Cellular maturation defects in Bruton's tyrosine kinase-deficient immature B cells are amplified by premature B cell receptor expression and reduced by receptor editing

In the mouse, Bruton's tyrosine kinase (Btk) is essential for efficient developmental progression of CD43(+)CD2(-) large cycling into CD43(-)CD2(+) small resting pre-B cells in the bone marrow and of IgM(high) transitional type 2 B cells into IgM(low) mature B cells in the spleen. In this study, we show that the impaired induction of cell surface changes in Btk-deficient pre-B cells was still noticeable in kappa(+) immature B cells, but was largely corrected in lambda(+) immature B cells. As lambda gene rearrangements are programmed to follow kappa rearrangements and lambda expression is associated with receptor editing, we hypothesized that the transit time through the pre-B cell compartment or receptor editing may affect the extent of the cellular maturation defects in Btk-deficient B cells. To address this issue, we used 3-83 mu delta transgenic mice, which prematurely express a complete B cell receptor and therefore manifest accelerated B cell development. In Btk-deficient 3-83 mu delta mice, the IgM(+) B cells in the bone marrow exhibited a very immature phenotype (pre-BCR(+)CD43(+)CD2(-)) and were arrested at the transitional type 1 B cell stage upon arrival in the spleen. However, these cellular maturation defects were largely restored when Btk-deficient 3-83 mu delta B cells were on a centrally deleting background and therefore targeted for receptor editing. Providing an extended time window for developing B cells by enforced expression of the antiapoptotic gene Bcl-2 did not alter the Btk dependence of their cellular maturation. We conclude that premature B cell receptor expression amplifies the cellular maturation defects in Btk-deficient B cells, while extensive receptor editing reduces these defects.     

Ig light chain receptor editing in anergic B cells

Receptor editing in the bone marrow (BM) serves to modify the Ag receptor specificity of immature self-reactive B cells, while anergy functionally silences self-reactive clones. Here, we demonstrate that anergic B cells in hen egg lysozyme Ig (HEL-Ig)/soluble HEL double transgenic mice show evidence of having undergone receptor editing in vivo, as demonstrated by the presence of elevated levels of endogenous kappa light chain rearrangements in the BM and spleen. In an in vitro IL-7-driven BM culture system, HEL-Ig BM B cells grown in the presence of soluble HEL down-regulated surface IgM expression and also showed induction of new endogenous kappa light chain rearrangements. Using a panel of soluble protein ligands with reduced affinity for the HEL-Ig receptor, the editing response was shown to correlate in a dose-dependent fashion with the strength of signaling through the B cell receptor. The finding that the level of B cell receptor cross-linking sufficient to induce anergy in B cells is also capable of engaging the machinery required for receptor editing suggests an intimate relationship between these two mechanisms in maintaining B cell tolerance.     

Receptor editing can lead to allelic inclusion and development of B cells that retain antibodies reacting with high avidity autoantigens

Receptor editing is a major B cell tolerance mechanism that operates by secondary Ig gene rearrangements to change the specificity of autoreactive developing B cells. In the 3-83Igi mouse model, receptor editing operates in every autoreactive anti-H-2K(b) B cell, providing a novel receptor without additional cell loss. Despite the efficiency of receptor editing in generating nonautoreactive Ag receptors, we show in this study that this process does not inactivate the autoantibody-encoding gene(s) in every autoreactive B cell. In fact, receptor editing can generate allelically and isotypically included B cells that simultaneously express the original autoreactive and a novel nonautoreactive Ag receptors. Such dual Ab-expressing B cells differentiate into transitional and mature B cells retaining the expression of the autoantibody despite the high avidity interaction between the autoantibody and the self-Ag in this system. Moreover, we find that these high avidity autoreactive B cells retain the autoreactive Ag receptor within the cell as a consequence of autoantigen engagement and through a Src family kinase-dependent process. Finally, anti-H-2K(b) IgM autoantibodies are found in the sera of older 3-83Igi mice, indicating that dual Ab-expressing autoreactive B cells are potentially functional and capable of differentiating into IgM autoantibody-secreting plasma cells under certain circumstances. These results demonstrate that autoreactive B cells reacting with ubiquitous membrane bound autoantigens can bypass mechanisms of central tolerance by coexpressing nonautoreactive Abs. These dual Ab-expressing autoreactive B cells conceal their autoantibodies within the cell manifesting a superficially tolerant phenotype that can be partially overcome to secrete IgM autoantibodies.     

