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Objective: B cells play a dominant role in the pathogenesis of several autoimmune diseases, including systemic lupus erythematosus. It is not well understood how B cell signaling contributes to autoantibody production. The goal of this study was to elucidate the role of CD72 in modulating B cell receptor (BCR)-mediated tolerogenic signaling and peripheral B cell tolerance., Methods: A mouse model utilizing hen egg lysozyme (HEL) "anergic" B cells was studied. CD72-deficient mice carrying the BCR-specific IgHEL and/or soluble HEL (sHEL) transgenes were generated by breeding IgHEL-transgenic MD4 mice and/or sHEL-transgenic ML5 mice with congenic, CD72-deficient C57BL/6J mice. Normal and anergic B cells were isolated for analyses of B cell signaling. Aged wild-type and CD72-deficient mice were also examined for autoimmune phenomena., Results: In the absence of CD72, anergic B cells inappropriately proliferated and survived in response to stimulation with self antigen. Biochemical analyses indicated that in anergic B cells, CD72 dominantly down-regulated BCR signaling to limit the antigen-induced elevation in [Ca2+]i and the activation of NFATc1, NF-kappaB, MAPK, and Akt. Mechanistically, CD72 was associated with, and regulated, the molecular adaptor Cbl-b in anergic B cells, suggesting that Cbl-b may play a role in mediating the negative effects of CD72 on BCR signaling. Moreover, in aged CD72-deficient mice, spontaneous production of antinuclear and anti-double-stranded DNA autoantibodies and features of lupus-like autoimmune disease were observed., Conclusion: CD72 is required to maintain B cell anergy and functions as a regulator of peripheral B cell tolerance. Thus, altered CD72 expression may play a role during the development of systemic lupus erythematosus.

It has long been known that ligation of the transmembrane CD72 glycoprotein delivers signals to B lymphocytes, with the outcome depending on context. Of particular interest is its ability to function as a counter-receptor/ ligand for the CD100 semaphorin protein. We have now obtained evidence that CD72 physically interacts on the lymphocyte membrane with Fcgamma receptor II (CD32). The association was first revealed with a new monoclonal antibody that recognizes polymorphic determinants on murine CD72. Although the specificity for CD72 was clear from immunoblotting, transfection and other experiments, staining with this reagent was inhibited when cells were pretreated with an Fc receptor-blocking antibody (CD16/CD32 specific). Furthermore, confocal microscopy revealed that the two molecules co-distributed on viable B cells. We also used the antibody to determine when CD72 becomes available to maturing lymphocytes. The marker is first acquired as large pre-B cells and enter the IL-7 independent phase of maturation within bone marrow. Subsequent interactions between CD72 and CD32 may cooperatively deliver negative signals that modulate humoral immune responses.

The pleiotropic functions of tumor necrosis factor-alpha (TNFalpha) have brought considerable attention in the past decade to its physiological and pathological roles in inflammatory and autoimmune diseases. However, little is known about how the production of TNFalpha is regulated at the transcriptional and translational levels in immune cells such as T and B lymphocytes. Our previous study demonstrated that unspliced "pre-mRNA" of TNFalpha is present in resting T cells. Initiation of splicing of TNFalpha pre-mRNA to mature mRNA requires T cell activation, which is unique and necessary for TNFalpha production when compared to its production in mononuclear phagocytes, including different lineages of macrophages (Mvarphi) and dendritic cells (DC). In this study, we further demonstrate that resting mouse B cells also contain pre-existing TNFalpha mRNA. The physiological process of B cell activation induced by (1) either the cross-linking of the B cell receptor (BCR) or CD40, (2) treatment with LPS, or PMA plus ionomycin, induces TNFalpha mRNA splicing in vitro. The kinetic response of TNFalpha splicing in B cells is much slower when compared to that in activated T cells. Studies using well-known kinase inhibitors demonstrated that MAP kinase kinase (MEK) and protein kinase C (PKC) are required for TNFalpha splicing upon stimulation through the BCR. These studies demonstrate that the production of TNFalpha in activated B cells is regulated differently than in activated T cells, and these differences may allow for the selective inhibition of TNFalpha in various autoimmune diseases depending on the mechanism of action of the selected anti-TNFalpha therapy.

The class IV semaphorin CD100/Sema4D differentially utilizes two distinct receptors: plexin-B1 in nonlymphoid tissues, such as brain and kidney, and CD72 in lymphoid tissues. We have generated CD100-deficient mice and demonstrated that they have functional defects in their immune system, without apparent abnormalities in other tissues. The number of CD5(+) B-1 cells was considerably decreased in the mutant mice, whereas conventional B cells and T cells appeared to develop normally. In vitro proliferative responses and immunoglobulin production were reduced in CD100-deficient B cells. The humoral immune response against a T cell-dependent antigen and in vivo priming of T cells were also defective in the mutant mice. These results demonstrate nonredundant and essential roles of CD100-CD72 interactions in the immune system.

