CD40-CD40L Interaction Research Articles

The CD4+ T-cell help signal is transmitted from APC to CD8+ T-cells via CD27–CD70 interactions

CD8(+) cytotoxic T lymphocytes are critical components of immunity against infectious pathogens, tumours, and in the case of pathogenic autoimmunity, normal self tissues. CD4(+) T (T(H)) cells provide 'help' to CD8(+) cytotoxic T lymphocytes during priming by first activating antigen-presenting cells via CD40-CD40L interactions. Here we show that, after immunization with either a noninflammatory, nonreplicating antigen or an overtly inflammatory replicating antigen, CD8(+) cytotoxic T lymphocytes prevented from receiving a signal through CD27 during priming subsequently exhibit a specific defect in their capacity for secondary expansion that can be rescued by the absence of TRAIL. Thus, the 'help message' is transmitted to CD8(+) T cells via CD70-CD27 signals, enabling them to undergo secondary expansion and avoid TRAIL-mediated apoptosis on re-stimulation. These findings complete our understanding of the cellular interactions through which T(H) is provided to CD8(+) cytotoxic T lymphocytes during priming.

Primed for Death: Hypoxia Sensitizes Primary CLL Cells towards Compounds Targeting Mitochondrial Integrity

Abstract 1767 Background:CLL cells prefer to remain in the microenvironment since they feel safe. CD40 ligand (CD40L)-CD40 interaction induces proliferative/anti-apoptotic genes in CLL cells, which protects them from apoptosis and most cytotoxic drugs by the microenvironment.Research interested in identifying novel drugs that effectively target CLL cells within microenvironmental niches has to consider further microenvironmental stimuli, especially hypoxia. Lymph nodes, especially those being infiltrated by malignant cells show low oxygen tension (1%). Prior CLL investigations never took this important factor into account. The impact of hypoxia on survival and drug-resistance is still unknown. Methods:Therefore we have established an in vitro model, which mimics hypoxic conditions and CD40L-CD40 interaction, in order to understand the molecular basis of drug resistance of CLL resident in the microenvironment. CLL cells were cultured on CD40L feeder cells and kept up to 96 hours in hypoxia (1% O2 tension) or normoxia (21% O2 tension).We applied several drugs under these conditions to investigate the differences between normoxia and hypoxia. The miRNA expression was determined by using Illumina Bead Chip Arrays compromising 752 miRNAs. Gene expression was analyzed via mRNA-based Illumina microchip array. Target miRNAs and mRNAs were validated by qRT-PCR. Apoptosis was determined by AnnexinV-7AAD and JC-1 staining (mitochondrial outer membrane permeabilization) and subsequent flow cytometry. Results:In solid cancers hypoxia is expected to protect malignant cells from chemotherapy. We made similar observations, since classical DNA-targeting drugs were inefficient to kill CLL cells cultured on CD40L feeder cells under hypoxia and normoxia. However, we identified ABT-737, which affect mitochondrial integrity, to be even more efficient under hypoxic conditions and CD40L interaction compared to CD40L stimulation and normoxia (74,1% vs. 52,1% apoptotic cells, n=15; p<0.001). Interestingly, overall survival of primary CLL cells during CD40L-CD40 interaction without any cytotoxic treatment was higher under hypoxia compared to normoxia. To understand this discrepancy, we investigated the expression of several mitochondrial localized anti-/pro-apoptotic genes on RNA and protein level. We identified, that the de-regulation of BCLXL and MCL1 is crucial for ABT-737 sensitivity during hypoxic conditions. This de-regulation was also detectable during CD40L interaction. BCLXL deregulation could be attributed to differential NF-κB expression, as determined by EMSA. Since MCL1 protein expression differs from its mRNA expression, we expected regulation prior to protein synthesis. Indeed, we could identify miRNAs, which were upregulated during hypoxia and CD40L stimulation and regulate MCL1 expression. These miRNAs were validated by luciferase expression assays. Conclusion:Here we investigated for the first time the impact of oxygen tension on therapeutic response of CLL cells. We assume that small molecules like ABT-737, which specifically target mitochondria, might be efficient in targeting CLL cells protected by CD40L-CD40 interaction within the microenvironment.Development of novel in vitro models will allow us to understand the specific molecular changes induced by microenvironmental stimuli and to develop novel therapeutic targets.L.P.F. and M.Hue. contributed equally to this work. Disclosures:No relevant conflicts of interest to declare.