Negative selection by IgM superantigen defines a B cell central tolerance compartment and reveals mutations allowing escape

To analyze B lymphocyte central tolerance in a polyclonal immune system, mice were engineered to express a superantigen reactive to IgM of allotype b (IgM(b)). IgM(b/b) mice carrying superantigen were severely B cell lymphopenic, but small numbers of B cells matured. Their sera contained low levels of IgG and occasionally high levels of IgA. In bone marrow, immature B cells were normal in number, but internalized IgM and had a unique gene expression profile, compared with those expressing high levels of surface IgM, including elevated recombinase activator gene expression. A comparable B cell population was defined in wild-type bone marrows, with an abundance suggesting that at steady state ～20% of normal developing B cells are constantly encountering autoantigens in situ. In superantigen-expressing mice, as well as in mice carrying the 3H9 anti-DNA IgH transgene, or 3H9 H along with mutation in the murine κ-deleting element RS, IgM internalization was correlated with CD19 downmodulation. CD19(low) bone marrow cells from 3H9;RS(-/-) mice were enriched in L chains that promote DNA binding. Our results suggest that central tolerance and attendant L chain receptor editing affect a large fraction of normal developing B cells. IgH(a/b) mice carrying the superantigen had a ～50% loss in follicular B cell numbers, suggesting that escape from central tolerance by receptor editing from one IgH allele to another was not a major mechanism. IgM(b) superantigen hosts reconstituted with experimental bone marrow were demonstrated to be useful in revealing pathways involved in central tolerance.     

Transitional B cells lose their ability to receptor edit but retain their potential for positive and negative selection

Ligation of B cell receptors on immature bone marrow B cells, either by an endogenous Ag or by an anti-B cell receptor Ab induces secondary V(D)J gene rearrangements, termed receptor editing. Whether the same signal induces receptor editing in transitional B cells is not clear. In this study, we examined the responses of immature and transitional B cells from V(H)12Vkappa1A Ig transgenic mice to stimulation with an anti-Igbeta Ab. Our results demonstrated that immature B cells stimulated with a low concentration of anti-Igbeta Ab, mimicking Ag stimulation, underwent receptor editing both in vivo and in vitro, as evidenced by the detection of dsDNA breaks at Jkappa recombination signal sequences, whereas transitional B cells did not. The lack of dsDNA breaks in transitional B cells contrasts with their increased expression of RAG1 and RAG2, suggesting a novel mechanism that may prevent rearrangements. Furthermore, treatment of transitional B cells with high concentrations of anti-Igbeta Abs induced apoptosis, whereas low concentrations induced differentiation. Our results support the idea that transitional B cells lose the capacity to edit, but are sensitive to positive and negative selection.     

Antigen receptor editing in anti-DNA transitional B cells deficient for surface IgM

In response to encounter with self-Ag, autoreactive B cells may undergo secondary L chain gene rearrangement (receptor editing) and change the specificity of their Ag receptor. Knowing at what differentiative stage(s) developing B cells undergo receptor editing is important for understanding how self-reactive B cells are regulated. In this study, in mice with Ig transgenes coding for anti-self (DNA) Ab, we report dsDNA breaks indicative of ongoing secondary L chain rearrangement not only in bone marrow cells with a pre-B/B cell phenotype but also in immature/transitional splenic B cells with little or no surface IgM (sIgM(-/low)). L chain-edited transgenic B cells were detectable in spleen but not bone marrow and were still found to produce Ab specific for DNA (and apoptotic cells), albeit with lower affinity for DNA than the unedited transgenic Ab. We conclude that L chain editing in anti-DNA-transgenic B cells is not only ongoing in bone marrow but also in spleen. Indeed, transfer of sIgM(-/low) anti-DNA splenic B cells into SCID mice resulted in the appearance of a L chain editor (Vlambdax) in the serum of engrafted recipients. Finally, we also report evidence for ongoing L chain editing in sIgM(low) transitional splenic B cells of wild-type mice.     

