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International audience; Background: Despite being quite effective, conventional cancer therapies have the major drawback of triggering numerous side effects. Currently, a challenging goal in this area is the development of innovative targeted antitumor immunotherapy with a long-term efficiency. In this context, our team took advantage of liposomal nanoparticle properties for the conception of cancer vaccines, which contain all the elements needed for the induction of an efficient antitumor immune response i) a peptide able to activate CD4+ T helper cells ii) a tumor peptide expressed by the cancer cells, recognized by CD8+ T cytotoxic cells and iii) Toll or Nod-Like receptor (TLR and NLR) ligand which act as adjuvants for the activation of the innate immune response. The aim of our project is to conceive liposomal vaccines, which would be effective even against tumors in later stage. Materials and methods: Different vaccines formulations containing the three types of components have been designed and compared for their capacity to induce a strong immune response associated with antitumor activity in vivo. C57BL/6J mice were injected in the tail vein with tumor cells (TC-1 cell line) expressing the E7 peptide, leading to the development of pulmonary tumor nodules. Then, injections of liposome-based vaccines were performed subcutaneously at different times after tumor implantation. One month later, lung tumor nodules are counted and the spleen and lymph nodes are harvested to perform an IFN-g ELISpot assay against tumor cell epitopes to measure the number of tumor specific T cell. Results: We compared the therapeutic potential of these three vaccinal candidates and analyzed the immunological mechanisms involved in their action. Liposomal vaccine containing MPLA as adjuvant (TLR4 liposomes), was the most effective treatment against TC-1. This vaccine induced a potent Th1-oriented antitumor immunity, which led to a significant reduction in tumor growth and a prolonged survival of mice, even when injected after pulmonary tumors apparition. TLR4 liposomes antitumor efficacy was dependent of CD8+ Tcells presence, whereas CD4+ cell depletion enhanced its therapeutic impact. Subcutaneous injection of this treatment induced the migration of CD103 skin DCs to draining lymph nodes. We also observed unexpected differences between TLR and NLR agonists activity. In the same way as TLR4 liposomes, TLR2/6 liposomes induced a Th1-immune response but weak and not sufficient for a prolonged antitumor activity. Surprisingly, whereas NOD1 liposomes treatment resulted in the control of early tumor growth, they did not extend survival. Conclusions: This study highlights the importance of immune monitoring of cancer vaccine therapy and shows that our modulable platform can be used for the strategical development of vaccines

International audience; Background: Platinum (II)-based drugs are widely used chemother-apeutic agents. However, as they have many side effects, numerous platinum (Pt) derivatives have been developed during the last years. Furthermore, some cancer treatments including Pt compounds are able to activate the antitumor immune response by inducing a particular cell death: the immunogenic cell death (ICD). This process is characterized by the exposure of the endoplasmic reticulum chaperone calreticulin (CRT) at the cell surface as well as the release of ATP and non-histone chromatin-binding protein high mobility group box 1 (HMGB1) which serve as immunostimulatory damage-associated molecular patterns (DAMPs) and increase the antitumor immune response. Based on these considerations, we decided to focus on N-het-erocyclic carbene Pt complexes associated with the transfection agent polyethylenimine (PEI) to create multivalent cationic platinum compounds (NHC-Pt(II)-PEI). Materials and methods: We investigated the cytotoxic activity of our derivatives in vitro by flow cytometry and in vivo in different mouse models. Then, we evaluated by an IFN-g ELISpot assay the potential implication of the immune response on the NHC-Pt(II)-PEI in vivo effect in immunocompetent mice bearing tumors treated with Pt particles. Moreover, we assessed if NHC-Pt(II)-PEI were able to induce ICD by analysing the exposure of CRT at the cell surface by flow cytometry. We also combined our Pt derivative with immune danger signals, like poly-inosinic: polycytidylic acid (polyI:C), to enhance this effect. Results: Our conjugate displayed a good cytotoxic activity in vitro on several cancer cell lines as well as an antitumor effect in vivo in immunodeficient [1] and immunocompetent mice, in the same range than clinical used Pt drugs, but with fewer side effects. First results showed exposure of CRT at the cell surface of cancer cells treated with NHC-Pt(II)-PEI, suggesting the induction of ICD. To improve the antitumor immune response, we associated NHC-Pt(II)-PEI with polyI:C and found that both compounds formed a complex and we are now evaluating its cytotoxicity and effect on the immune response. Conclusions: Altogether our results reveal the possibility of creating Pt derivatives that can be used as chemotherapeutic agents by killing tumor cells and as immunotherapeutic agents by triggering the anti-tumor immune response.