English

Replication-defective adenovirus vector without both E1 and E3 is one of the most popular tools in transgenic therapies. However, more attention should be paid to adenovirus vectors mediated-gene modified study on endothelial cells (ECs). To verify the possible danger in that process, we explored the effect of adenovirus on ECs in this study. By using western blot analysis, we showed that the level of both CD40 and CD40L on human umbilical vein endothelial cells (HUVECs) were upregraduated by adenovirus vector infection at 100 multiplicity of infection (MOI). The activation of ECs induced by adenovirus vector infection at MOI 100 can be partly inhibited by a blockade of CD40/CD40L interactions by using the recombinant adenovirus Ad-sCD40LIg or an anti-CD40L monoclonal antibody (mAb) in vitro. On ECs, blockade of CD40/CD40L decreased the expression of IL (interleukin)-6, IL-8 and intercellular adhesion molecule (ICAM) in adenovirus vector-induced cells. In electrophoretic mobility shift assay (EMSA), both Ad-sCD40LIg and anti-CD40L mAb can attenuate the activity of NF-kappaB (NF-κB) pathway contributing to the activation of ECs, which indicated that CD40-CD40L interactions played significant role in the activation of ECs induced by adenovirus vectors via an NF-κB pathway. Our study provide evidences for a supplementary mechanism of the ECs activation induced by adenovirus vector infection and suggests that CD40-CD40L interactions partly participate in the ECs activation induced by adenovirus vectors in an NF-κB-dependent manner. Key words : Adenovirus vector, CD40, CD40L, endothelial cells, NF-kappaB.

Noncollagenous 16A domain of type XVII collagen-reactive CD4+ T cells play a pivotal role in the development of active disease in experimental bullous pemphigoid model

Bullous pemphigoid (BP), the most common autoimmune blistering disease, is caused by autoantibodies against type XVII collagen (COL17). We recently demonstrated that CD4+ T cells were crucial for the production of anti-COL17 IgG and for the development of the BP phenotype by using a novel active BP mouse model by adoptively transferring immunized splenocytes into immunodeficient COL17-humanized mice. Noncollagenous 16A (NC16A) domain of COL17 is considered to contain the main pathogenic epitopes of BP, however, the pathogenicity of COL17 NC16A-reactive CD4+ T cells has never been elucidated. To address this issue, we modulated the immune responses against COL17 in active BP model by using anti-CD40 ligand (CD40L) monoclonal antibody MR1, an inhibitor of the CD40-CD40L interaction, in various ways. First, we show the essential role of CD4+ T cells in the model by showing that CD4+ T cells isolated from wild-type mice immunized with human COL17 enabled naïve B cells to produce anti-COL17 NC16A IgG in vivo. Second, we show that the activation of anti-COL17 NC16A IgG-producing B cells via CD40-CD40L interaction was completed within 5 days after the adoptive transfer of immunized splenocytes. Notably, a single administration of MR1 at day 0 was enough to inhibit the production of anti-COL17 NC16A IgG and to diminish skin lesions despite the presence of restored anti-COL17 IgG at the later stage. In contrast, the delayed administration of MR1 failed to inhibit the production of anti-COL17 NC16A IgG and the development of the BP phenotype. These results strongly suggest that COL17 NC16A-reactive CD4+ T cells play a pivotal role in the production of pathogenic autoantibodies and in the development of active disease in experimental BP model.

