Lymphoid cell growth and transformation are suppressed by a key regulatory element of the gene encoding PU.1.

Tight regulation of transcription factors, such as PU.1, is crucial for generation of all hematopoietic lineages. We previously reported that mice with a deletion of an upstream regulatory element (URE) of the gene encoding PU.1 (Sfpi1) developed acute myeloid leukemia. Here we show that the URE has an essential role in orchestrating the dynamic PU.1 expression pattern required for lymphoid development and tumor suppression. URE deletion ablated B2 cells but stimulated growth of B1 cells in mice. The URE was a PU.1 enhancer in B cells but a repressor in T cell precursors. TCF transcription factors coordinated this repressor function and linked PU.1 to Wnt signaling. Failure of appropriate PU.1 repression in T cell progenitors with URE deletion disrupted differentiation and induced thymic transformation. Genome-wide DNA methylation assessment showed that epigenetic silencing of selective tumor suppressor genes completed PU.1-initiated transformation of lymphoid progenitors with URE deletion. These results elucidate how a single transcription factor, PU.1, through the cell context-specific activity of a key cis-regulatory element, affects the development of multiple cell lineages and can induce cancer.

Immunoglobulin secretion by B1 cells: differential intensity and IRF4-dependence of spontaneous IgM secretion by peritoneal and splenic B1 cells.

Peritoneal B1 cells are typified by spontaneous, constitutive secretion of IgM natural antibody, detected by ELISPOT assay, among other means. Recently, this key characteristic has been called into question, a reason for which we evaluated the integrity of IgM(+) ELISPOT spots. We found that fixed B1 cells fail to produce ELISPOT spots, that interference with Golgi function inhibits ELISPOT spot formation, and that B1 cell-derived immunoglobulin in supernatant samples is EndoH-resistant. These findings indicate that spots produced by B1 cells on ELISPOT assay reflect secretory IgM actively exported by viable B1 cells. Current paradigms propose that interferon response factor 4 (IRF4) is required for plasma cell differentiation and immunoglobulin secretion. However, we found that IgM secretion by peritoneal B1 cells is not altered in IRF4-null mice. In contrast, spontaneous IgM secretion by splenic B1 cells, which amounts to much more IgM secreted per cell, is dramatically reduced in the absence of IRF4. These results indicate that peritoneal B1 cells spontaneously secrete low levels of IgM via an IRF4-independent non-classical pathway, and, considering the low level of serum IgM in IRF-null mice, further suggest that accumulation of serum immunoglobulin depends on IRF4-dependent secretion by splenic B1 cells.

B cell receptor crosstalk: B cells express osteopontin through the combined action of the alternate and classical BCR signaling pathways.

Classical BCR signaling requires a number of signalosome mediators that are bypassed when BCR signaling follows an alternate pathway produced by prior exposure of B cells to IL-4. The two pathways, classical and alternate, co-exist in IL-4-treated B cells. Here we report that operation of the IL-4-induced alternate pathway in combination with the classical pathway changes the nature of the B cell response to BCR engagement so that the cytokine, osteopontin (Opn), is produced and secreted. Although Opn expression by B cells has not previously been noted, anti-Ig-induced secretion by IL-4-treated B cells amounts to levels comparable to those secreted by activated T cells. However, unlike T cell Opn expression, B cell expression of Opn is not mediated by T-Bet. Because elevated levels of IL-4 occur in association with severe illness, and because Opn is strongly associated with autoimmunity, these results suggest that the IL-4-induced alternate BCR signaling pathway may participate in the pathophysiology of autoimmune dyscrasias.

Immunoglobulin kappa enhancers are differentially regulated at the level of chromatin structure.

