CD40 Deficiency Research Articles

Programmed death 1 ligand signaling regulates the generation of adaptive Foxp3 + CD4 + regulatory T cells

Although mature dendritic cells (DCs) are potent initiators of adaptive immune response, immature steady-state DCs contribute to immune tolerance. In this study, we show that ex vivo splenic DCs are capable of inducing conversion of naïve CD4(+) T cells to adaptive Foxp3(+)CD4(+) regulatory T cells (aTreg) in the presence of TGF-beta. In particular, when compared with splenic CD8alpha(-) DCs, the CD8alpha(+) DC subset were superior in inducing higher frequencies of conversion. This was not attributable to the difference in basal level of costimulation, because deficiency of CD40 or CD80/86 signaling did not diminish the differential induction of Foxp3. Conversion was regulated by DC maturation status. Further insights into the molecular mechanisms of conversion were gained by analyzing the contribution of several costimulatory and coinhibitory receptors. Costimulatory signals through GITR suppressed conversion, whereas coinhibitory signaling via programmed death 1 ligand (PD-L1) but not PD-L2 was required for conversion. Ex vivo PD-L1(-/-) DCs failed to support Foxp3 induction in the presence of TGF-beta. In vivo blocking PD-L1 signaling abolished conversion in a tumor-induced aTreg conversion model. Collectively, this study highlights the cellular and molecular parameters that might be exploited to control the de novo generation of aTregs and peripheral tolerance.

CD40 ligation mediates plaque-associated tau phosphorylation in β-amyloid overproducing mice

Neuritic dystrophy with amyloid burden and neurofibrillary tangles are pathological hallmarks of Alzheimer's disease. Genetic disruption of CD40 or CD40L alleviates amyloid burden, astrocytosis, and microgliosis in transgenic animal models of Alzheimer's disease. It has been reported that phosphorylated tau-positive dystrophic neurites are observed in transgenic mice over-expressing human mutant beta-amyloid precursor protein (Tg2576). Here, we studied the pattern of phosphorylated tau (labeled with AT8, CP13, PG5, and PHF1 antibodies) and plaques using immunohistochemical techniques. Phosphorylated tau-positive dystrophic neurites were exclusively associated with Congo red-positive plaques as previously reported. Further, we show that CD40L or CD40 deficiency reduces the mean ratio of dystrophic neurite area to congophilic plaque area and the level of expression of cdk5 and p35/p25 in mice. In addition, we show that in a human neuroblastoma cell line treated with CD40L, cdk5 and p35/p25 are increased. Together, our data suggest that CD40–CD40L interaction has an effect on tau phosphorylation independent of beta-amyloid pathology, and that this effect may occur through a decrease of cdk5 and p35/p25.

Negative Regulation of CD40-Mediated B Cell Responses by E3 Ubiquitin Ligase Casitas-B-Lineage Lymphoma Protein-B

It has been documented that CD40 is essential for B cell function. Casitas-B-lineage lymphoma protein-b (Cbl-b), an adapter protein and ubiquitin ligase, has been shown to regulate the activation of T and B cells through their Ag receptors. In this study, we report that CD40-induced B cell proliferation is significantly augmented in mice lacking Cbl-b. Furthermore, Cbl-b(-/-) mice display enhanced thymus-dependent Ab responses and germinal center formation, whereas introduction of CD40 deficiency abolishes these effects. Hyper thymus-dependent humoral response in Cbl-b(-/-) mice is in part due to an intrinsic defect in B cells. Mechanistically, Cbl-b selectively down-modulates CD40-induced activation of NF-kappaB and JNK. Cbl-b associates with TNF receptor-associated factor 2 upon CD40 ligation, and inhibits the recruitment of TNF receptor-associated factor 2 to the CD40. Together, our data suggest that Cbl-b attenuates CD40-mediated NF-kappaB and JNK activation, thereby suppressing B cell responses.

Alpha tumor necrosis factor contributes to CD8 + T cell survival in the transition phase

Cytokine and costimulation signals determine CD8 + T cell responses in proliferation phase. In this study, we assessed the potential effect of cytokines and costimulations to CD8 + T cell survival in transition phase by transferring in vitro ovalbumin (OVA)-pulsed dendritic cell-activated CD8 + T cells derived from OVA-specific T cell receptor transgenic OT I mice into wild-type C57BL/6 mice or mice with designated gene knockout. We found that deficiency of IL-10, IL-12, IFN-γ, CD28, CD40, CD80, CD40L, and 41BBL in recipients did not affect CD8 + T cell survival after adoptive transfer. In contrast, TNF-α deficiency in both recipients and donor CD8 + effector T cells significantly reduced CD8 + T cell survival. Therefore, our data demonstrate that the host- and T cell-derived TNF-α signaling contributes to CD8 + effector T cell survival and their transition to memory T cells in the transition phase, and may be useful information when designing vaccination.

