NKG2D ligands mediate immunosurveillance of senescent cells

Cellular senescence is a stress response mechanism that limits tumorigenesis and tissue damage. Induction of cellular senescence commonly coincides with an immunogenic phenotype that promotes self-elimination by components of the immune system, thereby facilitating tumor suppression and limiting excess fibrosis during wound repair. The mechanisms by which senescent cells regulate their immune surveillance are not completely understood. Here we show that ligands of an activating Natural Killer (NK) cell receptor (NKG2D), MICA and ULBP2 are consistently up-regulated following induction of replicative senescence, oncogene-induced senescence and DNA damage - induced senescence. MICA and ULBP2 proteins are necessary for efficient NK-mediated cytotoxicity towards senescent fibroblasts. The mechanisms regulating the initial expression of NKG2D ligands in senescent cells are dependent on a DNA damage response, whilst continuous expression of these ligands is regulated by the ERK signaling pathway. In liver fibrosis, the accumulation of senescent activated stellate cells is increased in mice lacking NKG2D receptor leading to increased fibrosis. Overall, our results provide new insights into the mechanisms regulating the expression of immune ligands in senescent cells and reveal the importance of NKG2D receptor-ligand interaction in protecting against liver fibrosis.

LOSS OF CD40 ENDOGENOUS S-NITROSYLATION DURING INFLAMMATORY RESPONSE IN ENDOTOXEMIC MICE AND PATIENTS WITH SEPSIS

Signal transduction through the surface molecule CD40 is critical for cellular activation in immunoinflammatory states such as sepsis. The mechanisms regulating this pathway are not completely understood. Because CD40 displays potentially regulatory cysteine residues and CD40 is probably exposed to NO in the inflammatory milieu, we hypothesized that S-nitrosylation, the interaction of NO with cysteines residues, acts as a post-translational modification on CD40, coregulating the signaling activity and, therefore, the level of cellular activation. As assessed by the biotin switch and the reduction/chemiluminescence S-nitrosylation detection techniques, CD40 was found to be S-nitrosylated endogenously and upon exposure to NO donors in both human and murine macrophages. S-nitrosylation of CD40 was associated with milder activation by its ligand (CD40L), leading to reduced in vitro cytokine (IL-1 beta, IL-12, and TNF-alpha) production, which was reversed in the presence of inhibitors of NO synthesis. S-nitrosylated CD40 was found in resting RAW 246.7 macrophages and BALB/c mice peritoneal macrophages, turning into the denitrosylated state upon in vitro or systemic exposure, respectively, to LPS. Moreover, monocytes from patients with sepsis displayed denitrosylated CD40 in contrast to the CD40 S-nitrosylation measured in healthy individuals. Finally, in an attempt to explain how S-nitrosylation regulates CD40 activation, we demonstrate that NO affects the redistribution of CD40 on the cell surface, which is a requirement for optimal signal transduction. Our results support a novel post-translational regulatory mechanism in which the CD40 signal may be, at least in part, dependent on cellular activation-induced receptor denitrosylation.

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.

Dominant Role of CD80-CD86 Over CD40 and ICOSL in the Massive Polyclonal B Cell Activation Mediated by LAT(Y136F) CD4(+) T Cells.

Coordinated interactions between T and B cells are crucial for inducing physiological B cell responses. Mutant mice in which tyrosine 136 of linker for activation of T cell (LAT) is replaced by a phenylalanine (Lat(Y136F)) exhibit a strong CD4(+) T cell proliferation in the absence of intended immunization. The resulting effector T cells produce high amounts of T(H)2 cytokines and are extremely efficient at inducing polyclonal B cell activation. As a consequence, these Lat(Y136F) mutant mice showed massive germinal center formations and hypergammaglobulinemia. Here, we analyzed the involvement of different costimulators and their ligands in such T-B interactions both in vitro and in vivo, using blocking antibodies, knockout mice, and adoptive transfer experiments. Surprisingly, we showed in vitro that although B cell activation required contact with T cells, CD40, and inducible T cell costimulator molecule-ligand (ICOSL) signaling were not necessary for this process. These observations were further confirmed in vivo, where none of these molecules were required for the unfolding of the LAT CD4(+) T cell expansion and the subsequent polyclonal B cell activation, although, the absence of CD40 led to a reduction of the follicular B cell response. These results indicate that the crucial functions played by CD40 and ICOSL in germinal center formation and isotype switching in physiological humoral responses are partly overcome in Lat(Y136F) mice. By comparison, the absence of CD80-CD86 was found to almost completely block the in vitro B cell activation mediated by Lat(Y136F) CD4(+) T cells. The role of CD80-CD86 in T-B cooperation in vivo remained elusive due to the upstream implication of these costimulatory molecules in the expansion of Lat(Y136F) CD4(+) T cells. Together, our data suggest that CD80 and CD86 costimulators play a key role in the polyclonal B cell activation mediated by Lat(Y136F) CD4(+) T cells even though additional costimulatory molecules or cytokines are likely to be required in this process.

