CD40-CD40 ligand: a multifunctional receptor-ligand pair.
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Amino Acid Sequence↗
Biomedical subjects
J Banchereau
Publications and source records attributed to J Banchereau.
Follicular dendritic cells and germinal centers.
Follicular dendritic cells (FDCs) are stromal cells unique to primary and secondary lymphoid follicles. Recirculating resting B cells migrate through the FDC networks, whereas antigen-activated B cells undergo clonal expansion within the FDC networks in a T cell-dependent fashion, thereby generating germinal centers. Here, B cells undergo somatic mutation, positive and negative selection, isotype switching and differentiation into high-affinity plasma cells and memory B cells. Since the discovery of FDCs by electron microscopy as long-term antigen-retaining cells 30 years ago isolation of FDCs and generation of FDC-like cells lines and of FDC-specific monoclonal antibodies have been achieved. FDCs express all three types of complement receptors as well as Ig-Fc receptors, through which antigen-antibody immune complexes are retained. However, the mechanism that prevents FDCs from internalizing the antigens and retaining them in native form for long periods of time remains obscure. Substantial evidence derived from cultures in vitro indicates that FDCs contribute directly to the survival and activation of peripheral B cells. The adhesion between FDCs and B cells is mediated by ICAM-1 (CD54)-LFA-1(CD11a) and VCAM-VLA-4. T cells may interact with FDCs in a CD40/CD40-ligand-dependent fashion. Whether FDCs originate from hematopoietic progenitors or from stromal elements is still a controversy. New evidence suggests the presence of two types of dendritic cells within human germinal centers: (i) the classic FDCs that express DRC-1, KiM4, and 7D6 antigens represent stromal cells; and (ii) the newly identified CD3-CD4-CD11c- germinal center dendritic cells (GCDC) represent hematopoietic cells that may be analogous to the antigen-transporting cells described in mice. Finally, FDCs appear to be involved in the growth of follicular lymphomas and in the pathogenesis of HIV infection.
Animals↗
Within germinal centers, isotype switching of immunoglobulin genes occurs after the onset of somatic mutation.
Human tonsillar B cells were separated into naive IgD+CD38-CD23- (Bm1) and IgD+CD38-CD23 (Bm2), germinal center IgD-CD38+CD23- centroblasts (Bm3) and IgD-CD38+CD77- centrocytes (Bm4) and memory IgD-CD38- (Bm5) subsets. Previous IgVH sequence analysis concluded that the triggering of somatic mutations occurs during the transition from Bm2 subset into the Bm3 subset. To determine the initiation of isotype switching, sterile transcript expression was analyzed by amplification, cloning, and sequencing. A selective sterile I gamma, I alpha, and I epsilon expression was observed at centrocyte (Bm4) stage, suggesting that isotype switch is triggered within germinal centers, after somatic mutation is initiated with centroblasts (Bm3). Finally, the high level of 5'S gamma-S mu 3' DNA switching circles observed in germinal center B cells indicates that within human tonsils, germinal center is a major location for isotype switching.
B-Lymphocytes↗
Normal human IgD+IgM- germinal center B cells can express up to 80 mutations in the variable region of their IgD transcripts.
Somatic hypermutation in immunoglobulin variable region genes occurs within germinal centers. Here, we describe a subset of germinal center dark zone centroblasts that express only sIgD and have accumulated up to 80 mutations per heavy chain variable region (IgVH delta gene). Over half of the hypermutated IgVH delta sequences were found to be clonally related. This level of mutation is not observed in either IgVH gamma transcripts from the same sample or IgVH delta transcripts from peripheral blood, suggesting that these cells neither undergo isotype switch nor mature into circulating memory B cells. Optimal growth of these cells in vitro depends on CD40 ligand, T cell cytokines, and a fibroblast stroma, a combination possibly mimicking the dark zone microenvironment. Our hypothesis is that these cells may be sequestered within germinal centers, where their somatic mutation machinery is triggered. The isolation of these hypermutated B cells may represent a critical step for studying both the biology and biochemistry of somatic hypermutation.
ADP-ribosyl Cyclase↗
IL-10-driven immunoglobulin production by B lymphocytes from IgA-deficient individuals correlates to infection proneness.