First report of successful stem cell transplantation in a child with CD40 deficiency

First report of successful stem cell transplantation in a child with CD40 deficiency

Negative Regulation of CD40-mediated B Cell Responses by E3 Ubiquitin Ligase Cbl-b (87.21)

Abstract CD40 is a TNF receptor (TNFR) superfamily member which is essential for B cell function. Cbl-b, an adapter protein and ubiquitin ligase, has been shown to regulate activation of T and B cells through their antigen receptors. In this study, we report that CD40-induced proliferation is significantly augmented in mice lacking Cbl-b. Furthermore, Cbl-b−/− mice display enhanced thymus–dependent (TD) Ab responses and germinal center (GC) formation, whereas introduction of CD40 deficiency abolishes the hyper-Ab responses and heightened GC formation in response to TD Ag challenge. Mechanistically, Cbl-b selectively down-modulates CD40-induced activation of NF-κB and JNK. Cbl-b associates with TNFR-associated factor 2 (TRAF-2) upon CD40 ligation, and inhibits the recruitment of TRAF-2 to the CD40. Together, our data suggest that Cbl-b attenuates CD40-mediated NF-κB and JNK activation, thereby suppressing B cell responses.

CD40 promotion of amyloid beta production occurs via the NF‐κB pathway

The CD40 receptor is a member of the tumor necrosis factor (TNF) super-family of trans-membrane receptors. Interaction of CD40 with its ligand CD40L mediates a broad range of immune and inflammatory responses in the periphery and in the central nervous system. Recently it has been suggested that CD40/CD40L interaction is involved in amyloid precursor protein (APP) processing and Alzheimer's disease (AD)-like pathology in transgenic mouse models of AD. We have previously shown that pharmacologically inhibiting CD40/CD40L interaction improves memory deficits in the PSAPP AD mouse model. We have also recently shown that CD40 deficiency mitigates amyloid deposition in APPsw and PSAPP mouse models. In the present report, using human embryonic kidney cells (HEK293) over-expressing both the APPsw mutation and CD40, we demonstrate that CD40/CD40L interaction directly increases the production of APP metabolites (Abeta 1-40, Abeta 1-42, CTFs, sAPPbeta and sAPPalpha). The results also show that CD40/CD40L interaction affects APP processing via the NF-kappaB pathway. Using NFkappaB inhibitors and SiRNAs to silence diverse elements of the NFkappaB pathway, we observe a reduction in levels of both Abeta 1-40 and Abeta 1-42. Taken together, our results further suggest that CD40L stimulation may be a key component in AD pathology and that elements of the NF-kappaB pathway may be suitable targets for therapeutic approaches against AD.

Blood-Borne, but Not Endothelial CD40 Promotes Experimental Lung Metastasis.