The kappa intronic and the kappa 3' enhancers synergize to regulate recombination and transcription of the Ig kappa locus. Although these enhancers have overlapping functions, the kappa i enhancer appears to predominate during receptor editing, while the kappa 3' enhancer may be more important for initiating Ig kappa germline transcription to target locus recombination and, later in development, somatic hypermutation. Changes in chromatin structure appear to regulate both enhancers, and previous reports suggest that both enhancers are packaged into an accessible chromatin structure only in B lineage cells. Why these enhancers cannot activate the demethylated, accessible, protein-associated Ig kappa allele in pro-B cells is not known. Furthermore, how the enhancers function to reactivate the locus for receptor editing or to quantitatively promote hypermutation in B cells is vague. Quantitative analysis of Ig enhancer chromatin structure in murine pro-, pre-and splenic B cells demonstrated that the kappa i enhancer maintains a highly accessible chromatin structure under a variety of conditions. This stable chromatin structure mirrored the highly accessible structure characterizing the Ig mu intronic enhancer, despite the fact that Ig mu is activated prior to Ig kappa during B cell development. Surprisingly, parallel analysis of the kappa 3' enhancer demonstrated its accessible chromatin structure is markedly unstable, as characterized by sensitivity to changes in environmental conditions. These data unexpectedly suggest that kappa locus regulation is compartmentalized along the gene in B lineage cells. Furthermore, these findings raise the possibility that environmentally dependent regulation of kappa 3' enhancer structure underlies changes in kappa activation during B cell development.

B-1 cells express transgelin 2: unexpected lymphocyte expression of a smooth muscle protein identified by proteomic analysis of peritoneal B-1 cells.

B-1 cells constitute a unique B cell subset that differs phenotypically, biochemically, and functionally from the predominant population of conventional B-2 cells. Functional differences include constitutive secretion of natural immunoglobulin and failure of BCR signaling to initiate proliferation. The origin of these differences remains uncertain. We hypothesized that unbiased analysis of differences in protein expression between highly pure populations of B-1 and B-2 cells might provide information not readily available through other means. To pursue this, we undertook 2D gel analysis of B-1 and B-2 cells combined with mass spectrometry. We identified the smooth muscle protein, transgelin 2, in peritoneal (but not splenic) B-1 cells and did not find it in splenic B-2 cells; these results were confirmed by Western blot analysis, which showed a more than 60-fold difference in transgelin 2 expression between peritoneal B-1 and splenic B-2 cells. In contrast, levels of transgelin 2 RNA differed to a much lesser extent (3-fold) in the two B cell populations, so transgelin 2 is an example of a molecule whose subset-specific expression is more readily detected by proteomic than transcriptomic analyses. Finally, transgelin 2 protein expression was induced in splenic B-2 cells; thus, transgelin 2 joins a number of other inducible molecules that are constitutively expressed by peritoneal B-1 but not splenic B-2 cells. Although the role of transgelin 2 in B-1 cell function remains uncertain, identification of this molecule demonstrates the value of examining protein expression in this B cell subset.

CD25 + B-1a Cells Express Aicda.

B-1a cells are innate-like B-lymphocytes producing natural antibodies. Activation-induced cytidine deaminase (AID), a product of the Aicda gene, plays a central role in class-switch recombination and somatic hypermutation in B cells. Although a role for Aicda in B-1a cells has been suggested on the basis of experiments with knock out (KO) mice, whether B-1a cells express Aicda, and if so, which B-1a cell subpopulation expresses Aicda, remains unknown. Here, we demonstrate that B-1 cells express Aicda, but at a level below that expressed by germinal center (GC) B cells. We previously reported that B-1a cells can be subdivided based on CD25 expression. We show here that B-1a cell Aicda expression is concentrated in the CD25+ B-1a cell subpopulation. These results suggest the possibility that previous studies of memory B cells identified on the basis of Aicda expression may have inadvertently included an unknown number of CD25+ B-1a cells. Although B-1a cells develop normally in the absence of Aicda, a competitive reconstitution assay reveals enhanced vigor for AID KO B-1a cell bone marrow (BM) progenitors, as compared with wild-type BM B-1 cell progenitors. These results suggest that AID inhibits the development of B-1a cells from BM B-1 cell progenitors in a competitive environment.

CD5+/Mac-1- peritoneal B cells: a novel B cell subset that exhibits characteristics of B-1 cells.