Definition of a novel cellular constituent of the bone marrow that regulates the response of immature B cells to B cell antigen receptor engagement

Previously we defined a Thy1(dull) bone marrow-derived cell population that regulated fate decisions by immature B cells after Ag receptor signaling. The microenvironmental signals provided by this cell population were shown to redirect the B cell Ag receptor -induced apoptotic response of immature B cells toward continued recombination-activating gene (RAG) expression and secondary light chain recombination (receptor editing). Neither the identity of the cell responsible for this activity nor its role in immature B cell development in vivo were addressed by these previous studies. Here we show that this protective microenvironmental niche is defined by the presence of a novel Thy1(dull), DX5(pos) cell that can be found in close association with immature B cells in vivo. Depletion of this cell eliminates the anti-apoptotic effect of bone marrow in vitro and leads to a significant decrease in the number and frequency of bone marrow immature B cells in vivo. We propose that, just as the bone marrow environment is essential for the survival and progression of pro-B and pre-B cells through their respective developmental checkpoints, this cellular niche regulates the progression of immature stage B cells through negative selection.     

B cell receptor basal signaling regulates antigen-induced Ig light chain rearrangements

BCR editing in the bone marrow contributes to B cell tolerance by orchestrating secondary Ig rearrangements in self-reactive B cells. We have recently shown that loss of the BCR or a pharmacologic blockade of BCR proximal signaling pathways results in a global "back-differentiation" response in which immature B cells down-regulate genes important for the mature B cell program and up-regulate genes characteristic of earlier stages of B cell development. These observations led us to test the hypothesis that self-Ag-induced down-regulation of the BCR, and not self-Ag-induced positive signals, lead to Rag induction and hence receptor editing. Supporting this hypothesis, we found that immature B cells from xid (x-linked immunodeficiency) mice induce re-expression of a Rag2-GFP bacterial artificial chromosome reporter as well as wild-type immature B cells following Ag incubation. Incubation of immature B cells with self-Ag leads to a striking reversal in differentiation to the pro-/pre-B stage of development, consistent with the idea that back-differentiation results in the reinduction of genes required for L chain rearrangement and receptor editing. Importantly, Rag induction, the back-differentiation response to Ag, and editing in immature and pre-B cells are inhibited by a combination of phorbol ester and calcium ionophore, agents that bypass proximal signaling pathways and mimic BCR signaling. Thus, mimicking positive BCR signals actually inhibits receptor editing. These findings support a model whereby Ag-induced receptor editing is inhibited by BCR basal signaling on developing B cells; BCR down-regulation removes this basal signal, thereby initiating receptor editing.     

In vivo assessment of the relative contributions of deletion, anergy, and editing to B cell self-tolerance

In normal B cell development, a large percentage of newly formed cells bear receptors with high levels of self-reactivity that must be tolerized before entry into the mature B cell pool. We followed the fate of self-reactive B cells expressing high affinity anti-hen egg lysozyme (HEL) Ag receptors exposed in vivo to membrane HEL in a setting in which the anti-HEL L chain was "knocked-in" at the endogenous L chain locus. These mice demonstrated extensive and efficient L chain receptor editing responses and had B cell numbers comparable to those found in animals lacking membrane Ag. BrdU labeling indicated that the time required for editing in response to membrane HEL was approximately 6 h. In mice transgenic for soluble HEL, anti-HEL B cells capable of editing showed evidence for both editing and anergy. These data identify receptor editing as a major physiologic mechanism by which highly self-reactive B cells are tolerized to membrane and soluble self-Ags.     

Development of myelin oligodendrocyte glycoprotein autoreactive transgenic B lymphocytes: receptor editing in vivo after encounter of a self-antigen distinct from myelin oligodendrocyte glycoprotein