During experimental lung metastasis, procoagulant tumor cells adhere to the microvascular endothelium and activate coagulation, which leads to thrombin generation and platelet activation. It is well established that hematogenous metastasis is supported by activated platelets. CD40 ligand (CD40L) expressed on, and subsequently released from, activated platelets can induce an inflammatory response, including the production of tissue factor, in endothelial cells and monocytes. Furthermore, CD40L has been shown to play an important role in platelet function and in vivo thrombus formation. These properties of CD40L may be mediated, at least in part, by CD40 signaling on platelets and vascular endothelial cells. We have previously shown that tumor cell-induced coagulation activation and experimental lung metastasis are markedly reduced in CD40L-deficient mice. In addition, we demonstrated significantly prolonged bleeding and PFA-100 closure times in mice deficient for CD40L or CD40. In the present study, we hypothesized that blood-borne (i.e. platelet-derived) CD40 is required for optimum hematogenous metastasis. Wild-type (WT) C57/BL6 mice and mice deficient for CD40 (CD40−/−) were injected via the tail vein with 2×105 B16 murine melanoma cells (n=13 per group), and tumor nodules were counted on lung surfaces 18 days after. The results revealed a significant (73%, P<0.001) reduction in lung metastasis in CD40−/− mice. To assess the specific contribution of blood-borne CD40 to experimental lung metastasis, bone marrow cells (4×106) from WT donor mice were injected (i.v.) into CD40−/− recipient mice 24 hrs after lethal irradiation (1000 rad in 1 hr). The transplanted mice (n=14) were designated CD40-BC, as they expressed CD40 only on blood cells, but not on endothelial cells. Irradiated WT mice receiving WT bone marrow (n=17) served as transplant controls. CD40-BC mice were phenotyped 9 weeks after transplantation by flow cytometric analysis of CD40 on peripheral B lymphocytes using a PE-labeled rat anti-mouse CD40 monoclonal antibody. All of the transplanted CD40−/− mice demonstrated the chimeric phenotype. There were no differences in platelet counts between CD40-BC and transplanted WT mice or between the non-transplanted groups (CD40−/− vs. WT). However, bone marrow transplantation (BMT) was associated with decreased platelet counts within the recipient strains: 471±63/nl in CD40-BC vs. 644±150/nl in CD40−/− mice (P=NS) and 472±12/nl in transplanted vs. 692±73/nl in non-transplanted WT mice (P<0.01). Compared to WT mice, the number of lung tumor nodules (median [IQR]) was significantly reduced in CD40−/− mice (11 [10–14] vs. 41 [26–49], P<0.001). However, compared to CD40−/− mice, lung metastasis was significantly increased in CD40-BC mice (49 [23–67] vs. 11 [10–14], P<0.001). No difference in lung seeding was observed between CD40-BC (median, 49), transplanted WT (48) and non-transplanted WT mice (41), indicating that, first, the protection against lung metastasis due to CD40 deficiency was completely reversed by selective expression of CD40 on peripheral blood cells and, second, BMT did not alter baseline susceptibility of C57/BL6 mice to B16 melanoma lung seeding. In summary, our results suggest an important contribution of CD40 to experimental lung metastasis. Furthermore, the data point to a selective role of CD40 expressed on peripheral blood cells (i.e. platelets) in this process.

Triggering CD40 on endothelial cells contributes to tumor growth

Inflammatory cells can either promote or inhibit tumor growth. Here we studied whether CD40, a key molecule for adaptive immune response, has any role in mammary carcinogenesis of BALB/NeuT transgenic tumor-prone mice. We transferred the HER2/neu oncogene into CD40-null background to obtain the CD40-KO/NeuT strain. CD40-KO/NeuT mice showed delayed tumor onset and reduced tumor multiplicity. BM (BM) transplantation experiments excluded a role of BM-derived cells in the reduced tumorigenicity associated with CD40 deficiency. Rather, CD40 expressed by endothelial cells (ECs) takes part to the angiogenic process. Accordingly, large vessels, well organized around the tumor lobular structures, characterize BALB/NeuT tumors, whereas tiny numerous vessels with scarce extracellular matrix are dispersed in the parenchyma of poorly organized CD40-KO/NeuT tumors.Activated platelets, which may interact with and activate ECs, are a possible source of CD40L. Their localization within tumor vessels prompted the idea of treating BALB/NeuT and CD40-KO/NeuT mice chronically with the anti-platelet drug clopidogrel, known to inhibit platelet CD40L expression. Treatment of BALB/NeuT mice reduced tumor growth to a level similar to CD40-deficient mice, whereas CD40-KO/NeuT mice treated or not showed the same attenuated tumor outgrowth, indicating that activated platelets are the likely source of CD40L in this model. Collectively, these data point to a participation of CD40/CD40L in the angiogenic processes associated with mammary carcinogenesis of BALB/NeuT mice.

CD40 deficiency mitigates Alzheimer's disease pathology in transgenic mouse models

We have previously shown that transgenic mice carrying a mutant human APP but deficient in CD40L, display a decrease in astrocytosis and microgliosis associated with a lower amount of deposited Aβ. Furthermore, an anti-CD40L treatment causes a diminution of Aβ pathology in the brain and an improved performance in several cognitive tasks in the double transgenic PSAPP mouse model. Although these data suggest a potential role for CD40L in Alzheimer's disease pathology in transgenic mice they do not cast light on whether this effect is due to inhibition of signaling via CD40 or whether it is due to the mitigation of some other unknown role of CD40L. In the present report we have generated APP and PSAPP mouse models with a disrupted CD40 gene and compared the pathological features (such as amyloid burden, astrocytosis and microgliosis that are typical of Alzheimer's disease-like pathology in these transgenic mouse strains) with appropriate controls. We find that all these features are reduced in mouse models deficient for CD40 compared with their littermates where CD40 is present. These data suggest that CD40 signaling is required to allow the full repertoire of AD-like pathology in these mice and that inhibition of the CD40 signaling pathway is a potential therapeutic strategy in Alzheimer's disease.

