14 research outputs found
Contribution à l'étude du monde d'action de deux adjuvants synthétiques ciblant TLR4, diC14-amidine et CRX-527
Une comprĂ©hension fine et dĂ©taillĂ©e ciblant les mĂ©canismes dâaction de nouvelles molĂ©cules adjuvantes sur notre systĂšme immunitaire vise de maniĂšre directe Ă lâĂ©laboration de nouveaux vaccins plus ciblĂ©s et plus efficaces, mais aussi Ă Ă©largir nos connaissances quant Ă lâinduction dâune rĂ©ponse immune protectrice.Au cours de cette thĂšse nous avons voulu comprendre les modes dâaction de deux molĂ©cules lipidiques distinctes.La premiĂšre est le lipide cationique diC14-amidine dont il avait Ă©tĂ© dĂ©montrĂ© une action sur les cellules dendritiques en culture par une voie qui restait Ă Ă©lucider. Ce lipide cationique s'organise sous forme de liposomes en milieu aqueux et peut s'associer Ă de nombreux antigĂšnes. La seconde est un analogue synthĂ©tique de l'adjuvant monophosphoryl lipide A (MPL), un dĂ©rivĂ© du LPS, nommĂ© CRX-527. Ă l'instar de sa molĂ©cule parente, le CRX-527 active le rĂ©cepteur TLR4 et est considĂ©rĂ© comme un adjuvant potentiel de vaccin ou comme immunostimulant isolĂ©.Au cours de notre travail, nous avons dĂ©montrĂ© que la diC14-amidine active les cellules cibles via le rĂ©cepteur TLR4. En effet, l'absence de ce rĂ©cepteur abolit les rĂ©ponses induites par le lipide cationique diC14-amidine et la transfection du gĂšne codant pour TLR4 rend rĂ©pondeuses des cellules qui n'exprimaient pas ce rĂ©cepteur. De plus, la diC14-amidine active et mature des cellules dendritiques, aussi bien de provenance murine qu'humaine, suggĂ©rant qu'elle puisse ĂȘtre utilisĂ©e en tant quâadjuvant. Il avait dâailleurs Ă©tĂ© prĂ©cĂ©demment dĂ©crit que l'injection d'un complexe diC14-amidine / allergĂšne chez la souris induisait une rĂ©ponse immune suffisante pour confĂ©rer une protection contre cet allergĂšne. Dans ce contexte, nous avons caractĂ©risĂ© au niveau cellulaire la rĂ©ponse induite suite Ă l'injection du complexe diC14-amidine / ovalbumine chez la souris. Cette rĂ©ponse se manifeste par une production d'IFNÎł lors d'une re-stimulation ex vivo par l'antigĂšne OVA. En ce qui concerne la molĂ©cule CRX-527, nous nous sommes particuliĂšrement focalisĂ©s sur le rĂŽle du co-rĂ©cepteur du TLR4, le CD14, dans les rĂ©ponses innĂ©es induites par le CRX-527. Nous avons Ă©tabli que, de maniĂšre inattendue et contrairement Ă la plupart des ligands TLR4, le CRX-527 induit la production de nombreuses cytokines et chimiokines en complĂšte absence de CD14, mĂȘme Ă faible dose. De plus, l'ajout de CD14 sous sa forme soluble ne modifie pas le niveau des rĂ©ponses associĂ©es Ă la voie de signalisation MyD88 / NF-ÎșB. Cependant, il semblerait que la stimulation de cellules par du CRX-527 en prĂ©sence de CD14 soluble recombinant, favorise plutĂŽt la voie TRIF / IRF3, comme le suggĂšre l'augmentation du taux de production d'IFNÎČ et d'activation d'IRF3. La molĂ©cule CD14 (membranaire et/ou soluble) ne serait donc pas qu'un simple transporteur de ligands, comme il l'a Ă©tĂ© dĂ©crit par le passĂ©, mais bien une protĂ©ine impliquĂ©e dans la modulation des rĂ©ponses induites lors de l'activation du TLR4. Le CD14 jouerait donc un rĂŽle, aussi bien au niveau de la discrimination des ligands, que celle des voies de signalisation activĂ©es.Doctorat en Sciences agronomiques et ingĂ©nierie biologiqueinfo:eu-repo/semantics/nonPublishe
