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Vav1 exhibits two signal transducing properties as an adaptor protein and a regulator of cytoskeleton organization through its Guanine nucleotide Exchange Factor module. Although the expression of Vav1 is restricted to the hematopoietic lineage, its ectopic expression has been unraveled in a number of solid tumors. In this study, we show that in lung cancer cells, as such in hematopoietic cells, Vav1 interacts with the Spleen Tyrosine Kinase, Syk. Likewise, Syk interacts with β-catenin and, together with Vav1, regulates the phosphorylation status of β-catenin. Depletion of Vav1, Syk or β-catenin inhibits Rac1 activity and decreases cell migration suggesting the interplay of the three effectors to a common signaling pathway. This model is further supported by the finding that in turn, β-catenin regulates the transcription of Syk gene expression. This study highlights the elaborated connection between Vav1, Syk and β-catenin and the contribution of the trio to cell migration.

// Stephane Saint-Georges 1,2 , Maude Quettier 1,2 , Marouane Bouyaba 3 , Stephanie Le Coquil 1,2 , Vanessa Lauriente 1,2 , Lionel Guittat 1,2 , Vincent Levy 3 , Florence Ajchenbaum-Cymbalista 1,2,4 , Nadine Varin-Blank 1,2 , Christine Le Roy 1,2,* and Dominique Ledoux 1,2,* 1 INSERM U978, Bobigny, France 2 Universite Paris 13, Sorbonne Paris Cite, Labex “Inflamex”, Bobigny, France 3 Assistance Publique-Hopitaux de Paris, Hopital Avicenne, Unite de Recherche Clinique, Bobigny, France 4 Assistance Publique-Hopitaux de Paris, Hopital Avicenne, Service d’Hematologie Biologique, Bobigny, France * These authors are co-senior authors Correspondence to: Christine Le Roy, email: // Nadine Varin-Blank, email: // Keywords : CLL, lymphadenopathy, B-cell receptor, CXCR4/CXCR5, protein kinase D Received : December 23, 2015 Accepted : April 16, 2016 Published : April 26, 2016 Abstract In Chronic Lymphocytic Leukemia (CLL), infiltration of lymph nodes by leukemic cells is observed in patients with progressive disease and adverse outcome. We have previously demonstrated that B-cell receptor (BCR) engagement resulted in CXCR4 down-regulation in CLL cells, correlating with a shorter progression-free survival in patients. In this study, we show a simultaneous down-regulation of CXCR4, CXCR5 and CD62L upon BCR triggering. While concomitant CXCR4 and CXCR5 down-regulation involves PKDs, CD62L release relies on PKC activation. BCR engagement induces PI3K-δ-dependent phosphorylation of PKD2 and 3, which in turn phosphorylate CXCR4 Ser 324/325 . Moreover, upon BCR triggering, PKD phosphorylation levels correlate with the extent of membrane CXCR4 decrease. Inhibition of PKD activity restores membrane expression of CXCR4 and migration towards CXCL12 in BCR-responsive cells in vitro . In terms of pathophysiology, BCR-dependent CXCR4 down-regulation is observed in leukemic cells from patients with enlarged lymph nodes, irrespective of their IGHV mutational status. Taken together, our results demonstrate that PKD-mediated CXCR4 internalization induced by BCR engagement in B-CLL is associated with lymph node enlargement and suggest PKD as a potential druggable target for CLL therapeutics.

The electrochemical behavior of complexes [FeMo(CO) 5 (κ 2 -dppe)(μ-pdt)] (1) and [FeMo(CO) 4 (MeCN)( κ 2 -dppe)(μ-pdt)] (2), in the absence and in the presence of acid, has been investigated. The reduction of 1 follows at slow scan rates, in CH 2 Cl 2 -[NBu 4 ][PF 6 ] and acid-free media, an EC rev E mechanism that is supported by cyclic voltammetry (CV) experiments and digital CV simulations. In MeCN-[NBu 4 ][PF 6 ], the electrochemical reduction of 1 is the same as in dichloromethane and follows an ECE mechanism at slow scan rates, but with a positive shift of the redox potentials. In contrast, the oxidation of 1 is strongly solvent-dependent. In dichloromethane, the oxidation of 1 is reversible and involves a single electron, while in acetonitrile, it is irreversible at moderate and slow scan rates (v ≤ ca. 1 V s -1 ), and some chemical reversibility is apparent at higher scan rates (v = 10 V s -1 ). Density functional theory calculations revealed that the chemical step in the EC rev E mechanism corresponds to the dissociation of one PPh 2 end of the diphosphine ligand and the transfer of the semibridging CO to the Fe atom, similarly to the mechanism observed in the FeFe analogue complex. However, in the case of 1, the subsequent coordination of the phosphine ligand to the other metal is an unfavorable process.

