Activation of dendritic cells from patients with epithelial ovarian cancer

Dendritic cells (DCs) are the most potent antigen-presenting cells for inducing immune responses to tumor cells. Lin−HLA-DR + DC populations in peripheral blood mononuclear cells (PBMCs) and in ascites mononuclear leukocytes (MNLs) of patients with epithelial ovarian cancer (EOC) are phenotypically immature. Lin−HLA-DR + DCs from PBMCs of normal subjects and EOC patients and MNLs from ascites cells of patients were examined for specific cell surface markers or indicators of differentiation or activation. Separating Lin− HLA-DR+ DCs into subsets based on their HLA-DR intensity provided an additional method for identifying the two major lineages of DCs, myeloid and plasmacytoid. The activation potential of these DCs following exposure to the maturation agents CD40 ligand (CD40L) and lipopolysaccharide (LPS) was examined by measurement of IL-12 and IL-10 concentrations in DC culture supernatants in addition to their ability to stimulate allogeneic T cells. DCs from PBMCs of normal subjects and EOC patients and DCs isolated from ascites MNLs of EOC patients were separated into subsets based on CD11c and CD123 cell surface marker expression identifying the major DC types. These subsets were then compared with cells sorted on the basis of HLA-DR intensity. The in vivo behavior of DCs and DC subsets in peripheral blood and ascites following treatment of peritoneal carcinoma patients with the growth factor fins-like tyrosine kinase 3 ligand (Flt3L) was also examined. Increases in proportions and total numbers of DCs from peripheral blood and ascites were associated with increased secretion of IL-12 and IL-10 following in vitro activation of cultured DCs. There were differences between DCs from PBMCs and ascites and between DC subsets in expression of cell surface markers, cytokine profile, and the ability of Lin−HLA-DR + cells to stimulate proliferation of allogeneic T cells from EOC patients. These Lin−HLA-DR+ cells have certain functional properties that suggest that they could have the potential to facilitate an adaptive anti-tumor immune response. ^

Trichostatin A reverses skewed expression of CD154, interleukin-10, and interferon-γ gene and protein expression in lupus T cells

In systemic lupus erythematosus (SLE), T helper cells exhibit increased and prolonged expression of cell-surface CD40 ligand (CD154), spontaneously overproduce interleukin-10 (IL-10), but underproduce interferon-gamma (IFN-γ). We tested the hypothesis that the imbalance of these gene products reflects skewed expression of CD154, IL-10, and IFN-γ genes. Here, we demonstrate that the histone deacetylase inhibitor, trichostatin A, significantly down-regulated CD154 and IL-10 and up-regulated IFN-γ gene expression in SLE T cells. This reversal corrected the aberrant expression of these gene products, thereby enhancing IFN-γ production and inhibiting IL-10 and CD154 expression. That trichostatin A can simultaneously reverse the skewed expression of multiple genes implicated in the immunopathogenesis of SLE suggests that this pharmacologic agent may be a candidate for the treatment of this autoimmune disease.

An essential role for Bruton's [corrected] tyrosine kinase in the regulation of B-cell apoptosis.

Mutations of the Bruton's tyrosine kinase (btk) gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immune deficiency (Xid) in mice. To establish the BTK role in B-cell activation we examined the responses of wild-type and Xid B cells to stimulation through surface IgM and CD40, the transducers of thymus independent-type 2 and thymus-dependent activation, respectively. Wild-type BTK was necessary for proliferation induced by soluble anti-IgM (a prototype for thymus independent-type 2 antigen), but not for responses to soluble CD40 ligand (CD40L, the B-cell activating ligand expressed on T-helper cells). In the absence of wild-type BTK, B cells underwent apoptotic death after stimulation with anti-IgM. In the presence of wild-type but not mutated BTK, anti-IgM stimulation reduced apoptotic cell death. In contrast, CD40L increased viability of both wild-type and Xid B cells. Importantly, viability after stimulation correlated with the induced expression of bcl-XL. In fresh ex vivo small resting B cells from wild-type mice there was only barely detectable bcl-XL protein, but there was more in the larger, low-density ("activated") splenic B cells and peritoneal B cells. In vitro Bcl-XL induction following ligation of sIgM-required BTK, was cyclosporin A (CsA)-sensitive and dependent on extracellular Ca2+. CD40-mediated induction of bcl-x required neither wild-type BTK nor extracellular Ca2+ and was insensitive to CsA. These results indicate that BTK lies upstream of bcl-XL in the sIgM but not the CD40 activation pathway. bcl-XL is the first induced protein to be placed downstream of BTK.