The peritoneal cavity of mice is enriched for B-1 B cells, a lymphocyte subset that differs from conventional B-2 cells phenotypically, functionally, and developmentally. According to current paradigms, all peritoneal B-1 cells express Mac-1 whereas B-2 cells do not and thus these populations are often purified by FACS sorting or magnetic bead isolation based on B cell expression of Mac-1 or lack thereof. However, in the course of studying B220+/Mac-1- peritoneal B-2 cells, we discovered that this population is actually heterogeneous, with approximately 30-40% of these B220+/Mac-1- cells expressing the B-1 cell marker CD5. It was unclear whether this B220+/CD5+/Mac-1- peritoneal B cell population represented aberrantly CD5 expressing B-2 cells or Mac-1- B-1 cells. To address this issue we tested CD5+/Mac-1- peritoneal B cells for several traits that distinguish B-1 and B-2 cells. We found that CD5+/Mac-1- peritoneal B cells resembled CD5+ B-1 cells and not B-2 cells in terms of expression of several additional surface markers (IgM, IgD, CD23, CD43, and CD80). Further, CD5+/Mac-1- peritoneal B cells expressed high levels of V(H)11 and V(H)12, two Ig variable genes that are expressed mainly by B-1 but not B-2 cells. In addition, CD5+/Mac-1- peritoneal B cells responded to PMA, a mitogen that stimulates B-1 cells but not B-2 cells, and not to anti-Ig, that stimulates B-2 cells but not B-1 cells. ELISPOT analyses of freshly isolated CD5+/Mac-1- peritoneal B cells revealed that they secreted IgM constitutively, like B-1 cells and unlike B-2 cells. These results indicate that CD5+/Mac-1- peritoneal B cells are a new subset of B-1 cells, here termed B-1c, and stress the importance of using multiple surface markers to identify and purify specific B cell populations.

Characteristic features of B cells in murine cervical lymph nodes.

CONCLUSION: B cells in cervical lymph nodes correspond to typical conventional B cells (B-2). OBJECTIVE: The special status of cervical lymph nodes in relation to the oropharynx, and the need to maintain the integrity of the oropharnygeal mucosal barrier, suggest the possibility that cervical lymph node B cells located in the oropharynx may behave differently from B cells located elsewhere. In this study we examined the symmetry or lack thereof between cervical lymph node B cells and other B-cell subsets. MATERIAL AND METHODS: We isolated B cells from murine cervical lymph node tissue and evaluated them in vitro according to several criteria. RESULTS: We found that cervical lymph node B cells expressed typical B-cell phenotypic markers and proliferated normally in response to mitogenic stimulation. They did not spontaneously secrete immunoglobulin and, in keeping with this, did not express elevated levels of either CD138 (Syndecan-1), a marker for plasma cells, or BLIMP-1, a putative master regulator of B-cell differentiation.

A CD25⁻ positive population of activated B1 cells expresses LIFR and responds to LIF.

B1 B cells defend against infectious microorganisms by spontaneous secretion of broadly reactive "natural" immunoglobulin that appears in the absence of immunization. Among many distinguishing characteristics, B1 B cells display evidence of activation that includes phosphorylated STAT3. In order to identify the origin of pSTAT3 we examined interleukin-2 receptor (IL-2R) expression on B1 cells. We found that some (about 1/5) B1a cells express the IL-2R α chain, CD25. Although lacking CD122 and unresponsive to IL-2, B1a cells marked by CD25 express increased levels of activated signaling intermediates, interruption of which results in diminished CD25. Further, CD25⁺ B1a cells contain most of the pSTAT3 found in the B1a population as a whole. Moreover, CD25⁺ B1a cells express leukemia inhibitory factor receptor (LIFR), and respond to LIF by upregulating pSTAT3. Together, these results define a new subset of B1a cells that is marked by activation-dependent CD25 expression, expresses substantial amounts of activated STAT3, and contains a functional LIFR.

Continual signaling is responsible for constitutive ERK phosphorylation in B-1a cells.

B-1a cells constitutively express phosphorylated, activated ERK, but the origin of pERK in B-1 cells has not been determined. To address this issue, we examined specific mediators of intracellular signaling in unmanipulated B-1a cells. We found that constitutive pERK was rapidly lost from B-1a cells following addition of metabolic inhibitors that block src kinase, Syk, PI-3K, and PLC function. We examined Syk and PLC in more detail and found rapid accumulation of phosphorylated forms of these molecules in B-1a cells, but not B-2 cells, when phosphatase activity was inhibited, and this change occurred in the majority of B-1a cells. Further, we showed that inhibition of src kinase activity eliminated "downstream" pSyk and pPLC accumulation in phosphatase-inhibited B-1a cells, indicating a pathway connection. CD86 expression is greater on B-1 than B-2 cells and plays a role in antigen presentation by B-1 cells to T cells. We found that when Syk or PI-3K was inhibited, CD86 expression was diminished in a reversible fashion. All together, these results indicate that continual activation of intracellular signaling leads to constitutive activation of ERK in B-1 cells, with attendant consequences for co-stimulatory molecule expression.

