A novel mechanism for complement activation at the surface of B cells following antigen binding

Coligation of CD21 with BCR on the surface of B cells provides a costimulatory signal essential for efficient Ab responses to T-dependent Ags. To achieve this, Ag must be directly linked to C3 fragments, but how this occurs in vivo is not fully understood. Using BCR transgenic mice, we demonstrated that C3 was deposited on the surface of B cells following both high- and moderate-affinity Ag binding. This was dependent on the specific binding of IgM to the BCR-bound Ag and can occur independently of soluble immune complex formation. Based on these data, we propose a novel model in which immune complexes can form directly on the surface of the B cell following Ag binding. This model has implications for our understanding of B lymphocyte activation.     

Signaling through surface IgM in tolerance-susceptible immature murine B lymphocytes. Developmentally regulated differences in transmembrane signaling in splenic B cells from adult and neonatal mice.

During the course of B lymphocyte development, newly emerging surface Ig+ B cells pass through a stage when Ag-Ag receptor interactions lead not to immune responsiveness but to a state of functional tolerance. We have explored the molecular basis of antigenic nonresponsiveness and tolerance susceptibility using tolerance-susceptible surface Ig+ splenic B lymphocytes from neonatal mice and anti-mu chain antibodies as a polyclonal ligand. In this population of cells, surface IgM is uncoupled from the inositol phospholipid (PI)-hydrolysis pathway at a point proximal to the receptor; anti-mu antibodies did not stimulate inositol phosphate generation despite the fact that PI-hydrolysis was observed after treatment with A1F4, implicating the existence of a functional G protein and phospholipase C. Further evidence for a difference early in the signal transduction pathway stems from the finding that anti-mu stimulation does not induce the expression of two immediate/early PKC-linked genes egr-1 and c-fos. This appears to be the primary signaling difference between the mature and immature B cells from the neonatal mouse splenic population, as these cells undergo a G0-G1 cell cycle phase transition when surface IgM is bypassed using phorbol diester and calcium ionophore. Interestingly, despite undetectable levels of PI-hydrolysis, we observed equivalent receptor-mediated changes in intracellular calcium when comparing the immature and mature populations. These results indicate incomplete coupling of surface IgM to the signal transduction machinery operative in mature, immunocompetent B cells and suggests a molecular mechanism accounting for the differential processing of surface IgM signals into activation vs tolerogenic responses observed in these two stages of B cell development.     

Neonatal suppression with anti-Ia antibody. III. In vivo responses to the type 2 antigen TNP-Ficoll

We studied the response to thymus-independent type 2 (type 2) Ag in mice suppressed from birth with anti-Ia antibody. Although these mice have significantly reduced numbers of surface IgM+ cells and reduced or absent levels of Ia-restricted Th cell activity, their IgM antibody response to the type 2 Ag TNP-Ficoll was unaffected whereas that to the prototypic thymus-dependent Ag SRBC was predictably eliminated. These data suggest that an in vivo antibody response can be made to type 2 Ag in the absence of Ia-dependent cellular interactions. The surface IgM+IgD-Ia- B cells that are found in the anti-Ia antibody-suppressed mouse may represent an expanded population of Ia-independent, type 2 Ag-sensitive B cells normally present as a smaller proportion of the splenic lymphocyte population. Thymus-dependent responses, which have been shown to have an absolute requirement for an Ia-dependent interaction, are absent in these animals.     

Transitional B lymphocyte subsets operate as distinct checkpoints in murine splenic B cell development

Signaling through the Ag receptor is required for peripheral B lymphocyte maturation and maintenance. Defects in components of the B cell receptor (BCR) signalosome result in developmental blocks at the transition from immature (heat-stable Ag (HSA)(high)) to mature (HSA(low)) B cells. Recent studies have subdivided the immature, or transitional, splenic B cells into two subsets, transitional 1 (T1) and transitional 2 (T2) cells. T1 and T2 cells express distinct surface markers and are located in distinct anatomic locations. In this report, we evaluated the BCR signaling capacity of T1 and T2 B cell subsets. In response to BCR engagement, T2 cells rapidly entered cell cycle and resisted cell death. In contrast, T1 cells did not proliferate and instead died after BCR stimulation. Correlating with these results, T2 cells robustly induced expression of the cell cycle regulator cyclin D2 and the antiapoptotic factors A1/Bfl-1 and Bcl-x(L) and exhibited activation of Akt. In contrast, T1 cells failed to up-regulate these markers. BCR stimulation of T2 cells also led to down-regulation of CD21 and CD24 (HSA) expression, resulting in a mature B cell phenotype. In addition, T2 cells from Bruton's tyrosine kinase-deficient Xid mice failed to generate these proliferative and survival responses, suggesting a requirement for the BCR signalosome specifically at the T2 stage. Taken together, these data clearly demonstrate that T2 immature B cells comprise a discrete developmental subset that mediates BCR-dependent proliferative, prosurvival, and differentiation signals. Their distinct BCR-dependent responses suggest unique roles for T1 vs T2 cells in peripheral B cell selection.     