The tumor necrosis factor-inducible zinc finger protein A20 interacts with TRAF1/TRAF2 and inhibits NF-kappaB activation.

TRAF1 and TRAF2 form an oligomeric complex that associates with the cytoplasmic domains of various members of the tumor necrosis factor (TNF) receptor superfamily. TRAF2 action is required for activation of the transcription factor NF-kappaB triggered by TNF and the CD40 ligand. Here we show that TRAF1 and TRAF2 interact with A20, a zinc finger protein, whose expression is induced by agents that activate NF-kappaB. Mutational analysis revealed that the N-terminal half of A20 interacts with the conserved C-terminal TRAF domain of TRAF1 and TRAF2. In cotransfection experiments, A20 blocked TRAF2-mediated NF-kappaB activation. A20 also inhibited TNF and IL-1-induced NF-kappaB activation, suggesting that it may inhibit NF-kappaB activation signaled by diverse stimuli. The ability of A20 to block NF-kappaB activation was mapped to its C-terminal zinc finger domain. Thus, A20 is composed of two functionally distinct domains, an N-terminal TRAF binding domain that recruits A20 to the TRAF2-TRAF1 complex and a C-terminal domain that mediates inhibition of NF-kappaB activation. Our findings suggest a possible molecular mechanism that could explain A20's ability to negatively regulate its own TNF-inducible expression.

Increased mononuclear cell activation and apoptosis early after human liver transplantation is associated with a reduced frequency of acute rejection

Experimental models of orthotopic liver transplantation (OLT) have shown that the very early events post-OLT are critical in distinguishing immunogenic and tolerogenic reactions. In rodents, increased leukocyte apoptosis and cytokine expression have been demonstrated in tolerogenic strain combinations. Information from human OLT recipients is less abundant. The aim of this study was to determine the amount of early leukocyte activation and apoptosis following human OLT, and to correlate this with subsequent rejection status. Peripheral blood mononuclear cells (PBMC) were isolated from 76 patients undergoing OLT - on the day prior, 5 hrs after reperfusion (day 0), and 18-24 hrs post-OLT (day 1). The mean level of apoptotic PBMCs on post OLT day 1 was higher than healthy recipients (0.9% +/- 0.2 vs. 0.2% +/- 0.1, p = 0.013). Apoptosis was greater in nonrejecting (NR) (1.1% +/- 0.3) compared with acutely-rejecting (R) (0.3% +/- 0.1, p = 0.021) patients. On day 1, PBMC from NR patients had increased expression of IFN-gamma (p = 0.006), IL-10 (p = 0.016), and CD40 ligand (p = 0.02) compared with R. Donor cell chimerism on day 1 did not differ between the groups indicating that this was unlikely to account for increased PBMC apoptosis in the NR group. Interestingly, the level of chimerism on day 0 was significantly higher in NR (3.8% +/- 0.6) compared with R (1.2% +/- 0.4, p = 0.004) patients and there was a close correlation between chimerism on day 0 and cytokine expression on day 1. These results imply that similar mechanisms are occurring in the human liver to promote graft acceptance as in the experimental models of liver transplantation and suggest that strategies that promote liver transplant acceptance in rodents might be applicable to humans.