CD40L-Containing Virus-Like Particle as a Candidate HIV-1 Vaccine Targeting Dendritic Cells

The central role of dendritic cell (DC) in mounting an immune response to a novel antigen is now well established. We sought to demonstrate the use of a particular vaccine strategy based on directing HIV-1 Gag proteins to DCs in conjunction with an activation signal. CD40L was expressed on the surface of virus-like particles (VLPs) to target HIV-1 Gag antigens to the CD40 receptor on DCs, whereas CD40L-CD40 interaction would also result in cellular activation. Multiple CD40L VLP constructs were made and evaluated in vitro and in vivo. Indeed, one VLP that expressed CD40L to the highest level showed greatest capacity to activate DCs in vitro. Correspondingly, this CD40L-VLP also proved to be most immunogenic in mice in raising both humoral and cellular responses to HIV-1 Gag. Confirmatory studies were performed to demonstrate the increased immunogenicity of CD40L-VLP is no longer observed when tested in CD40-/- mice. Our findings lend support to the belief that vaccine strategies that both target and activate DCs could yield a superior immune response.

Allograft survival is prolonged in the absence of NKG2D (169.19)

Abstract NKG2D is expressed by Natural Killer (NK cells) and subsets of T cells. On NK cells, NKG2D functions as a stimulatory receptor that induces effector functions. We have demonstrated increased expression of NKG2D ligands on rejecting grafts and enhanced infiltration of NKG2D-expressing cells in allografts during rejection. To further examine the role of NKG2D in graft survival we utilized Klrk1 mice that are deficient for NKG2D as recipients of cardiac or small intestine allografts. Groups (n=4-6) of C57BL/6 (H-2b) or NKG2D deficient Klrk1 mice (H-2b) received heterotopic donor hearts or vascularized, orthotopic small intestine grafts from BALB/c (H-2d) or BALB/c x C57BL/6 F1 mice (H-2d/b). Some groups of mice also received 200μg (heart) or 500μg (intestine) anti-CD40L mAb (MR1) at transplant. Cardiac allograft survival was similar in wild-type and Klrk1 mice although intestinal allograft survival was prolonged in Klrk1 mice. Treatment with MR1 prolonged cardiac allograft survival that was further enhanced in the absence of NKG2D. Treatment with MR1 did not prolong survival of intestine allografts in wild-type recipients. Quite dramatically, however, treatment with MR1, significantly (p=0.007) prolonged intestinal allograft survival in Klrk1 mice. Prolongation of intestinal graft survival in the absence of NKG2D and CD40-CD40L interactions suggest that the addition of therapeutics to block NKG2D has great potential for improving costimulation blockade based therapies

Signal pathways in astrocytes activated by cross-talk between of astrocytes and mast cells through CD40-CD40L