Extreme skewing of annexin II and S100A6 expression identified by proteomic analysis of peritoneal B-1 cells.

B-1 cells differ phenotypically, biochemically and functionally from conventional B-2 cells. The origin of these differences remains uncertain. We hypothesized that unbiased analysis of differences in protein expression between B-1 and B-2 cells might provide information not otherwise available, and thus undertook 1-dimensional (1D) gel analysis combined with mass spectrometry. We identified annexin II and S100A6 in peritoneal B-1 cells (B-1P) but not in splenic B-2 cells (B-2S); these results were confirmed by western blot analysis and reflected in mRNA expression. Further analysis of mRNA indicated that elevated expression levels of annexin II and S100A6 were unique to B-1P among several naive lymphoid populations. However, expression of annexin II and S100A6 protein was up-regulated in mitogenically stimulated B-2S. In both naive B-1 cells and stimulated B-2 cells, annexin II and S100A6 formed Ca++-sensitive complexes. These results confirm that the emerging field of proteomics detects differentially expressed molecules independently of RNA screening methods. These results identify two proteins (annexin II and S100A6) that are unexpectedly differentially expressed in B-1 cells and, although members of larger families, may fulfill unique, subset-specific functions. These results also validate 1D GE/LC-MS/MS as a reliable screening tool in identifying final protein product expression differences between B-1P and B-2S.

PD-L2 expression extends beyond dendritic cells/macrophages to B1 cells enriched for V(H)11/V(H)12 and phosphatidylcholine binding.

B1 B cells are the major source of natural antibody that is essential for innate immunity. The B1 repertoire is skewed toward production of phosphatidylcholine (PtC)-binding V(H)11 and V(H)12 immunoglobulin that plays a key role in immune defense against bacterial infection. Programmed death-ligand 2 (PD-L2) is a ligand for the immunosuppressive receptor programmed death-1 (PD-1). It has been reported that expression of PD-L2 is restricted to dendritic cells and macrophages in mice. Here we show that 50-70% of resting peritoneal B1 cells express PD-L2, which is not present or inducible on conventional B2 B cells or PD-L2(-) B1 cells. Although PD-L2(+) and PD-L2(-) B1 cells are similar in proliferative responses and spontaneous immunoglobulin secretion, PD-L2(+) B1 cells are highly enriched for expression of V(H)11 and V(H)12 genes and encompass the bulk of PtC-binding B1 cells. These findings extend the range of known PD-L2 expression to B cells and show that B1 cells identified by this marker express a specific repertoire associated with anti-bacterial immunity.

Functional outcome of B cell activation by chromatin immune complex engagement of the B cell receptor and TLR9.

We have previously shown that rheumatoid factors produced by Fas-deficient autoimmune-prone mice typically bind autologous IgG2a with remarkably low affinity. Nevertheless, B cells representative of this rheumatoid factor population proliferate vigorously in response to IgG2a/chromatin immune complexes through a mechanism dependent on the sequential engagement of the BCR and TLR9. To more precisely address the role of both receptors in this response, we analyzed the signaling pathways activated in AM14 B cells stimulated with these complexes. We found that the BCR not only serves to direct the chromatin complex to an internal compartment where it can engage TLR9 but also transmits a suboptimal signal that in combination with the signals emanating from TLR9 leads to NF-kappaB activation and proliferation. Importantly, engagement of both receptors leads to the up-regulation of a group of gene products, not induced by the BCR or TLR9 alone, that include IL-2. These data indicate that autoreactive B cells, stimulated by a combination of BCR and TLR9 ligands, acquire functional properties that may contribute to the activation of additional cells involved in the autoimmune disease process.