T cells signaled by NF-kappa B- dendritic cells are sensitized not anergic to subsequent activation

Paradoxically, while peripheral self-tolerance exists for constitutively presented somatic self Ag, self-peptide recognized in the context of MHC class II has been shown to sensitize T cells for subsequent activation. We have shown that MHC class II(+)CD86(+)CD40(-) DC, which can be generated from bone marrow in the presence of an NF-kappaB inhibitor, and which constitutively populate peripheral tissues and lymphoid organs in naive animals, can induce Ag-specific tolerance. In this study, we show that CD40(-) human monocyte-derived dendritic cells (DC), generated in the presence of an NF-kappaB inhibitor, signal phosphorylation of TCRzeta, but little proliferation or IFN-gamma in vitro. Proliferation is arrested in the G(1)/G(0) phase of the cell cycle. Surprisingly, responding T cells are neither anergic nor regulatory, but are sensitized for subsequent IFN-gamma production. The data indicate that signaling through NF-kappaB determines the capacity of DC to stimulate T cell proliferation. Functionally, NF-kappaB(-)CD40(-)class II+ DC may either tolerize or sensitize T cells. Thus, while CD40(-) DC appear to prime or prepare T cells, the data imply that signals derived from other cells drive the generation either of Ag-specific regulatory or effector cells in vivo.

Either interleukin-12 or interferon-gamma can correct the dendritic cell defect induced by transforming growth factor beta(1) in patients with myeloma

The poor response to immunotherapy in patients with multiple myeloma (MM) indicates that a better understanding of any defects in the immune response in these patients is required before effective therapeutic strategies can be developed. Recently we reported that high potency (CMRF44(+)) dendritic cells (DC) in the peripheral blood of patients with MM failed to significantly up-regulate the expression of the B7 co-stimulatory molecules, CD80 and CD86, in response to an appropriate signal from soluble trimeric human CD40 ligand. This defect was caused by transforming growth factor beta(1) (TGFbeta(1)) and interleukin (IL)-10, produced by malignant plasma cells, and the defect was neutralized in vitro with anti-TGFbeta(1). As this defect could impact on immunotherapeutic strategies and may be a major cause of the failure of recent trials, it was important to identify a more clinically useful agent that could correct the defect in vivo. In this study of 59 MM patients, the relative and absolute numbers of blood DC were only significantly decreased in patients with stage III disease and CD80 up-regulation was reduced in both stage I and stage III. It was demonstrated that both IL-12 and interferon-gamma neutralized the failure to stimulate CD80 up-regulation by huCD40LT in vitro. IL-12 did not cause a change in the distribution of DC subsets that were predominantly myeloid (CD11c+ and CDw123-) suggesting that there would be a predominantly T-helper cell type response. The addition of IL-12 or interferon-gamma to future immunotherapy trials involving these patients should be considered.

Generation and maturation of dendritic cells for clinical application under serum-free conditions

Monocyte-derived dendritic cells (MoDCs) in clinical use for cancer immunotherapy are ideally generated in serum-free medium (SFM) with inclusion of a suitable maturation factor toward the end of the incubation period. Three good manfacturing practice (GMP) grade SFMs (AIM-V, X-VIVO 15, and X-VIVO 20) were compared with RPMI-1640, supplemented with 10% fetal bovine serum or 10% human serum. DCs generated for 7 days in SFM were less mature and secreted less interleukin (IL) 12p70 and IL-10 than DCs generated in 10% serum. DC yield was comparable in SFMs, and a greater proportion of cells was viable after maturation. Toll-like receptor (TLR) ligands were compared for their ability to induce cytokine secretion under serum-free conditions in the presence of interferon (IFN) gamma. With the exception of Poly I:C, TLR ligands stimulated high levels of IL-10 secretion. High levels of IL-12p70 were induced by two TLR4-mediated stimuli, lipopolysaccharide and Ribomunyl, a clinical-grade bacterial extract. When T-cell responses were compared in allogeneic mixed leukocyte reaction, DCs stimulated with Ribomunyl induced higher levels of IFN gamma than DCs stimulated with the cytokine cocktail: tumor necrosis factor-alpha, IL-1 beta, IL-6, and prostaglandin E-2. In the presence of IL-10 neutralizing antibodies, DC IL-12p70 production and T-cell IFN gamma were increased in vitro. Similarly, DCs stimulated with Ribomunyl, IFN gamma, and anti-IL-10 induced high levels of tetanus toxoid-specific T-cell proliferation and IFN gamma secretion. Thus, MoDCs generated ill SFM efficiently stimulate T-cell IFN gamma production after maturation in the presence of a clinical-grade TLR4 agonist and IL-10 neutralization.

