22 research outputs found
Ătude de lâimplication de la force du signal transmis par le rĂ©cepteur des cellules T dans le dĂ©veloppement et la survie des lymphocytes T mĂ©moires
Suite Ă la rencontre dâun antigĂšne (Ag) prĂ©sentĂ© Ă la surface des cellules prĂ©sentatrice de lâAg (CPA), les lymphocytes T naĂŻfs, ayant un rĂ©cepteur des cellules T (RCT) spĂ©cifique de lâAg, vont prolifĂ©rer et se diffĂ©rencier en LT effecteurs (1). Suite Ă lâĂ©limination de lâAg la majoritĂ© des LTe vont mourir par apoptose alors que les restants vont se diffĂ©rencier en LT mĂ©moire (LTm) protĂ©geant lâorganisme Ă long terme. Les mĂ©canismes qui permettent la diffĂ©renciation des LTe en LTm sont encore inconnus.
Pour comprendre comment les LTm CD8+ sont gĂ©nĂ©rĂ©s Ă partir des LTe, nous avons Ă©mis lâhypothĂšse que la densitĂ© de lâAg prĂ©sentĂ© par les CPA peut avoir un impact sur la sĂ©lection des LT CD8+ rĂ©pondant lâAg Ă se diffĂ©rencier en LTm. De maniĂšre intĂ©ressante, nos rĂ©sultats montrent quâune immunisation avec des cellules dendritiques (DCs) exprimant un haut niveau de complexe CMH/peptide Ă sa surface permet le dĂ©veloppement de LTm. Ă lâinverse, le dĂ©veloppement des LTm est fortement rĂ©duit (10-20X) lorsque les souris sont immunisĂ©es avec des DCs exprimant un niveau faible de complexes CMH/peptide Ă leur surface. De plus, la quantitĂ© dâAg nâa aucune influence ni sur lâexpansion des LT CD8+ ni sur lâacquisition de leurs fonctions effectrices, mais affecte de maniĂšre critique la gĂ©nĂ©ration des LTm. Nos rĂ©sultats suggĂšrent que le nombre de RCT engagĂ© lors de la reconnaissance de lâAg est important pour la formation des LTm. Pour cela nous avons observĂ© par vidĂ©o-microscopie le temps dâinteraction entre des LTn et des DCs. Nos rĂ©sultats montrent que le temps et la qualitĂ© de lâinteraction sont dĂ©pendants de la densitĂ© dâAg prĂ©sentĂ© par les DCs. Effectivement, nous observons une diminution dans le pourcentage de LT faisant une interaction prolongĂ©e avec les DCs quand le niveau dâAg est faible. De plus, nous observons des variations de lâexpression des facteurs de transcription clefs impliquĂ©s dans la diffĂ©renciation des LTm tels quâEomes, Bcl-6 et Blimp-1. Par ailleurs, la densitĂ© dâAg fait varier lâexpression du Neuron-derived orphan nuclear receptor 1 (Nor-1). Nor-1 est impliquĂ© dans la conversion de Bcl-2 en molĂ©cule pro-apoptotique et contribue Ă la mort par apoptose des LTe pendant la phase de contraction. Notre modĂšle propose que la densitĂ© de lâĂ©pitope contrĂŽle la gĂ©nĂ©ration des CD8+ LTm. Une meilleure comprĂ©hension des mĂ©canismes impliquĂ©s dans la gĂ©nĂ©ration des LTm permettra le dĂ©veloppement de meilleures stratĂ©gies pour la gĂ©nĂ©ration de vaccin.
Dans un second temps, nous avons Ă©valuĂ© le rĂŽle du signal RCT dans lâhomĂ©ostasie des LTm. Pour ce faire, nous avons utilisĂ© un modĂšle de souris transgĂ©nique pour le RCT dont son expression peut ĂȘtre modulĂ©e par un traitement Ă la tĂ©tracycline. Ce systĂšme nous a permis dâabolir lâexpression du RCT Ă la surface des LTm. De maniĂšre intĂ©ressante, en absence de RCT exprimĂ©, les LTm CD8+ peuvent survivre Ă long terme dans lâorganisme et rester fonctionnels. De plus, une sous population des LTm CD4+ a la capacitĂ© de survivre sans RCT exprimĂ© dans un hĂŽte lymphopĂ©nique alors que lâautre sous population nĂ©cessite lâexpression du RCT.Following antigen (Ag) encounter presented at surface of antigen presenting cell (APC), naĂŻve T lymphocytes, which express a T cell receptor (TCR) specific for Ag, undergo massive proliferation and differentiate into effector T cells (1). After elimination of the pathogen, most effector T cells die, while the remaining differenciates into memory T cells (LTm) which are responsible for long-term protection of the organism. The mechanism that promotes the differentiation of effectors T cells into memory T cells is still largely unknown.