BackgroundAstrocytes, which play an active role in chronic inflammatory diseases like multiple sclerosis, exist close to mast cells with which they share perivascular localization. We previously demonstrated the possibility that astrocytes and mast cells interact in vitro and in vivo. This study aimed to investigate the signaling pathways and the role for astrocytes in the interaction of astrocytes and mast cells.MethodsWe co-cultured human U87 glioblastoma (U87) and human mast cell-1 (HMC-1) cell lines, and mouse cerebral cortices-derived astrocytes and mouse bone marrow-derived mast cells (BMMCs). Intracellular Ca2+ ([Ca2+]i) was measured by confocal microscopy; CD40 siRNA by Silencer Express Kit; small GTPases by GTP-pull down assay; PKCs, MAPKs, CD40, CD40L, Jak1/2, STAT1, TNF receptor 1 (TNFR1) by Western blot; NF-κB and AP-1 by EMSA; cytokines by RT-PCR. An experimental allergic encephalomyelitis (EAE) model was induced using myelin oligodendrocyte glycoprotein (MOG) peptide and pertussis toxin in mice. Co-localization of TNFR1 and astrocytes in EAE brain tissues was determined by immunohistochemistry.ResultsEach astrocyte co-culture had increases in [Ca2+]i levels, release of cytokines and chemokines; activities of Rho-family GTPases, NF-κB/AP-1/STAT1727, and Jack1/2, STAT1701. These effects were inhibited by anti-CD40 antibody or CD40 siRNA, and signaling pathways for Jak1/2 were inhibited by anti-TNFR1 antibody. EAE score, expression of TNFR1, and co-localization of TNFR1 and astrocytes were enhanced in brain of the EAE model. Anti-CD40 antibody or 8-oxo-dG pretreatment reduced these effects in EAE model.ConclusionsThese data suggest that astrocytes activated by the CD40-CD40L interaction in co-culture induce inflammatory cytokine production via small GTPases, and the secreted cytokines re-activate astrocytes via Jak/STAT1701 pathways, and then release more cytokines that contribute to exacerbating the development of EAE. These findings imply that the pro-inflammatory mediators produced by cell-to-cell cross-talk via interaction of CD40-CD40L may be as a promising therapeutic target for neurodegenerative diseases like MS.

Abrogation of pathogenic IgG autoantibody production in CD40L gene-deleted lupus-prone New Zealand Black mice

New Zealand Black (NZB) mice spontaneously develop a lupus-like autoimmune disease. Since CD40-CD40L interactions are important for B cell class-switch recombination and germinal center formation, we sought to understand the impact of these interactions on the immune abnormalities in NZB CD40L gene-deleted (CD40L(-/-)) mice in vivo. NZB.CD40L(-/-) mice demonstrated abrogation of all IgG autoantibodies tested and attenuated kidney disease. However, polyclonal B cell activation in vivo and B cell proliferation and class-switching in response to TLR ligands in vitro were preserved in the absence of CD40L in NZB mice. Although, plasmacytoid dendritic cell expansion and elevated BAFF production were unaffected by the absence of CD40L, there was some evidence that IFN-α-induced gene expression was reduced in the bone marrow of NZB.CD40L(-/-) mice. Our results suggest that CD40-CD40L interactions play an important role in promoting pathogenic IgG autoantibody production and kidney disease in NZB mice.

Role of CD40 ligand in transfusion-related acute lung injury

Transfusion-related acute lung injury (TRALI) is an important problem in the critically ill. CD40L has been implicated as a cofactor or even the cause of TRALI [1]. (1) We hypothesized that blocking of the CD40-CD40L interaction protects against lung injury in a murine model of TRALI. (2) Furthermore, we hypothesized that plasma sCD40 levels are elevated in TRALI patients compared with controls.

Ileal Immune Dysregulation in Necrotizing Enterocolitis: Role of CD40/CD40L in the Pathogenesis of Disease

CD40, a co-stimulatory molecule, plays a critical role in coordinating enteric inflammatory immune responses. In necrotizing enterocolitis (NEC), upregulation of IL-10, a CD40-modulated cytokine, has been described, but the role of the IL-10 receptor (IL-10Rβ), CD40, and its ligand CD40L in disease pathogenesis is unknown. The study herein investigates ileal expression of CD40, CD40L, and IL-10R in a rat model of NEC. NEC was induced in newborn rats using established methods of formula feeding, asphyxia, and cold stress. Expression of CD40, CD40L, IL-10Rβ, and other proinflammatory molecules, including Toll-like receptor-4 (TLR-4) and IL-18, was assessed by immunoblotting. Tissue infiltration by macrophages, monocytes, and T cells was examined by confocal immunohistochemistry. Ileum from rat pups with NEC showed increased expression of TLR-4, IL-18, and IL-10Rβ. Sections from both NEC and control pups demonstrated preservation of ileal cells expressing CD40/CD40L. The distal ileum of controls expressed both CD40 and CD40L; conversely, neither molecule was detected in ileal tissue from NEC pups. Additional studies showed that treatment with epidermal growth factor (EGF), previously shown to ameliorate the severity of NEC in an animal model, did not restore CD40 expression. Ileal cytokine dysregulation, manifested by decreased CD40/CD40L and increased IL-10Rβ expression, may be involved in the pathogenesis of NEC. Dampened CD40 signaling may be related to enhanced IL-10 expression and a suppressed T-cell response to injury. We speculate that augmenting CD40-CD40L interactions may achieve a protective effect in this NEC model.