Differential peptide binding to CD40 evokes counteractive responses.

The antigen-presenting cell-expressed CD40 is implied in the regulation of counteractive immune responses such as induction of pro-inflammatory and anti-inflammatory cytokines interleukin (IL)-12 and IL-10, respectively. The mechanism of this duality in CD40 function remains unknown. Here, we investigated whether such duality depends on ligand binding. Based on CD40 binding, we identifed two dodecameric peptides, peptide-7 and peptide-19, from the phage peptide library. Peptide-7 induces IL-10 and increases Leishmania donovani infection in macrophages, whereas peptide-19 induces IL-12 and reduces L. donovani infection. CD40-peptide interaction analyses by surface plasmon resonance and atomic force microscopy suggest that the functional differences are not associated with the studied interaction parameters. The molecular dynamic simulation of the CD40-peptides interaction suggests that these two peptides bind to two different places on CD40. Thus, we suggest for the first time that differential binding of the ligands imparts functional duality to CD40.

Cysteine-rich Domain 1 of CD40 Mediates Receptor Self-assembly.

The activation of CD40 on B cells, macrophages, and dendritic cells by its ligand CD154 (CD40L) is essential for the development of humoral and cellular immune responses. CD40L and other TNF superfamily ligands are noncovalent homotrimers, but the form under which CD40 exists in the absence of ligand remains to be elucidated. Here, we show that both cell surface-expressed and soluble CD40 self-assemble, most probably as noncovalent dimers. The cysteine-rich domain 1 (CRD1) of CD40 participated to dimerization and was also required for efficient receptor expression. Modelization of a CD40 dimer allowed the identification of lysine 29 in CRD1, whose mutation decreased CD40 self-interaction without affecting expression or response to ligand. When expressed alone, recombinant CD40-CRD1 bound CD40 with a KD of 0.6 μm. This molecule triggered expression of maturation markers on human dendritic cells and potentiated CD40L activity. These results suggest that CD40 self-assembly modulates signaling, possibly by maintaining the receptor in a quiescent state.

