CD83 is a regulator of murine B cell function in vivo

The transmembrane glycoprotein CD83 has been described as a specific maturation marker for dendritic cells and several lines of evidence suggest that CD83 regulates thymic T cell maturation as well as peripheral T cell activation. Here we show for the first time that CD83 is involved also in the regulation of B cell function. CD83 is up-regulated on activated B cells in vivo, specifically in the draining lymph nodes of Leishmania major-infected mice. The ubiquitous transgenic (Tg) expression of CD83 interferes with Leishmania-specific T cell-dependent and with T cell-independent antibody production. This defect is restricted to the B cell population since the antigen-specific T cell response of CD83Tg mice to L. major infection is unchanged. The defective immunoglobulin (Ig) response is due to Tg expression of CD83 on the B cells because wild-type B cells display normal antigen-specific responses in CD83Tg hosts and CD83Tg B cells do not respond to immunization in a mixed wild-type/CD83Tg bone marrow chimera. Finally, the treatment of non-Tg C57BL/6 mice with anti-CD83 mAb induces a dramatic increase in the antigen-specific IgG response to immunization, thus demonstrating a regulatory role for naturally induced CD83 on wild-type B cells.     

Induction of B cell costimulatory function by recombinant murine CD40 ligand

T cell-dependent regulation of B cell growth and differentiation involves an interaction between CD40, a B cell surface molecule, and the CD40 ligand (CD40L) which is expressed on activated CD4⁺ T cells. In the current study, we show that recombinant membrane-bound murine CD40L induces B cells to express costimulatory function for the proliferation of CD4⁺ Tcells. CD40L- or lipopolysaccharide (LPS)-activated, but not control-cultured B cells were strong costimulators of anti-CD3 or alloantigen-dependent T cell responses. The molecular interactions responsible for the increased costimulatory functions were examined by analyzing the activated B cells for changes in the expression of two costimulatory molecules, B7 and heat-stable antigen (HSA), as well as by the use of antagonists of B7 and HSA (CTLA4.Fc and 20C9, respectively). The expression of both B7 and HSA was enhanced on B cells activated with LPS. As observed in previous studies, the costimulatory activity of the LPS-activated B cells was dependent on both B7 and HSA and was completely inhibited in the presence of a combination of CTLA4.Fc and 20C9. In contrast, activation of B cells with CD40L induced the expression of B7 but did not enhance the expression of HSA. In addition the costimulatory activity of the CD40L-activated B cells was partially, but not completely, inhibited by the combination of CTLA4.Fc and 20C9. These results demonstrate that CD40L regulates costimulatory function of B cells in part by inducing the expression of B7 and suggest that CD40L-activated B cells express an additional costimulatory activity that is not associated with LPS-activated B cells.     

Antigen-induced T cell death is regulated by CD4 expression

Activation induced cell death (AICD) is a major physiologic pathway that regulates T cell homeostasis. In CD4 T cells, AICD is mediated mainly through Fas/FasL interactions. Although TCR occupancy triggers AICD, the contribution of its tightly associated CD4 coreceptor to the process that leads to AICD is not known. Here we show that CD4 molecule plays an essential regulatory role of TCR dependent AICD. Loss of CD4 rendered activated 5kc T cell hybridoma resistant to AICD. The resistance of CD4 negative 5kc T cells to AICD was due to selective inhibition of FasL expression and it could be reversed by addition of recombinant FasL. Furthermore, a direct functional link between CD4 and FasL was demonstrated by induction of FasL upon CD4 crosslinking in a TCR independent fashion. The importance of CD4 interaction with MHC/peptide complex in mediating AICD was also evident in normal T cells that could survive chronic stimulation with anti-CD3 but died after short period of proliferation after stimulation with MHC/peptide. Thus it appears that AICD is controlled by the CD4 molecule via regulation of FasL expression. These findings have important implications for our understanding of mechanisms of peripheral tolerance as well as pathogenesis of autoimmune diseases.     