We have identified the lymphocyte semaphorin CD100/Sema4D as a CD40-inducible molecule by subtractive cDNA cloning. CD100 stimulation significantly enhanced the effects of CD40 on B cell responses. Administration of soluble CD100 markedly accelerated in vivo antigen-specific antibody responses. CD100 receptors with different binding affinities were detected on renal tubular cells (K(d) = approximately 1 x 10(-9)M) and lymphocytes (K(d) = approximately 3 x 10(-7)M). Expression cloning revealed that the CD100 receptor on lymphocytes is CD72, a negative regulator of B cell responsiveness. CD72 thus represents a novel class of semaphorin receptors. CD100 stimulation induced tyrosine dephosphorylation of CD72 and dissociation of SHP-1 from CD72. Our findings indicate that CD100 plays a critical role in immune responses by the novel mechanism of turning off negative signaling by CD72.

The ability of lymphocytes to respond to antigenic or mitogenic stimulation is regulated not only by specific receptor proteins, but also by both positive and negative regulatory proteins that set or fine-tune the threshold for responsiveness. CD72 is one such regulatory protein on B lymphocytes. It is a member of the C-type lectin superfamily and is expressed on the surface of B cells from the pro-B through the mature B-cell stage. Studies with anti-CD72 antibodies have suggested a positive regulatory role for CD72 in B-cell activation. However, the cytoplasmic tail of CD72 contains two potential immunoreceptor tyrosine-based inhibitory motifs, one of which has been shown to recruit the tyrosine phosphatase SHP- 1. These features suggest a negative regulatory role for CD72. We have generated CD72-deficient mice to elucidate the physiological role of CD72 in B-lymphocyte development and activation. Our analyses of these mice and their B-cell compartment demonstrate that CD72 is a nonredundant regulator of B-cell development and a negative regulator of B-cell responsiveness.

CD72 is a 45-kDa transmembrane glycoprotein that is predominantly expressed on cells of the B lineage except plasma cells. Previously, we identified the 255-bp minimal mouse CD72 promoter capable of tissue-specific and developmental stage-specific expression. DNase I footprinting analysis of the 255-bp CD72 promoter revealed three protected elements, footprint (FP) I, FP II, and FP III. FP II, which extends from nucleotide -189 to -169 of the mouse CD72 promoter, exhibited both tissue-specific and developmental stage-specific activity that was reflective of the activity of the CD72 gene in vivo. In this report, we show that FP II is specifically recognized by the transcription factor B cell-specific activator protein (BSAP). Mutations eliminating the binding of BSAP in reporter constructs also eliminated the increase of reporter activity in B cells. In addition, cotransfections with reporter constructs plus different amounts of expression plasmids for BSAP showed that CD72 promoter activity was up-regulated by BSAP in plasmacytoma cells and T cells in a dose-dependent manner. Moreover, the expression level of CD72 decreased 10-fold on normal plasma cells. Compared with the presence of BSAP binding in mature B cells, the binding of BSAP was undetectable in those plasma cells. This study strongly suggests that BSAP-FP II interaction plays a critical role in determining the cell-type specificity of the CD72 promoter. The absence of positive factors such as BSAP accounts for at least part of the loss of mouse CD72 expression in plasma cells and thus might be common for the down-regulation of many molecules at the plasma cell stage.

CD72 is a 45-kDa glycoprotein that is predominantly expressed on cells of the B lineage, except for plasma cells. Its expression pattern is representative of many B cell-specific proteins, which are essential for B cell development and activation but are down-regulated after B cells become terminally differentiated plasma cells. We have examined the promoter region of the mouse CD72 gene to identify sequences responsible for this regulatory pattern. The CD72 gene does not have an obvious TATAA box. Primer extension assays identified multiple transcription initiation sites. Deletion analyses have identified the 255-bp minimal promoter required for tissue-specific and developmental stage-specific expression. DNase I footprinting analysis of the CD72 minimal promoter revealed three protected elements: FP I, FP II, and FP III. Sequences corresponding to FP I or III gave increased reporter gene activity specifically in B cells, but not in T cells or NIH-3T3 cells. Sequences corresponding to FP II gave increased reporter gene activity in mature B cells, but not in plasma cells or non-B cells. Electrophoretic mobility shift assays and DNase I protection analyses revealed that FP I was bound by the transcription factor PU.1/Spi-1. Transient reporter analyses with plasmid bearing the mutated PU.1 binding site showed that binding of PU.1 is necessary for the increase in CD72 promoter activity in B cells. These results suggest that the 255-bp CD72 promoter confers both tissue specificity and developmental stage specificity, and that the B cell and macrophage-specific transcription factor PU.1 is essential for regulating the tissue specificity of the mouse CD72 promoter.