We explored mechanisms involved in B cell self-tolerance against brain autoantigens in a double-transgenic mouse model carrying the Ig H-chain (introduced by gene replacement) and/or the L-chain kappa (conventional transgenic) of the mAb 8.18C5, specific for the myelin oligodendrocyte glycoprotein (MOG). Previously, we demonstrated that B cells expressing solely the MOG-specific Ig H-chain differentiate without tolerogenic censure. We show now that double-transgenic (THkappa(mog)) B cells expressing transgenic Ig H- and L-chains are subjected to receptor editing. We show that in adult mice carrying both MOG-specific Ig H- and L-chains, the frequency of MOG-binding B cells is not higher than in mice expressing solely the transgenic Ig H-chain. In fact, in THkappa(mog) double-transgenic mice, the transgenic kappa(mog) L-chain was commonly replaced by endogenous L-chains, i.e., by receptor editing. In rearrangement-deficient RAG-2(-) mice, differentiation of THkappa(mog) B cells is blocked at an immature stage (defined by the B220(low)IgM(low)IgD(-) phenotype), reflecting interaction of the autoreactive B cells with a local self-determinant. The tolerogenic structure in the bone marrow is not classical MOG, because back-crossing THkappa(mog) mice into a MOG-deficient genetic background does not lead to an increase in the proportion of MOG-binding B cells. We propose that an as yet undefined self-Ag distinct from MOG cross-reacts with the THkappa(mog) B cell receptor and induces editing of the transgenic kappa(mog) L-chain in early immature B cells without affecting the pathogenic potential of the remaining MOG-specific B cells. This phenomenon represents a particular form of chain-specific split tolerance.     

Modification of ligand-independent B cell receptor tonic signals activates receptor editing in immature B lymphocytes

Maturation of B lymphocytes strictly depends on the signaling competence of the B cell antigen receptor (BCR). Autoreactive receptors undergo negative selection and can be replaced by receptor editing. In addition, the process of maturation of non-self B cells and migration to the spleen, referred to as positive selection, is limited by the signaling competence of the BCR. Using 3-83Tg mice deficient of CD19 we have shown that signaling incompetence not only blocks positive selection but also activates receptor editing. Here we study the role of ligand-independent BCR tonic tyrosine phosphorylation signals in activation of receptor editing. We find that editing, immature 3-83Tg B cells deficient of CD19 have elevated BCR tonic signals and that lowering these tonic signals effectively suppresses receptor editing. Furthermore, we show that elevation of BCR tonic signals in non-editing, immature 3-83Tg B cells stimulates significant receptor editing. We also show that positive selection and developmental progression from the bone marrow to the spleen are limited to cells capable of establishing appropriate tonic signals, as in contrast to immature cells, splenic 3-83Tg B cells deficient of CD19 have BCR tonic signals similar to those of the control 3-83Tg cells. This developmental progression is accompanied by activation of molecules signaling for growth and survival. Hence, we suggest that ligand-independent BCR tonic signals are required for promoting positive selection and suppressing the receptor-editing mechanism in immature B cells.     

Differential c-Myc responsiveness to B cell receptor ligation in B cell-negative selection

Responsiveness of c-Myc oncogene to B cell receptor ligation has been implicated in the induction of apoptosis in transformed and normal immature B cells. These studies provided compelling evidence to link the c-Myc oncogene with the process of negative selection in B-lymphocytes. However, in addition to apoptosis, B cell-negative selection has been shown to occur by secondary Ig gene rearrangements, a mechanism called receptor editing. In this study, we assessed whether differential c-Myc responsiveness to B cell receptor (BCR) ligation is associated with the mechanism of negative selection in immature B cells. Using an in vitro bone marrow culture system and an Ig-transgenic mouse model (3-83) we show here that c-Myc is expressed at low levels throughout B cell development and that c-Myc responsiveness to BCR ligation is developmentally regulated and increased with maturation. Furthermore, we found that the competence to mount c-Myc responsiveness upon BCR ligation is important for the induction of apoptosis and had no effect on the process of receptor editing. Therefore, this study suggests an important role of c-Myc in promoting and/or maintaining B cell development and that compartmentalization of B cell tolerance may also be developmentally regulated by differential c-Myc responsiveness.     