Regulation of murine B cell growth and differentiation by CD30 ligand

A ligand for CD30 has been recently cloned, and has been shown to have sequence homology with the tumor necrosis factor family of cytokines. CD30 ligand (CD30L) was found to be induced on helper T cell clones, and its receptor was expressed on freshly isolated and activated murine B cells. Recombinant murine CD30L was found to share many functional properties with CD40 ligand (CD40L) in the regulation of murine B cell growth and differentiation in vitro. CD30L stimulated B cell proliferation, antigen-specific antibody production, and polyclonal immunoglobulin secretion in a cytokine-dependent manner. In particular, the stimulation of B cell proliferation by CD30L required interleukin (IL)-4 and IL-5, induction of anti-sheep red blood cell antibody-secreting B cells by CD30L required IL-2 and IL-5, and optimal induction of polyclonal immunoglobulin secretion required IL-4 and IL-5. Under these conditions, the polyclonal secretion of IgGl, IgA, IgG3 and IgE was induced. The induction of immunoglobulin secretion by CD30L was independent of CD40L, as B cells from CD40L deficient-mice responded normally to CD30L treatment. We conclude that CD30L is a potent mediator of B cell growth and differentiation in vitro and may play a role in cognate T cell-B cell interactions.     

Homotypic aggregation of murine B lymphocytes is independent of CD23

CD23 is a low-affinity receptor for IgE (Fc?RII). Functions attributed to CD23 not involving IgE suggest that it interacts with ligands other than IgE. CD21 has recently been described as a counter ligand for CD23. A number of lines of evidence have implicated CD23 as an adhesion molecule in human B cells. We have investigated the role of CD23 in homotypic B cell aggregation in the mouse, using lipopolysaccharide plus interleukin-4-induced aggregation as a model system. In this system high levels of aggregation are accompanied by a massive up-regulation of CD23 expression. However, in contrast to what has been observed in human B cells, we find no evidence of a role for CD23 in homotypic adhesion of murine B cells.     

Functional interaction between B cell subpopulations defined by CD23 expression

Using the CD23 monoclonal antibody (mAb) MHM6 and sheep anti-mouse Ig bound to magnetic beads we have obtained highly purified populations of MHM6+ and MHM6- tonsil B cells. We have found that the increased expression of MHM6 reactivity seen on B cells after activation results from up-regulation of antigen on cells already weakly positive and not from expression of new antigen on the previously negative population. The strong proliferative responses of MHM6+ cells seen in the presence of anti-IgM (alpha mu) and interleukin 4 (IL4) or the CDw40 mAb G28-5, and with Staphylococcus aureus Cowan I (SAC), and to a lesser extent with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), resemble that seen among unfractionated B cells. In contrast, the MHM6- population cultured alone responds only weakly to alpha mu + G28-5 or SAC and exhibits virtually no response to alpha mu + IL4 or TPA. With all these mitogenic stimuli, tritiated thymidine uptake by the MHM6- population is augmented three- to sixfold by the addition of mitomycin C (MC)-treated MHM6+ cells. Pretreatment of cells with anti-leukocyte functional antigen 1 mAb has little effect on the subsequent proliferation of the MHM6- population but shows cell contact to be critical for the proliferation of MHM6+ cells. Such pretreatment has revealed that the functional interaction observed between MHM6+ and MHM6- cells is dependent on both cell contact and the presence of an MHM6+ cell-derived soluble component. We have found that addition of soluble CD23, purified from Epstein-Barr virus-transformed lymphoblastoid cell line supernatant, increases the proliferative response of MHM6- tonsil B cells to mitogenic stimuli in the presence of inactivated MHM6+ cells but has no effect on proliferation when MHM6+ cells are absent. By way of contrast to normal B lymphocytes, we have examined functional responses of prolymphocytic leukemia (PLL) B cells. Although these cells, when freshly isolated, show comparable levels of CD23 expression to normal B cells, this expression is not increased upon activation. In addition, in contrast to normal B cells, the PLL MHM6- population cultured alone shows a strong proliferative response to various mitogenic stimuli, comparable to that of MHM6+ or unfractionated cells, and this response is not augmented by the addition of MC-treated MHM6+ cells. Thus, a novel functional interaction is described between normal, but not leukemic, B cell populations defined by their expression of CD23.(ABSTRACT TRUNCATED AT 400 WORDS)     

Administration of an anti-IgE antibody inhibits CD23 expression and IgE production in vivo.