On/Off TLR Signaling Decides Proinflammatory or Tolerogenic Dendritic Cell Maturation upon CD1d-Mediated Interaction with Invariant NKT Cells

Invariant NKT (iNKT) cells play an effector/adjuvant function during antimicrobial and antitumoral immunity and a regulatory role to induce immune tolerance and prevent autoimmunity. iNKT cells that differentially modulate adaptive immunity do not bear a unique phenotype and/or specific cytokine secretion profile, thus opening questions on how a single T cell subset can exert opposite immunological tasks. In this study, we show that iNKT cells perform their dual roles through a single mechanism of action relying on the cognate interaction with myeloid dendritic cells (DCs) and leading to opposite effects depending on the presence of other maturation stimuli simultaneously acting on DCs. The contact of murine purified iNKT cells with immature autologous DCs directly triggers the tolerogenic maturation of DCs, rendering them able to induce regulatory T cell differentiation and prevent autoimmune diabetes in vivo. Conversely, the interaction of the same purified iNKT cells with DCs, in the presence of simultaneous TLR4 stimulation, significantly enhances proinflammatory DC maturation and IL-12 secretion. The different iNKT cell effects are mediated through distinct mechanisms and activation of different molecular pathways within the DC: CD1d signaling and activation of the ERK1/2 pathway for the tolerogenic action, and CD40-CD40L interaction and NF-κB activation for the adjuvant effect. Our data suggest that the DC decision to undergo proinflammatory or tolerogenic maturation results from the integration of different signals received at the time of iNKT cell contact and could have important therapeutic implications for exploiting iNKT cell adjuvant/regulatory properties in autoimmune diseases, infections, and cancer.

Involvement of CD40–CD40 Ligand in Uncomplicated and Refractory Celiac Disease

Cognate interaction between CD40 on antigen-presenting cells and CD40 ligand (CD40L) on T cells is a crucial costimulatory signal in T-cell activation. In this study, we investigated CD40-CD40L expression in the duodenum of uncomplicated and refractory celiac disease patients, and explored the ex vivo effects of CD40L blockade on cytokine production and the T-helper cell type 1-specific transcription factor T-bet. CD40L and colocalization of CD40 with the dendritic cell markers CD11c and CD123 were investigated by confocal microscopy on tissue sections of duodenal biopsy samples obtained from 14 uncomplicated celiac patients before and after 12 months of gluten-free diet, 5 refractory celiac patients, and 12 controls. CD40 was also analyzed by flow cytometry on single cell suspension of mucosal biopsies. Treated celiac biopsies were stimulated with peptic-tryptic digest of gliadin (PT-gliadin) with or without an anti-CD40L-neutralizing antibody. Interferon (IFN)-γ and interleukin (IL)-17 were measured by ELISA (enzyme-linked immunosorbent assay). T-bet, CD40, and CD40L were determined by immunoblotting. CD40 and CD40L expression was higher in uncomplicated untreated and refractory celiac patients than in controls; the expression returned to normal after gluten-free diet in uncomplicated patients. Flow cytometric analysis confirmed that most CD40(+) cells were dendritic cells. The addition of the anti-CD40L antibody to treated celiac biopsies significantly inhibited the PT-gliadin-induced production of IFN-γ and IL-17, and mucosal T-bet. Our results indicate that the CD40-CD40L pathway has a key role in celiac disease. Disruption of CD40-CD40L interaction may offer a therapeutic alternative in refractory celiac disease.

