The human antibody response to pneumococcal capsular polysaccharides is dependent on the CD40-CD40 ligand interaction

Protection against infections with Streptococcus pneumoniae is mediated by antibodies against the capsular polysaccharides (caps-PS). Here we show that in in vitro experiments CD4+ T lymphocytes stimulate and CD8+ T lymphocytes inhibit the human anti-caps-PS antibody response. Using antagonistic anti-CD40 and antagonistic anti-CD40 ligand (CD40L) monoclonal antibodies, we showed that the CD4+ T lymphocyte-mediated stimulation is dependent on the CD40-CD40L interaction. The role of CD40L was further illustrated by the observation that CD4+ T lymphocytes obtained from a patient with hyper-IgM syndrome were unable to enhance the immune response to caps-PS. Furthermore, CD4+ T lymphocytes from cord blood, which did not express CD40L in response to stimulation with caps-PS, failed to stimulate the antibody response of adult B lymphocytes to caps-PS. These in vitro findings were confirmed by in vivo experiments in which SCID/SCID mice were reconstituted with human mononuclear cells. Furthermore, we showed that caps-PS induce production of IL-4, IL-6, IL-10, and IFN-gamma, and that this enhanced production was inhibited by blocking the CD40-CD40L interaction. This is the first demonstration that the human immune response to caps-PS, which is markedly regulated by T lymphocytes, is dependent on the CD40-CD40L interaction.     

CD40/CD40 ligand interactions are required for T cell-dependent production of interleukin-12 by mouse macrophages

We have previously shown that T cell receptor-activated mouse T helper (Th)1 clones induce the production of interleukin (IL)-12 by splenic antigen-presenting cells (APC). Here, we show that the expression of CD40L by activated T cells is critical for T cell-dependent IL-12 production by mouse macrophages. IL-12 was produced in cultures containing alloreactive Th1 clones stimulated with allogeneic peritoneal macrophages, or in cultures of splenocytes stimulated with anti-CD3. Anti-CD40L monoclonal antibodies (mAb) inhibited the production of IL-12, but not IL-2, in these cultures by ?90% and had dramatic inhibitory effects on antigen-dependent proliferation of Th1 clones. In addition, both activated T cells and a Th1 clone derived from CD40L knockout mice failed to induce IL-12 production from splenic APC or peritoneal macrophages. Finally, macrophages cultured in the absence of T cells produced IL-12 upon stimulation with soluble recombinant CD40L in combination with either supernatants from activated Th1 clones or with interferon-γ and granulocyte/macrophage colony-stimulating factor. Thus, both CD40L-dependent and cytokine-mediated signals from activated T cells are required to induce the production of IL-12 by macrophages. A blockade at the level of IL-12 production may explain, at least in part, the dramatic ability of anti-CD40L mAb to inhibit disease in animal models that are dependent upon the generation of a cell-mediated immune response. Moreover, a defect in T cell-dependent induction of IL-12 may contribute to the immune status of humans that lack functional CD40L.     

Cleavage of membrane-bound CD40 ligand is not required for inducing B cell proliferation and differentiation

The phycical interaction between the B cell surface molecule CD40 and its ligand, CD40L, is known to be crucial in the development and maintenance of humoral immunity. Recently it has been shown that the CD40L is processed and that its soluble cleavage products are released into the extracellular environment. To study the functions of soluble and membrane-bound human CD40L on human B cells, we generated an uncleavable CD40L cDNA deletion mutant. The activities of transfectants expressing either mutated or wild-type CD40L were then compared on human B cells. Both the soluble and the uncleavable membrane-bound CD40L were able to induce, in conjunction with interleukin-4, B cell proliferation and IgE synthesis. Therefore, membrane-bound and soluble CD40L exhibit the same pattern of activities on B cells and membrane CD40L cleavage is not a prerequisite for its function.     