Leishmania (L.) amazonensis inibe a maturação e a função ativadora das células de Langerhans da pele tratadas com TNF-α e anti-CD40 in vitro

Leishmania amazonensis é um dos principais agentes etiológicos em um amplo espectro de formas clínicas da Leishmaniose Tegumentar Americana. De modo geral, a resistência frente às leishmanioses decorre do desenvolvimento de uma resposta imune celular eficiente, porém muitos estudos têm demonstrado que citocinas específicas ou combinações de citocinas podem ser fatores de resistência ou suscetibilidade à infecção por L. amazonensis. Estudos recentes sugerem a participação das células de Langerhans (LCs) nas resposta anti-Leishmania, porém os mecanismos envolvidos durante esta interação são ainda pouco estudados. Objetivos: Estudar o papel do TNF-α e anti-CD40 nas interações in vitro entre as LCs e L. amazonensis, observando o perfil de citocinas produzidas e a expressão de moléculas de superfície, bem como verificar a capacidade destas células em ativar a produção de IFN-γ e IL-4 por células do linfonodo. Metodologia: As LCs foram isoladas da epiderme de camundongos BALB/c e incubadas com promastigotas de L. amazonensis, TNF-α e/ou anti- CD40. Após 24h, as LCs foram co-cultivadas com células obtidas de linfonodos por 72h. As citocinas IL-6, IL-12, IFN-γ e IL-4 foram dosadas por ensaio imunoenzimático (ELISA) e as moléculas de superfície foram analisadas por citometria de fluxo. Resultados: Os níveis de IL- 6 e IL-12p70 produzidos pela LCs foram significativamente reduzidos após interação com L. amazonensis, mesmo após o tratamento das LCs com TNF-α ou anti-CD40. Em relação às moléculas de superfície, não houve diferença na expressão de CD207 em nenhum dos grupos, porém a presença de L. amazonensis promoveu uma redução significativa na expressão de CD40 nas LCs tratadas com TNF-α ou anti-CD40, e aumentou a expressão de CD86 em todos os grupos. Na presença de L. amazonensis, as células do linfonodo apresentaram uma produção diminuída de IFN-γ e não houve alteração na produção de IL-4. Quando cocultivadas com LCs estimuladas previamente com L. amazonensis, a produção de IFN-γ também foi reduzida, mesmo na presença dos estímulos TNF-α e/ou anti-CD40. Não foram observadas alterações significativas na produção de IL-4 pelas células do linfonodo cocultivadas nas mesmas condições experimentais. Conclusão: L. (L.) amazonensis exerce um efeito imunomodulador sobre a resposta imune mediada por LCs, inibindo a produção de IL-6 e IL-12p70 e expressão de CD40, além de impedir a ativação da produção de IFN-γ por células do linfonodo co-cultivadas com LCs, mesmo após tratamento com TNF-α e anticorpo anti-CD40.

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.

CD40 ligation protects bronchial epithelium against oxidant-induced caspase-independent cell death

CD40 and its ligand regulate pleiotropic biological responses, including cell proliferation, differentiation, and apoptosis. In many inflammatory lung diseases, tissue damage by environmental or endogenous oxidants plays a major role in disease pathogenesis. As the epithelial barrier is a major target for these oxidants, we postulated that CD40, the expression of which is increased in asthma, plays a role in the regulation of apoptosis of bronchial epithelial cells exposed to oxidants. Using 16HBE 14o- cells exposed to oxidant stress, we found that ligation of CD40 (induced by G28-5 monoclonal antibodies) enhanced cell survival and increased the number of cells in G2/M (interphase between DNA synthesis and mitosis) of the cell cycle. This was associated with NF-kappaB and activator protein-1 activation and increased expression of the inhibitor of apoptosis, c-IAP1. However, oxidant stress-induced apoptosis was found to be caspase- and calpain-independent implicating CD40 ligation as a regulator of caspase-independent cell death. This was confirmed by the demonstration that CD40 ligation prevented mitochondrial release and nuclear translocation of apoptosis inducing factor. In conclusion, we demonstrate a novel role for CD40 as a regulator of epithelial cell survival against oxidant stress. Furthermore, we have identified, for the first time, an endogenous inhibitory pathway of caspase-independent cell death.

CD30/TNF receptor-associated factor interaction: NF-kappa B activation and binding specificity.