Direct reduction of antigen receptor expression in polyclonal B cell populations developing in vivo results in light chain receptor editing

Secondary Ab V region gene segment rearrangement, termed receptor editing, is a major mechanism contributing to B lymphocyte self-tolerance. However, the parameters that determine whether a B cell undergoes editing are a current subject of debate. We tested the role that the level of BCR expression plays in the regulation of receptor editing in a polyclonal population of B cells differentiating in vivo. Expression of a short hairpin RNA for κ L chain RNA in B cells resulted in reduction in levels of this RNA and surface BCRs. Strikingly, fully mature and functional B cells that developed in vivo and efficiently expressed the short hairpin RNA predominantly expressed BCRs containing λ light chains. This shift in L chain repertoire was accompanied by inhibition of development, increased Rag gene expression, and increased λ V gene segment-cleavage events at the immature B cell stage. These data demonstrated that reducing the translation of BCRs that are members of the natural repertoire at the immature B cell stage is sufficient to promote editing.     

Split tolerance in peripheral B cell subsets in mice expressing a low level of Igkappa-reactive ligand

Peripheral B cell tolerance differs from central tolerance in anatomic location, in the stage of B cell development, and in the diversity of Ag-responsive cells. B cells in secondary lymphoid organs are heterogeneous, including numerous subtypes such as B-1, marginal zone, transitional, and follicular B cells, which likely respond differently from one another to ligand encounter. We showed recently that central B cell tolerance mediated by receptor editing was induced in mice carrying high levels of a ubiquitously expressed kappa-macroself Ag, a synthetic superantigen reactive to Igkappa. In this study, we characterize a new transgenic line that has a distinctly lower expression pattern from those described previously; the B cell tolerance phenotype of these mice is characterized by the presence of significant numbers of immature kappa+ B cells in the spleen, the loss of mature follicular and marginal zone B cells, the persistence of kappa+ B-1 cells in the peritoneal cavity, and significant levels of serum IgM,kappa. These findings suggest distinct signaling thresholds for tolerance among peripheral B cell subsets reactive with an identical ligand.     

Basal B cell receptor-directed phosphatidylinositol 3-kinase signaling turns off RAGs and promotes B cell-positive selection

PI3K plays key roles in cell growth, differentiation, and survival by generating the second messenger phosphatidylinositol-(3,4,5)-trisphosphate (PIP3). PIP3 activates numerous enzymes, in part by recruiting them from the cytosol to the plasma membrane. We find that in immature B lymphocytes carrying a nonautoreactive Ag receptor, PI3K signaling suppresses RAG expression and promotes developmental progression. Inhibitors of PI3K signaling abrogate this positive selection. Furthermore, immature primary B cells from mice lacking the p85alpha regulatory subunit of PI3K suppress poorly RAG expression, undergo an exaggerated receptor editing response, and, as in BCR-ligated cells, fail to progress into the G1 phase of cell cycle. Moreover, immature B cells carrying an innocuous receptor have sustained elevation of PIP3 levels and activation of the downstream effectors phospholipase C (PLC)gamma2, Akt, and Bruton's tyrosine kinase. Of these, PLCgamma2 appears to play the most significant role in down-regulating RAG expression. It therefore appears that when the BCR of an immature B cell is ligated, PIP3 levels are reduced, PLCgamma2 activation is diminished, and receptor editing is promoted by sustained RAG expression. Taken together, our results provide evidence that PI3K signaling is an important cue required for fostering development of B cells carrying a useful BCR.     

Resolution of three nonproliferative immature splenic B cell subsets reveals multiple selection points during peripheral B cell maturation.

Although immature/transitional peripheral B cells may remain susceptible to selection pressures before full maturation, the nature and timing of these selection events remain unclear. We show that correlated expression of surface (s) IgM (sIgM), CD23, and AA4 defines three nonproliferative subpopulations of immature/transitional peripheral B cells. We designate these populations transitional (T) 1 (AA4(+)CD23(-)sIgM(high)), T2 (AA4(+)CD23(+)sIgM(high)), and T3 (AA4(+)CD23(+)sIgM(low)). Cells within all three subsets are functionally immature as judged by their failure to proliferate following sIgM cross-linking in vitro, and their rapid rate of turnover in vivo as assessed by 5-bromo-2'-deoxyuridine labeling. These labeling studies also reveal measurable cell loss at both the T1-T2 and T2-T3 transitions, suggesting the existence of multiple selection points within the peripheral immature B cell pool. Furthermore, we find that Btk-deficient (xid) mice exhibit an incomplete developmental block at the T2-T3 transition within the immature B cell pool. This contrasts markedly with lyn(-/-) mice, which exhibit depressed numbers but normal ratios of each immature peripheral B cell subset and severely reduced numbers of mature B cells. Together, these data provide evidence for multiple selection points among immature peripheral B cells, suggesting that the B cell repertoire is shaped by multiple unique selection events that occur within the immature/transitional peripheral B cell pool.     