CD72 is a 45 000 Mr mouse B-cell surface glycoprotein involved in B-cell proliferation and differentiation. Expression of mouse CD72 is thought to be restricted to the B-cell lineage. We recently demonstrated that the monoclonal antibodies K10.6 and B9.689, previously defined as recognizing the mouse lymphocyte alloantigens Ly-19.2 and Ly-32.2, respectively, recognize specific alleles of CD72. Early studies using antibody-mediated cytotoxicity assays demonstrated that K10.6 and B9.689 react with B cells, several T-cell lines, and a subset of peripheral T cells. These findings led us to consider the possibility that CD72 might also be expressed on a subset of T cells. In this report we demonstrate that CD72 is constitutively expressed on a fraction of peripheral T cells isolated from strains of mice expressing the CD72(b) allele, but not the CD72(a) or CD72(c) alleles. Three days after activating T cells with concanavalin A or plate-bound CD3-specific mAb, CD72 is expressed on a larger fraction of peripheral T cells as well as a fraction of thymocytes from mouse strains expressing the CD72(b) allele. CD72 is expressed on both the CD4(+) and CD8(+) thymocyte and peripheral T-cell subsets. No CD72 expression is detected on activated thymocytes or peripheral T cells from mouse strains expressing the CD72(a) or CD72(c) alleles. Expression of CD72(b) on peripheral T cells was confirmed by northern blot analysis demonstrating CD72 mRNA expression. These results demonstrate that CD72 expression is not restricted to B lineage cells in mouse strains expressing the CD72(b) allele; instead, a population of T lineage cells in these mice also expresses CD72.

CD5 is a 67-kDa glycoprotein expressed on the cell surface membrane of all T lymphocytes and on a small proportion of B lymphocytes. The physiologic role of this Ag is still unknown. Structural and functional studies of CD5 suggest that it might act as a receptor for a positive signal. CD5-specific mAb augment CD3- or mitogen-induced T cell proliferation, IL-2 secretion, and IL-2R expression and induce a rise in intracellular [Ca2+]. In this report, we describe the purification of mouse CD5 protein (mCD5) and its use as a probe to search for the ligand of CD5. We demonstrate that mCD5 specifically interacts with the mouse B cell differentiation Ag CD72/Lyb-2. Three serologically defined allelic forms of mouse CD72/Lyb-2 can all interact with mCD5. We further show that mCD5 can interact with human CD72/Lyb-2, and similarly, that human CD5 can interact with mouse CD72/Lyb-2. These studies may have major implications for the mechanisms of T-B cell communication.

The glycoprotein CD5 is expressed on the surface membrane of all mature T cells and a small proportion of B lymphocytes. Its exact role in immune interactions is still unknown. Studies indicate that CD5 functions both in mice and humans as a receptor, delivering co-stimulatory signals to T cells in a manner similar to CD2 (ref. 11) and CD28 (ref. 12). Anti-CD5 antibodies stimulate both T-cell proliferation mediated by CD3 in association with the T-cell receptor and secretion of interleukin-2 and expression of its receptor, as well as inducing an increase in intracellular Ca2+ concentration (refs 5-10). To identify the ligand for CD5 we purified the human CD5 protein, labelled it with biotin and used it as a probe. Here we report that CD5 specifically interacts with the cell-surface protein CD72 exclusive to B cells. This interaction is blocked by anti-CD72 antibodies, but not by any other anti-B-cell antibodies. Moreover, non-B cells (mouse L-cell fibroblasts and human Jurkat T cells) expressing a transfected human CD72 complementary DNA could bind to the CD5-biotin conjugate. The results demonstrate that the B-cell surface protein CD72 (Lyb-2 in mice) is the ligand for CD5.

We have isolated and sequenced cDNA clones encoding the human homolog of the mouse Lyb-2 B cell differentiation Ag. Previous data suggest that Lyb-2 might represent a growth factor or lymphokine receptor. Human Lyb-2 mRNA is expressed in normal human tonsils and bone marrow cells, in the pre-B cell line REH, in three Burkitt lymphoma cell lines, and in some EBV-transformed B cell lines, but not in antibody-secreting myeloma cell lines, T cell lines, or a promyelocytic leukemia cell line. These data indicate that expression of human Lyb-2 is restricted to B lineage cells and turned off in antibody-secreting plasma cells. A polyclonal mouse antiserum was raised against human Lyb-2 and immunoprecipitates a Mr 42,000 protein from REH, Raji, and Daudi cells and from mouse L(tk) cells transfected with the human Lyb-2 cDNA in an expression vector. The human Lyb-2 protein is related to both the asialoglycoprotein receptor and CD23, the B cell-specific FcR for IgE. These data demonstrate that human B cells express a previously undescribed cell surface protein that is homologous to mouse Lyb-2 and has a similar pattern of expression during B cell development.

Lyb-2 is a mouse B-cell differentiation antigen expressed on the surface of pre-B cells and B cells but not on terminally differentiated antibody-secreting plasma cells. Lyb-2 has been shown to play a role in B-cell activation and differentiation and may be a receptor for a B-cell growth factor or lymphokine. We have isolated and sequenced cDNA encoding the Lyb-2.1 allele. Lyb-2 mRNA is expressed only in B-lineage cells and is absent from antibody-secreting cell lines. The predicted protein contains 354 amino acids and is lacking an amino-terminal signal peptide. The protein is shown to be oriented with its carboxyl terminus external to the cell. Sequence comparisons demonstrate that Lyb-2 is homologous to the asialoglycoprotein receptor and to CD23, the B-cell-specific Fc receptor for IgE, both of which are oriented with their carboxyl termini external to the cell. These molecules, therefore, constitute a gene superfamily of cell surface receptors with inverted membrane orientation.