Knowing that several CD4 mAbs may delay allograft rejection in the absence of circulating CD4+ lymphocyte depletion in vivo, we investigated the mechanisms whereby CD4 mAbs can interfere with the development of alloreactive T cells in the mixed lymphocyte reaction (MLR). In agreement with previous reports, CD4 mAbs of different species (mouse, rat, humanized), isotypes (IgG1, IgG2a, and IgG2b) and different epitope specificities decreased 3H-TdR incorporation in MLR, using monocyte-depleted or CD4+ T lymphocyte-enriched blood mononuclear cells as responders. Those effects were achieved at nonsaturating mAb concentration and were still demonstrable upon delayed addition of CD4 mAbs. However, CD4 mAbs decreased neither the number of blast cells nor the expression of CD25 (the alpha chain of IL-2 receptor), indicating that initial activation events leading to blast transformation were not affected. Determination of cytokine gene expression by non competitive quantitative RT-PCR and measurement of protein concentration in supernatants demonstrated that CD4 mAbs did not decrease IFN-gamma induced by alloactivation. However IL-2 concentration was decreased in all supernatants whereas IL-2 mRNA expression, only slightly decreased at 24 hr, and dropped after 72 hr. IL-5 and IL-10 mRNAs, equally expressed by stimulated or nonstimulated responder cells, were not affected by CD4 mAbs. IL-4 mRNA was not detectable. Furthermore, addition of rIL-2, rIFN-gamma or rIL-4 did not overcome proliferation inhibition. The data provide a novel insight into the mechanisms of CD4 mAbs immunosuppresssion that associates a decrease of IL-2 expression with an IL-2 resistant blockade of the progression of activated CD4+ T cells from the G1 to the S phases of the cell cycle.

Proteins recognized by antibodies from patients with autoimmune diseases have been intensively studied over the two past decades since cDNAs encoding autoantigens have become available. Identity of many of them has been defined, and specific structural motifs or post-translational modifications, which may be important to explain the generation of such antibodies during the autoimmune process, have been pointed out. Immunological analysis of sera from autoimmune patients with recombinant fragments and with short peptides has revealed the presence of dominant epitopes along proteins; some of them are targeted by antibodies from patients with specific diseases or disease subsets. Innovative technologies such as peptide arrays and biosensors as well as the exploitation of large peptides libraries have recently open up new perspectives. Peptides bearing natural modifications, peptide analogues, as well as mimotopes of protein or non-protein antigens (DNA, RNA, sugar) have been developed and might advantageously replace native antigens in routine immunoassays. Although numerous conformational epitopes have not yet been identified, and cannot be identified by the approaches classically used in epitope mapping studies, such peptides and peptide analogues may represent efficient probes to detect the presence of circulating autoantibodies in the serum of autoimmune patients and help for establishing specific and sensitive early diagnostic tests. They may also lead to the design of high-affinity ligands for purifying autoantibodies. These different aspects are discussed and epitope mapping studies of a number of autoantigens (e.g. histones, sn and hnRNP proteins and Ro proteins) are summarized.