CD30 is a member of the tumor necrosis factor (TNF) receptor superfamily. CD30 is expressed on normal activated lymphocytes, on several virally transformed T- or B-cell lines and on neoplastic cells of Hodgkin's lymphoma. The interaction of CD30 with its ligand induces pleiotropic effects on cells resulting in proliferation, differentiation, or death. The CD30 cytoplasmic tail interacts with TNF receptor-associated factors (TRAFs), which have been shown to transduce signals mediated by TNF-R2 and CD40. We demonstrate here that TRAF2 also plays an important role in CD30-induced NF-kappa B activation. We also show that TRAF2-mediated activation of NF-kappa B plays a role in the activation of HIV transcription induced by CD30 cross-linking. Detailed site-directed mutagenesis of the CD30 cytoplasmic tail reveals that there are two independent binding sites for TRAF, each interacting with a different domain of TRAF. Furthermore, we localized the TRAF-C binding site in CD30 to a 5-7 amino acid stretch.

CD40 and CD86 upregulation with divergent CMRF44 expression on blood dendritic cells in inflammatory bowel diseases

OBJECTIVE: Dendritic cells (DC) are the only antigen-presenting cells that can activate naive T lymphocytes and initiate a primary immune response. They are also thought to have a role in immune tolerance. DC traffic from the blood to peripheral tissue where they become activated. They then present antigen and the costimulating signals necessary to initiate an immune response. In this study, we investigated the number, subsets, and activation pattern of circulating and intestinal DC from patients with clinically mild ulcerative colitis (UC) or Crohn's disease. METHODS: Patients were recruited, if they were not taking immunosuppressive therapy, and were assessed for clinical severity of their disease using for UC, the Clinical Activity Index, and for Crohn's disease, the Crohn's Disease Activity Index. Blood CD11c(+) and CD11c(-) DC subsets, expression of costimulatory antigens, CD86 and CD40, and the early differentiation/activation antigen, CMRF44, were enumerated by multicolor flow cytometry of lineage negative (lin(-) = CD3(-), CD19(-), CD14(-), CD16(-)) HLA-DR+ DC. These data were compared with age-matched healthy and the disease control groups of chronic noninflammatory GI diseases (cGI), acute noninflammatory GI diseases (aGI), and chronic non-GI inflammation (non-GI). In addition, cryostat sections of colonoscopic biopsies from healthy control patients and inflamed versus noninflamed gut mucosa of inflammatory bowel disease (IBD) patients were examined for CD86(+) and CD40(+)lin(-) cells. RESULTS: Twenty-one Crohn's disease and 25 UC patients, with mean Crohn's Disease Activity Index of 98 and Clinical Activity Index of 3.1, and 56 healthy controls, five cGI, five aGI, and six non-GI were studied. CD11c(+) and CD11c(-) DC subsets did not differ significantly between Crohn's, UC, and healthy control groups. Expression of CD86 and CD40 on freshly isolated blood DC from Crohn's patients appeared higher (16.6%, 31%) and was significantly higher in UC (26.6%, 46.3%) versus healthy controls (5.5%, 25%) (p = 0.004, p = 0.012) and non-GI controls (10.2%, 22.8%) (p = 0.012, p = 0.008), but not versus cGI or aGI controls. CD86(+) and CD40(+) DC were also present in inflamed colonic and ileal mucosa from UC and Crohn's patients but not in noninflamed IBD mucosa or normal mucosa. Expression of the CMRF44 antigen was low on freshly isolated DC, but it was upregulated after 24-h culture on DC from all groups, although significantly less so on DC from UC versus Crohn's or healthy controls (p = 0.024). The CMRF44(+) antigen was mainly associated with CD11c(+) DC, and in UC was inversely related to the Clinical Activity Index (r = -0.69, p = 0.0002). CONCLUSIONS: There is upregulation of costimulatory molecules on blood DC even in very mild IBD but surprisingly, there is divergent expression of the differentiation/activation CMRF44 antigen. Upregulation of costimulatory molecules and divergent expression of CMRF44 in blood DC was also apparent in cGI and aGI but not in non-GI or healthy controls, whereas intestinal CD86(+) and CD40(+) DC were found only in inflamed mucosa from IBD patients. Persistent or distorted activation of blood DC or divergent regulation of costimulatory and activation antigens may have important implications for gut mucosal immunity and inflammation. (Am J Gastroenterol 2001;96:2946-2956. (C) 2001 by Am. Coll. of Gastroenterology).