Expression and purification of soluble murine CD40L monomers and polymers in yeast Pichia pastoris

The anti-murine CD40L monoclonal antibody MR1 has been widely used in immunology research to block the CD40-CD40L interaction for induction of transplantation tolerance and to abrogate autoimmune diseases. The availability of recombinant CD40L with high binding capacity for MR1 would provide a valuable immunologic research tool. In this study, we constructed the single chain murine soluble CD40L monomer, dimer, trimer and successfully expressed them in yeast Pichia pastoris under the control of the alcohol oxidase promoter. The secreted single chain murine soluble CD40L monomers, dimers, and trimers were initially enriched through histidine tag capture by Ni-Sepharose 6 fast flow resin and further purified on a cation exchange resin. Purity reached more than 95% for the monomer and dimer forms and more than 90% for the trimer. Protein yield following purification was 16 mg/L for the monomer and dimer, and 8 mg/L for the trimer. ELISA analysis demonstrated that the CD40L dimers and trimers correctly folded in conformations exposing the MR1 antigenic determinant.

Role of vascular endothelial growth factor receptor in the pro-proliferation activity of CD40-CD40L in AGS gastric cancer cells

Abstract Background: CD40 is a type α-membrane protein of the tumor necrosis factor receptor super-family, and CD40- induced responses may mediate growth and angiogenesis in carcinoma cells. Objectives: Define the effect of CD40 ligation on AGS gastric cancer cell line and the role of vascular endothelial growth factor/vascular endothelial growth factor receptor (VEGF/VEGFR) signals in this process. Methods: We treated AGS cells with 1 μg/mL soluble CD40 ligand (sCD40L) with or without pre-incubation of either anti-VEGF mAb (MAB293) or VEGFR tyrosine kinase inhibitor (SU5416). We determined the growth effects by cell counts or [3H]-thymidine incorporation assay and VEGF levels in cell-free supernatant using enzymelinked immunosorbent assays. Results: The engagement of CD40-induced AGS cells proliferation accompanied by a significant increase autocrine VEGF through PI3K activation (p &lt;0.05), and exogenous VEGF alone had no effect on spontaneous cell growth. SU5416 with a concentration of 8 μM lead to a dramatic decrease in cell survival induced by sCD40L (p &lt;0.05), whereas MAB293 did not have the similar effect (p &gt;0.05). Conclusion: CD40-CD40L interaction promoted AGS cancer cell line proliferation through a VEGFR-dependent signal pathway in the presence of an internal autocrine loop.

Ethylenecarbodiimide-Treated Splenocytes Carrying Male CD4 Epitopes Confer Histocompatability Y Chromosome Antigen Transplant Protection by Inhibiting CD154 Upregulation

In humans and certain strains of laboratory mice, male tissue is recognized as nonself and destroyed by the female immune system via recognition of histocompatibility Y chromosome Ag (Hya). Male tissue destruction is thought to be accomplished by CTLs in a helper-dependent manner. We show that graft protection induced with the immunodominant Hya-encoded CD4 epitope (Dby) attached to female splenic leukocytes (Dby-SPs) with the chemical cross-linker ethylenecarbodiimide significantly, and often indefinitely, prolongs the survival of male skin graft transplants in an Ag-specific manner. In contrast, treatments with the Hya CD8 epitopes (Uty-/Smcy-SPs) failed to prolong graft survival. Dby-SP-tolerized CD4(+) T cells fail to proliferate, secrete IFN-gamma, or effectively prime a CD8 response in recipients of male grafts. Ag-coupled splenocyte treatment is associated with defective CD40-CD40L interactions as demonstrated by the observation that CD4 cells from treated animals exhibit a defect in CD40L upregulation following in vitro Ag challenge. Furthermore, treatment with an agonistic anti-CD40 Ab at the time of transplantation abrogates protection from graft rejection. Interestingly, anti-CD40 treatment completely restores the function of Dby-specific CD4 cells but not Uty- or Smcy-specific CD8 cells.