To understand how Tm cells are generated from effectors, we hypothesized that the density of antigen on the APC could have an impact on the selection of CD8+ T cell responders differentiating into memory. Very interestingly, our results show that immunization of mice with dendritic cells (DCs) expressing high levels of peptide-MHC complexes on their surface allow a strong development of LTm. In contrast, the development of memory T cells was strongly reduced (10-20X) when mice were immunized with DCs expressing two-fold less level of peptide-MHC complexes. In agreement with the results described above, the amount of Ag does not have any influence on T cell expansion and acquisition of effector functions, but critically affects memory T cell generation. Our data suggest that the numbers of TCR engaged in MHC/peptide recognition are important for the formation of memory T cells. To do that, we evaluated by time-lapse videomicroscopy the time of interaction between LTn and DCs. Effectively, we observed a significant reduction in the percentage of cells making prolonged interaction with DCs when the level of Ag is decreased. Moreover, we observed a modification in the expression of key transcription factors involved in the differentiation of Tm cells, such as Eomes, Bcl6 and Blimp-1. Further analysis reveals that the Ag density influences the expression of Neuron-derived orphan nuclear receptor 1 (Nor1). Nor-1 is involved in the conversion of Bcl-2 into a pro-apoptotic molecule and contributes to effector death by apoptosis during contraction phase. Our model proposes that density of Ag controls the generation of LTm. A better understanding of the role of TCR signals in the generation of LTm will help to develop better vaccination strategies.
Second time, we have evaluated the role of TCR signals in Tm cell homeostasis. To do that, we have used a tetracycline-inducible expression system of the TCR in mice. This system allows us to abolish TCR expression on Tm cells. Interestingly, we show that the ablation of TCR expression did not influence the survival and functionnality of Ag-specific CD8+ LTm cells. Furthermore, our results show that a subset of CD4 Tm cells can survive in the absence of TCR expression in nonlymphopenic hosts while another subset requires the TCR expression to survive
Epitope Density Influences CD8+ Memory T Cell Differentiation
The generation of long-lived memory T cells is critical for successful vaccination but the factors controlling their differentiation are still poorly defined. We tested the hypothesis that the strength of T cell receptor (TCR) signaling contributed to memory CD8(+) T cell generation.We manipulated the density of antigenic epitope presented by dendritic cells to mouse naĂŻve CD8(+) T cells, without varying TCR affinity. Our results show that a two-fold decrease in antigen dose selectively affects memory CD8(+) T cell generation without influencing T cell expansion and acquisition of effector functions. Moreover, we show that low antigen dose alters the duration of the interaction between T cells and dendritic cells and finely tunes the expression level of the transcription factors Eomes and Bcl6. Furthermore, we demonstrate that priming with higher epitope density results in a 2-fold decrease in the expression of Neuron-derived orphan nuclear receptor 1 (Nor-1) and this correlates with a lower level of conversion of Bcl-2 into a pro-apoptotic molecule and an increased number of memory T cells.Our results show that the amount of antigen encountered by naĂŻve CD8(+) T cells following immunization with dendritic cells does not influence the generation of functional effector CD8(+) T cells but rather the number of CD8(+) memory T cells that persist in the host. Our data support a model where antigenic epitope density sensed by CD8(+) T cells at priming influences memory generation by modulating Bcl6, Eomes and Nor-1 expression
Improving the efficacy of hormone therapy in breast cancer: The role of cholesterol metabolism in SERM-mediated autophagy, cell differentiation and death