In search for a possible explanation of the phenotypic heterogeneity in IgA deficiency, we studied the function of B cells from IgA-deficient (IgAd) individuals. Two groups of IgAd individuals, one frequently infected and one clinically apparently healthy, as well as normal controls, were studied. Peripheral blood mononuclear cells (PBMC) and B cells from IgAd individuals and controls were cultured with Staphylococcus aureus Cowan I strain and with anti-CD40 MoAb presented on the CD32-transfected fibroblast cell line in the presence of IL-10. In this experimental system PBMC and B cells from the infection-prone IgAd individuals produced only minute amounts of IgA. In contrast, PBMC and B cells from healthy IgAd subjects secreted significantly more IgA1 and IgA2 in comparison with infection-prone IgAd patients (P < 0.05). These data suggest that the abnormalities of B cell differentiation in IgAd could be of heterogeneous origin. Thus, whereas in healthy IgAd subjects IgA production may be efficiently up-regulated in vitro by addition of IL-10 to CD40-activated B cell culture, the corresponding B cell differentiation does not occur in infection-prone IgAd patients. These observations provide a conceptual framework for phenotypic heterogeneity in IgAd subjects.
Adult↗
Identification of human IgG autoantibodies specific for IL-10.
Since autoantibodies to IL-1alpha, interferon-alpha (IFN-alpha) and IL-6 have been described, this study concentrated on the search for autoantibodies to hIL-10 using an assay based on the precipitation of 125I-hIL-10 autoantibody complexes using Protein G-Sepharose. Among 1860 tested sera, only seven were found to specifically precipitate IL-10, thus indicating the rare occurrence of such autoantibodies. Four of those seven anti-IL-10 autoantibody sera were specific for hIL-10, two recognized both human and viral IL-10, while the last one recognized human, viral and murine IL-10, thus suggesting the existence of at least three different epitopic specificities. The purification of anti-IL-10 autoantibody from one serum demonstrated the existence of a single (IgG1, lambda) autoantibody that neutralized IL-10 biological activity. Thus, autoantibodies to IL-10 may represent natural antagonists to IL-10.
Antibody Specificity↗
The functional CD40 antigen of fibroblasts may contribute to the proliferation of rheumatoid synovium.
This paper demonstrates that CD40 is expressed on rheumatoid synovial pannus and primary fibroblast cell lines established from rheumatoid and osteoarthritic synovium as well as normal skin. Among various tested cytokines, interferon-gamma (IFN-gamma) and to a lower extent, tumour necrosis factor-alpha (TNF-alpha) were found to upregulate CD40 expression on fibroblasts. Synovial and skin fibroblasts cultured over CD40 Ligand transfected L cells (L-CD40 L) demonstrate a CD40 specific increase of DNA synthesis as measured by tritiated thymidine incorporation. Cell-cycle analysis and enumeration of viable cells further show that CD40 induced fibroblast proliferation. Costimulation with L-CD40 L and IFN-gamma resulted in maximal proliferation. Engagement of fibroblasts CD40 increased the IL-1-induced production of granulocyte macrophage-colony stimulating factor and macrophage inflammatory protein-1 alpha MIP-1 alpha. CD40 L activated fibroblasts showed decreased levels of CD40, but only marginal alterations of other cell-surface antigens. Taken together, the present results indicate that fibroblasts express functional CD40 and suggest a possible role of CD40 L expressing cells, such as activated T cells and mast cells, in the development of synovium hyperplasia.
Animals↗
Delayed IgG2 humoral response in infants is not due to intrinsic T or B cell defects.