CD40 signalling in platelet function

Le CD40 est un membre de la famille des récepteurs du facteur de nécrose tumorale ("Tumour necrosis factor", TNF), initialement identifié sur des cellules de carcinome de la vessie. L'interaction du CD40 avec son ligand (CD40L) est d'une importance cruciale pour le développement des cellules B et de la commutation d'isotype au cours de la réponse immunitaire acquise. L'expression du complexe CD40/CD40L était initialement cru d'être limiter aux cellules du système immunitaire, mais aujourd'hui il est bien connu que ce complexe est également exprimé sur les cellules du système circulatoire et vasculaire, et est impliqué dans diverses réactions inflammatoires; de sorte que le CD40L est maintenant considéré comme une molécule thrombo-inflammatoire prédictive des événements cardiovasculaires. Les plaquettes expriment constitutivement le CD40, alors que le CD40L n'est exprimé que suite à leur l'activation. Il est ensuite clivé en sa forme soluble (sCD40L) qui représente la majorité du sCD40L en circulation. Il fut démontré que le sCD40L influence l'activation plaquettaire mais son effet exact sur la fonction plaquettaire, ainsi que les mécanismes cellulaires et moléculaires sous-jacents à son action demeurent inconnus. Ainsi, ce projet a été entrepris dans le but d’adresser les objectifs spécifiques suivants: 1) évaluer les effets in vitro du sCD40L sur l'activation et l'agrégation plaquettaire; 2) identifier les récepteurs plaquettaires impliqués dans l’action du sCD40L; 3) élucider les voies signalétiques intracellulaires induits par le sCD40L; 4) évaluer les effets du sCD40L sur la formation de thrombus in vivo. Nous avons trouvé que le sCD40L augmente fortement l'activation et l'agrégation des plaquettes en réponse à de faibles concentrations d'agonistes. Les plaquettes humaines traitées avec une forme mutante du sCD40L qui n'interagit pas avec le CD40, et les plaquettes de souris déficientes en CD40 ne furent pas en mesure d'induire de telles réponses, indiquant que le récepteur principal du sCD40L au niveau des plaquettes est le CD40. En plus, nous avons identifié la présence de plusieurs membres de la famille du facteur associé du récepteur du TNF ("TNF receptor-associated factor", TRAF) dans les plaquettes et nous avons montré que seulement le TRAF2 s'associe avec le CD40 suite à la stimulation par le sCD40L. Nos résultats indiquent aussi que le sCD40L agisse sur les plaquettes au repos par l'entremise de deux voies signalétiques distinctes. La première voie implique l'activation de la petite GTPase Rac1 et de sa cible en aval, soit la protéine kinase p38 activée par le mitogène ("p38 mitogen-activated protein kinase", p38 MAPK ), menant au changement de forme plaquettaire et à la polymérisation de l'actine; alors que la deuxième voie implique l'activation de la cascade signalétique du NF-kB. Par ailleurs, à la suite d'une lésion artérielle induite par le chlorure de fer, le sCD40L exacerbe la formation de thrombus et l'infiltration leucocytaire au sein du thrombus dans les souris du type sauvage, mais pas chez les souris déficientes en CD40. En conclusion, ce projet a permis d'identifier pour la première fois deux voies signalétiques distinctes en aval du CD40 plaquettaire et a permis d'établir leur implication dans l'activation et l'agrégation plaquettaire en réponse au sCD40L. De manière plus importante, ce projet nous a permis d'établir un lien direct entre les niveaux élevés du sCD40L circulant et la formation de thrombus in vivo, tout en soulignant l'importance du CD40 dans ce processus. Par conséquent, l'axe CD40/CD40L joue un rôle important dans l'activation des plaquettes, les prédisposant à une thrombose accrue en réponse à une lésion vasculaire. Ces résultats peuvent expliquer en partie la corrélation entre les taux circulants élevés du sCD40L et l'incidence des maladies cardiovasculaires.