CD40-CD40 ligand costimulation is not required for initiation and maintenance of a Th1-type response to Leishmania major infection

Although previous studies demonstrated a requirement for CD40-CD40 ligand (CD40L) interaction in the development of resistance to Leishmania infection, we recently showed that mice lacking the gene for CD40L (CD40L(-/-) mice) can control Leishmania major infection when they are infected with reduced numbers of parasites. In this study, we examine the cytokine pattern in healing versus nonhealing CD40L(-/-) mice and investigated whether CD40 activation is required for resistance to reinfection. We observed that CD4(+) cells in healed CD40L(-/-) mice produce high levels of gamma interferon compared to cells from nonhealing, high-dose-inoculated mice. In addition, we observed a higher frequency of interleukin-12 (IL-12)- producing cells and a reduced number of IL-4-producing cells in mice infected with reduced numbers of parasites. Importantly, we found that healed CD40L(-/-) mice are highly resistant to reinfection with a large parasite inoculum. In addition, by comparing the cytokine patterns at an early and late stage of infection in nonhealing CD40L(-/-) mice, we demonstrated that nonhealing CD40L(-/-) mice produce a weak Th1-type response during the early stage of infection, but this response wanes as a Th2-type response emerges during late stages of infection. Anti-IL-4 antibody treatment, starting either at the beginning of infection or at week 4 postinfection enabled CD40L(-/-) mice to control a high-dose infection. Together, these results show that CD40-CD40L interaction, although important for IL-12 production in high-dose infections, is not required for either the development or maintenance of resistance in mice infected with reduced numbers of parasites.     

CD40-CD40 ligand

CD40 is a cell surface receptor that belongs to the tumor necrosis factor-R (TNF-R) family, and that was first identified and functionally characterized on B lymphocytes. Its critical role in T cell-dependent humoral immune responses was demonstrated by patients with the hyper-IgM syndrome, as well as by gene targeting in mice. However, in recent years it has become clear that CD40 is expressed much more broadly, including expression on monocytes, dendritic cells, endothelial cells, and epithelial cells. In addition, the CD40-ligand (CD40-L/CD154), a member of the TNF family, is also expressed more widely than activated CD4+ T cells only. Therefore it is now thought that CD40-CD40-L interactions play a more general role in immune regulation. Collectively these studies have culminated in pre-clinical and clinical studies that are in progress. This article reviews recent developments in this field of research, with main emphasis on (1) structure and expression of CD40 and its ligand; (2) CD40 signal transduction; (3) in vitro function of CD40 on different cell types; and (4) in vivo functions of CD40/CD40-L interactions.     

The CD40/CD40 ligand interactions exert pleiotropic effects on bone marrow granulopoiesis

CD40 is a member of the TNFR family and upon interaction with its cognate ligand (CD40L), induces diverse biologic responses related to cell survival/growth. As altered CD40/CD40L interactions have been associated with neutropenia, we investigated the role of CD40/CD40L on human granulopoiesis using immunomagnetically sorted CD34(+), CD34(-)/CD33(+), and CD34(-)/CD33(-)/CD15(+) BM cells, which represent sequential stages of the granulocytic development, the KG-1 cells that constantly express CD34 and CD33, and LTBMCs that mimic the BM microenvironment. CD40 and CD40L were minimally expressed on CD34(+), CD34(-)/CD33(+), and CD34(-)/CD33(-)/CD15(+) cells, but CD40 was substantially induced in the presence of TNF-α. Cross-linking of CD40 in the above cell populations resulted in induction of apoptosis that was enhanced further in the presence of FasL. CD40 activation in primary as wells as in KG-1 cells resulted in Fas up-regulation, providing a mechanism for the CD40-mediated apoptosis. Addition of CD40L in clonogenic assays resulted in a significant decrease in the colony-forming capacity of BMMCs from patients with chronic neutropenia, presumably expressing high levels of CD40 in the progenitor cells, and this effect was reversed upon CD40 blockade. CD40 was constitutively expressed on LTBMC stromal cells and upon activation, resulted in an increase in G-CSF and GM-CSF production. These data show that CD40/CD40L interactions may promote granulopoiesis under steady-state conditions by inducing the stromal release of granulopoiesis-supporting cytokines, whereas under inflammatory conditions, they may affect the granulocytic progenitor/precursor cell survival by accelerating the Fas-mediated apoptosis.     

