Biomedical subjects

Nicolas Blanchard

Publications and source records attributed to Nicolas Blanchard.

At least 19 recordsLinked to original sources

PubMed2006

T cell-dependent activation of dendritic cells requires IL-12 and IFN-gamma signaling in T cells.

Patients presenting with genetic deficiencies in IFNGR1, IFNGR2, IL-12B, and IL-12RB1 display increased susceptibility to mycobacterial infections. We analyzed in this group of patients the cross-talk between human CD4+ T lymphocytes and dendritic cells (DCs) that leads to maturation of DC into producers of bioactive IL-12 and to activation of T cells into IFN-gamma producers. We found that this cross-talk is defective in all patients from this group. Unraveling the mechanisms underlying this deficiency, we showed that IL-12 signaling in T cells is required to induce expression of costimulatory molecules and secretion of IL-12 by DCs and that IFNGR expression is required on both DCs and CD4+ T cells to induce IL-12 secretion by DCs. These data suggest that CD4+ T cell-mediated activation of DCs plays a critical role in the defense against mycobacterial infections in humans.

Cells, Cultured↗

PubMed2006

Dendritic cells can turn CD4+ T lymphocytes into vascular endothelial growth factor-carrying cells by intercellular neuropilin-1 transfer.

Neuropilin-1 (NRP1) is a transmembrane protein expressed on neuronal and endothelial cells where it plays a crucial role in guiding axons and regulating angiogenesis. We have recently shown that NRP1 also is expressed on dendritic cells (DC) in the human immune system and have proposed a role for NRP1 in the first stages of the immune response. In these studies, we show that NRP1 can be transferred with a high efficiency from human DC to T lymphocytes by trogocytosis. The NRP1 transfer can occur independently of T lymphocyte activation; the amount of NRP1 transferred depends on the NRP1 expression level on APC and is enhanced when T cells are activated through the TCR. Moreover, the NRP1 transfer occurs between specific donor and recipient cells, because no NRP1 transfer is observed between endothelial cells and T lymphocytes or between APCs and CD34(+) hemopoietic cells. Finally, we show that a major NRP1 ligand, vascular endothelial growth factor (VEGF)(165), is secreted by mature human DCs and binds to NRP1 captured by T lymphocytes. These results show that NRP1 transfer to T lymphocytes during the immune synapse can convert T lymphocytes into VEGF(165)-carrying cells. Together with the enhanced signaling of VEGF-R2 on endothelial cells in the presence, in trans, of the NRP1-VEGF(165) complex, our results suggest that the intercellular transfer of NRP1 might participate in the Ag-independent remodelling of the endothelial vessels in secondary lymphoid organs during inflammation.

Antigen Presentation↗

PubMed2006

Functional implication of cellular prion protein in antigen-driven interactions between T cells and dendritic cells.

The cellular prion protein (PrPC) is a host-encoded, GPI-anchored cell surface protein, expressed on a wide range of tissues including neuronal and lymphoreticular cells. PrPC may undergo posttranslational conversion, giving rise to scrapie PrP, the pathogenic conformer considered as responsible for prion diseases. Despite intensive studies, the normal function of PrPC is still enigmatic. Starting from microscope observations showing an accumulation of PrPC at the sites of contact between T cells and Ag-loaded dendritic cells (DC), we have studied the contribution of PrPC in alloantigen and peptide-MHC-driven T/DC interactions. Whereas the absence of PrPC on the DC results in a reduced allogeneic T cell response, its absence on the T cell partner has no apparent effect upon this response. Therefore, PrPC seems to fulfill different functions on the two cell partners forming the synapse. In contrast, PrPC mobilization by Ab reduces the stimulatory properties of DC and the proliferative potential of responding T cells. The contrasted consequences, regarding T cell function, between PrPC deletion and PrPC coating by Abs, suggests that the prion protein acts as a signaling molecule on T cells. Furthermore, our results show that the absence of PrPC has consequences in vivo also, upon the ability of APCs to stimulate proliferative T cell responses. Thus, independent of neurological considerations, some of the evolutionary constraints that may have contributed to the conservation of the Prnp gene in mammalians, could be of immunological origin.

Amino Acid Sequence↗

PubMed2006

Daucus carota L. mediated bioreduction of prochiral ketones.

