Baseline autoantibody profiles predict normalization of complement and anti-dsDNA autoantibody levels following rituximab treatment in systemic lupus erythematosus.

B cells are thought to play a major role in the pathogenesis of systemic lupus erythematosus (SLE). Rituximab (RTX), a chimeric anti-CD20 mAb, effectively depletes CD20( +) peripheral B cells. Recent results from EXPLORER, a placebo-controlled trial of RTX in addition to aggressive prednisone and immunosuppressive therapy, showed similar levels of clinical benefit in patients with active extra-renal SLE despite effective B cell depletion. We performed further data analyses to determine whether significant changes in disease activity biomarkers occurred in the absence of clinical benefit. We found that RTX-treated patients with baseline autoantibodies (autoAbs) had decreased anti-dsDNA and anti-cardiolipin autoAbs and increased complement levels. Patients with anti-dsDNA autoAb who lacked baseline RNA binding protein (RBP) autoAbs showed increased complement and decreased anti-dsDNA autoAb in response to RTX. Other biomarkers, such as baseline BAFF levels or IFN signature status did not predict enhanced effects of RTX therapy on complement or anti-dsDNA autoAb levels. Finally, platelet levels normalized in RTX-treated patients who entered the study with low baseline counts. Together, these findings demonstrate clear biologic activity of RTX in subsets of SLE patients, despite an overall lack of incremental clinical benefit with RTX in the EXPLORER trial.

Cellular ITAM-containing proteins are oncoproteins in nonhematopoietic cells.

Immunoreceptor tyrosine-based activation motifs (ITAMs) are involved in the transduction of signals necessary for activation, differentiation, and survival in hematopoietic cells. Several viruses have been shown to encode ITAM-containing transmembrane proteins. Although expression of these viral proteins has in some cases been shown to transform nonhematopoietic cells, a causal role for a functional ITAM in this process has not been elucidated. To examine the potential transforming properties of ITAM-containing proteins, a recombinant protein consisting of ITAM-containing cytoplasmic regions of the B-cell antigen receptor was expressed in immortalized murine mammary epithelial and fibroblast cells. Mammary epithelial cells expressing this construct exhibited depolarized morphology in three-dimensional cultures. This transformed phenotype was characterized by a loss of anchorage dependence and hallmarks of epithelial to mesenchymal transition. Fibroblasts expressing this ITAM construct also lost contact inhibition and anchorage dependence. The transformed phenotype seen in both cell types was abrogated upon tyrosine to phenylalanine substitutions of the ITAMs. Inhibition of Syk tyrosine kinase, which associates with the ITAM, also prevented cell transformation. Our results indicate that expression of a nonviral ITAM-containing protein is sufficient for cell transformation. Despite lacking intrinsic enzymatic activity, ITAM-containing proteins can function as potent oncoproteins by scaffolding downstream mediators.

Ligand-independent signaling functions for the B lymphocyte antigen receptor and their role in positive selection during B lymphopoiesis.

Signal transduction through the B cell antigen receptor (BCR) is determined by a balance of positive and negative regulators. This balance is shifted by aggregation that results from binding to extracellular ligand. Aggregation of the BCR is necessary for eliciting negative selection or activation by BCR-expressing B cells. However, ligand-independent signaling through intermediate and mature forms of the BCR has been postulated to regulate B cell development and peripheral homeostasis. To address the importance of ligand-independent BCR signaling functions and their regulation during B cell development, we have designed a model that allows us to isolate the basal signaling functions of immunoglobulin (Ig)alpha/Igbeta-containing BCR complexes from those that are dependent upon ligand-mediated aggregation. In vivo, we find that basal signaling is sufficient to facilitate pro-B --> pre-B cell transition and to generate immature/mature peripheral B cells. The ability to generate basal signals and to drive developmental progression were both dependent on plasma membrane association of Igalpha/Igbeta complexes and intact immunoregulatory tyrosine activation motifs (ITAM), thereby establishing a correlation between these processes. We believe that these studies are the first to directly demonstrate biologically relevant basal signaling through the BCR where the ability to interact with both conventional as well as nonconventional extracellular ligands is eliminated.

Role for transcription Pax5A factor in maintaining commitment to the B cell lineage by selective inhibition of granulocyte-macrophage colony-stimulating factor receptor expression.