The physiologically low or absent IgG2 responses of infants have been attributed to T or B cell functional immaturity. We have analyzed the capacity of adult and neonatal T lymphocytes to secrete IgG2 switch factor (IgG2-SF) and the capacity of neonatal B cells to respond to such factors. The IgG2-SF capacity was assessed on CD40-activated naive B cells, measuring IgG2 by ELISA in supernatants of cultures performed in the presence of IL-10. T cells secreted IgG2-SF together with IL-2 and IFN-gamma, after activation with a combination of anti-CD2, anti-CD28 and phorbol myristate acetate (Th1-like activation). In contrast, activation with anti-CD3 and anti-CD28, which yielded IL-4 and IL-10 but neither IL-2 nor IFN-gamma (Th2-like activation), did not result in the secretion of IgG2-SF. The supernatant of activated neonatal T cells contained IgG2-SF. Neonates' B cells produced almost as much IgG2 as did naive adult B cells. The effect of IgG2-SF was further demonstrated by its ability to induce 3-15% of CD40-activated naive B cells to express cytoplasmic IgG2 regardless of the presence of IL-10. This study demonstrates that: (i) IgG2 switch can be T cell dependent in humans, (ii) IgG2-SF is produced with Th1-like cytokines and (iii) low IgG2 responses in infants do not result from either an inability of T cells to produce IgG2-SF or an inability of B cells to undergo IgG2 switch in vitro.
Adult↗
Biochemical and morphological characterization of vascular and lymphocytic interleukin-4 receptors.
The distribution of the interleukin (IL)-4 receptor in normal human and common marmoset (Callithrix jacchus) tissues was examined by immunofluorescence and flow cytometry using monoclonal antibodies specific for the human IL-4 receptor to gain further insight into IL-4-mediated inflammatory and immunological events. IL-4 receptor positivity was unequivocally demonstrated on lymphocytes, predominantly T cells, and on blood vessels in many tissues. Vascular IL-4 receptor immunofluorescence consisted of a strong smooth muscle cell positivity and weaker positive staining of capillary and venular endothelial cells. Subnanomolar concentrations of IL-4 induced a genistein-sensitive up-regulation of VCAM-1 in vascular cell cultures. Tumor necrosis factor-alpha induced a genistein-resistant up-regulation of VCAM-1. IL-4 strongly induced expression of the IL-4 receptor on splenocytes (T lymphocytes) but not on vascular smooth muscle or endothelial cell cultures. Receptor cross-linking to [125I]IL-4 revealed a 65- to 75-kDa accessory receptor subunit consistent with a recently cloned IL-13 receptor associated with the IL-4 receptor on both vascular endothelial and smooth muscle cells. The demonstration of a vascular distribution pattern for the IL-4 receptor in addition to expression on lymphocytes suggests that vascular functional alterations, transduced through a unique IL-4 receptor complex (the type II IL-4 receptor), may be of importance during immunological and allergic inflammatory events.
Animals↗
[B lymphocytes of patients with complete IgA deficiency secrete IgA in response to interleukin 10].
We have previously shown that human B lymphocytes cultured in the CD40 system, composed of an anti-CD40 mAb presented by a CD32-transfected fibroblastic cell line, proliferate but do not secrete immunoglobulins (Igs). However, the addition of particles of Staphylococcus aureus Cowan (SAC) induces B cell to secrete considerable amounts of Igs even in the absence of exogenous cytokines (CD40/SAC system). Additionally, B lymphocytes cultured in the CD40 system in the presence of human IL-10, produce high level of IgM, IgG and IgA, which are further increased by addition of SAC. Here, we have studied the capacity of peripheral blood lymphocytes from patients with IgA deficiency (IgA-D) to secrete Igs, particularly IgA after CD40 triggering. Peripheral blood mononuclear cells (PBMNC) from IgA-D patients cultured in the CD40/SAC system produced IgM and IgG, but no IgA. The addition of IL-10 to the cultures, enhanced the production of IgM and IgG and most strikingly induced the production of high amounts of IgA. The addition of IL-10 to PBMNC from IgA-D patients activated through CD40 alone resulted in the production of IgA. Thus, IL-10 can remove the block in B cell differentiation and allows B cells from IgA-D patients to differentiate into IgA secreting cells.
Adult↗
Presence of Epstein-Barr virus viral interleukin-10 in the serum of patients with non-human-immunodeficiency-virus-related diffuse large-cell non-Hodgkin's lymphomas.
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Antineoplastic Combined Chemotherapy Protocols↗
Human dendritic Langerhans cells generated in vitro from CD34+ progenitors can prime naive CD4+ T cells and process soluble antigen.
