Inhibition of human B-cell lymphoma growth by CD40 stimulation
Satoshi FunakoshiDL LongoMargaret BeckwithDK ConleyGalia TsarfatyIlan TsarfatyR J ArmitageWC FanslowMK SpriggsW. H. Murphy
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In this thesis aspects of peripheral B lymphocyte differentiation were studied. Through in vitro experiments, B lymphocyte activation was assessed. B lymphocytes express immunoglobulin (Ig) specific for a certain antigen on the cell surface, and when surface Ig is crosslinked by an antigen, the cell is activated. Antigens giving rise to a T cell dependent immune response are internalized by the B cell, degraded, and presented on MHC II. In order to differentiate, an activated B cell has to interact with a T cell bearing a T cell receptor specific for the same antigen. During B cell/T cell interaction a crosstalk mediated by cell surface molecules is initiated. For example, the CD40/CD40 ligand (CD40L) receptor interaction plays a critical role in B cell activation; genetically engineered animals deficient for either the CD40L or CD40, lack germinal centres, Ig switch and immunological memory. When B cells were activated with anti-CD40 monoclonal antibodies (mAbs) together with anti-CD21 mAbs coupled to Sepharose differentiation to Ig secretion was detected. Addition of anti-CD40 mAbs to lipopolysaccharide (LPS) stimulated B cells was, however, shown to inhibit B cell differentiation in the same manner as addition of anti-Ig mAbs to LPS treated cultures. Further, B cells prestimulated with anti-CD40 were shown to undergo apoptosis when restimulated with anti-Ig. Because of the pivotal role of the CD40L/CD40 interaction in T cell dependent immune responses, the CD40L promoter was analyzed. The CD40L promoter and deletants of that promoter were cloned into an expression vector containing the structural gene for luciferase, as a reporter gene. Upon transient transfection into Jurkat T cells CD40L promoter function was shown to be dependent on signaling via the T cell receptor and CD28. A 270 bp region upstream of the translational start was shown to suffice for full promoter activity. A putative CD28 responsive DNA element distinct from the one in the IL-2 promoter was identified. Streptococcal Ig-binding surface proteins L, M1, and H were shown to bind various cells of the hematopoetic lineage; proteins L and H coupled to Sepharose induced B cell proliferation, whereas protein H coupled to Sepharose also induced B cell differentiation. Soluble protein H was shown to be taken up by T and B cells and transported to the nucleus. Protein H was found to interact with nucleophosmin/B23 (NPM), a protein previously shown to traffic from the cytoplasm to the nucleus, but which was isolated from a membrane preparation from Jurkat T cells. In the nucleus protein H was found to interact with hnRNP A2/B1 and the SET protein. Affinities of protein H with NPM and a nuclear extract prepared from Jurkat T cells were determined. Lastly, when murine B cells were stimulated with LPS, addition of protein H was shown to exert a cytostatic effect. (Less)
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Male BXSB mice, unlike female BXSB, develop a severe early onset lupus-like disease that has been linked to an intrinsic B cell defect. In investigating this B cell defect the present study showed that male, but not female, BXSB contained a higher percentage of large, activated splenic B cells that were more responsive to anti-CD40 mAb-induced proliferation. The hyperactivity of the large B cells from the male mice was also observed in the absence of anti-CD40 mAb or any other stimuli. In examining the mechanism of the B cell hyperactivity, it was found that 20% of unstimulated large B cells from male mice, unlike large B cells from female mice, expressed CD40 ligand (CD40L), a molecule normally expressed on activated CD4+ cells. The percentage of large B cells from the male BXSB that expressed CD40L was increased to 43% by stimulation with LPS. A functional role for CD40L expression on B cells was confirmed by showing that CD40-Ig blocked the spontaneous proliferation of the large B cells from male mice. In addition, the stimulatory capacity of the large B cells from the male mice was demonstrated by their ability to induce DNA synthesis in small B cells in a CD40L-dependent manner. These results demonstrated that large B cells from male BXSB expressed functionally active CD40L. It is likely that the B cell CD40L expression and increased susceptibility to CD40 signaling due to an intrinsic B cell hyperactivity promotes autoimmune disease in BXSB mice.
