Jean M. Kanellopoulos

Activation of the NLRP3 inflammasome in dendritic cells induces IL-1β–dependent adaptive immunity against tumors

The therapeutic efficacy of anticancer chemotherapies may depend on dendritic cells (DCs), which present antigens from dying cancer cells to prime tumor-specific interferon-γ (IFN-γ)–producing T lymphocytes. Here we show that dying tumor cells release ATP, which then acts on P2X7 purinergic receptors from DCs and triggers the NOD-like receptor family, pyrin domain containing-3 protein (NLRP3)-dependent caspase-1 activation complex (inflammasome), allowing for the secretion of interleukin-1β (IL-1β). The priming of IFN-γ–producing CD8+ T cells by dying tumor cells fails in the absence of a functional IL-1 receptor 1 and in Nlpr3-deficient (Nlrp3−/−) or caspase-1–deficient (Casp-1−/−) mice unless exogenous IL-1β is provided. Accordingly, anticancer chemotherapy turned out to be inefficient against tumors established in purinergic receptor P2rx7−/− or Nlrp3−/− or Casp1−/− hosts. Anthracycline-treated individuals with breast cancer carrying a loss-of-function allele of P2RX7 developed metastatic disease more rapidly than individuals bearing the normal allele. These results indicate that the NLRP3 inflammasome links the innate and adaptive immune responses against dying tumor cells.

Chemotherapy-triggered cathepsin B release in myeloid-derived suppressor cells activates the Nlrp3 inflammasome and promotes tumor growth

Chemotherapeutic agents are widely used for cancer treatment. In addition to their direct cytotoxic effects, these agents harness the hosts immune system, which contributes to their antitumor activity. Here we show that two clinically used chemotherapeutic agents, gemcitabine (Gem) and 5-fluorouracil (5FU), activate the NOD-like receptor family, pyrin domain containing-3 protein (Nlrp3)-dependent caspase-1 activation complex (termed the inflammasome) in myeloid-derived suppressor cells (MDSCs), leading to production of interleukin-1β (IL-1β), which curtails anticancer immunity. Chemotherapy-triggered IL-1β secretion relied on lysosomal permeabilization and the release of cathepsin B, which bound to Nlrp3 and drove caspase-1 activation. MDSC-derived IL-1β induced secretion of IL-17 by CD4+ T cells, which blunted the anticancer efficacy of the chemotherapy. Accordingly, Gem and 5FU exerted higher antitumor effects when tumors were established in Nlrp3−/− or Casp1−/− mice or wild-type mice treated with interleukin-1 receptor antagonist (IL-1Ra). Altogether, these results identify how activation of the Nlrp3 inflammasome in MDSCs by 5FU and Gem limits the antitumor efficacy of these chemotherapeutic agents.

Extracellular ATP Is a Danger Signal Activating P2X7 Receptor in Lung Inflammation and Fibrosis

RATIONALEnPulmonary fibrosis is a devastating as yet untreatable disease. We previously investigated the endogenous mediators released on lung injury and showed that uric acid is a danger signal activating Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in lung inflammation and fibrosis (Gasse et al., Am J Respir Crit Care Med 2009;179:903-913).nnnOBJECTIVESnHere we address the role of extracellular adenosine triphosphate (eATP) in pulmonary inflammation and fibrosis.nnnMETHODSnATP was quantified in bronchoalveolar lavage fluid (BALF) of control subjects and patients with idiopathic pulmonary fibrosis. The contribution of eATP as a danger signal was assessed in a murine model of lung fibrosis induced by airway-administered bleomycin (BLM), an intercalating agent that causes DNA strand breaks.nnnMEASUREMENTS AND MAIN RESULTSnFibrotic patients have elevated ATP content in BALF in comparison with control individuals. In mice, we report an early increase in eATP levels in BALF on BLM administration. Modulation of eATP levels with the ATP-degrading enzyme apyrase greatly reduced BLM-induced inflammatory cell recruitment, lung IL-1β, and tissue inhibitor of metalloproteinase (TIMP)-1 production, while administration of ATP-γS, a stable ATP derivative, enhanced inflammation. P2X(7) receptor-deficient mice presented dramatically reduced lung inflammation, with reduced fibrosis markers such as lung collagen content and matrix-remodeling proteins TIMP-1 and matrix metalloproteinase-9. The acute inflammation depends on a functional pannexin-1 hemichannel protein. In vitro, ATP is released by pulmonary epithelial cells on BLM-induced stress and this is partly dependent on the presence of functional P2X(7) receptor and pannexin-1 hemichannel.nnnCONCLUSIONSnATP released from BLM-injured lung cells constitutes a major endogenous danger signal that engages the P2X(7) receptor/pannexin-1 axis, leading to IL-1β maturation and lung fibrosis.