During early B lymphopoiesis, developing B cells maintain lineage commitment despite the local presence of myeloid lineage-promoting cytokines such as GM-CSF and IL-3. Previous observations suggest that the B cell-specific transcription factor Pax5A (paired box 5A transcription factor) plays a role in maintaining B cell lineage commitment by limiting expansion and survival of early IL-3/GM-CSF-dependent myeloid lineage cells. To define a mechanism by which Pax5A can exert these inhibitory effects on myeloid lineage differentiation, an inducible form of the Pax5A protein was expressed in the myeloid cell line FDC-P1. This cell line models myeloid progenitors in that it responds to the survival and growth-potentiating effects of IL-3 and GM-CSF. We observed that enforced expression of Pax5A selectively suppressed proliferation in response to GM-CSF, but not IL-3. This effect was associated with specific down-regulation of GM-CSFR alpha-chain, but not beta-chain expression. These data provide a molecular mechanism to enforce commitment to the B cell lineage despite the presence of GM-CSF, a factor that has been shown to convert early developing B cells to the myeloid lineage. Furthermore, they indicate a role for B cell Pax5A expression in maintaining rather than directing commitment to the B cell lineage.

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.

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.

Ligand-dependent and -independent processes in B-cell-receptor-mediated signaling.

The B cell antigen receptor (BCR) is a protein complex expressed on the surface of immature and mature B cells. After ligand-induced aggregation, this complex generates signals that lead to a variety of biological outcomes, including survival, proliferation and differentiation. During B cell development intermediate forms of the BCR are expressed on the surface. The composition of these pro- and preBCR complexes reflects the ordered assembly of the BCR complex and they exist to generate signals for positive selection at defined developmental checkpoints. Because these receptors lack the ability to bind conventional ligands, the pro- and preBCR have been postulated to signal via ligand-independent processes. This ligand-independent or constitutive signal may also play a role in the survival of peripheral mature B cells. Here we discuss the evidence for ligand-independent functions for the BCR and postulate how it may be regulated and linked to biological processes associated with B cell development and survival.

Immunobiology of the immature B cell: plasticity in the B-cell antigen receptor-induced response fine tunes negative selection.

The immature and transitional immature B-cell stages define an important window in B-cell development, as it is at this point that cells committed to the B-cell lineage first express the clonotypic B-cell antigen receptor (BCR) and cells expressing self-reactive specificities may be identified and eliminated. The intrinsic susceptibility of the immature B cell to negative selection following BCR engagement distinguishes these cells functionally from mature-stage B cells in which BCR cross-linking leads to activation. Our laboratory has been interested in determining the molecular events responsible for the distinct and disparate responses of immature and mature B cells to antigen receptor signaling in order to understand the molecular basis of negative selection of developing B cells. These studies have indicated that developmentally regulated mechanisms, intrinsic to the B cell, regulate the differential responsiveness of the immature and mature stage B cell to antigen. However, the "hard-wired" BCR-induced apoptotic response of the immature B cell can be modified by the microenvironmental context in which the antigen is encountered. This plasticity fine tunes the BCR-induced response of the immature B cell by regulating the mechanism of negative selection and, under defined circumstances, allowing for recruitment into an immune response.

Evidence that immunoglobulin specificities of AIDS-related lymphoma are not directed to HIV-related antigens.

Chronic B-cell stimulation may be a predisposing event in the early pathogenesis of the acquired immunodeficiency syndrome (AIDS)-related lymphoma (ARL). ARL-derived immunoglobulin (Ig) genes are significantly diversified from germline, suggesting that antigenic stimulation via Ig receptors may occur prior to malignant transformation. We have evaluated 6 ARL-derived antibodies for binding to human immunodeficiency virus (HIV) and cell surface epitopes. Five cases expressed IgM, and 1 case expressed IgG. Expressed V genes were significantly diversified (3%-15%) from known germline V genes. A non-Ig producing mouse myeloma cell line was transfected with expression vectors containing the lymphoma-derived V genes. By enzyme-linked immunosorbent assay and Western blot assay, the lymphoma-derived Ig's showed no reactivity against HIV recombinant proteins. Also, no specific HIV reactivity was observed by flow cytometry with lymphoma-derived Ig's against the T-cell line infected with T-tropic HIV-1 or peripheral blood mononuclear cells infected with M-tropic HIV strains, indicating lack of binding to native HIV epitopes. However, 2 of the lymphoma-derived Ig's (ARL-7 and ARL-14) bound strongly to non-HIV-infected cells of various tissue origins. Thus, these findings suggest that the transformed B cells of AIDS-associated lymphomas may not arise from the pool of anti-HIV specific B cells but, rather, may develop from B cells responding to other antigens, including self-antigens. (Blood. 2000;95:1393-1399)

BSAP/Pax5A expression blocks survival and expansion of early myeloid cells implicating its involvement in maintaining commitment to the B-lymphocyte lineage.