CD40-CD40配体相互作用对内皮细胞促凝活性的影响

目的: 探讨CD40-CD40配体(CD40L)相互作用对内皮细胞促凝活性的影响。方法: 建立内皮细胞与巨噬细胞膜共孵育体系(细胞比例1∶1),观察内皮细胞促凝活性、组织因子(TF)及其抑制物(TFPI)表达的变化,以及阻断CD40-CD40L相互作用对上述效应的影响。结果: 与巨噬细胞膜共孵育6h后,内皮细胞促凝活性增强10.9倍(P＜0.01),TF蛋白的表达亦显著增强。共孵育3h后,TFmRNA表达升高7.5倍,而TFPImRNA仅升高0.99倍。抗CD40L抗体可分别抑制促凝活性和TFmRNA表达变化的66.6%和51.0%(P＜0.01)。结论: 巨噬细胞通过CD40-CD40L相互作用增强内皮细胞促凝活性及TF表达,可能是动脉粥样硬化斑块局部血栓形成的原因之一。     

CD40/CD40L interaction is essential for the induction of EAE in the absence of CD28-mediated co-stimulation

CD28 provides a co-stimulatory signal critical for optimal T cell activation. We and others have shown that the B7/CD28 co-stimulatory pathway is a major regulatory pathway for the control of immune responses. Experimentally induced models of autoimmunity have been shown to be prevented or reduced in intensity in mice deficient for CD28. Here, we show that EAE and accompanying neuroantigen-specific immune responses are drastically reduced in the absence of CD28. However, we go on to show that EAE can be induced in CD28-deficient mice following two immunizations. After re-immunization, CD28-deficient mice develop severe EAE with myelin-specific responses equal to those of wildtype controls, and extensive demyelination in the spinal cord. Treatment of CD28-deficient mice with anti-CD40L at the time of immunization significantly reduced DTH responses and prevented the development of EAE following two immunizations, indicating a critical role for CD40/CD40L signaling in the absence of CD28. Taken together, our results indicate that CD28-mediated co-stimulation does not regulate immunological anergy. Instead, CD28 appears to adjust the threshold for activation and expansion of autoreactive cells.     

CD40 ligand is functionally expressed on human eosinophils

CD40 ligand (CD40L), a surface molecule which can be expressed by T cells, mast cells and basophils, has been shown to be involved in the control of B cell proliferation, immunoglobulin class switching as well as in the activation of monocytes and T cells. We demonstrate that CD40L can also be expressed constitutively by eosinophils from an hypereosinophilic patient or, upon activation, by the eosinophilic cell line EOL-3 and normal blood eosinophils. Eosinophils were able to induce, in conjunction with IL-4, CD40L-dependent B cell proliferation in vitro. These results suggest that CD40L could play a role in the inflammatory processes during which eosinophil infiltration and activation are observed.     

CD40 ligand is a T cell growth factor

CD40 ligand (CD40L) is a 33-kDa type II membrane glycoprotein induced on T cells upon activation. CD40L has previously been shown to induce proliferation of resting B cells, immunoglobulin (Ig) secretion from B cells cultured with cytokines and cytokine secretion and tumoricidal activity from monocytes. In this report CD40L is shown to be stimulatory for human T cells, inducing CD25 (p55 IL-2R) and CD40L expression on resting peripheral blood T cells, enhanced expression of these molecules and CD69 on CD3-activated cells and secretion of interferon-y, tumor necrosis factor-a and interleukin (IL)-2 from T cells cultured in the presence of a sub-mitogenic concentration of phytohemagglutinin A (PHA). Furthermore, stimulation with CD40L induces proliferation of CD3- or PHA-activated T cells of blood, tonsillar or thymic origin. A similar proliferative response is observed with CD4^? and CD8⁺ T cells and this effect is largely IL-2 independent. A soluble construct of the extracellular domain of the CD40L has similar activity to that of membrane-expressed ligand in the induction of T cell surface antigens and proliferation. The results presented here taken together with the various activities ascribed for CD40L on B cells and monocytes demonstrate that CD40L has pleiotropic biological activity for cells of the hemopoietic lineage.     