Palmitoylation of the P2X7 receptor, an ATP-gated channel, controls its expression and association with lipid rafts

The P2X7 receptor (P2X7R) is an ATP‐gated cationic channel expressed by hematopoietic, epithelial, and neuronal cells. Prolonged ATP exposure leads to the formation of a nonselective pore, which can result in cell death. We show that P2X7R is associated with detergent‐resistant membranes (DRMs) in both transfected human embryonic kidney (HEK) cells and primary macrophages independently from ATP binding. The DRM association requires the posttranslational modification of P2X7R by palmitic acid. Treatment of cells with the palmitic acid analog 2‐bromopalmitate as well as mutations of cysteine to alanine residues abolished P2X7R palmitoylation. Substitution of the 17 intracellular cysteines of P2X7R revealed that 4 regions of the carboxyl terminus domain are involved in palmitoylation. Palmitoylation‐defective P2X7R mutants showed a dramatic decrease in cell surface expression because of their retention in the endoplasmic reticulum and proteolytic degradation. Taken together, our data demonstrate that P2X7R palmitoylation plays a critical role in its association with the lipid microdomains of the plasma membrane and in the regulation of its half‐life.— Gonnord, P., Delarasse, C., Auger, R., Benihoud, K., Prigent, M., Cuif, M. H., Lamaze, C., Kanellopoulos, J. M. Palmitoylation of the P2X7 receptor, an ATP‐gated channel, controls its expression and association with lipid rafts. FASEB J. 23, 795–805 (2009)

Neural progenitor cell death is induced by extracellular ATP via ligation of P2X7 receptor

J. Neurochem. (2009) 109, 846–857.

Persistence of autoreactive myelin oligodendrocyte glycoprotein (MOG)-specific T cell repertoires in MOG-expressing mice

Experimental autoimmune encephalomyelitis, an experimental murine model for multiple sclerosis, is induced by stimulation of myelin‐specific Tu2004lymphocytes. Myelin oligodendrocyte glycoprotein (MOG), a minor component of myelin proteins, is a potent autoantigen which contributes extensively to the anti‐myelin response. In the present work, immunoscope analyses and sequencing of the oligoclonal expansions revealed anti‐MOGu2004Vα and Vβ public repertoires in lymphocytes infiltrating the CNS of wild‐type (WT) mice. Moreover, a subset of CNS‐infiltrating CD4+ Tu2004lymphocytes bearing the public Vβ8.2 segment have an inflammatory phenotype strongly suggesting that it is encephalitogenic. We then observed that, in lymph node cells of MOG‐deficient and WT animals, the Vα and Vβ public repertoires expressed by MOG‐specific Tu2004cells are identical in both strains of mice and correspond to those found in the CNS of WT animals. These findings indicate that the MOG immunodominant determinant is unable to induce tolerance by deletion, and public anti‐MOG Tu2004cell repertoires are selected for, regardless of the presence of MOG in the thymus and peripheral organs.

Vα and Vβ Public Repertoires Are Highly Conserved in Terminal Deoxynucleotidyl Transferase-Deficient Mice

T cell repertoires observed in response to immunodominant and subdominant peptides include private, i.e., specific for each individual, as well as public, i.e., common to all mice or humans of the same MHC haplotype, Vα-Jα and Vβ-Dβ-Jβ rearrangements. To measure the impact of N-region diversity on public repertoires, we have characterized the αβ TCRs specific for several CD4 or CD8 epitopes of wild-type mice and of mice deficient in the enzyme TdT. We find that V, (D), J usage identified in public repertoires is strikingly conserved in TdT°/° mice, even for the CDR3 loops which are shorter than those found in TdT+/+ animals. Moreover, the 10- to 20-fold decrease in αβ T cell diversity in TdT°/° mice did not prevent T cells from undergoing affinity maturation during secondary responses. A comparison of the CDR3β in published public and private repertoires indicates significantly reduced N-region diversity in public CDR3β. We interpret our findings as suggesting that public repertoires are produced more efficiently than private ones by the recombination machinery. Alternatively, selection may be biased in favor of public repertoires in the context of the interactions between TCR and MHC peptide complexes and we hypothesize that MHCα helices are involved in the selection of public repertoires.