Earlier studies have concluded that fresh Langerhans cells (LC) are able to capture and process native Ags, whereas cultured LC have lost these functions while acquiring the capacity to prime naive T cells. Herein we studied the functions of human dendritic/Langerhans cells (d-Lc) generated in vitro by culturing CD34+ hemopoietic progenitor cells in the presence of granulocyte-macrophage CSF (GM-CSF) + TNF-alpha. Less than 50 d-Lc were found to strongly stimulate the proliferation of 2.5 x 10(4) allogeneic naive CD4+ T cells. Furthermore, six to 50 d-Lc induced half-maximal proliferation of naive syngeneic CD4+ cord blood T cells, in the presence of picomolar concentrations of superantigens. During the alloreaction, the CD4+ T cells were expanded up to 100-fold within two successive stimulation cycles with the same d-Lc, and the recovered T cells were specific for the d-Lc alloantigen. HLA-matched tetanus toxoid (TT)-specific T cell clones were found to proliferate in response to TT presented by CD1a+ d-Lc. Finally, electron microscopy demonstrated that CD1a+ d-Lc were able to capture an Ag (gold-labeled Igs) through receptor-mediated endocytosis. Thus, in vitro generated d-Lc can prime naive T cells and process native Ags, a property that might eventually prove useful for priming Ag-specific naive T cells for cellular immunotherapy.
Adult↗
Fas ligation induces apoptosis of CD40-activated human B lymphocytes.
Since CD40/CD40 ligand (CD40Lig) interactions are essential in vivo for the generation of germinal center B cells that express Fas (Apo-1/CD95), we explored whether CD40 engagement may modulate Fas expression and function on human B lymphocytes. Resting tonsil B cells, isolated by density gradient centrifugation, express either absent or low levels of Fas. They could be induced to promptly express Fas after ligation of their CD40, however, using either a recombinant human CD40Lig or a cross-linked anti-CD40 mAb. In contrast, engagement of the B cell antigen receptor by immobilized anti-kappa and -lambda antibodies did not turn on Fas expression. Addition of anti-Fas mAb CH11 inhibited the later phases of CD40-induced B cell growth as a result of apoptotic cell death. Furthermore, Fas ligation inhibited proliferation and Ig secretion of CD40-activated B cells in response to recombinant cytokines such as interleukin (IL)-2, IL-4, and IL-10, as well as a cytokine-rich supernatant of phytohemagglutinin-activated T cells, indicating that none of those B cell tropic factors were able to prevent the Fas-induced death. Taken together, the present results show that engagement of CD40 antigen on B cells induces Fas expression and sensitizes them to Fas-mediated apoptosis. The delayed functional response to Fas ligation after CD40 activation may represent a way to limit the size of a specific B cell clone that is generated during T-B cell interactions.
Animals↗
Functional expression of CD40 antigen on human epidermal Langerhans cells.
It is now well established that interactions of CD40 on the B cells, along with its ligand (CD40-L) on the T cells, regulate B cell proliferation and differentiation. However, the functional significance of CD40 expression on cells known for most efficient Ag-presenting function, i.e., dendritic cells, is not so clear. In this study, we demonstrate that CD40 is expressed on human dendritic Langerhans cells (LC) freshly isolated from epidermis. Using CD40-L transfected cells, CD40 triggering was found to enhance LC viability when cultured and to result in phenotypic alterations. Thus, a 2-day CD40 activation induced up-regulation of CD54 and CD86 at the LC surface, while it did not significantly affect the levels of HLA-DR, CD1a, CD58, and CD80 expression. These phenotypic changes correlate with enhanced LC allostimulatory property, as shown by the use of paraformaldehyde-fixed LC. Furthermore, mAbs against CD40, as well as CD40-L, strongly inhibit the primary T cell response to allogeneic LC. Collectively, these data support a role for CD40/CD40-L pair in the development of normal T cell functions.
Binding Sites, Antibody↗
Human follicular dendritic cells enhance cytokine-dependent growth and differentiation of CD40-activated B cells.