Contribution à l'étude du monde d'action de deux adjuvants synthétiques ciblant TLR4, diC14-amidine et CRX-527
Une comprĂ©hension fine et dĂ©taillĂ©e ciblant les mĂ©canismes dâaction de nouvelles molĂ©cules adjuvantes sur notre systĂšme immunitaire vise de maniĂšre directe Ă lâĂ©laboration de nouveaux vaccins plus ciblĂ©s et plus efficaces, mais aussi Ă Ă©largir nos connaissances quant Ă lâinduction dâune rĂ©ponse immune protectrice.Au cours de cette thĂšse nous avons voulu comprendre les modes dâaction de deux molĂ©cules lipidiques distinctes.La premiĂšre est le lipide cationique diC14-amidine dont il avait Ă©tĂ© dĂ©montrĂ© une action sur les cellules dendritiques en culture par une voie qui restait Ă Ă©lucider. Ce lipide cationique s'organise sous forme de liposomes en milieu aqueux et peut s'associer Ă de nombreux antigĂšnes. La seconde est un analogue synthĂ©tique de l'adjuvant monophosphoryl lipide A (MPL), un dĂ©rivĂ© du LPS, nommĂ© CRX-527. Ă l'instar de sa molĂ©cule parente, le CRX-527 active le rĂ©cepteur TLR4 et est considĂ©rĂ© comme un adjuvant potentiel de vaccin ou comme immunostimulant isolĂ©.Au cours de notre travail, nous avons dĂ©montrĂ© que la diC14-amidine active les cellules cibles via le rĂ©cepteur TLR4. En effet, l'absence de ce rĂ©cepteur abolit les rĂ©ponses induites par le lipide cationique diC14-amidine et la transfection du gĂšne codant pour TLR4 rend rĂ©pondeuses des cellules qui n'exprimaient pas ce rĂ©cepteur. De plus, la diC14-amidine active et mature des cellules dendritiques, aussi bien de provenance murine qu'humaine, suggĂ©rant qu'elle puisse ĂȘtre utilisĂ©e en tant quâadjuvant. Il avait dâailleurs Ă©tĂ© prĂ©cĂ©demment dĂ©crit que l'injection d'un complexe diC14-amidine / allergĂšne chez la souris induisait une rĂ©ponse immune suffisante pour confĂ©rer une protection contre cet allergĂšne. Dans ce contexte, nous avons caractĂ©risĂ© au niveau cellulaire la rĂ©ponse induite suite Ă l'injection du complexe diC14-amidine / ovalbumine chez la souris. Cette rĂ©ponse se manifeste par une production d'IFNÎł lors d'une re-stimulation ex vivo par l'antigĂšne OVA. En ce qui concerne la molĂ©cule CRX-527, nous nous sommes particuliĂšrement focalisĂ©s sur le rĂŽle du co-rĂ©cepteur du TLR4, le CD14, dans les rĂ©ponses innĂ©es induites par le CRX-527. Nous avons Ă©tabli que, de maniĂšre inattendue et contrairement Ă la plupart des ligands TLR4, le CRX-527 induit la production de nombreuses cytokines et chimiokines en complĂšte absence de CD14, mĂȘme Ă faible dose. De plus, l'ajout de CD14 sous sa forme soluble ne modifie pas le niveau des rĂ©ponses associĂ©es Ă la voie de signalisation MyD88 / NF-ÎșB. Cependant, il semblerait que la stimulation de cellules par du CRX-527 en prĂ©sence de CD14 soluble recombinant, favorise plutĂŽt la voie TRIF / IRF3, comme le suggĂšre l'augmentation du taux de production d'IFNÎČ et d'activation d'IRF3. La molĂ©cule CD14 (membranaire et/ou soluble) ne serait donc pas qu'un simple transporteur de ligands, comme il l'a Ă©tĂ© dĂ©crit par le passĂ©, mais bien une protĂ©ine impliquĂ©e dans la modulation des rĂ©ponses induites lors de l'activation du TLR4. Le CD14 jouerait donc un rĂŽle, aussi bien au niveau de la discrimination des ligands, que celle des voies de signalisation activĂ©es.Doctorat en Sciences agronomiques et ingĂ©nierie biologiqueinfo:eu-repo/semantics/nonPublishe
DiC14-amidine confers new anti-inflammatory properties to phospholipids.