Early B lymphopoiesis is marked by plasticity between the myeloid and B lineages. An attractive model for B-lineage development is that commitment to this lineage is partly determined by the ordered expression of genes that prohibit switching to the myeloid lineage. In this regard, whereas the role of the B-cell-specific transcription factor BSAP/Pax5A in regulating B-lymphoid-restricted gene expression has been well-established, its role in maintaining B-lineage commitment is unclear. Thus, BSAP/Pax5A was constitutively expressed in the multipotent EML cell line, which can be directed toward the myeloid lineage by culture with interleukin-3 (IL-3) and retinoic acid. EML cells expressing BSAP/Pax5A successfully acquired the myeloid lineage markers CD11b and F4/80 in response to IL-3 and retinoic acid, indicating differentiation to the myeloid lineage. However, these early myeloid cells failed to expand in culture with granulocyte-macrophage colony-stimulating factor and were directed instead toward an apoptotic pathway. In parallel, primary bone marrow stem cells transduced with retrovirus constitutively expressing BSAP/Pax5A began myeloid cell differentiation, but like the transformed EML model failed to expand in response to myeloid growth factors. These studies identify a role for BSAP/Pax5A in suppressing the response to myeloid growth factors, which may be a component of the regulatory processes that limit plasticity of early B-lymphoid progenitors.

Negative selection of immature B cells by receptor editing or deletion is determined by site of antigen encounter.

Immature B cells that encounter self-antigen are eliminated from the immune repertoire by negative selection. Negative selection has been proposed to take place by two distinct mechanisms: deletion by apoptosis or alteration of the antigen receptor specificity by receptor editing. While convincing evidence exists for each, the two models are inherently contradictory. In this paper, we propose a resolution to this contradiction by demonstrating that the site of first antigen encounter dictates which mechanism of negative selection is utilized. We demonstrate that the bone marrow microenvironment provides signals that block antigen-induced deletion and promote RAG reinduction. In the periphery, the absence of these signals allows the immature B cell to default to apoptosis as a result of BCR engagement.

Antigen receptor-induced signal transduction imbalances associated with the negative selection of immature B cells.

Signals transduced through the B cell Ag receptor (BCR) drive B cell development. However, BCR-induced responses are developmentally regulated; immature B cells are tolerized following antigenic exposure while mature B cells are triggered to proliferate and differentiate. This differential responsiveness allows for the negative selection of self-reactive immature B cells while simultaneously allowing for clonal expansion of mature B cells in response to foreign Ags. Intrinsic differences in BCR-induced signal transduction at various stages of development may account for this functional dichotomy. We had previously demonstrated that the BCR-induced proliferation of mature B cells is accompanied by an increase in intracellular calcium levels and polyphosphoinositide bis phosphate (PIP2) hydrolysis. In contrast, immature B cells that undergo BCR-induced apoptosis increase intracellular calcium in the relative absence of PIP2 hydrolysis. Since PIP2 hydrolysis leads to the generation of diacylglycerol, a cofactor for protein kinase C (PKC) activation, these data suggested that an "imbalance" in BCR-induced signal transduction resulting from a relative inability to activate PKC may play a role in the susceptibility of immature B cells to BCR-induced apoptosis. In support of this hypothesis, we demonstrate that PKC activation can rescue immature B cells from BCR-induced apoptosis. Furthermore, the susceptibility of immature B cells to BCR-induced apoptosis is recapitulated in mature B cells that are either PKC depleted or are stimulated in the presence of PKC inhibitors, suggesting that an uncoupling of PKC activation from BCR-induced signaling is responsible for the apoptotic response of immature B cells.

B cell receptor-induced apoptosis in primary transitional murine B cells: signaling requirements and modulation by T cell help.