Expansion of CD22lo B cells in the spleen of autoimmune-prone flaky skin mice

Similar to murine models with compromised CD22/SHP-1 function, flaky skin (fsn) mutant mice exhibit lymphocyte hyperactivation and an autoimmune phenotype characterized by circulating autoantibodies to dsDNA and glomerulonephritis. Immunophenotyping of fsn/fsn splenic B cells was performed to determine if abnormalities in CD22 expression contributed to the phenotype. We identified an expansion of an IgM(bright) CD22lo population consistent with immature B-lymphocytes. While normal B-lymphocytes require IL-4 to achieve down-modulation of CD22 expression in response to BCR cross-linking, culture with anti-IgM alone led to reduced CD22 expression in fsn/fsn mice. Furthermore, when IL-4 was added to fsn/fsn cultures, no further reduction in CD22 expression was observed. This suggested that fsn/fsn B cells were pre-activated in vivo by chronic IL-4 exposure. A portion of these CD22lo cells expressed the B-1 surface marker CD11b. We contend that decreased activation thresholds among CD22lo B-lymphocytes contributes to the expansion of immature and B-1 B cell populations and to the development of autoimmune pathology in fsn/fsn mice.     

Expression of a V region-less B cell receptor confers a tolerance-like phenotype on transgenic B cells

Neoplastic B cells from H chain disease patients express a truncated B cell receptor (BCR), comprising a membrane Ig that lacks part of its extracellular domain. It has been speculated that deletion of the Ag binding domain would confer a constitutive activity on the BCR, as it has been shown for oncogenic growth factor receptors. A V region-less BCR has constitutive activity, because in transgenic mice it causes inhibition of endogenous H chain gene rearrangements and relieves the requirement for surrogate L chain in pre-B cell development. However, it has been speculated that normal Ag receptors also display constitutive activity. Here we show that transgenic B cells expressing a membrane H chain disease protein on their surface are phenotypically and functionally similar to B cells developing in the presence of their cognate Ag and that cells with normal levels of mutant BCR are eliminated in spleen via a bcl-2 sensitive pathway while progressing toward the mature stage. In contrast, cells with lower levels of mutant receptors develop as mature B cells. These findings support the view that the truncated BCR has a constitutive activity that mimics ligand binding, in analogy to what has been shown for oncogenic growth factor receptors.     

CD72 down-modulates BCR-induced signal transduction and diminishes survival in primary mature B lymphocytes

CD72, a 45-kDa type II transmembrane glycoprotein carrying an ITIM motif, is believed to be an inhibitory coreceptor of the BCR. Mature B cells lacking CD72 show enhanced Ca(2+) mobilization and are hyperproliferative in response to BCR ligation. However, the signal transduction pathways downstream of BCR signaling that transmit the inhibitory effect of CD72 in mature B cells remain unknown. To address this question, we used hen egg lysozyme-specific BCR transgenic mice to elucidate the differential cell signaling between wild-type and CD72-deficient B cells in response to hen egg lysozyme Ag stimulation. Our results demonstrate that CD72 predominantly down-regulates the major signal transduction pathways downstream of the BCR, including NF-AT, NF-kappaB, ERK, JNK, p38-MAPK, and PI3K/Akt in mature B cells. CD72 ligation with anti-CD72 Ab (K10.6), which mimics the binding of CD100 (a natural ligand for CD72) to release the inhibitory function of CD72, augments cell proliferation, Ca(2+) flux, IkappaBalpha activation, and ERK MAPK activity upon Ag stimulation in wild-type B cells. In addition, we show direct evidence that CD72 promotes cell cycle arrest and apoptosis after Ag stimulation in mature B cells. Taken together, our findings conclude that CD72 plays a dominant role as a negative regulator of BCR signaling in primary mature B lymphocytes.     