International audienceBreast cancer (BC) is one of the most common female cancers in the world, with estrogen receptor (ER)-positive BC the most frequent subtype. Tamoxifen (Tam) is an effective drug that competitively binds to the ER and is routinely used for the treatment of ER-positive BC. However, a number of ER-positive BC do not respond to Tam treatment and acquired resistance is often observed, constituting a major challenge for extending patient life expectancy. The mechanisms responsible for these treatment failures remain unclear, indicating the requirement for other targets and better predictors for patient response to Tam. One of Tam's off-targets of interest is the microsomal antiestrogen binding site (AEBS), a multiproteic complex made up of the cholesterol-5,6-epoxide hydrolase (ChEH) enzymes that are involved in the late stages of cholesterol biosynthesis. Tam and other selective ER modulators stimulate oxidative stress and inhibit the ChEH subunits at pharmacological doses, triggering the production and accumulation of cholesterol-5,6-epoxide metabolites responsible for BC cell differentiation and death. However, inhibition of the cholesterogenic activity of the AEBS subunits also induces the accumulation of sterol precursors, which triggers a survival autophagy to impair Tam's efficacy. Altogether, these studies have highlighted the involvement of cholesterol metabolism in the pharmacology of Tam that has provided new clues on how to improve its therapeutic efficacy in both BC and other cancers as well as offering a new rationale for developing more efficient drugs for BC treatment
Lysine-Dendrimer, a New Non-Aggressive Solution to Rebalance the Microbiota of Acne-Prone Skin
Acne is a chronic inflammatory skin disease that affects the quality of life of patients. Several treatments exist for acne, but their effectiveness tends to decrease over time due to increasing resistance to treatment and associated side effects. To circumvent these issues, a new approach has emerged that involves combating the pathogen Cutibacterium acnes while maintaining the homeostasis of the skin microbiome. Recently, it was shown that the use of a G2 lysine dendrigraft (G2 dendrimer) could specifically decrease the C. acnes phylotype (IAI) involved in acne, compared to non-acne-causing C. acnes (phylotype II) bacteria. In the present study, we demonstrate that the efficacy of this technology is related to its 3D structure, which, in contrast to the linear form, significantly decreases the inflammation factor (IL-8) linked to acne. In addition, our in-vitro data confirm the specific activity of the G2 dendrimer: after treatment of bacterial cultures and biofilms, the G2 dendrimer affected neither non-acneic C. acnes nor commensal bacteria of the skin (Staphylococcus epidermidis, S. hominis, and Corynebacterium minutissimum). In parallel, comparative in-vitro and in-vivo studies with traditional over-the-counter molecules showed G2âs effects on the survival of commensal bacteria and the reduction of acne outbreaks. Finally, metagenomic analysis of the cutaneous microbiota of volunteers who applied a finished cosmetic product containing the G2 dendrimer confirmed the ability of G2 to rebalance cutaneous acne microbiota dysbiosis while maintaining commensal bacteria. These results confirm the value of using this G2 dendrimer to gently prevent the appearance of acne vulgaris while respecting the cutaneous microbiota
Improvement of 5,6α-epoxycholesterol, 5,6ÎČ-epoxycholesterol, cholestane-3ÎČ,5α,6ÎČ-triol and 6-oxo-cholestan-3ÎČ,5α-diol recovery for quantification by GC/MS
International audience5,6α-epoxycholesterol (5,6α-EC) and 5,6ÎČ-epoxycholesterol (5,6ÎČ-EC) are oxysterols involved in the anticancer pharmacology of the widely used antitumor drug tamoxifen. They are both metabolized into cholestane-3ÎČ,5α,6ÎČ-triol (CT) by the cholesterol-5,6-epoxide hydrolase (ChEH) enzyme, and CT is metabolized by an as-yet uncharacterized enzyme into 6-oxo-cholestan-3ÎČ,5α-diol (OCDO). A recent feasibility study showed that the 5,6-ECs may represent surrogate markers of tamoxifen activity in breast cancer patients undergoing endocrine therapy, thus there is a growing interest in their accurate quantification. These oxysterols are usually quantified by gas-liquid chromatography coupled to mass spectrometry (GC/MS), using an isotope dilution methodology with the corresponding deuterated oxysterol. This method is considered to be relative quantitative since all of the standards used are deuterated oxysterols, however it is not known whether the preparation of each oxysterol