Using 3H-Tyr-d-Ala-Gly-Phe-d-Leu-OH (3H-DADLE) as a radioligand, δ-opioid binding sites on the IRD 98 rat epithelial cell line were identified. These sites were found to be reversible, saturable, specific and displayed high affinity for DADLE. Scatchard analysis revealed a dissociation constant (K d) of 4.9±0.5 nmol/l, a maximum binding capacity (B max) of 1.7 pmol/mg protein, and 5×105 binding sites per cell. The presence of opioid receptors suggests the possibility that enkephalins directly control ion transport in enterocytes. In order to verify this hypothesis, investigations were designed to determine whether these receptors are functional and whether enkephalins can inhibit the stimulation of adenosine 3',5' cyclic monophosphate (cAMP) synthesis induced by cholera toxin. The increase in cAMP synthesis induced by cholera toxin was inhibited in a dose-dependent manner by H-Tyr-d-Ser-Gly-Phe-Leu-Thr-OH (DSLET), a δ-agonist. The enkephalinase inhibitor thiorphan potentiated this effect on IRD 98 cells, which contain enkephalinase. The action of DSLET was increased by 40% in the presence of this inhibitor. This effect was reversed by naltrindole, a potent δ-antagonist. Enkephalins can regulate intestinal secretion by acting directly on enterocytes; they thus have an antidiarrheal role, especially in the presence of an enkephalinase inhibitor.

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 non-covalent 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 non-covalent 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 signalling, possibly by maintaining the receptor in a quiescent state.

In addition to their inhibitory action on gastric acid secretion, prostaglandins may exert part of their protective effect on the gastrointestinal mucosa by specifically maintaining the cellular integrity of the intestinal epithelium. This in vitro study investigated the cytoprotective effect conferred on intestinal epithelial cell lines IRD 98 and IEC 17 against ethanol injury by the synthetic prostaglandin enprostil and compared effects with those of the histamine H2-receptor blocker cimetidine. Exposure to 3% ethanol (652 mM) reduced cell viability and increased the cAMP and membrane fluidity of both cell lines. Our results demonstrate that: (i) enprostil exerts a significant cytoprotective effect against damage by ethanol; (ii) cimetidine has no cytoprotective effect; (iii) IRD 98 cells are more sensitive to enprostil than IEC 17 cells.

Clonal expansion of activated T and B cells is controlled by homeostatic mechanisms resulting in apoptosis of a large proportion of activated cells, mostly through interaction between CD95 (Fas or Apo-1) receptor and its ligand CD95-L. CD2, which is considered as a CD3/TCR alternative pathway of T cell activation, may trigger activation-induced cell death, but the role of CD95/CD95-L interaction in CD2-mediated apoptosis remains controversial. We show here that the CD2R mAb YTH 655.5, which does not induce comitogenic signals when associated with another CD2 mAb, triggers CD95-L expression by preactivated but not resting T cells, resulting in CD95/CD95-L-mediated apoptosis. The critical role of CD95/CD95-L interaction was supported by complete inhibition in the presence of the antagonist CD95 mAb ZB4 and by blocking CD95-L synthesis and surface expression by cycloheximide, cyclosporin A, EGTA, or cytochalasin B. YTH 655.5 was shown to stimulate p56lck phosphorylation and enzymatic activity. However, p56lck activation is not sufficient to trigger apoptosis, because other CD2R and CD4 mAbs that activate p56lck do not induce apoptosis. In conclusion, CD2 can mediate nonmitogenic signals, resulting in CD95-L expression and apoptosis of CD95+ cells.

International audience

Chronic malnutrition results in severe metabolic imbalance in man as the body modifies its modes of regulation of different nutrients, and in particular lipids. This study of the modifications in lipid metabolism induced by 15 days of enteral renutrition include: 1. 1. A prospective investigation of the effects of refeeding in malnourished patients by evaluation of enterocytic HMG CoA (HMG-CoA) reductase activity and lipid synthesis in intestinal organ culture. 2. 2. Use of the same parameters to compare the effects of a diet enriched in ω3 fatty acid (FA) with those of a standard diet. 12 malnourished patients (global nutritional deficit (GND)