Characterization of B-cell populations bearing Fc epsilon receptor II

We have characterized the B-cell population that expresses low affinity Fc receptors for IgE (Fc epsilon RII). Fc epsilon RII+ B cells from normal adult BALB/c mice expressed high levels of surface IgD and low/medium levels of surface IgM and constituted the majority of mature splenic B cells. Fc epsilon RII+ splenic B cells expressed high levels of class II MHC antigens and medium to high levels of B220, and consisted of approximately equal numbers of J11dhigh and J11dlow cells. CD5+ B cells did not appear to be Fc epsilon RII+, and interleukin 4 did not induce Fc epsilon RII expression on CD5+ B cells. Fc epsilon RII was not expressed by B cells that had differentiated to secrete immunoglobulin but was expressed on activated B cells. These data suggest that Fc epsilon RII is a differentiation marker expressed on mature and activated B lymphocytes in the major B-cell lineage.     

Progressive surface B cell antigen receptor down-regulation accompanies efficient development of antinuclear antigen B cells to mature, follicular phenotype

Previous studies have suggested that B cell Ag receptor (BCR) down-regulation by potentially pathological autoreactive B cells is associated with pathways leading to developmental arrest and receptor editing, or anergy. In this study we compare the primary development of B cells in two strains of mice expressing transgenic BCRs that differ by a single amino acid substitution that substantially increases reactivity for nuclear autoantigens such as DNA. Surprisingly, we find that both BCRs promote efficient development to mature follicular phenotype, but the strongly autoreactive BCR fails to promote marginal zone B cell development. The follicular B cells expressing the strongly autoreactive BCR do not appear to be anergic, as they robustly respond to polyclonal stimuli in vitro, are not short-lived, and can participate in germinal center reactions. Strikingly however, substantial and progressive down-modulation of surface IgM and IgD takes place throughout their primary development in the BM and periphery. We propose that BCR-autoantigen interactions regulate this pathway, resulting in reduced cellular avidity for autoantigens. This process of "learned ignorance" could allow autoreactive B cells access to the foreign Ag-driven memory B cell response, during which their self-reactivity would be attenuated by somatic hypermutation and selection in the germinal center.     

Reduced dosage of Bruton's tyrosine kinase uncouples B cell hyperresponsiveness from autoimmunity in lyn-/- mice

The development of autoimmunity is correlated with heightened sensitivity of B cells to B cell Ag receptor (BCR) cross-linking. BCR signals are down-regulated by Lyn, which phosphorylates inhibitory receptors. lyn(-/-) mice have reduced BCR signaling thresholds and develop autoantibodies, glomerulonephritis, splenomegaly due to myeloid hyperplasia, and increased B-1 cell numbers. Bruton's tyrosine kinase (Btk), a critical component of BCR signaling pathways, is required for autoantibody production in lyn(-/-) mice. It is unclear whether Btk mediates autoimmunity at the level of BCR signal transduction or B cell development, given that lyn(-/-)Btk(-/-) mice have a severe reduction in conventional B and B-1 cell numbers. To address this issue, we crossed a transgene expressing a low dosage of Btk (Btk(low)) in B cells to lyn(-/-)Btk(-/-) mice. Conventional B cell populations were restored to levels similar to those in lyn(-/-) mice. These cells were as hypersensitive to BCR cross-linking as lyn(-/-) B cells as measured by proliferation, Ca(2+) flux, and activation of extracellular signal-regulated kinase and Akt. However, lyn(-/-)Btk(low) mice did not produce anti-ssDNA, anti-dsDNA, anti-histone, or anti-histone/DNA IgM or IgG. They also lacked B-1 cells and did not exhibit splenomegaly. Thus, B cell hyperresponsiveness is insufficient for autoimmunity in lyn(-/-) mice. These studies implicate B-1 and/or myeloid cells as key contributors to the lyn(-/-) autoimmune phenotype.     

