitive to antigen than are mature B cells. We have used newly improved methods for measuring signaling reactions in individual cells from immune organs to compare the sensitivity of the various developmental stages of B cells in the spleen and bone marrow for two key BCR-induced signaling responses, the elevation of intracellular free calcium and the activation of the Erk MAP kinase. These results confirm that T1 B cells are highly sensitive to BCR engagement and that follicular B cells are distinctly less sensitive. Moreover, we have shown that this change in signaling sensitivity of the BCR is due to a maturation-associated increase in the ability of the Lyn-CD22-SHP-1 pathway to attenuate BCR signaling. We propose that the low level of function of this inhibitory mechanism in immature B cells serves to permit efficient engagement of tolerance-promoting programs in these cells whereas its greater activity in mature follicular B cells serves to create a threshold for activation that minimizes the activation of B cells in response to self-antigens encountered in the periphery.
Previous studies have also compared the BCR responsiveness of immature and mature B cells, but have disagreed as to whether immature B cells are more sensitive(23
), equally sensitive(25
) or less sensitive(26
) to BCR stimulation relative to mature B cells. Our studies simultaneously analyzed immature and mature B cell populations taken immediately ex-vivo
from the BM or spleen of unmanipulated mice and used staining conditions that did not perturb signaling, and thus, we feel they are likely to represent the responses of these cell types in a reliable way. The difference in sensitivity of BCR signaling that we observed did not appear to result primarily from changes in the expression of mIgM and mIgD that occur as B cells mature in the spleen. T1 cells have high levels of mIgM and low levels of mIgD, whereas mature B cells have high levels of mIgD and diverse levels of mIgM, ranging from low levels to the high levels seen in T1 B cells. We found that there was still a considerable difference in sensitivity to anti-IgM treatment between T1 B cells and a gated subpopulation of follicular B cells that had levels of mIgM comparable to the expression level on T1 cells (data not shown). Moreover, when we used the antigen lysozyme to engage the BCR on MD4 anti-lysozyme Ig transgenic T1 and follicular B cells, we again found substantially greater sensitivity of the former compared to the latter. Similarly, Wen et al(23
), demonstrated that immature anti-Thy1 Ig transgenic B cells were more sensitive to Thy1 antigen than their mature B cell counterparts using techniques similar to ours. These findings indicate that mechanisms other than changes in surface Ig expression regulate B cell sensitivity to BCR engagement during maturation.
Genetic evidence indicates that this reduction in BCR sensitivity during B cell maturation is due to an increase in the efficacy of the inhibitory pathway mediated by Lyn, CD22 and SHP-1. BCR signaling of T1 and T2 B cells was only modestly affected by Lyn-deficiency, CD22-deficiency, or SHP-1 mutation, indicating that this inhibitory pathway is relatively inactive in immature B cells of the bone marrow or spleen. In contrast, there was a dramatic enhancement in BCR signaling of mature B cells by Lyn- and CD22-deficiencies demonstrating the importance of this inhibitory pathway in limiting BCR sensitivity once B cells mature. One contributor to this developmental change in the ability of the Lyn-CD22-SHP-1 pathway to inhibit BCR signaling is likely to be the 2.5-fold upregulation of CD22 during B cell maturation in the spleen. Follicular B cells from cd22+/−
mice, which express CD22 levels similar to those of wild type T1 B cells, exhibited enhanced calcium responses compared to WT follicular B cells but were distinctly less sensitive than WT T1 B cells. This indicates that other factors are present that mediate the developmental reduction in BCR sensitivity, besides upregulation of CD22. Regulation of SHP-1, CD22 or Lyn activity could contribute to the developmental change in inhibitory signaling. For example, Sialic acid acetylesterase has recently been shown to facilitate CD22 suppression of BCR signaling(42
) and is upregulated during B cell development(43
). In contrast, Lyn levels did not change significantly between T1 and mature follicular B cells. However, we did see a higher level of Lyn activity in unstimulated mature B cells compared to unstimulated T1 B cells, as evidenced by phosphorylation of the active site Y397 residue. Although regulation of the Lyn Y397 site is not well understood, phosphorylation of Y397 might be enhanced in a lipid raft environment(44
), and mature B cells appear to have a higher lipid raft content than transitional B cells(45
). Interestingly, Lyn phosphorylation of Y397 was substantially decreased in unstimulated cd22−/−
mature B cells, which suggests that increasing CD22 levels during B cell development promotes Lyn activity, in addition to providing more substrate for Lyn.
Mice deficient in Lyn or CD22, as well as mice with a B cell-specific deletion of SHP-1 (36
), are known to spontaneously produce autoantibodies against nuclear antigens, but the mechanism leading to this autoantibody production is unknown. We found that lyn−/−
mice had increased rag1
mRNA expression in newly formed B cells and more modest increases in Igλ+ B cell frequencies and RS rearrangements compared to their wild-type counterparts. These data suggest that Lyn deficiency does not compromise tolerance-related responses of immature B cells to self-antigen and may even enhance them. Consistent with this conclusion, it has been shown that Lyn-deficiency does not cause a significant change in the number of VH
DNA-reactive B cells in the spleen(46
). Another study demonstrated that Lyn-deficiency minimally affected the number of anti-HEL transgenic B cells in the bone-marrow of HEL-expressing mice, but did enhance the loss of anti-HEL transgenic mature B cells in the spleens of these mice(5
). The implication of these results is that the Lyn–CD22–SHP-1 inhibitory pathway is not necessary for the establishment of tolerance to self during B cell development, in agreement with the slightly enhanced level of BCR signaling seen in immature B cells from mice genetically missing this pathway. In contrast, genetic mutations causing reductions in BCR signaling, and therefore reduced sensitivity to antigen, lead to enrichment of the B cell repertoire with autoreactive specificities in mice(47
) and humans(48
). Thus, it appears that Lyn-deficiency leads to autoimmunity not through a defect in central tolerance induction but rather due to a defect in the maintenance of tolerance in the periphery. In agreement with this idea, lyn−/−
mature follicular B cells exhibited substantially enhanced sensitivity to BCR-induced calcium release, Erk activation and proliferation relative to their wild type counterparts, which indicates that there is a reduction in the threshold of B cell activation in the absence of effective inhibition by the Lyn–CD22–SHP-1 pathway. This reduced threshold of activation could allow the recruitment of autoreactive B cells into an immune response, and perhaps in combination with abnormal myeloid cell function from Lyn-deficiency, result in autoantibody production.
In summary, we have found that during maturation of B cells in the spleen, the sensitivity of the BCR to antigen stimulation was down-regulated. This developmentally acquired down-regulation in BCR sensitivity required Lyn and CD22, which act together with SHP-1 in an inhibitory pathway to suppress BCR signaling selectively in mature follicular B cells. Surprisingly, the Lyn–CD22–SHP-1 inhibitory pathway had a minor role in the regulation of BCR signaling in immature B cells. In agreement with the signaling results, Lyn-deficiency had little effect on receptor editing-related events in immature B cells, which suggests that central tolerance mechanisms do not require this inhibitory pathway. On the basis of these observations, we propose that modulation of B cell sensitivity to antigen by the Lyn–CD22–SHP-1 inhibitory pathway during the course of B cell maturation is a critical factor in the maintenance of peripheral B cell tolerance to self-antigens.