CD137 Is Required for M Cell Functional Maturation but Not Lineage Commitment

Mucosal immune surveillance depends on M cells that reside in the epithelium overlying Peyer's patch and nasopharyngeal associated lymphoid tissue to transport particles to underlying lymphocytes. M cell development is associated with B lymphocytes in a basolateral pocket, but the interactions between these cells are poorly understood. In a cell culture model of M cell differentiation, we found lymphotoxin/tumor necrosis factor alpha induction of CD137 (TNFRSF9) protein on intestinal epithelial cell lines, raising the possibility that CD137 on M cells in vivo might interact with CD137L expressed by B cells. Accordingly, while CD137-deficient mice produced UEA-1+ M cell progenitors in nasopharyngeal associated lymphoid tissue and Peyer's patch epithelium, they showed an abnormal morphology, including the absence of basolateral B cell pockets. More important, CD137-deficient nasopharyngeal associated lymphoid tissue M cells were defective in microparticle transcytosis. Bone marrow irradiation chimeras confirmed that while induction of UEA-1+ putative M cell precursors was not CD137-dependent, full M cell transcytosis function required expression of CD137 by radioresistant stromal cells as well as by bone marrow-derived cells. These results are consistent with a two-step model of M cell differentiation, with initial CD137-independent commitment to the M cell lineage followed by a CD137-CD137L interaction of M cells with CD137-activated B lymphocytes or dendritic cells for functional maturation.

Production and characterization of agonistic monoclonal antibodies against chicken CD40

CD40 is mainly expressed by professional antigen-presenting cells (APCs). Its ligand, CD40L, is transiently expressed on activated CD4 + T-cells. CD40–CD40L interactions mediate T-cell help to APCs and provide crucial signals for affinity maturation and B-cell class switching. In mammals, agonistic monoclonal anti-CD40 antibodies (mAbs) mimic the effects of CD40L on APCs, leading to enhanced T-cell priming and expansion, increased antibody production and class switching. In this study, we describe agonistic anti-chicken CD40 mAb 2C5. This mAb detected CD40 on primary chicken B-cells and macrophages, DT40 B-cells, and HD11 macrophages, induced NO synthesis in HD11 macrophages, and stimulated DT40 B-cell proliferation. These observations demonstrated at least partial functional equivalence of 2C5 to chicken CD154. This mAb may therefore constitute a new tool to study the role of CD40 in the chicken immune system, and its agonistic effects suggest that it could also be used as an adjuvant.

Signaling Pathways in the Activation of Mast Cells Cocultured with Astrocytes and Colocalization of Both Cells in Experimental Allergic Encephalomyelitis

Mast cells in the CNS participate in the pathophysiology of chronic neurodegenerative inflammatory diseases. This study aimed to investigate the signaling pathway of mast cells activated in an environment cocultured with astrocytes and to explore the role of their colocalization in brain of experimental allergic encephalomyelitis. Human mast cell line-1 cells and human U87 glioblastoma cell lines (U87) or mouse bone marrow-derived mast cells and mouse cerebral cortices-derived astrocytes were cocultured. Intracellular Ca(2+) was measured by confocal microscopy; histamine by fluorometric analyzer; leukotrienes by ELISA; small GTPases, protein kinase Cs, MAPK, c-kit, CD40, and CD40L by Western blot; NF-kappaB and AP-1 by EMSA; cytokines by RT-PCR; and colocalization of mast cells and astrocytes in brain by immunohistochemistry. Mast cells cocultured with astrocytes showed time-dependent increases in intracellular Ca(2+) levels, release of histamine and leukotrienes, and cytokine production. Mast cells or astrocytes showed enhanced surface expression of CD40L and CD40, respectively, during coculture. Mast cells cocultured with astrocytes induced small GTPases (Rac1/2, cdc42), protein kinase Cs, MAPK, NF-kappaB, and AP-1 activities. These changes were blocked by anti-CD40 Ab pretreatment or CD40 small interfering RNA. Mast cells increased in the thalamus of experimental allergic encephalomyelitis model, particularly colocalized with astrocytes in the thalamic border region of the habenula. In conclusion, the data suggest that activation of mast cells cocultured with astrocytes induces release of mediators by small GTPases/Ca(2+) influx through CD40-CD40L interactions to participate in the pathophysiology of chronic neurodegenerative inflammatory diseases, such as multiple sclerosis.