High IgE responder BDF1 mice were immunized intraperitoneally (i.p.) with dinitrophenol4 (DNP4)-ovalbumin (OVA) in alum concomitant with intravenous (i.v.) administration of an anti-IgE monoclonal antibody (mAb). IgE levels were undetectable in mice treated with the anti-IgE antibody, whereas mice treated with isotype-matched irrelevant mAb had IgE levels comparable to that of untreated, immunized mice. Subsequent antigen challenges with DNP4-OVA, either at weekly or monthly intervals, failed to evoke an IgE response for greater than 2 months in mice treated with anti-IgE during the primary sensitization, even though the terminal half-life of the anti-IgE antibody was 7 days. This inhibition was specific for DNP4-OVA since the DNP4-OVA-suppressed mice were able to respond to keyhole limpet haemocyanin (KLH). To investigate the effects of antibody treatment at the cellular level, passive transfer experiments were performed. The primary DNP-specific IgE response of adoptive transfer recipient mice was the same whether the donor cells were from mice treated with IgG or anti-IgE. Transfer of enriched T- or B-cell populations indicated that T-cell help was not compromised by administration of the anti-IgE mAb. However, splenocytes from the anti-IgE-treated mice failed to synthesize IgE in vitro, and flow cytometric analysis of B cells from anti-IgE-treated mice showed a dose-dependent decrease in CD23+ cells following antibody treatment, which correlated with decreased serum IgE levels. Taken together, the results of these studies suggest that anti-IgE treatment suppresses IgE responses via effects on B cells rather than T cells, possibly through effects on CD23-dependent pathways.     

CD40 expression and function in murine B cell ontogeny

The CD40 antigen, a member of the nerve growth factor/tumornecrosis factor receptor family, is expressed on all matureB lymphocytes and plays a crucial role in B cell activation,T cell- dependent antigen-driven isotype switching and germinalcenter formation. We have analyzed C040 expression and functionduring mouse B cell development by examining B cell precursorsin normal mice and in transgenic animals in which B cell developmentis frozen at discrete stages. These models included RAG-2 ^-/-mice, and transgenlc littermates that express µ heavychain and/ or the bcl-2 proto-oncogene transgene. CD40 was undetectableat the pro-B cell stage, but was expressed, although at lowlevels, on pre-B cells. However, pre-B cells failed to respondto CD40 triggering either by expression of CD23 or by proliferationin the presence of lL-4. Overexpression of bcl-2 increased thedensity of CD40 expression on pre-B cells: these cells respondto CD40 ligation by expressing CD23 and by proliferating inthe presence of IL-4.     

Soluble CD40 ligand induces expression of CD25 and CD23 in resting human tonsillar B lymphocytes

In this report, we describe the dose-dependent increase in both CD25 and CD23 levels on resting human B cells in response to CD40 ligation, as mediated by soluble CD40 ligand (sCD40L) or anti-CD40 antibody. In combination with interleukin (IL)-4, sCD40L had limited additive effects on CD25 expression, but significantly enhanced CD23 expression on tonsillar B cells. Interferon-γ (IFN-γ) exerted no inhibitory effect upon increases in CD25 or CD23 driven by CD40 ligation with sCD40L or anti-CD40 antibody. These data suggest that the induction of CD25 and CD23 genes by IL-4 is mediated, at least in part, by an IFN-γ-sensitive component, whereas gene activation driven via CD40 ligation involves signaling pathways which are not sensitive to IFN-γ.     