CD40L, but not CD40, is required for allergen-induced bronchial hyperresponsiveness in mice

Asthma is characterized by immunoglobulin (Ig) E production, infiltration of the respiratory mucosa by eosinophils (EOSs) and mononuclear cells, and bronchial hyperresponsiveness (BHR). Interaction of CD40 on B cells and antigen presenting cells, with its ligand (CD40L) expressed transiently on activated T cells, is known to augment both T cell-driven inflammation and humoral immune responses, especially IgE production. Considering both the prominent role of inflammation in asthma and the association of the disease with IgE, we hypothesized that CD40-CD40L interactions would be important in pathogenesis. To test this hypothesis, we subjected wild-type (WT) mice and animals lacking either CD40 or CD40L to repeated inhalation of Aspergillus fumigatus (Af ) antigen. Af-treated WT mice displayed elevated IgE levels, bronchoalveolar lavage and pulmonary tissue eosinophilic inflammation, and BHR. IgE production was markedly suppressed in both the CD40 -/- and CD40L -/- strains. However, pulmonary inflammation did not appear to be inhibited by either of these mutations. Paradoxically, development of BHR was prevented by the lack of CD40L but not by the absence of CD40. We conclude that CD40/CD40L interactions, although critical in the induction of IgE responses to inhaled allergen, are not required for the induction of EOS-predominant inflammation. CD40L, but not CD40, is necessary for the development of allergen-induced BHR.     

CD40 is required for the optimal induction of protective immunity to Mycobacterium avium

C57Bl/6 mice and mice deficient in the CD40 molecule were infected with three strains of Mycobacterium avium. Two of the M. avium strains proliferated more extensively in CD40-deficient (CD40-/-) mice than in control mice. The increased susceptibility to infection of CD40-/- mice was associated with the generation of poorer interleukin-12 (IL-12) p40 and interferon-gamma (IFN-gamma) responses as compared to the controls, suggesting a role for CD40 in the development of protective immunity. In contrast, direct triggering of CD40 on infected macrophages failed to induce any anti-mycobacterial activity in infected macrophages.     

Defective Th1 and Th2 cytokine synthesis in the T-T cell presentation model for lack of CD40/CD40 ligand interaction

In this study, T or NK cell clones used as antigen-presenting cells (T- or NK-APC) were shown to be significantly less efficient than professional APC in inducing Th1 and Th2 cytokines by antigen-specific T cell clones. This phenomenon was not related to a limited engagement of TCR by T-APC, since comparable thresholds of TCR down-regulation were shown when antigen was presented by either T-APC or professional APC. Rather, the stimulatory T-APC weakness was due to their inability, because they are CD40⁻, to provide the appropriate co-stimuli to responder T cells both indirectly via IL-12, and partially via direct CD40L triggering on T cells. Indeed, the simultaneous addition of IL-12 and reagents directly engaging CD40L on responder T cells restored T cell cytokine synthesis when antigen was presented by T-APC. In addition, either IL-12 production or blocking of T cell cytokine synthesis by anti-IL-12 p75 antibodies was evident only when professional APC were used in our antigen-specific system. The down-regulation of cytokine synthesis in the system of T-T cell presentation could represent a novel mechanism of immune regulation, which may intervene to switch off detrimental Th1- or Th2-mediated responses induced by antigen presentation among activated T cells infiltrating inflamed tissues.     

Disruption of CD40/CD40 ligand interaction with cleavage of CD40 on human gingival fibroblasts by human leukocyte elastase resulting in down-regulation of chemokine production

CD40 is a crucial element in the process of fibroblast activation. We demonstrated that treatment of human gingival fibroblast (HGF) with human leukocyte elastase (HLE), a neutrophil serine protease, down-regulated the expression of CD40 and binding to the CD40 ligand (CD40L) using flow cytometry. The other neutrophil serine proteases, cathepsin G and proteinase 3, exhibited markedly less activity for CD40 reduction. The CD40 reduction by HLE was also observed in skin and lung fibroblasts, but not in monocytes, macrophages, and dendritic cells. The reduction resulted from direct proteolysis by HLE on the cell surface, because HLE reduced CD40 on fixed HGF and also on cell lysates and membranes. HLE treatment of HGF decreases interleukin (IL)-8 and macrophage chemoattractant protein-1 production by HGF when stimulated by CD40L, but not by IL-1alpha, suggesting that HLE inhibited a CD40-dependent cell activation. These results suggest that HLE possesses an anti-inflammatory effect for the HGF-mediated inflammatory process.     