is affected in the same way by the extraction, pre-purification by solid phase extraction (SPE) and trimethylsilylation steps, particularly when using biological samples that contain many other reactive compounds. Thus, in this study we investigated the yield of the 5,6-ECs, CT and OCDO recovery from patient serum samples at different stages of their work-up and trimethylsilylation prior to GC/MS analysis, using [14C]-labeled analogs to follow these oxysterols at each step. We measured a 40 to 60% loss of material for the 5,6-ECs and OCDO, however we also describe the conditions that improved their recovery. Our data also show that the use of deuterated 5,6α-EC, 5,6ÎČ-EC, CT and OCDO is an absolute requirement for their accurate quantification
Selective Defect in Antigen-Induced TCR Internalization at the Immune Synapse of CD8 T Cells Bearing the ZAP-70(Y292F) Mutation
Full House: Microbial and Nonmicrobial Volatile Organic Compounds Competing for âResidencyâ in Indoor Environments at Cycling Relative Humidity
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Quantitative analysis of the tumor suppressor dendrogenin A using liquid chromatography tandem mass spectrometry
International audienceDendrogenin A (DDA) was recently identified as a mammalian cholesterol metabolite that displays tumor suppressor and neurostimulating properties at low doses. In breast tumors, DDA levels were found to be decreased compared to normal tissues, evidencing a metabolic deregulation of DDA production in cancers. DDA is an amino-oxysterol that contains three protonatable nitrogen atoms. This makes it physico-chemically different from other oxysterols and it therefore requires specific analytical methods We have previously used a two-step method for the quantification of DDA in biological samples: 1) DDA purification from a Bligh and Dyer extract by RP-HPLC using a 250Ă4.6mm column, followed by 2) nano-electrospray ionization mass spectrometry (MS) fragmentation to analyze the HPLC fraction of interest. We report here the development a liquid chromatography tandem mass spectrometry method for the analysis of DDA and its analogues. This new method is fast (10min), resolving (peak width <4s) and has a weak carryover (<0.01%). We show that this technique efficiently separates DDA from its C17 isomer and other steroidal alkaloids from the same family establishing a proof of concept for the analysis of this family of amino-oxysterols
Limit of detection: Phantom with TFA.
<p>(A) Concentration in M of TFA per well. (B) Equivalent number of <sup>19</sup>F atoms per well. (C) Axial view of the tubes. Upper panels: <sup>19</sup>F images acquired; lower panels: <sup>19</sup>F and <sup>1</sup>H overlaid images. Voxel volume: 0.63mm<sup>3</sup>. (D) Coronal view of the tubes, <sup>1</sup>H images. (E) Mean SNR in the region of interest (ROI) with respect to <sup>19</sup>F atoms/voxel. Each line corresponds to the acquisition of a slice as shown in panel D.</p
In-vivo protocol description.
<p>Overview of the time scale of the different experimental procedures. At day 0 (D0) eight CD45.1 C57BL/6 mice received 10<sup>6</sup> B16-F10 melanoma cells by subcutaneous injection in order to induce a malignant melanoma. On the same day SP were prepared from OT-1 mice and two different protocols were applied to generate T<sub>act</sub> or T<sub>OVA-act</sub> (as described in Materials and Methods, T cells isolation and activation section). At day 8 (D8), PFC was added in the cell culture medium for 18h in order to label SP, T<sub>act</sub> and T<sub>OVA-act</sub> with <sup>19</sup>F. Then, at day 9 (D9) the <sup>19</sup>F-labeled cells were injected IV: 2 control mice (with no tumors) received 50 x 10<sup>6</sup> SP, 3 mice received 20 to 50 x 10<sup>6</sup> T<sub>act</sub> and 5 mice received 20 to 40 x 10<sup>6</sup> T<sub>OVA-act</sub>. Finally, 9 mice were imaged at day 10 (D10; 1 T<sub>act</sub> injected mouse was not imaged) and all mice were immediately sacrificed for subsequent analysis of the organs (liver, lungs, spleen and tumor) by flow cytometry (all mice) and high resolution in-vitro NMR spectroscopy (2 SP injected mice, 3 T<sub>OVA-act</sub> injected mice and 1 T<sub>act</sub> injected mouse). The study protocol was performed in a total of 10 animals. In black: in-vivo part; in blue: cell preparation.</p