The follicular versus marginal zone B lymphocyte cell fate decision is regulated by Aiolos, Btk, and CD21

Most splenic B cells in mice that lack Aiolos are mature IgD(hi)IgM(lo) follicular lymphocytes, suggesting that maturation signals delivered via the BCR are enhanced in the absence of Aiolos. The enhanced maturation of follicular B cells is accompanied by the absence of MZ B lymphocytes and the downregulation of CD21 expression in follicular B cells, all of which depend on the generation of signals via Btk, which is in epistasis to Aiolos. The inverse relationship between the strength of BCR signaling and MZ B cell development is supported by an examination of MZ B cells in CD21 null mice. These data support the view that antigens (in contrast to "tonic" signals) drive the development of naive B cells.     

Anti-CD48 (murine CD2 ligand) mAbs suppress cell mediated immunity in vivo

With the identification of murine CD48 as a homolog of the humanCD2 ligand LFA-3 (CD58) and as a ligand itself for murine CD2,the antl-murine CD48 mAb HM48-1 was administered intravenouslyto investigate the role of CD48 In cell mediated immunity invivo. Antl-CD48 mAb diminished the contact sensitivity responseto the hapten trlnltrophenol (TNP). mAb also inhibited in vivopriming for the subsequent generation of secondary, TNP-speclflc,cytotoxic T lymphocytes (CTL) in vitro. The inhibitory effectwas most effective in the afferent or inductive phase of immunityfor CTL, while antl-CD48 mAb was most inhibitory for the efferentor ellcltatlve phase of contact sensitivity. Addition of antl-CD48mAb directly to secondary CTL cultures also completely inhibitedCTL generation, while addition to the lytic assay showed onlyminimal inhibition of CTL activity. Combining cells from mAbtreated and untreated animals showed no evidence for suppressorcells. Further experiments revealed that mAb administered invivo, as well as to culture, Inhibited development of primary,alloantlgen-speclflc CTL in vitro. Mixed lymphocyte reactionand phytohemagglutlnln proliferation were partially suppressedby mAb administered in vivo or in vitro, whereas other mltogenlcresponses remained unaffected. Flow cytometrlc analysis revealeda moderate down modulation of CD48, CD3 and CD8 after treatmentwith anti-CD48. However, this did not represent T cell depletionsince CD2, Thy-1.2 and ig expression did not change. These resultssupport a major unrecognized role for CD48 In diverse aspectsof cell mediated immunity, affecting both CD4⁺ and CD8⁺ effectorT cell function. The anti-CD48 mAb functions not by depletingrelevant T cell populations, but rather by altering the arrayof cell surface receptors, and subsequent responses to primaryand secondary antlgenlc challenge.     

Epitope‐dependent synergism and antagonism between CD40 antibodies and soluble CD40 ligand for the regulation of CD23 expression and IgE synthesis in human B cells

BACKGROUND: The induction of IgE synthesis in naive B cells requires two T-cell-derived signals: one delivered through CD40 and the other via interleukin-4 (IL-4). The natural counterstructure to CD40 is the CD40 ligand (CD40L). We have asked about the interplay between CD40L and CD40 mAb that recognize distinct epitopes in delivering signals for regulating IL-4-dependent IgE synthesis and the expression of CD23, the low-affinity IgE receptor, in resting B cells. METHODS: After culture of purified human tonsillar B cells with CD40 agonists and IL-4, surface CD23 was determined by flow cytometric analysis. CD23 levels in cell lysates and supernatants were quantified by ELISA, as were those of secreted IgE. RESULTS: With regard to both induction of CD23 and IgE production, soluble CD40L trimer (sCD40LT) showed synergistic interaction with two mAb to CD40 which bind to epitopes located outside the ligand binding site (EA5 and 5C3), but not with a mAb (G28-5) which effectively competes for CD40L binding to CD40. Each of the two noncompeting mAb to CD40 was able to cooperate strongly with sCD40LT in promoting high-level induction of CD23 even in the absence of IL-4, an effect mirrored in the promotion of strong homotypic clustering and high-rate DNA synthesis. G28-5, uniquely, induced a down-regulation in IL-4-induced CD23 expression with time, a change that was accompanied by an increase in the amount of soluble CD23 detected. While the two noncompeting mAb consistently synergized with sCD40LT for the promotion of IL-4-dependent IgE synthesis, sCD40LT and G28-5 (which, by itself, was the most potent of the CD40 mAb at inducing IL-4-dependent IgE production) exhibited mutual antagonism in this regard, the level of which could be quite profound. CONCLUSIONS: This study demonstrates that appropriate targeting of CD40 can modulate IgE synthesis either positively or negatively.     