Peritoneal B-2 cells comprise a distinct B-2 cell population with B-1b-like characteristics.

B-1 and B-2 cells are lymphocyte populations that differ in development, surface marker expression, tissue localization, and function. Though mainly found in the spleen, lymph nodes, and circulation of mice, small numbers of B-2 cells are found in the peritoneal cavity, a site predominantly populated by B-1 cells. Here, we characterized peritoneal B-2 cells, and determined their relationship to B-1 cells. We found that peritoneal B-2 cells appear to be intermediate between splenic B-2 and peritoneal B-1 cells in terms of surface marker expression of B220, CD80, and CD43, expression of several marker genes, and in vitro viability and IgM secretion. Adoptive transfer of peritoneal B-2 cells into severe combined immunodeficiency mice resulted in the acquisition of a phenotype reminiscent of B-1b cells, as shown by up-regulation of Mac-1 and CD43, and down-regulation of CD23. Moreover, adoptively transferred peritoneal B-2 cells recapitulated B-1 cell function by producing natural IgM in recipient mice. These data suggest that peritoneal B-2 cells express some characteristics of B-1b cells and that this similarity increases with additional time in the peritoneal cavity.

Disruption of cyclin D3 blocks proliferation of normal B-1a cells, but loss of cyclin D3 is compensated by cyclin D2 in cyclin D3-deficient mice.

Peritoneal B-1a cells differ from splenic B-2 cells in the molecular mechanisms that control G(0)-S progression. In contrast to B-2 cells, cyclin D2 is up-regulated in a rapid and transient manner in phorbol ester (PMA)-stimulated B-1a cells, whereas cyclin D3 does not accumulate until late G(1) phase. This nonoverlapping expression of cyclins D2 and D3 suggests distinct functions for these proteins in B-1a cells. To investigate the contribution of cyclin D3 in the proliferation of B-1a cells, we transduced p16(INK4a) peptidyl mimetics (TAT-p16) into B-1a cells before cyclin D3 induction to specifically block cyclin D3-cyclin-dependent kinase 4/6 assembly. TAT-p16 inhibited DNA synthesis in B-1a cells stimulated by PMA, CD40L, or LPS as well as endogenous pRb phosphorylation by cyclin D-cyclin-dependent kinase 4/6. Unexpectedly, however, cyclin D3-deficient B-1a cells proliferated in a manner similar to wild-type B-1a cells following PMA or LPS stimulation. This was due, at least in part, to the compensatory sustained accumulation of cyclin D2 throughout G(0)-S progression. Taken together, experiments in which cyclin D3 was inhibited in real time demonstrate the key role this cyclin plays in normal B-1a cell mitogenesis, whereas experiments with cyclin D3-deficient B-1a cells show that cyclin D2 can compensate for cyclin D3 loss in mutant mice.

B-1 cells are deficient in Lck: defective B cell receptor signal transduction in B-1 cells occurs in the absence of elevated Lck expression.

B-1 cells constitute a unique B cell subset that is primarily responsible for producing nonimmune Ig. This natural Ig acts as a principal line of defense against infection. A key feature of B-1 cells is the failure of BCR-triggered signal transduction. Recently, defective BCR signaling in B-1 cells has been attributed to elevated expression of the canonical T cell src kinase, Lck. In the present study, we re-examined Lck expression in normal B-1 cells. We found that B-1 cells expressed less Lck at both the protein and RNA levels than did B-2 cells. The same B-1 cells manifested defective BCR-mediated induction of IKKbeta phosphorylation, IkappaBalpha degradation, and intracellular Ca(2+) mobilization. Thus, the failure of BCR signaling in B-1 cells does not relate to subset-specific elevation of Lck.

Spontaneously Ig-secreting B-1 cells violate the accepted paradigm for expression of differentiation-associated transcription factors.