Reprogrammed quiescent B cells provide an effective cellular therapy against chronic experimental autoimmune encephalomyelitis

Activated B cells can regulate immunity and have been envisaged as a potential cell‐based therapy for treating autoimmune diseases. However, activated human B cells can also propagate immune responses, and the effects resulting from their infusion into patients cannot be predicted. This led us to consider resting B cells, which in contrast are poorly immunogenic, as an alternative cellular platform for the suppression of unwanted immunity. Here, we report that resting B cells can be directly engineered with lentiviral vectors to express antigens in a remarkably simple, rapid, and effective way. Notably, this neither required nor induced activation of the B cells. With this approach we were able to produce reprogrammed resting B cells that inhibited antigen‐specific CD4+ T cells, CD8+ T cells, and B cells upon adoptive transfer in mice. Furthermore, resting B cells engineered to ectopically express myelin oligodendrocyte glycoprotein antigen protected recipient mice from severe disability and demyelination in EAE, and even induced complete remission from disease in mice lacking functional natural Tregs, which otherwise developed chronic paralysis. In conclusion, our study introduces reprogrammed quiescent B cells as a novel tool for suppressing undesirable immunity.

Long-term exposure to LPS enhances the rate of stimulated exocytosis and surfactant secretion in alveolar type II cells and upregulates P2Y2 receptor expression

Bacterial LPS is a potent proinflammatory molecule. In the lungs, LPS induces alterations in surfactant pool sizes and phospholipid (PL) contents, although direct actions of LPS on the alveolar type II cells (AT II) are not yet clear. For this reason, we studied short- and long-term effects of LPS on basal and agonist-stimulated secretory responses of rat AT II by using Ca(2+) microfluorimetry, a microtiter plate-based exocytosis assay, by quantitating PL and (3)H-labeled choline released into cell supernatants and by using quantitative PCR and Western blot analysis. Long term, but not short term, exposures to LPS led to prolonged ATP-induced Ca(2+) signals and an increased rate in vesicle fusions with an augmented release of surfactant PL. Most notably, the stimulatory effect of LPS was ATP-dependent and may be mediated by the upregulation of the purinergic receptor subtype P2Y(2). Western blot analysis confirmed higher levels of P2Y(2), and suramin, a P2Y receptor antagonist, was more effective in LPS-treated cells. From these observations, we conclude that LPS, probably via Toll-like receptor-4, induces a time-dependent increase in P2Y(2) receptors, which, by yet unknown mechanisms, leads to prolonged agonist-induced Ca(2+) responses that trigger a higher activity in vesicle fusion and secretion. We further conclude that chronic exposure to endotoxin sensitizes AT II to increase the extracellular surfactant pool, which aids in the pulmonary host defense mechanisms.

T Cell Repertoire Diversity Is Required for Relapses in Myelin Oligodendrocyte Glycoprotein-Induced Experimental Autoimmune Encephalomyelitis

Comparison of TCRαβ repertoires of myelin oligodendrocyte glycoprotein (MOG)-specific T lymphocytes in C57BL/6 and TdT-deficient littermates (TdT−/−) generated during experimental autoimmune encephalomyelitis (EAE) highlights a link between a diversified TCRαβ repertoire and EAE relapses. At the onset of the disease, the EAE-severity is identical in TdT+/− and TdT−/− mice and the neuropathologic public MOG-specific T cell repertoires express closely similar public Vα-Jα and Vβ-Jβ rearrangements in both strains. However, whereas TdT+/+ and TdT+/− mice undergo successive EAE relapses, TdT−/− mice recover definitively and the lack of relapses does not stem from dominant regulatory mechanisms. During the first relapse of the disease in TdT+/− mice, new public Vα-Jα and Vβ-Jβ rearrangements emerge that are distinct from those detected at the onset of the disease. Most of these rearrangements contain N additions and are found in CNS-infiltrating T lymphocytes. Furthermore, CD4+ T splenocytes bearing these rearrangements proliferate to the immunodominant epitope of MOG and not to other immunodominant epitopes of proteolipid protein and myelin basic protein autoantigens, excluding epitope spreading to these myelin proteins. Thus, in addition to epitope spreading, a novel mechanism involving TCRαβ repertoire diversification contributes to autoimmune progression.