Recent advances on the role of CD40 and dendritic cells in immunity and tolerance

CD40 is a key signaling pathway for the function of B cells, monocytes, and dendritic cells in the immune system, and plays an important role in inflammatory pathways of nonhemopoietic cells. The NFkappaB family of transcription factors is a critical mediator in inflammation. NFkappaB is involved both in the regulation of CD40 expression and in cell signaling after CD40 ligation. This positive feedback loop linking NFkappaB and CD40 plays an important role in the control of the adaptive immune response, with fundamental implications for immunity and tolerance in vivo.

Enhanced effector and memory CTL responses generated by incorporation of receptor activator of NF-kappa B(RANK)/RANK ligand costimulatory molecules into dendritic cell immunogens expressing a human tumor-specific antigen

The outcome of dendritic cell (DC) presentation of Ag to T cells via the TCR/MHC synapse is determined by second signaling through CD80/86 and, importantly, by ligation of costimulatory ligands and receptors located at the DC and T cell surfaces. Downstream signaling triggered by costimulatory molecule ligation results in reciprocal DC and T cell activation and survival, which predisposes to enhanced T cell-mediated immune responses. In this study, we used adenoviral vectors to express a model tumor Ag (the E7 oncoprotein of human papillomavirus 16) with or without coexpression of receptor activator of NF-kappaB (RANK)/RANK ligand (RANKL) or CD40/CD40L costimulatory molecules, and used these transgenic DCs to immunize mice for the generation of E7-directed CD8(+) T cell responses. We show that coexpression of RANK/RANKL, but not CD40/CD40L, in E7-expressing DCs augmented E7-specific IFN-gamma-secreting effector and memory T cells and E7-specific CTLs. These responses were also augmented by coexpression of T cell costimulatory molecules (RANKL and CD40L) or DC costimulatory molecules (RANK and CD40) in the E7-expressing DC immunogens. Augmentation of CTL responses correlated with up-regulation of CD80 and CD86 expression in DCs transduced with costimulatory molecules, suggesting a mechanism for enhanced T cell activation/survival. These results have generic implications for improved tumor Ag-expressing DC vaccines, and specific implications for a DC-based vaccine approach for human papillomavirus 16-associated cervical carcinoma.

Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo-C Interaction in a Biological System

J. Am. Chem. Soc., 2009, 131 (23), pp 7990–7998 DOI: 10.1021/ja809448r

Therapeutic Blockade of LIGHT Interaction With Herpesvirus Entry Mediator and Lymphotoxin β Receptor Attenuates In Vivo Cytotoxic Allogeneic Responses.

BACKGROUND: Tumor necrosis factor/tumor necrosis factor receptor superfamily members conform a group of molecular interaction pathways of essential relevance during the process of T-cell activation and differentiation toward effector cells and particularly for the maintenance phase of the immune response. Specific blockade of these interacting pathways, such as CD40-CD40L, contributes to modulate the deleterious outcome of allogeneic immune responses. We postulated that antagonizing the interaction of LIGHT expression on activated T cells with its receptors, herpesvirus entry mediator and lymphotoxin β receptor, may decrease T cell-mediated allogeneic responses. METHODS: A flow cytometry competition assay was designed to identify anti-LIGHT monoclonal antibodies capable to prevent the interaction of mouse LIGHT with its receptors expressed on transfected cells. An antibody with the desired specificity was evaluated in a short-term in vivo allogeneic cytotoxic assay and tested for its ability to detect endogenous mouse LIGHT. RESULTS: We provide evidence for the first time that in mice, as previously described in humans, LIGHT protein is rapidly and transiently expressed after T-cell activation, and this expression was stronger on CD8 T cells than on CD4 T cells. Two anti-LIGHT antibodies prevented interactions of mouse LIGHT with its two known receptors, herpesvirus entry mediator and lymphotoxin β receptor. In vivo administration of anti-LIGHT antibody (clone 10F12) ameliorated host antidonor short-term cytotoxic response in wild type B6 mice, although to a lesser extent than that observed in LIGHT-deficient mice. CONCLUSION: The therapeutic targeting of LIGHT may contribute to achieve a better control of cytotoxic responses refractory to current immunosuppressive drugs in transplantation.

CD40, autophagy and Toxoplasma gondii

Toxoplasmagondii represents a pathogen that survives within host cells by preventing the endosomal-lysosomal compartments from fusing with the parasitophorous vacuoles. The dogma had been that the non-fusogenic nature of these vacuoles is irreversible. Recent studies revealed that this dogma is not correct. Cell-mediated immunity through CD40 re-routes the parasitophorous vacuoles to the lysosomal compartment by a process called autophagy. Autophagosome formation around the parasitophorous vacuole results in killing of the T. gondii. CD40-induced autophagy likely contributes to resistance against T. gondii particularly in neural tissue.