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Abstract Immunological memory comprising of antigen‐specific B and T cells contributes to the acquisition of long‐term resistance to pathogens. Interactions between CD40 on B cells and CD40L on T cells are responsible for several aspects of acquired immune responses including generation of memory B cells. In order to gain insights into events leading to memory B cell formation, we analyzed the genome‐wide expression profile of murine naive B cells stimulated in the presence of anti‐CD40. We have identified over 8,000 genes whose expression is altered minimally 1.5‐fold at least at one time point over a 3‐day time course. The array analysis indicates that changes in expression level of maximum number of these genes occur within 24 h of anti‐CD40 treatment. In parallel, we have studied the events following CD40 ligation by examining the expression of known regulators of naive B cell to plasma cell transition, including Pax5 and BLIMP1. The expression profile of these regulatory genes indicates firstly, that CD40 signaling activates naïve B cells to a phenotype that is intermediate between the naive and plasma cell stages of the B cell differentiation. Secondly, the major known regulator of plasma cell differentiation, BLIMP1, gets irreversibly downregulated upon anti‐CD40 treatment. Additionally, our data reveal that CD40 signaling mediated BLIMP1 downregulation occurs by non‐Pax5/non‐Bcl6 dependent mechanisms, indicating novel mechanisms at work that add to the complexity of understanding of B cell master regulatory molecules like BLIMP1 and Pax5. J. Cell. Physiol. 229: 1387–1396, 2014. © 2014 Wiley Periodicals, Inc.
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Abstract We report the capacity of CD40 ligand (CD40L)‐negative T cell clones to activate human B cells. CD40L‐negative T cells induce a level of B cell proliferation 10–20% of that seen with normal T cells. The signal provided by the negative clones is synergistic with that derived from a CD40L transfectant, and restores B cell proliferation to normal levels, showing that CD40L‐negative T cell clones are not inherently inhibitory for B cells. Although their capacity to induce proliferation was much reduced, CD40L‐negative T cell clones were still strong inducers of B cell differentiation to plasma cells. This differentiation to plasma cells was inhibited by a CD40L transfectant. The data are discussed with regard to the normal in vivo mechanism for maintaining B cell memory and memory antibody responses to T‐dependent antigens.
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In mice, memory B (Bmem) cells can be divided into two subpopulations: CD80hi Bmem cells, which preferentially differentiate into plasma cells; and CD80lo Bmem cells, which become germinal center (GC) B cells during a recall response. We demonstrate that these distinct responses can be B-cell-intrinsic and essentially independent of B-cell receptor (BCR) isotypes. Furthermore, we find that the development of CD80hi Bmem cells in the primary immune response requires follicular helper T cells, a relatively strong CD40 signal and a high-affinity BCR on B cells, whereas the development of CD80lo Bmem cells does not. Quantitative differences in CD40 stimulation were enough to recapitulate the distinct B cell fate decisions in an in vitro culture system. The quantity of CD40 signaling appears to be translated into NF-κB activation, followed by BATF upregulation that promotes Bmem cell differentiation from GC B cells.