Self-reactive immature B cells may be eliminated in the bone marrow (BM) after B cell receptor (BCR) engagement in a process known as negative selection. Immature B cells emigrating from the BM, the so-called transitional cells, remain sensitive to negative selection and are likely to be important targets of tolerance towards peripheral antigens. Transitional cells are deleted through apoptosis after BCR cross-linking in vitro. Using anti-Ig as a surrogate antigen, we determined the signaling requirements for the induction of apoptosis in transitional cells. Treatment with anti-Ig for only 20 min causes most cells to be apoptotic 16 h later. Furthermore, apoptosis of transitional cells is induced with low doses of anti-Ig while mature cell proliferation requires extended culture at 30-fold higher concentrations. For both populations of B cells, total surface Ig expression is equivalent, therefore indicating that the threshold of BCR signaling required to elicit these responses is different. T cell help can modulate B cell tolerance. However, specific help may not be available when apoptosis is triggered by a peripheral antigen. The opportunity to reverse apoptosis of transitional cells is surprisingly long. Even 8 h after anti-Ig treatment, IL-4 or anti-CD40 antibody can block apoptosis. The upper time limit of protection is concurrent with irreversibility of apoptosis as measured by DNA fragmentation. These findings indicate that B cell negative selection is more easily triggered than activation, and that the induction of apoptosis and its reversal by T cell help can be events that occur in distinct microenvironments.

Antigen receptor-initiated signals for B cell development and selection.

The B cell antigen receptor or signaling components of this receptor play varying roles in determining the fate of the B cell at different stages of B cell development. Signals generated through this receptor complex or its various components determine the survival, progression, expansion, and activation of the B cell. Factors that determine the fate of the B cell following antigen receptor engagement include the developmental stage, coreceptor expression, degree and stability of ligand-receptor engagement, and the availability of T cell-derived secondary signals. The author's laboratory is interested in defining the molecular processes linking B cell antigen receptor signaling to these specific cellular responses in the context of antigen-independent and dependent B cell development. Here our current progress and thinking with regard to the determination between negative selection and activation as it pertains to the transition from the immature- to the mature-stage B cell will be discussed.

Characterization of anti-single-stranded DNA B cells in a non-autoimmune background.

Anti-DNA Abs are prevalent in the serum of systemic lupus erythematosus (SLE) patients and in the MRL-lpr/lpr mouse model of SLE, but are generally absent in normal individuals. We have studied the regulation of anti-ssDNA B cells in a non-autoimmune (BALB/c) background by using Ig transgenes (Tgs) encoding anti-DNA Abs. In one case, they are present with other non-DNA-binding B cells (the VH3H9 Tg with endogenous light chains); in the other, they are present as an essentially monospecific population (VH3H9/Vkappa8). We have previously observed that serum anti-ssDNA levels in these Tg mice were no higher than those of non-Tg mice, despite the fact that anti-ssDNA B cells dominate the peripheral B cell repertoire. These results suggested that the anti-ssDNA Tg B cells present are functionally inactivated. In this paper, we isolate B cells from VH3H9/Vkappa8 Tg mice to show that this is indeed the case and go on to further define this state. We demonstrate that VH3H9/Vkappa8 Tg B cells have diminished Ig secretion in response to both T-independent and T-dependent stimuli compared with non-Tg controls. VH3H9/Vkappa8 Tg B cells also show suboptimal proliferation in response to anti-IgM F(ab)'2 fragments and LPS, and are phenotypically distinct in expressing decreased total surface Ig. Despite their functional defects, however, VH3H9/Vkappa8 Tg B cells have an in vivo turnover rate comparable to non-Tg B cells, suggesting that they are long lived.

Decreased immunoglobulin deposition in tumors and increased immature B cells in p53-null mice.

Recent studies have hinted that there may be a relationship between p53 and the immune response. In preliminary experiments, we found significantly decreased levels of immunoglobulin deposition in 13 of 16 p53-null tumors compared with 2 of 17 tumors derived from p53 +/- mice. We further explored the effect of p53 on B-cell development and function. p53-null mice contained more splenic white pulp and more immature B cells in the bone marrow compared with p53 +/- mice. p53-null B cells were hyperresponsive to proliferative challenge but were not more resistant to signal-induced apoptosis. Several p53 DNA-binding sites were localized to the regulatory regions of immunoglobulin heavy and light chain genes, including the KII site, which serves as an enhancer for rearrangement of the mouse kappa chain J cluster genes. Levels of p53 protein and the kappa chain sterile transcript increased after exposure of pre-B cells to the DNA damaging agents etoposide and Adriamycin. Our observations suggest that p53 may be involved in B-cell maturation and may relay certain stress signals to affect B-cell function.