The inflammatory effect of unmethylated CpG DNA sequences represents a major obstacle to the use of cationic lipids for in vivo gene therapy. Although the mechanism of CpG-induced inflammatory response is rather well understood nowadays, few solutions have been designed to circumvent this effect in gene therapy experiments. Our previous work has shown that a refractory state towards inflammation can be elicited by preinjecting cationic liposomes. Here, we present evidence that diC14-amidine liposomes confer new anti-inflammatory properties to phospholipids from low-density lipoprotein (LDL) and even to synthetic phospholipids for which such an observation has not been reported so far. Whereas oxidation of LDL lipids was a prerequisite for any anti-inflammatory activity, lipid oxidation is no longer required in our experiments, suggesting that cationic lipids transport phospholipids through a different route and affect different pathways. This opens up new possibilities for manipulating inflammatory responses in gene therapy protocols but also in a general manner in immunological experiments.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe
Free diC14-amidine liposomes inhibit the TNF-alpha secretion induced by CpG sequences and lipopolysaccharides: role of lipoproteins.
It has been shown that a preinjection of diC14-amidine cationic liposomes decreased TNF-alpha secretion induced by lipoplexes intravenous injection. We showed here that free cationic liposomes inhibit CpG sequences- or lipopolysaccharides-induced TNF-alpha secretion by macrophages. Surprisingly, this effect was strictly dependent on serum. Free cationic liposomes alone did not reveal any anti-inflammatory activity. Low-density lipoproteins and triglyceride-rich lipoproteins were identified as the serum components that confer to the liposomes an anti-inflammatory activity. Lipid fractions of these lipoproteins were able to reproduce the effect of the total lipoproteins and could inhibit, in association with diC14-amidine liposomes, the CpG-induced TNF-alpha secretion. Serum components confer to cationic liposomes new properties that can be used to modulate the inflammatory response directed against CpG sequences and lipopolysaccharides.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe
Exhaustion of tumor-specific CD8âș T cells in metastases from melanoma patients.
In chronic viral infections, CD8âș T cells become functionally deficient and display multiple molecular alterations. In contrast, only little is known of self- and tumor-specific CD8âș T cells from mice and humans. Here we determined molecular profiles of tumor-specific CD8âș T cells from melanoma patients. In peripheral blood from patients vaccinated with CpG and the melanoma antigen Melan-A/MART-1 peptide, we found functional effector T cell populations, with only small but nevertheless significant differences in T cells specific for persistent herpesviruses (EBV and CMV). In contrast, Melan-A/MART-1-specific T cells isolated from metastases from patients with melanoma expressed a large variety of genes associated with T cell exhaustion. The identified exhaustion profile revealed extended molecular alterations. Our data demonstrate a remarkable coexistence of effector cells in circulation and exhausted cells in the tumor environment. Functional T cell impairment is mediated by inhibitory receptors and further molecular pathways, which represent potential targets for cancer therapy
Inhibitory receptor expression by Melan-A specific CD8 T-cells depending on vaccination.