Stereoselective reductions of ketones to secondary alcohols are of the utmost importance in organic synthesis. Very high selectivities are observed with traditional reducing agents, mainly based on boron or transition metals, complexed with chiral ligands. Bioreductions mediated by intact cells from cut plants, vegetables and fruits are attractive alternatives and could facilitate transition towards a more biobased economy. This emerging area highlights the recent results obtained in the aqueous bioreduction of prochiral ketones using carrot roots. The applications of this methodology to asymmetric protonation, dynamic kinetic resolution and the synthesis of biologically relevant targets are presented.

Alcohols↗

PubMed2006

ERAAP synergizes with MHC class I molecules to make the final cut in the antigenic peptide precursors in the endoplasmic reticulum.

The major histocompatibility complex class I molecules display peptides (pMHC I) on the cell surface for immune surveillance by CD8(+) T cells. These peptides are generated by proteolysis of intracellular polypeptides by the proteasome in the cytoplasm and then in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with antigen processing (ERAAP). To define the unknown mechanism of ERAAP function in vivo, we analyzed naturally processed peptides in cells with or without appropriate MHC I and ERAAP. In the absence of MHC I, ERAAP degraded the antigenic precursors in the ER. However, MHC I molecules could bind proteolytic intermediates and were essential for generation of the final peptide by ERAAP. Thus, ERAAP synergizes with MHC I to generate the final pMHC I repertoire.

Amino Acid Sequence↗

PubMed2005

Intermolecular nitroso Diels-Alder cycloaddition of alpha-acetoxynitroso derivatives in aqueous medium.

The Diels-Alder cycloadditions of the alpha-acetoxynitroso dienophile in water are reported. The rapid and high yielding synthesis of structurally diverse 3,6-dihydro-1,2-oxazines complements the straightforward elaboration of aminoalcohols obtained from the alpha-acetoxynitroso derivative in anhydrous medium. A rationale for this solvent-dependent product distribution is proposed.

Journal Article↗

PubMed2004

Strong and durable TCR clustering at the T/dendritic cell immune synapse is not required for NFAT activation and IFN-gamma production in human CD4+ T cells.

The exact function of TCR clustering and organized macromolecular patterns at the immune synapse between APCs and T lymphocytes is unclear. Using human immature or mature dendritic cells (DCs) and autologous CD4(+) effector T cells, we demonstrate that, within a given conjugate, mature DCs induce strong and long-lasting TCR clustering and protein kinase C-theta translocation in a superantigen dose-dependent manner. Moreover, mature DCs promote CD43 exclusion in a dose-independent manner. In contrast, immature DCs are less potent at inducing these molecular rearrangements. Using these models to correlate T cell functions with the frequency, the intensity, and the duration of TCR clustering, we show, in Jurkat T cells, that weak and transient TCR clustering is sufficient to promote TCR down-modulation, protein kinase C-theta translocation at the synapse, and substantial NFAT transcriptional activation. Moreover, we show, in CD4(+) T cell blasts, that strong TCR clustering is required for neither TCR down-modulation nor optimal IFN-gamma production. Together, our results demonstrate that some CD4(+) functional responses, such as cytokine production, are independent of central supramolecular activation cluster formation.

CD4-Positive T-Lymphocytes↗

PubMed2004

Total synthesis of formamicin.

The enantioselective total synthesis of the cytotoxic plecomacrolide natural product formamicin (1) is described. Key aspects of this synthesis include the efficient transacetalation reactions of MOM ethers 28 and 38 to form the seven-membered formyl acetals 29 and 39, a late-stage Suzuki cross-coupling reaction of the highly functionalized vinyl boronic acid 6 and vinyl iodide 7, a highly beta-selective glycosidation reaction of beta-hydroxy ketone 4 with 2,6-dideoxy-2-iodoglucopyranosyl fluoride 3, and the global desilylation of penultimate intermediate 77 mediated by in situ generated Et(3)N.2HF.

Glycosylation↗

PubMed2004

Total synthesis of zincophorin and its methyl ester.

A total synthesis of the naturally occurring ionophore zincophorin has been realized. The route features an intramolecular oxymercuration of a cyclopropanemethanol and a Carroll-Claisen rearrangement for the respective elaboration of the C1-C12 and C13-C25 subunits, which have been assembled by using a highly diastereoselective titanium-mediated aldol condensation.

Anti-Bacterial Agents↗

PubMed2004

Lewis acid-promoted hetero Diels-Alder cycloaddition of alpha-acetoxynitroso dienophiles.