Unique signaling properties of B cell antigen receptor in mature and immature B cells: implications for tolerance and activation

Immature B cells display increased sensitivity to tolerance induction compared with their mature counterparts. The molecular mechanisms underlying these differences are poorly defined. In this study, we demonstrate unique maturation stage-dependent differences in B cell Ag receptor (BCR) signaling, including BCR-mediated calcium mobilization responses. Immature B cells display greater increases in intracellular calcium concentrations following Ag stimulation. This has consequences for the induction of biologically relevant responses: immature B cells require lower Ag concentrations for activation than mature B cells, as measured by induction of receptor editing and CD86 expression, respectively. BCR-induced tyrosine phosphorylation of CD79a, Lyn, B cell linker protein, and phospholipase Cgamma2 is enhanced in immature B cells and they exhibit greater capacitative calcium entry in response to Ag. Moreover, B cell linker protein, Bruton's tyrosine kinase, and phospholipase Cgamma2, which are crucial for the induction of calcium mobilization responses, are present at approximately 3-fold higher levels in immature B cells, potentially contributing to increased mobilization of calcium. Consistent with this possibility, we found that the previously reported lack of inositol-1,4,5-triphosphate production in immature B cells may be explained by enhanced inositol-1,4,5-triphosphate breakdown. These data demonstrate that multiple mechanisms guarantee increased Ag-induced mobilization of calcium in immature B cells and presumably ensure elimination of autoreactive B cells from the repertoire.     

Anergic responses characterize a large fraction of human autoreactive naive B cells expressing low levels of surface IgM

B cell anergy represents an important mechanism of peripheral immunological tolerance for mature autoreactive B cells that escape central tolerance enforced by receptor editing and clonal deletion. Although well documented in mice, the extent of its participation in human B cell tolerance remains to be fully established. In this study, we characterize the functional behavior of strictly defined human naive B cells separated on the basis of their surface IgM (sIgM) expression levels. We demonstrate that cells with lower sIgM levels (IgM(lo)) are impaired in their ability to flux calcium in response to either anti-IgM or anti-IgD cross-linking and contain a significantly increased frequency of autoreactive cells compared with naive B cells with higher levels of sIgM. Phenotypically, in healthy subjects, IgM(lo) cells are characterized by the absence of activation markers, reduction of costimulatory molecules (CD19 and CD21), and increased levels of inhibitory CD22. Functionally, IgM(lo) cells display significantly weaker proliferation, impaired differentiation, and poor Ab production. In aggregate, the data indicate that hyporesponsiveness to BCR cross-linking associated with sIgM downregulation is present in a much larger fraction of all human naive B cells than previously reported and is likely to reflect a state of anergy induced by chronic autoantigen stimulation. Finally, our results indicate that in systemic lupus erythematosus patients, naive IgM(lo) cells display increased levels of CD95 and decreased levels of CD22, a phenotype consistent with enhanced activation of autoreactive naive B cells in this autoimmune disease.     

Cutting edge: B cell antigen receptor signaling occurs outside lipid rafts in immature B cells

B cell Ag receptor (BCR) signaling changes dramatically during B cell development, resulting in activation in mature B cells and apoptosis, receptor editing, or anergy in immature B cells. BCR signaling in mature B cells was shown to be initiated by the translocation of the BCR into cholesterol- and sphingolipid-enriched membrane microdomains that include the Src family kinase Lyn and exclude the phosphatase CD45. Subsequently the BCR is rapidly internalized into the cell. Here we show that the BCR in the immature B cell line, WEHI-231, does not translocate into lipid rafts following cross-linking nor is the BCR rapidly internalized. The immature BCR initiates signaling from outside lipid rafts as evidenced by the immediate induction of an array of phosphoproteins and subsequent apoptosis. The failure of the BCR in immature B cells to enter lipid rafts may contribute to the dramatic difference in the outcome of signaling in mature and immature B cells.