Germinal centers constitute microanatomic subunits within secondary follicles where B cells undergo somatic mutations, isotype switch and affinity selection. This allows the generation of memory B cells and plasma cells, whose Igs bind to the eliciting Ag with a high affinity. T cells and follicular dendritic cells (FDCs) are thought to play key roles in the germinal center reaction. To study effects of FDCs on B cell growth and differentiation, we have isolated FDC-lymphocyte clusters from human tonsils by enzymatic digestion and centrifugation of the resulting cell suspension through BSA gradient. Irradiated FDC-lymphocyte clusters induced moderate proliferation of autologous B cells. IL-2 was the only cytokine able to enhance B cell proliferation cocultured with FDCs. When B cells were activated by soluble anti-CD40 Ab with or without IL-2, IL-3, IL-4, IL-10 or IL-13, addition of FDCs increased B cell proliferation. In the presence of FDCs, maximal B cell proliferation was observed in anti-CD40 stimulated cultures supplemented with either IL-4 + IL-10 or IL-2 + IL-10. Cultures performed in the presence of IL-2 and IL-10 resulted in high levels of Ig production in the presence of FDCs. In conclusion, the present study demonstrates that freshly isolated human FDCs can enhance the growth and differentiation of CD40-activated B cells.
B-Lymphocytes↗
Contribution of IL-1, CD14, and CD13 in the increased IL-6 production induced by in vitro monocyte-synoviocyte interactions.
Rheumatoid synovitis is characterized by an infiltration of mononuclear cells and by the proliferation of synoviocytes. Monocytes and synoviocytes are major producers of cytokines, growth factors, and enzymes that contribute to the rheumatoid arthritis (RA) process. Since they are in close contact in vivo, we engaged in an in vitro study of the functional consequences of their interactions. Coculture of unstimulated elutriated normal blood monocytes over RA synoviocytes resulted in a synergistic increase of the production of IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), and IL-8, when compared with their respective production in culture alone. In contrast, cytokines such as IL-10, IL-1 beta, IL-1 alpha, and TNF-alpha could not be detected. The IL-6 production in coculture was further increased by the addition of IL-1 beta, GM-CSF, IFN-gamma, or TNF-alpha, but was inhibited by the addition of IL-10, IL-4, IL-13, or IL-1Ra, an effect reverted by the addition of IL-1 beta. Moreover, an inhibition was also observed with anti-CD14 mAb and newly raised mAbs directed against RA synoviocytes. Under reducing conditions, the mAb SY12 precipitated a 150-kDa surface membrane protein, identified as amino-peptidase N (CD13/AP-N). Collectively, these results indicate that 1) monocytes and synoviocytes interact with each other to produce proinflammatory cytokines, 2) pro- and antiinflammatory cytokines have opposite effects on IL-6 production, and 3) molecules such as IL-1, CD14, and CD13 are involved.
Arthritis, Rheumatoid↗
Molecular evidence that in vivo isotype switching occurs within the germinal centers.
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Antigen Presentation↗
Distribution of carboxypeptidase M on lymphoid and myeloid cells parallels the other zinc-dependent proteases CD10 and CD13.
Monoclonal antibody (MoAb) M27 was generated after immunization of mice with the human B-lineage acute lymphoblastic leukemia cell line Pre-ALP. Under reducing conditions, MoAb M27 precipitated a 60-kD surface-membrane molecule from Pre-ALP cells. Expression cloning of Pre-ALP cDNA showed that M27 recognizes carboxypeptidase M (CPM), a cell-surface, zinc-dependent protease known to cleave off basic C-terminal amino acids from peptide hormones. Using M27 antibody, CPM was detected only at discrete B lymphocyte developmental stages, namely on committed precursors and on germinal center cells. CPM was also expressed on mature T cells, mainly after activation. These results provide the first description of a carboxy-peptidase on lymphoid cells. In addition, CPM was found on granulocytes and monocytes, but not on their progenitors. Strikingly, CPM was present only on CD38+ cells, irrespective of lineage affiliation. Of interest, CPM displayed a largely overlapping distribution with the CD10 and CD13 peptidases, with which it shares common substrates (enkephalins, bradykinin). Collectively, the present data show a previously unrecognized distribution pattern of CPM on lymphoid and myeloid cells and suggest that CPM may cooperate with CD10 and CD13 to regulate biologic activity of peptide hormones on leukocytes.
ADP-ribosyl Cyclase↗