Recruitment of a distinct but related set of VH sequences into the murine CD21hi/CD23- marginal zone B cell repertoire to that seen in the class-switched antibody response

Development and maintenance of cells in the murine follicular and marginal zone compartments is thought to involve differing levels of stimulation of the BCR, although it is still not clear which BCR ligands mediate these events. How the delineation between naive and Ag experienced B cell populations relates to cell phenotype and how precise or blurred this delineation is, is also not well understood. In this study, using PCR to analyze the Ab response to phenyl-oxazolone in the mouse, we show that the Ab repertoire of CD21(hi)/CD23(-) marginal zone B cells shows persistent increase in levels of particular IgM after immunization with foreign Ag. Further, we show that these IgMs have different but related VH/CDR3 sequences from those seen in the class-switched response to oxazolone that we have also analyzed. We also detect an effect of Ag on the follicular B cell repertoire that is less persisting. These results provide evidence consistent with the signal-strength model of mature B cell development being extended to include stimulation by foreign Ag, and also further the known zone of influence of foreign Ag on the B cell compartment.     

Cutting edge: differential sequestration of plasma membrane-associated B cell antigen receptor in mature and immature B cells into glycosphingolipid-enriched domains

Glycosphingolipid-enriched domains (GEDs) are believed to act as platforms for transduction of B cell Ag receptor (BCR)-induced signals from the cell surface. We sought to study whether differential sequestration of BCR into GEDs may contribute to the described intrinsic signaling differences between mature and immature B cells. In this study we found that mature B cells copolarize the BCR with GEDs following BCR aggregation, whereas transitional immature B cells do not. Although anti-BCR treatment leads to receptor aggregation by immature stage B cells, the aggregated complexes do not colocalize with GEDs. We found this difference to be independent of the isotype of the receptor, thereby associating this difference in BCR-GED colocalization to the developmental stage of the B cell. These findings suggest a structural basis for the developmentally regulated differences observed in Ag receptor-mediated signal transduction.     

Severely impaired B lymphocyte proliferation, survival, and induction of the c-Myc:Cullin 1 ubiquitin ligase pathway resulting from CD22 deficiency on the C57BL/6 genetic background

Understanding the molecular mechanisms through which CD22 regulates B lymphocyte homeostasis, signal transduction, and tolerance is critical to defining normal B cell function and understanding the role of CD22 in autoimmunity. Therefore, CD22 function was examined in vivo and in vitro using B cells from CD22-deficient (CD22(-/-)) mice. Backcrossing of founder CD22(-/-) mice onto the C57BL/6 (B6) genetic background from a B6/129 mixed background resulted in a dramatically reduced B cell proliferative response following IgM ligation, characterized by a paucity of lymphoblasts and augmented apoptosis. Also, the phenotype of splenic B6 CD22(-/-) B cells was uniquely HSA(high) and IgD(low)/CD21(low) with intermediate levels of CD5 expression, although the percentages of mature and transitional B cells were normal. That B6 CD22(-/-) B cells predominantly underwent apoptosis following IgM ligation correlated with this unique tolerant phenotype, as well as defective induction of the c-Myc:Cullin 1 (CUL1) ubiquitin ligase pathway that is necessary for progression to the S phase of cell cycle. CD40 ligation compensated for CD22 deficiency by restoring lymphoblast development, proliferation, c-Myc and CUL1 expression, and protein ubiquitination/degradation in IgM-stimulated B6 CD22(-/-) B cell cultures. Thereby, this study expands our current understanding of the complex role of CD22 during B cell homeostasis and Ag responsiveness, and reveals that the impact of CD22 deficiency is dictated by the genetic background on which it is rendered. Moreover, this study defines CD22 and CD40 as the first examples of lymphocyte coreceptors that influence induction of the c-Myc:CUL1 ubiquitin ligase pathway.     

The c-Abl tyrosine kinase is regulated downstream of the B cell antigen receptor and interacts with CD19

c-Abl is a nonreceptor tyrosine kinase that we have recently linked to growth factor receptor signaling. The c-Abl kinase is ubiquitously expressed and localizes to the cytoplasm, plasma membrane, cytoskeleton, and nucleus. Thus, c-Abl may regulate signaling processes in multiple subcellular compartments. Targeted deletion or mutation of c-Abl in mice results in a variety of phenotypes, including splenic and thymic atrophy and lymphopenia. Additionally, lymphocytes isolated from specific compartments of c-Abl mutant mice have reduced responses to a variety of stimuli and an increased susceptibility to apoptosis following growth factor deprivation. Despite these observations, little is known regarding the signaling mechanisms responsible for these phenotypes. We report here that splenic B cells from c-Abl-deficient mice are hyporesponsive to the proliferative effects of B cell Ag receptor (BCR) stimulation. The c-Abl kinase activity and protein levels are elevated in the cytosol following activation of the BCR in B cell lines. We show that c-Abl associates with and phosphorylates the BCR coreceptor CD19, and that c-Abl and CD19 colocalize in lipid membrane rafts. These data suggest a role for c-Abl in the regulation of B cell proliferation downstream of the BCR, possibly through interactions with CD19.     