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Abstract Two discrete mechanisms of T‐B cell collaboration appear to exist. In cognate recognition, B cell triggering results from a direct recognition of antigen and MHC determinants at the B cell surface. Alternatively, B cells can be triggered by transstimulation, in which the T h cell is activated by an antigen‐presenting cell to produce soluble factors which in turn trigger the B cell. This report addresses the question of whether antigen recognition at the B cell surface in association with Ia determinants delivers a signal to the B cell, which is qualitatively different from the signals delivered by the soluble mediators released by the activated T h cell. Previous reports from a number of laboratories suggest that cognate recognition is obligatory for the triggering of small resting B cells and B cells of the Lyb‐5 − phenotype, whereas enlarged B cell blasts and the Lyb‐5 + subset can be triggered solely by soluble mediators. Contrary to these findings, the experiments described here indicate that B cells isolated in different states of activation from normal spleens on the basis of their bouyant density in Percoll density gradients, or unfractionated B cells from mice differing genetically due to the xid defect [Lyb‐5 − B cells from (CBA/N × BALB/c)F 1 male mice], do not discriminate between the two modes of T h cell function. In both stimulation modes, the high density B cells, and the B cells from xid mice made very poor immunoglobulin secretory responses measured in terms of reverse plaque formation on protein A‐coupled erythrocytes. When the responses of different density fractions of B cells were compared under conditions where stimulation occurred either directly or indirectly via transstimulation, the following hierarchy of responsiveness in both the proliferative and plaque‐forming cell (PFC) responses was observed in the density fractions 60% > 65% > 70% > 75%. The hierarchy was the same in both modes of interaction and the deficiency of the high density, small B cells was far more marked in the PFC assay than in the proliferative assay. We conclude that the initial proliferative response of the resting B cell can be triggered comparably in vitro under conditions of direct or transstimulation. Thus, recognition of B cell surface Ia by T h cells is not obligatory for B cell activation and does not transfer an essential transmembrane signal to the B cell.
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The delivery of T‐cell help to B cells is antigen‐specific, MHC‐restricted, and CD40L (CD154) dependent. It has been thought that when a T cell recognizes an antigen‐presenting B cell, CD40L expressed on the T‐cell surface engages with CD40 on the surface of B cells as long as the cells remain conjugated. By adding fluorescently labeled anti‐CD40L antibody during overnight incubation of antigen‐presenting B cells with antigen‐specific T cells, we discovered that CD40L does not remain on the surface of the T cell, but it is transferred to and endocytosed by B cells receiving T‐cell help. In the presence of anti‐CD40L antibody, transferred CD40L is nearly absent on bystander B cells that are not presenting antigen, and the bystander cells do not become activated. Because transfer of CD40L to B cells correlates with B‐cell activation, we speculate that persistence of helper T‐cell‐derived CD40L on or in B cells could permit sustained CD40 signaling enabling survival and proliferation of antigen‐presenting B cells following brief interactions with helper T cells in vivo in germinal centers.
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Signals from CD4+ T cells induce two opposite fates in B cells: clonal proliferation of B cells that bind specifically to foreign antigens and clonal deletion of equivalent B cells that bind self-antigens. This B cell fate decision is determined by the concerted action of two surface proteins on activated T cells, CD40- and Fas-ligands (CD40L and FasL), whose effects are switched by signals from the B cell antigen receptor (BCR). Foreign antigens that stimulate the BCR acutely cause CD40L and FasL to promote clonal proliferation. CD40L and FasL trigger deletion, however, when the BCRs become desensitized by chronic stimulation with self-antigens or when BCRs have not bound an antigen. The need for both Fas and CD40L to correctly regulate self-reactive B cell fate may explain the severe autoantibody disorders in Fas- or CD40L-deficient children.
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Summary Dendritic cells (DC) have recently been shown to play an important role in B‐cell function. We have previously shown that DC can capture and retain unprocessed antigen in vitro and in vivo , and can transfer this antigen to naive B cells to initiate antigen‐specific antibody responses. We also demonstrated that DC were providing B cells with isotype‐switch signals independent of T cells but that T‐cell help was essential for antibody production. In this study, using B cells and DC from wild type (WT) and CD40 knockout (CD40KO) mice we show that DC initiate proliferation of B cells independently of CD40, because WT or CD40KO DC could induce proliferation of WT or CD40KO B cells, but proliferation was greater in the absence of CD40. DC also provide B cells with survival signals as WT DC improved viability of B cells after a 5‐day culture but survival was reduced in the absence of CD40 expression.
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