[reaction: see text] alpha-Acetoxynitroso compound 3 has been prepared as a new stable, isolable, and reactive dienophile in nitroso Diels-Alder reactions. The yield of the [4 + 2] cycloaddition of alpha-acetoxynitroso dienophile with 1,3-dienes could be enhanced in the presence of 20 mol % Lewis acid. An unexpected retro hetero-Michael reaction from 26 was observed, leading to the cleavage of the N-O bond of the cycloadduct. This tandem nitroso Diels-Alder/retro hetero-Michael sequence has been used with cyclic and acyclic 1,3-dienes.

Journal Article↗

PubMed2004

Dynamic recruitment of the adaptor protein LAT: LAT exists in two distinct intracellular pools and controls its own recruitment.

The integral membrane adaptor protein linker for activation of T cells (LAT) couples the T-cell receptor (TCR) with downstream signalling and is essential for T-cell development and activation. Here, we investigate the dynamic distribution of LAT-GFP fusion proteins by time-lapse video imaging of live T lymphocytes interacting with antigen-presenting cells. We show that LAT forms two distinct cellular pools, one at the plasma membrane and one that co-distributes with transferrin-labelled intracellular compartments also containing the TCR/CD3-associated zeta chain. The distribution of LAT between these two pools is dependent on LAT intracytoplasmic residues. Whereas plasma membrane-associated LAT is recruited to immune synapses after a few seconds of cell conjugate formation, the intracellular pool is first polarized and then recruited after a few minutes. We further show that LAT intracytoplasmic amino acid residues, particularly the Tyr136, 175, 195 and 235 residues, are required for its own recruitment to the immune synapse and that a herein-identified juxtamembrane LAT region (amino acids 32-104) is involved in the localization of LAT in intracellular pools and in T-cell signalling. Altogether, our results demonstrate that LAT controls its own recruitment at the immune synapse, where it is required as a scaffold protein for the signalling machinery. The results also suggest that the intracellular pool of LAT might be required for T-cell activation.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Total synthesis of zincophorin methyl ester.

[reaction: see text]. A convergent total synthesis of the methyl ester of zincophorin, an ionophore antibiotic, has been realized relying on a diastereoselective titanium-mediated aldol coupling between the C1-C12 and C13-C25 subunits. The latter fragment was prepared by using a Carroll-Claisen rearrangement.

Journal Article↗

PubMed2003

Total synthesis of the formamicin aglycon, formamicinone.

[structure: see text] The total synthesis of formamicinone (2), the aglycone of formamicin (1), has been accomplished via the late-stage Suzuki cross-coupling of fragments 5 and 6, the macrolactonization of seco ester 14, and the Mukaiyama aldol reaction of aldehyde 3 and methyl ketone 4. An efficient and highly stereoselective second generation synthesis of vinyl iodide 6 is also described.

Anti-Bacterial Agents↗

PubMed2003

2-Deoxy-2-iodo-beta-glucopyranosyl fluorides: mild and highly stereoselective glycosyl donors for the synthesis of 2-deoxy-beta-glycosides from beta-hydroxy ketones.

2-Deoxy-2-iodo-beta-glucopyranosyl fluoride 14 is a highly stereoselective glucopyranosyl donor that may be activated under mild conditions. Application of this new glycosyl donor to the glycosidation reactions of a variety of acceptors including beta-hydroxy ketones affords beta-glycosides with high efficiency and stereoselectivity. [reaction--see text]

Fluorides↗

PubMed2003

Stereoselective synthesis of polypropionate units and heterocyclic compounds by cyclopropylcarbinol ring-opening with mercury(II) salts.

The mercury(II)-mediated electrophilic ring-opening reaction of various cyclopropylcarbinol derivatives bearing adjacent stereocenters and a remote nucleophilic functional group provides a useful strategy for synthesizing compounds bearing several contiguous stereocenters. These highly diastereoselective reactions occur with anchimeric assistance by the internal nucleophilic moiety and afford synthetically valuable building blocks such as polypropionate units or heterocyclic compounds. The application of cyclopropylcarbinol ring-opening for the preparation of functionalized oxygen heterocycles in natural product synthesis is also outlined.

Journal Article↗

PubMed2002

TCR/CD3 down-modulation and zeta degradation are regulated by ZAP-70.