B-1 cells spontaneously secrete natural Ig that acts as a primary line of defense against infection. A major shortfall in our understanding of this key process centers on the molecular mechanisms regulating natural Ab secretion by B-1 cells. Herein, we demonstrate that secreting B-1 cells use some aspects of the recently recognized plasmacytic differentiation program but deviate from it in important ways. Specifically, we show that key repressors of the plasmacytic program, B cell leukemia/lymphoma-6 and paired box gene 5, are reduced in spontaneously secreting B-1 B cells, as in stimulated differentiated B-2 cells. Surprisingly, we find that key promoters of the plasmacytic program, B lymphocyte inducer of maturation program 1 and X-box binding protein 1, are not up-regulated in secreting B-1 cells, in contrast to secreting B-2 cells. These data demonstrate that B-1 cells operate under a differentiation program that is unique and differs from the paradigm associated with Ig-secreting B-2 cells.

IL-6 rescues the hyporesponsiveness of c-Rel deficient B cells independent of Bcl-xL, Mcl-1, and Bcl-2.

The hematopoietically restricted member of the NF-kappaB/Rel family, c-Rel, is essential for B cell survival and proliferation. Here we demonstrate that the production of the interleukins 6, 10, and 15 (IL-6, IL-10, and IL-15) are diminished in c-Rel(-/-) B lymphocytes. In a manner similar to that seen in IL-6(-/-) B cells, resultant STAT activation is reduced in c-Rel(-/-) B cells following B cell receptor (BCR) ligation. Addition of either exogenous IL-6 or IL-10, but not IL-15, partially restores proliferation, and this occurs through enhanced cell survival rather than promoting cell cycle progression. This increase in viability occurs independently of Bcl-xL and Mcl-1 expression though, two survival genes reported to be downstream of IL-6 signaling. Nonetheless, transgenically expressed Bcl-xL, a direct c-Rel target gene in B cells, corrects not only the survival defect of c-Rel deficiency, but also partially ameliorates hypoproliferation. Together IL-6 and Bcl-xL are additive but incomplete in the restoration of proliferation. Known deficits in the induction of several key cell cycle components in c-Rel(-/-)B cells are not corrected upon treatment with exogenous cytokine. Together, these data demonstrate that IL-6 enhances B cell responses by employing multiple survival factors.

Peritoneal and splenic B-1 cells are separable by phenotypic, functional, and transcriptomic characteristics.

B-1 cells constitute a distinct B cell population with unique phenotypic and functional characteristics. Although the origin of B-1 cells remains controversial, B-1 cells in different locations are generally considered to be part of the same pool. To determine the validity of this assumption, we examined peritoneal and splenic B-1 cells isolated by flow cytometric cell sorting from normal mice for several features. We found that splenic B-1 cells differ from peritoneal B-1 cells in terms of surface antigen expression, viability ex vivo, immunoglobulin secretion in vitro, stimulated cell cycle progression, and expression of Notch family, Notch-dependent, and Notch-associated genes. These results indicate that splenic and peritoneal B-1 cells are not the same and thus dispute the notion that B-1 cells are uniform, and may suggest that different subpopulations of B-1 cells arise separately, home individually, and/or are heavily influenced by local environmental factors.

E mu-BRD2 transgenic mice develop B-cell lymphoma and leukemia.

Transgenic mice with lymphoid-restricted overexpression of the double bromodomain protein bromodomain-containing 2 (Brd2) develop splenic B-cell lymphoma and, upon transplantation, B-cell leukemia with leukemic infiltrates in liver and lung. Brd2 is a nuclear-localized transcription factor kinase that is most closely related to TATA box binding protein-associated factor, 250 kDa (TAF(II)250) and the Drosophila developmental protein female sterile homeotic. Constitutive expression of BRD2 in the lymphoid compartment increases cyclin A transcription, "priming" transgenic B cells for proliferation. Mice stochastically develop an aggressive B-cell lymphoma with the features of B-1 cells, including CD5 and surface IgM expression. The B-cell lymphoma is monoclonal for immunoglobulin gene rearrangement and is phenotypically stable. The lymphoblasts are very large and express a transcriptome that is similar to human non-Hodgkin lymphomas. Both a wild-type BRD2 transgene and a kinase-null point mutant drive lymphomagenesis; therefore we propose that, rather than kinase activity, Brd2-mediated recruitment of E2 promoter binding factors (E2Fs) and a specific histone acetyltransferase to the cyclin A promoter by both types of transgene is a mechanistic basis for neoplasia. This report is the first to describe a transgenic mouse model for constitutive expression of a protein with more than one bromodomain.