Hyper immunoglobulin M syndrome due to CD40 deficiency: clinical, molecular, and immunological features

CD40 is a member of the tumor necrosis factor receptor family, which is expressed by a variety of cells including B cells, macrophages, dendritic cells, and other nonimmune cell types. CD40 activation is critical for B-cell proliferation, immunoglobulin (Ig)-isotype switching, and germinal center formation. In physiological conditions, the activation of CD40 occurs by binding to its natural ligand, CD154, which is expressed on activated T cells. The in vivo critical role of CD40-CD154 interaction on B-cell differentiation and isotype switching is provided by the discovery that mutations in either CD40 or CD154 gene cause the hyper IgM syndrome, termed HIGM3 or HIGM1, respectively, characterized by very low levels of serum IgG, IgA, and IgE, with normal or elevated IgM, associated with a defective germinal center formation. Originally considered humoral primary immunodeficiencies, the clinical features and the defect of T-cell priming, resulting from a defective T-B cell or dendritic cell interaction, is now considered as combined immunodeficiencies. In this article, we present a comprehensive overview of the clinical, genetic, and immunological features of patients with hyper IgM syndrome due to CD40 mutations.

Functional defects of dendritic cells in patients with CD40 deficiency

AbstractWe have recently identified 2 patients with a rare autosomal recessive form of hyper IgM disease, known as HIGM3, caused by mutations in theCD40 gene. These patients had opportunistic infections observed on X-linked hyper IgM syndrome (HIGM), suggesting that the CD40-CD40 ligand interaction is important for promoting T-cell-mediated immunity. To evaluate whether innate immunity signals may substitute CD154 for inducing the maturation of dendritic cells (DCs), we analyzed monocyte-derived DCs in these patients. Monocyte-derived DCs of HIGM3 subjects on ex vivo stimulation with tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS) combined with interferon-γ (IFN-γ) normally express all the markers of mature DCs, such as CD83 and DC-LAMP. However, cell surface levels of HLA-DR in mature DCs are reduced, as is costimulatory activity of these cells for allogeneic naive T cells. In addition, CD40-deficient DCs secrete lower amounts of interleukin-12 (IL-12) but larger quantities of IL-10 than control subjects. Finally, analysis of circulating plasmacytoid DCs demonstrates a normal percentage of this subset in CD40-deficient cells, but IFN-α secretion in response to herpes simplex virus 1 (HSV-1) infection is severely reduced in patients. These observations suggest that the severe impairment of DC maturation may contribute to the defect of T-cell-mediated immunity observed in HIGM3 patients. (Blood. 2003;102:

The immunological synapse for B-cell memory: the role of the ICOS and its ligand for the longevity of humoral immunity.

The ICOS is one of the many costimulatory molecules of T cells. Recently, the authors discovered that the homozygous loss of the ICOS in humans presents as common variable immunodeficiency, the most prevalent treated primary immunodeficiency in man. Therefore, the ICOS is after CD40 and CD40-ligand deficiency the third costimulatory molecule for which a human disease has been described. The description of ICOS deficiency made it necessary to review the vast amount of knowledge from experimental models in the light of a new clinical immunodeficiency phenotype. To this end, after introducing the ICOS and its ligand and comparing the murine and human 'knockout', the role of the ICOS in parasitic and viral infections, asthma, autoimmunity, transplantation immunology, and tumor surveillance will be discussed. The focus of this review is the interaction between the ICOS and its ligand, which is essential for T cell dependent B cell responses.

Disseminated cryptosporidium infection in an infant with hyper-IgM syndrome caused by CD40 deficiency

We report the case of an infant with severe respiratory infections, chronic diarrhea, failure to thrive, and disseminated Cryptosporidium parvum infection. Laboratory investigations disclosed a diagnosis of hyper-IgM syndrome caused by CD40 deficiency.

The revolution in molecular biology leads to new understanding of the clinical expression of immunodeficiencies

The revolution in molecular biology leads to new understanding of the clinical expression of immunodeficiencies

Inhibition of CD40 expression and CD40-mediated dendritic cell function by tumor-derived IL-10.