TCR down-modulation following binding to MHC/peptide complexes is considered to be instrumental for T cell activation because it allows serial triggering of receptors and the desensitization of stimulated cells. We studied CD3/TCR down-modulation and zeta degradation in T cells from two ZAP-70-immunodeficient patients. We show that, at high occupancy of the TCR, down-modulation of the CD3/TCR is comparable whether T cells express or do not express ZAP-70. However, if TCR occupancy was low, we found that CD3/TCR was down-regulated to a lesser extent in ZAP-70-negative than in ZAP-70-positive T cells. We studied CD3/TCR down-modulation in P116 (a ZAP-70-negative Jurkat cell-derived clone) and in P116 transfected with genes encoding the wild-type or a kinase-dead form of ZAP-70. Down-modulation of the TCR at high occupancy did not require ZAP-70, whereas at low TCR occupancy down-modulation was markedly reduced in the absence of ZAP-70 and in cells expressing a dead kinase mutant of ZAP-70. Thus, the presence of ZAP-70 alone is not sufficient for down-modulation; the kinase activity of this molecule is also required. The degradation of zeta induced by TCR triggering is also severely impaired in T cells from ZAP-70-deficient patients, P116 cells, and P116 cells expressing a kinase-dead form of ZAP-70. This defect in TCR-induced zeta degradation is observed at low and high levels of TCR occupancy. Our results identify ZAP-70, a tyrosine kinase known to be crucial for T cell activation, as a key player in TCR down-modulation and zeta degradation.

Down-Regulation↗

PubMed2002

TCR activation of human T cells induces the production of exosomes bearing the TCR/CD3/zeta complex.

We show in this study that human T cells purified from peripheral blood, T cell clones, and Jurkat T cells release microvesicles in the culture medium. These microvesicles have a diameter of 50-100 nm, are delimited by a lipidic bilayer membrane, and bear TCR beta, CD3epsilon, and zeta. This microvesicle production is regulated because it is highly increased upon TCR activation, whereas another mitogenic signal, such as PMA and ionomycin, does not induce any release. T cell-derived microvesicles also contain the tetraspan protein CD63, suggesting that they originate from endocytic compartments. They contain adhesion molecules such as CD2 and LFA-1, MHC class I and class II, and the chemokine receptor CXCR4. These transmembrane proteins are selectively sorted in microvesicles because CD28 and CD45, which are highly expressed at the plasma membrane, are not found. The presence of phosphorylated zeta in these microvesicles suggests that the CD3/TCR found in the microvesicles come from the pool of complexes that have been activated. Proteins of the transduction machinery, tyrosine kinases of the Src family, and c-Cbl are also observed in the T cell-derived microvesicles. Our data demonstrate that T lymphocytes produce, upon TCR triggering, vesicles whose morphology and phenotype are reminiscent of vesicles of endocytic origin produced by many cell types and called exosomes. Although the exact content of T cell-derived exosomes remains to be determined, we suggest that the presence of TCR/CD3 at their surface makes them powerful vehicles to specifically deliver signals to cells bearing the right combination of peptide/MHC complexes.

Apoptosis↗

PubMed2002

The immunological synapse: the more you look the less you know...

Before T cells of the immune system can recognize pathogens, antigen presenting cells (APCs) must process pathogen-derived peptides and present them together with major histocompatibility complex molecules (MHC) to T lymphocytes. T lymphocytes then scan the surface of APCs and antigen-specific activation of the T cell will happen after interaction of T cell antigen receptor (TCR) with MHC-peptide complexes expressed at the membrane of APCs. This interaction takes place in a nanometer scale gap between the two cells, referred to as an immunological synapse. Recent three-dimensional fluorescence analysis of this synapse revealed a dynamic spatial organization of membrane receptors, cytoskeleton and intracellular signaling complexes on the T cell side showing specific patterns, which depend on the nature of the T cell:APC pair. Although it is obvious that establishment of an intimate contact between T cells and APCs will facilitate cell:cell communication it is not clear what is the role, if any, of this receptors patterning. This molecular reorganization has long been thought to enhance and/or sustain TCR signaling and thus T cell activation, but this is now a matter of controversy. Moreover, mechanisms controlling immunological synapse formation are still unraveled. Segregation of proteins may occur spontaneously as proposed by mathematical modeling taking into account membrane fluidity, protein size and receptor/ligand affinity. Alternatively patterning of the molecules at the cell:cell interface could be driven by active processes involving T cell signaling and/or specific features of the APC. These different questions will be discussed herein.

Animals↗