The role of CD40L and VEGF in the modulation of angiogenesis and inflammation

Recently, there has been growing interest in deciphering the role of angiogenesis in the progression of atherogenesis. Importantly, CD40–CD40L interactions are of significant relevance because of their involvement in both angiogenesis and atherosclerotic development. Previously, we have shown that recombinant soluble CD40 ligand (rsCD40L) stimulates auto-inflammatory CD40L synthesis and reactive oxygen species (ROS) generation in vascular cells. In the current study, we demonstrate that redox-mediated CD40–CD40L interaction can enhance vascular endothelial growth factor (VEGF)-induced angiogenesis, endothelial migration, and actin polymerization processes. Interestingly, the addition of exogenous VEGF leads to cleavage of de novo CD40L produced in endothelial cells following rsCD40L treatment. Using inhibitor and silencing RNA-based experiments, it was observed that VEGF-induced protease, calpain 2, was responsible for the cleavage of de novo CD40L. While our in vivo experiments using a matrigel plug assay indicate a VEGF and CD40L induced enhancement of angiogenesis, our studies also identify a novel mechanism by which VEGF can abrogate CD40L-mediated inflammation. Together, these studies reveal a new pathway by which VEGF–CD40L interactions can regulate the angiogenic and inflammatory process depending on the specific environment.

CD4+ T Cells Elicit Host Immune Responses to MHC Class II− Ovarian Cancer through CCL5 Secretion and CD40-Mediated Licensing of Dendritic Cells

T cell adoptive transfer strategies that have produced clinical remissions against specific tumors have so far produced disappointing results against ovarian cancer. Recent evidence suggests that adoptively transferred CD4(+) T cells can trigger endogenous immune responses in particular patients with ovarian cancer through unknown mechanisms. However, conflicting reports suggest that ovarian cancer-infiltrating CD4(+) T cells are associated with negative outcomes. In this study, we elucidate the phenotypic attributes that enable polyclonal CD4(+) T cells briefly primed against tumor Ags to induce therapeutically relevant endogenous antitumor immune responses. Our results unveil a therapeutic mechanism whereby tumor-primed CD4(+) T cells transferred into ovarian cancer-bearing mice secrete high levels of CCL5, which recruits endogenous CCR5(+) dendritic cells to tumor locations and activate them through CD40-CD40L interactions. These newly matured dendritic cells are then able to prime tumor-specific endogenous CD8(+) T cells, which mediate long-term protection. Correspondingly, administration of tumor-primed CD4(+) T cells significantly delayed progression of MHC class II(-) ovarian cancers, similarly to CD8(+) T cells only, and directly activated wild-type but not CD40-deficient dendritic cells recruited to the tumor microenvironment. Our results unveil a CCL5- and CD40L-dependent mechanism of transferring immunity from exogenously activated CD4(+) T cells to tumor-exposed host cells, resulting in sustained antitumor effects. Our data provide a mechanistic rationale for incorporating tumor-reactive CD4(+) T cells in adoptive cell transfer immunotherapies against ovarian cancer and underscore the importance of optimizing immunotherapeutic strategies for the specific microenvironment of individual tumors.