Lipopolysaccharide desensitizes monocytes-macrophages to CD40 ligand stimulation

Polymicrobial sepsis induces the suppression of macrophage function as determined by a reduction of pro-inflammatory cytokine production upon re-exposure to lipopolysaccharide (LPS) in vitro. Here, we examined whether macrophages were refractory to only LPS or if they were unable to respond to other stimuli such as CD40 ligand (CD40L). Monocytic cells exposed in vitro to LPS showed a dose-dependent reduction of their ability to produce interleukin-12 and tumour necrosis factor-alpha upon subsequent CD40L stimulation, as compared to cells stimulated with CD40L alone. Similarly, LPS interfered with the up-regulation of CD40, CD80 and CD86 induced by CD40L in monocytic cells. The effect of LPS on the response of monocytes to CD40L was similar whether these cells were directly exposed to LPS or cocultured with LPS-pretreated cells, indicating that soluble factors released by LPS stimulation could mediate tolerance to CD40L. We also show that the functional alterations induced by LPS in monocytes can be reversed by indomethacin, thus suggesting a role for inducible cyclooxygenase in mediating the LPS-induced hyporesponsive state of monocytes to CD40L. In conclusion, we propose that in vitro CD40L tolerance may be an appropriate model of monocyte alteration observed during septic immunosuppression and may help in the development of novel therapeutic strategies.     

T lymphocyte and monocyte interaction by CD40/CD40 ligand facilitates a lymphoproliferative response and killing of Cryptococcus neoformans in vitro

This study explored the role of CD40 / CD40 ligand (CD40L) in the induction of a lymphoproliferative response and killing of Cryptococcus neoformans in vitro. In our experimental system, monocytes exposed to C. neoformans were used as antigen-presenting cells (APC) and co-cultured with autologous T cells. The results showed that CD40 / CD40L strongly regulated the blastogenic response of T cells to C. neoformans. The fungus up-regulated CD40 expression on APC. An acapsular strain appeared to be a better inducer than an encapsulated strain. Time course experiments showed optimal regulation of CD40 expression at 48 h of incubation. Blocking the interaction of CD40 on APC with CD40L on T cells using mAb to CD40L resulted in a significant inhibition of IFN-gamma production. The anti-cryptococcal activity of monocytes was greatly influenced by the CD40 / CD40L interaction, and a positive correlation was found between nitric oxide secretion and enhanced killing of C. neoformans. Finally, the CD40 / CD40L interaction was critical for induction of optimal secretion of pro-inflammatory cytokines such as TNF-alpha and IL-1beta. These results indicate an important role for CD40 / CD40L interaction in inducing activation of T cells. Such cell-to-cell contact promotes anti-cryptococcal activity as well as secretion of pro-inflammatory cytokines by monocytes.     

CD40 ligand is selectively expressed on CD4+ T cells and platelets: implications for CD40-CD40L signalling in atherosclerosis

Atherosclerosis is a degenerative inflammatory disease of the vascular system. Endothelial cells (ECs), smooth muscle cells, and macrophages, key elements in atherosclerosis, all have the potential to express the CD40 receptor and are thus susceptible to potent pro-inflammatory signals by CD40 ligand (CD40L)-bearing cells. CD40L is a TNF-alpha-related membrane protein originally identified on activated T cells. The recent recognition of platelets as an abundant source of CD40L led to a reassessment of the involvement of CD40L in atherosclerosis. In the present report, CD40L(+) T cells were identified in the intima of atherosclerotic tissues within macrophage infiltrates and in areas of neovascularization. These CD40L(+) T cells were CD4(+), CD69(+), but negative for CD8, CD25, CD28, and ICOS. In some specimens, CD40L(+) platelets were identified in the intima and in plaque ruptures. Contrary to previous reports, CD40L was not observed on ECs, smooth muscle cells, and macrophages in atherosclerotic tissues or in vitro at the protein and mRNA levels. Functionally, flow chamber experiments demonstrated that stimulation of ECs via CD40 is sufficient to recruit neutrophils and T cells from whole blood to ECs and suggested that CD40L(+) platelets contribute significantly to the recruitment of inflammatory cells to damaged endothelium in vivo. However, due to the short half-life of platelet CD40L, the chronic CD40L-driven inflammatory component can only be sustained by activated CD4(+) T cells. Contrary to current understanding, the contribution of CD40L to chronic inflammation in atherosclerosis is thus antigen-driven and MHC-dependent. This conclusion has significant therapeutic implications.