Le rôle du CD40 homodimère dans la réponse immunitaire

Le CD40 est une glycoprotéine transmembranaire de type I, appartenant à la famille des TNFRs, exprimée à la surface des cellules immunitaires, hématopoïétiques, vasculaires, épithéliales, et d’autres types de cellules, y compris les cellules tumorales. Le CD40 ne possédant pas de domaine kinase, pour induire un signal il interagit directement ou indirectement avec des protéines adaptatrices telles que les TRAFs et les JAKs. L’interaction du CD40 avec son principal ligand, le CD154, joue un rôle primordial dans la régulation de la réponse immunitaire et le maintien de l’homéostasie. L’activation du CD40 à la surface des cellules B augmente leur capacité de présentation d’antigène, en plus d’induire la prolifération, la commutation isotypique et l’apoptose. Les patients souffrant de mutations au niveau du gène codant pour le CD40 ou de son ligand sont immunosupprimés et sensibles à des infections opportunistes. Des études ont montré que le CD40 comme d’autres membres de la famille des TNFRs est capable de former des homodimères. Plus récemment, on a montré que la formation du CD40 homodimère est le résultat de son engagement sur les cellules B. En plus, cette homodimérisation du CD40 est importante pour la phosphorylation de l’Akt. L’interaction CD40/CD154 peut avoir un rôle direct dans l’immunothérapie par l’induction de l’apoptose de certaines cellules cancéreuses ou un rôle indirect en activant les cellules présentatrices d’antigènes (CPA) afin d'augmenter l’efficacité de l’activation des cellules T cytotoxiques. Nos résultats montrent que l’induction de la mort cellulaire par le CD40 requiert la perméabilisation du lysosome, la libération de la cathepsine B, la présence de ROS et une interaction avec le TRAF6, cette mort cellulaire programmée est plus importante en présence de la forme monomérique du CD40, muté au niveau de la cystéine 238. Par ailleurs, l’homodimérisation du CD40 requerrait sa translocation vers les radeaux lipidiques et nécessiterait la présence des ROS. Cette homodimérisation du CD40 semble être importante pour l’activation des cellules B par le biais de l’induction de l’expression du CD23, CD69 et CD80. De plus, nos résultats montrent pour la première fois une implication du CD40 homodimère dans l’induction du CD23 par le biais du TLR4. Nos résultats soulignent l’importance du CD40 homodimère dans certaines voies de signalisation. Ainsi, ils mettent en évidence le rôle de la Cys-238 dans la coopération entre des récepteurs de la réponse immunitaire innée et adaptative. Toutes ces données permettraient une meilleure compréhension de certaines voies de signalisation impliquées dans plusieurs maladies auto-immunes et faisant objet de plusieurs essais thérapeutiques.

Intraperitoneal Echinococcus multilocularis infection in C57BL/6 mice affects CD40 and B7 costimulator expression on peritoneal macrophages and impairs peritoneal T cell activation

One of the most important immunopathological consequence of intraperitoneal alveolar echinococcosis (AE) in the mouse is suppression of T cell-mediated immune responses. We investigated whether and how intraperitoneal macrophages (MØs) are, respectively, implicated as antigen-presenting cells (APCs). In a first step we showed that peritoneal MØs from infected mice (AE-MØs) exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells. In a subsequent step, AE-MØs as well as naïve MØs (positive control) proved their ability to uptake and process C-Ova fluorescein isthiocyanate (FITC). Furthermore, in comparison with naïve MØs, the surface expression of Ia molecules was up-regulated on AE-MØs at the early stage of infection, suggesting that AE-MØs provide the first signal via the antigen-Ia complex. To study the accessory activity of MØs, AE-MØs obtained at the early and late stages of infection were found to decrease Con A-induced proliferation of peritoneal naïve T cells as well as of AE-sensitized peritoneal T cells, in contrast to stimulation with naïve MØs. The status of accessory molecules was assessed by analysing the expression level of costimulatory molecules on AE-MØs, with naïve MØs as controls. It was found that B7-1 (CD80) and B7-2 (CD86) expression remained unchanged, whereas CD40 was down-regulated and CD54 (= ICAM-1) was slightly up-regulated. In a leucocyte reaction of AE-MØs with naïve or AE-T cells, both types of T cells increased their proliferative response when CD28 - the ligand of B7 receptors - was exposed to anti-CD28 in cultures. Conversely to naïve MØs, pulsing of AE-MØs with agonistic anti-CD40 did not even partially restore their costimulatory activity and failed to increase naïve or AE-T cell proliferation. Neutralizing anti-B7-1, in combination with anti-B7-2, reduced naïve and AE-T cell proliferation, whereas anti-CD40 treatment of naïve MØs increased their proliferative response to Con A. These results point at the key role of B7 receptors as accessory molecules and the necessity of the integrity of CD40-expression by naïve MØs to improve their accessory activity. Taken together, the obstructed presenting-activity of AE-MØs appeared to trigger an unresponsiveness of T cells, contributing to the suppression of their clonal expansion during the chronic phase of AE-infection.