<p>(A) Co-expression of KLRG-1, TIM-3, PD-1 and CD160, and of LAG-3, BTLA, 2B4 and CTLA-4 by Melan-A specific CD8 T-cells. Blood samples from healthy donors (HD) or from patients before immunotherapy (before vacc.) or after peptide+IFA vaccination with or without CpG-ODN 7909 were enriched for CD8 T-cells using magnetic beads. Melan-A-specific CD8 T-cells were identified by staining with CD8-specific antibody and tetramer. Positivity for inhibitory receptors was defined respective to isotype controls. nâ=â4 for HD; nâ=â3 for before vacc.; nâ=â9 for after vaccination without CpG-ODN and nâ=â11 for after vaccination with CpG-ODN. Colors of the pie arcs depict the expression of individual inhibitory receptors, while the color in the pie depicts the number of co-expressed inhibitory receptors. Co-expression was analyzed with SPICE 5.2. p-values of the permutation test are shown in tables next to the corresponding pie charts. (B) Hierarchical clustering based on co-expression data of the eight inhibitory receptors shown in A, including the four differentiation subsets (N, CM, EM, EMRA) of total CD8 T-cells. (C) Mean expression and SD of four inhibitory receptors upregulated on Melan-A-specific T-cells with vaccination. Data from HD and from patients before vaccination were pooled for the group without vaccination (no vacc.). nâ=â7 for no vacc.; nâ=â9 for vaccination with CpG-ODN.</p
Expression profiles of inhibitory receptors with differentiation.
<p>(A) CD8 T-cell subsets were defined depending on expression of CCR7 and CD45RA, namely naive (N), central memory (CM), effector memory (EM) and effector memory RA<sup>+</sup> (EMRA) cells. Gates used for inhibitory receptor analysis are shown in the four quadrants. (B) Mean values of inhibitory receptor expression in relation to the differentiation status. Individual values are shown in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030852#pone.0030852.s001" target="_blank">Figure S1B</a></i>. nâ=â31 for âstaining 1â (KLRG-1, TIM-3, PD-1 and CD160); nâ=â21 for âstaining 2â (LAG-3, BTLA, 2B4 and CTLA-4); four samples of staining 1 were from healthy donors, the remaining from melanoma patients. (C) Co-expression of KLRG-1, TIM-3, PD-1 and CD160 (staining 1) and of LAG-3, BTLA, 2B4 and CTLA-4 (staining 2). Colors of the pie arcs depict the expression of individual inhibitory receptors, while the color in the pie depicts the number of co-expressed inhibitory receptors. p-values of the permutation test are shown in tables next to the corresponding pie charts. Co-expression was analyzed with SPICE 5.2.</p
Extended co-expression of inhibitory receptors by human CD8 T-cells depending on differentiation, antigen-specificity and anatomical localization.
Inhibitory receptors mediate CD8 T-cell hyporesponsiveness against cancer and infectious diseases. PD-1 and CTLA-4 have been extensively studied, and blocking antibodies have already shown clinical benefit for cancer patients. Only little is known on extended co-expression of inhibitory receptors and their ligands. Here we analyzed the expression of eight inhibitory receptors by tumor-antigen specific CD8 T-cells. We found that the majority of effector T-cells simultaneously expressed four or more of the inhibitory receptors BTLA, TIM-3, LAG-3, KRLG-1, 2B4, CD160, PD-1 and CTLA-4. There were major differences depending on antigen-specificity, differentiation and anatomical localization of T-cells. On the other hand, naive T-cells were only single or double positive for BTLA and TIM-3. Extended co-expression is likely relevant for effector T-cells, as we found expression of multiple ligands in metastatic lesions of melanoma patients. Together, our data suggest that naive T-cells are primarily regulated by BTLA and TIM-3, whereas effector cells interact via larger numbers of inhibitory receptors. Blocking multiple inhibitory receptors simultaneously or sequentially may improve T-cell based therapies, but further studies are necessary to clarify the role of each receptor-ligand pair
Expression of ligands of inhibitory receptors in melanoma metastases and by melanoma cell lines.
<p>(A,B) Paraffin-embedded tumor sections from 16 to 18 tumors were stained by immunohistochemistry for seven inhibitory receptors and CD8. (A) Representative stainings (magnification Ă200) for each ligand investigated. (B) Summary of immunohistochemical stainings represented as percent of positive samples. Low (<10%), intermediate (int; 10â50%) and high (>50%) expression is indicated in a color scale. infilt: infiltration of CD8 T-cells in tumor cell nests; sec: secreted i.e. intra- and extracellular presence of galectin-9. (C) Summary of expression by melanoma cell lines on the surface or intracellular (ic) as percent of positive cell lines.</p