Activation-induced expression of murine CD83 on T cells and identification of a specific CD83 ligand on murine B cells

Human CD83 is a cell surface protein expressed predominantly by dendritic cells (DC) and lymphoid cells. So far, there exists no information on the function and distribution of mCD83. Here we demonstrate that mCD83 is moderately expressed on resting T cells and DC, but strongly increases in its expression on T cells following activation with antigenic peptides or T cell receptor-specific mAb. When returning to the resting state, T cells down-regulate CD83 again. Ig fusion proteins which express the extracellular part of the mCD83 molecule (mCD83-Ig) specifically inhibit antigen-specific T cell proliferation and IL-2 secretion in spleen cell cultures from DO11.10 T cell receptor transgenic mice. Staining of spleen cells from BALB/c, XID and mu MT (B cell) knockout mice with mCD83-Ig proteins reveals the presence of a CD83 ligand predominantly expressed most likely by B220(+) cells since spleen cells from mu MT knockout mice do not bind mCD83-Ig. CD83, besides its established expression on human dendritic cells, thus, also represents a new marker molecule on activated T cells which with its specific ligand is involved in the regulation of T cell responses.     

CD19 is essential for B cell activation by promoting B cell receptor-antigen microcluster formation in response to membrane-bound ligand

Here we describe the spatiotemporal architecture, at high molecular resolution, of receptors and signaling molecules during the early events of mouse B cell activation. In response to membrane-bound ligand stimulation, antigen aggregation occurs in B cell antigen receptor (BCR) microclusters containing immunoglobulin (Ig) M and IgD that recruit the kinase Syk and transiently associate with the coreceptor CD19. Unexpectedly, CD19-deficient B cells were significantly defective in initiation of BCR-dependent signaling, accumulation of downstream effectors and cell spreading, defects that culminated in reduced microcluster formation. Hence, we have defined the dynamics of assembly of the main constituents of the BCR 'signalosome' and revealed an essential role for CD19, independent of the costimulatory molecule CD21, in amplifying early B cell activation events in response to membrane-bound ligand stimulation.     

Enhanced murine CD4+ T cell responses induced by the CD2 ligand CD48

The physiological functions of murine CD2 and its ligand CD48 are uncertain. We have examined the role of the CD2-CD48 interaction in murine T cell activation using a series of Chinese hamster ovary (CHO) cell transfectants. CHO cells expressing I-A^d together with CD48 induced more potent activation of OVA-specific, I-A^d -restricted DO11.10-transgenic T cells than CHO cells expressing I-A^d alone. CD48 augmented proliferation and IL-2 production in response to antigen. The enhancing effect of CD48 was of the same magnitude as that seen for CD80 (B7-1). Conjugate assays revealed the ability of CD48 to increase adhesion between T cells and CHO transfectants. The enhancing effects of CD48 on T cell-antigen-presenting cell adhesion and T cell activation were inhibited by anti-CD2 monoclonal antibody. This report provides the first evidence that the CD2 ligand CD48 contributes to the interactions of murine CD4⁺ T cells with antigen-presenting cells.