As CD40 plays a key role in both antitumor immunity and DC maturation, we have studied the regulation of its expression during DC hematopoiesis (dendropoiesis) in vitro and in vivo in the tumor microenvironment. Using MC38 colon adenocarcinoma tumor models, we have demonstrated that DCs generated in vitro from bone marrow precursors obtained from tumor-bearers have significantly lower expression of CD40 molecules compared to DCs generated from tumor-free mice. Furthermore, CD40 expression on DCs isolated from the spleens of tumor-bearing mice was also significantly reduced, suggesting that tumor-derived factors inhibit CD40 expression on DCs during dendropoiesis both in vitro and in vivo. Interestingly, CD40 ligation on DCs generated from tumor-bearers did not result in inducible expression of IL-12 protein or IL-12 p40 mRNA. However, Staphylococcus aureus-induced IL-12 production by DCs was not altered in tumor-bearers, confirming that inhibition of IL-12 production by DCs generated in vitro from tumor-bearing mice was due to reduced expression of CD40 on DCs. We have also shown that MC38 tumor cells produce IL-10 and that exogenous IL-10 causes downregulation of CD40 expression on DCs. In addition, endogenous IL-10 produced by colon carcinoma cells inhibited CD40-dependent IL-12 production by DCs since tumor-induced inhibition of IL-12 production was abrogated by neutralizing anti-IL-10 antibody. Finally, systemic administration of FLT3L and/or CD40L reversed CD40 and IL-12 (p40) deficiency of DCs in tumor-bearing mice in vivo. These findings thus demonstrate that tumor-derived factors, including IL-10, inhibit CD40 expression on DCs and DC precursors and suppress their maturation and function.

Dendritic cell longevity and T cell persistence is controlled by CD154-CD40 interactions.

Inflammatory mediators facilitate the maturation of dendritic cells (DC), enabling them to induce the activation, proliferation and differentiation of cognate T cells. The role of CD40 on DC and CD154 on T cells has been studied by the co-adoptive transfer of antigen-pulsed DC and TCR-transgenic (Tg) T cells in vivo. It is shown that in the absence of CD40-CD154 interactions, initial Tg T cell expansion occurs in vivo, but over time, T cell expansion cannot be sustained. The basis for the demise of the T cell population is likely due to the disappearance of the antigen-pulsed DC in the draining lymph nodes when CD154-CD40 interactions are interrupted. These findings show that both T cell and DC persistence in vivo is dependent on CD40-CD154 interactions. In addition to the physical persistence of the DC, CD40 triggering of DC also greatly increases the period for which they can productively present antigen to Tg T cells. Hence DC persistence and antigen-presenting cell capacity are both dependent on CD40 signaling. While TNF-alpha can mature DC as measured by a variety of criteria, the unique capacity of CD40 signaling to sustain T cell responses and induce DC maturation is underscored by the inability of TNF-alpha to rescue the immune deficiency of CD40(-/-) DC. Hence, the profound impact of CD154 deficiency on cell-mediated immunity may be due to its ability to limit the duration of antigen presentation in vivo and cause the premature demise of antigen-specific T cells.

Profound reduction of mature B cell numbers, reactivities and serum Ig levels in mice which simultaneously carry the XID and CD40 deficiency genes.

It has been known for some time that single mutant nude or CD40T mice have apparently normal numbers of cells in the precursor compartments of bone marrow and the mature B cell compartments of the periphery. X-linked immunodeficiency (XID) mice are deficient only in some of the sIgM+sIgD+ B cells. We have investigated further the contributions of the xid mutation, of the T cell deficiency of nude of the inability of CD40T cells to cooperate with T cells in the generation of the precursor and the mature B cell compartments in mice. Double mutant XID/nu and XID/CD40T mice have precursor B cell compartments that are no more deficient than the single mutant XID mice. However, the peripheral B cell compartments of both XID/mu and XID/CD40T are even more deficient than those of single mutant XID mice. While 10% of the peripheral B cells of wild-type or CD40T, one-third of XID and half of XID/nu mice turn over rapidly, as many as three-quarters of those in XID/CD40T are short-lived. Total numbers of sIgM+sIgD+ B cells in the spleen are at best 10-15% of normal mice at 6-8 weeks of age in XID, XID/nu and XID/CD40T mice. They remain that low at 3 months of age in XID/CD40T mice, while in XID mice these peripheral B cells slowly build up in numbers with age. As expected, double mutant XID/CD40T mice do not respond to the T-dependent antigen keyhole limpet hemocyanin. Only the responses to the T-independent type I antigen, TNP-lipopolysaccharide (LPS), appear to be normal. In vitro, their splenic B cells respond poorly to LPS or to IgM-specific antibody in either the absence or presence of cytokines. Most notably, serum IgM, IgG2b or IgG3 levels are severely depressed, while IgG1, IgG2a and IgA levels are < 10 micrograms/ml. These results suggest a model of mature B cell development in which the peripheral, mature B cell compartments are generated in two parallel, not tandemly organized pathways. They could be selected and/or stimulated at the transition from immature to mature B cells: in btk controlled or in CD40 controlled ways.