B cell maintenance in aged mice reflects both increased B cell longevity and decreased B cell generation

In aged mice the population of mature peripheral B cells is maintained despite a severalfold decrease in the population of bone marrow B cell progenitors. The analysis of the rate of accumulation of 5'-bromo-2-deoxyuridine (BrdU)-labeled splenic B cells in mice fed BrdU for 8 days to 8 wk demonstrated a severalfold increase in the half-life of mature B cells in aged mice. Consistent with a role for decreased B cell turnover in maintaining the mature B cell population of aged mice, several findings indicate that fewer newly generated B cells enter the spleen from the bone marrow in aged vs young adult mice. These include 1) a fourfold decrease in the population of relatively immature splenic B cells, defined as cells that express high levels of heat-stable Ag and accumulate BrdU within 8 wk of labeling; and 2) an equivalent decrease in the population of bone marrow cells representative of later stages of B cell maturation (sIgD-sIgM(int-high)). Surprisingly, despite a four- to sixfold decrease in pre-B cells, the population of least mature bone marrow B cells (IgD-sIgM(very low)) remains intact. Because this population accumulates BrdU-labeled cells more slowly in aged mice than in younger mice, and bone marrow B cells at more mature developmental stages are diminished, it appears that in aged mice B cell development beyond the sIgM(very low) stage may be retarded and that cells, therefore, accumulate within this population.     

Transgenic expression of Helios in B lineage cells alters B cell properties and promotes lymphomagenesis

Helios, a member of the Ikaros family of DNA-binding proteins, is expressed in multipotential lymphoid progenitors and throughout the T lineage. However, in most B lineage cells, Helios is not expressed, suggesting that its absence may be critical for B cell development and function. To test this possibility, transgenic mice were generated that express Helios under the control of an Ig mu enhancer. Commitment to the B cell lineage was unaltered in Helios transgenic mice, and numbers of surface IgM(+) B cells were normal in the bone marrow and spleen. However, both bone marrow and splenic B cells exhibited prolonged survival and enhanced proliferation. B cells in Helios transgenic mice were also hyperresponsive to Ag stimulation. These alterations were observed even though the concentration of ectopic Helios in B lineage cells, like that of endogenous Helios in thymocytes, was well below the concentration of Ikaros. Further evidence that ectopic Helios expression contributes to B cell abnormalities was provided by the observation that Helios transgenic mice developed metastatic lymphoma as they aged. Taken together, these results demonstrate that silencing of Helios is critical for normal B cell function.     

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.     

Antinuclear antigen B cells that down-regulate surface B cell receptor during development to mature, follicular phenotype do not display features of anergy in vitro

We previously demonstrated that B cells expressing a transgenic BCR with "dual reactivity" for the hapten arsonate and nuclear autoantigens efficiently complete development to follicular phenotype and stably reside in follicles in vivo. These B cells express very low levels of surface IgM and IgD, suggesting that they avoid central deletion and peripheral anergy by reducing their avidity for autoantigen via surface BCR (sBCR) down-regulation. Since a variety of states of B cell anergy have been previously described, a thorough examination of the functional capabilities of these B cells was required to test this hypothesis. In this study, we show that surface Ig cross-linking induces amounts of proximal BCR signaling in these B cells commensurate with their reduced sBCR levels. Functionally, however, they are comparable to nonautoreactive B cells in cell cycle progression, up-regulation of activation and costimulatory molecules, and Ab-forming cell differentiation when treated with a variety of stimuli in vitro. In addition, these B cells can efficiently process and present Ag and are capable of undergoing cognate interaction with naive TCR-transgenic T cells, resulting in robust IL-2 production. Together, these data reveal a lack of intrinsic anergy involving any known mechanism, supporting the idea that this type of antinuclear Ag B cell becomes indifferent to cognate autoantigen by down-regulating sBCR.