Only a minority of chronic lymphocytic leukemia (CLL) patients harboring a positive direct antiglobulin test (DAT) will develop autoimmune hemolytic anemia (AIHA). In a single institution cohort of 378 CLL patients, 56 patients (14.8%) had at least one positive DAT during the course of the disease, either at diagnosis or later. We found no relationship between the time of the first positive DAT and overall survival (OS). However, patients with a positive DAT who did not develop AIHA had the same adverse outcome as patients who developed AIHA. Of the patients who were in Binet stage A at diagnosis, those with a positive DAT had a significantly shorter OS, regardless of their IGHV mutational status, however, there was a strong association with VH1-69. By multivariate analysis, a positive DAT was found to be an independent adverse prognostic factor for OS. Thus, DAT represents a strong adverse prognostic factor and its determination should be repeated during follow-up.

International audience; Dimolybdenum thiolate-bridged complexes [Mo2Cp2(μ-SMe)2(μ-SCH2CH2E)] (E = O (2) or NH (4)) with a proton-dependent protecting device have been synthesized by reaction of [Mo2Cp2(μ-SMe)2(μ-Cl)2] (1) with SCH2CH2EH. The reactivity of the resultant quadruply bridged complexes with acid was investigated in the absence and in the presence of a potential ligand (N2, MeCN, RNC). While the protonation of complexes 2 and 4 under N2 in dichloromethane produced only the oxidized derivatives instead of the desired diazenido compound, ligand binding was observed in MeCN or in the presence of RNC (R = t-Bu, Xyl). Whereas acetonitrile loss from [Mo2Cp2(μ-SMe)2(μ-SCH2CH2OH)(MeCN)2]+ (8+) prevented the isolation and characterization of this species, the t-BuNC analogue (6+) could be characterized by an X-ray crystal structure. The electrochemistry of 2 and 2+ was investigated in CH2Cl2 and in MeCN, both in the absence and in the presence of acid. While the addition of HBF4·Et2O to a dichloromethane solution of 2 only produced 2+ (and presumably H2), 8+ was the major product of the protonation in MeCN.

B-cell antigen receptor (BCR)–mediated signaling plays a critical role in chronic lymphocytic leukemia (CLL) pathogenesis and gives an in vitro survival advantage to B cells isolated from patients with unfavorable prognostic factors. In this study, we undertook to elucidate the signaling intermediates responsible for this biologic alteration. In responding cells only, in vitro BCR engagement triggers global phosphorylation of Syk, activation of phospholipase Cγ2, and intracellular calcium mobilization, reflecting competency of BCR signaling. The calcium–calcineurin-dependent transcription factor NFAT2 is up-regulated and to some extent constitutively activated in all CLL B cells. In contrast, its DNA-binding capacity is enhanced on IgM stimulation in responding cells only. NFAT inhibition using the VIVIT peptide prevents induction of CD23 target gene and IgM-induced survival, converting responding cells to unresponsive status. At the opposite, ionomycin-induced NFAT activity allows survival of nonresponding cells. These results demonstrate that the functional heterogeneity relies on variability of protein levels establishing BCR-dependent thresholds and NFAT-dependent activation. Finally, status of the BCR-NFAT pathway for each patient reveals its relevance for CLL clinical outcome and points out to BCR-NFAT intermediates as promising functional therapeutic targets.

Common in ageing patient, the solar lentigo is a macular hyperpigmented skin lesion that results from chronic exposure to ultraviolet irradiations. Despite sharing numerous features with other pigmented spots, the diagnostic of this benign lesion is well characterized at the tissue level. Recent studies shed lights on several factors and their pathogenic mechanisms involved in the development of the solar lentigo. This review summarizes how diverse experimental approaches allowed the identification of several biomarkers, which contribute to a better understanding on the initiation and the maintenance of this pigmentary disorder.

Mixed μ-phosphido or μ-thiolato μ-halo-dimolybdenum(III) compounds [Mo 2 Cp 2 (μ-SMe) 2 (μ-X)(μ-Y)] (X = PPh 2 , Y = Cl ( 1 ); X = SCH 3 , Y = Br ( 3 ), I( 4 )) have been studied. Syntheses and X-ray structures of the new bromo and iodo analogues of [Mo 2 Cp 2 (μ-SMe) 3 (μ-Cl)] ( 2 ) are reported. While preparing 3 a side-product 5 was obtained and structurally characterised as the Mo 5 system [{Mo 2 Cp 2 Br(μ-O)(μ-SMe) 2 } 2 (μ-MoO 4 )], containing a {Mo IV –Mo IV –O–(Mo VI O 2 )–O–Mo IV –Mo IV } unit. The influence of the bridging groups on the structures and electrochemical behaviour of the complexes [Mo 2 Cp 2 (μ-SMe) 2 (μ-X)(μ-Y)] 1 – 4 has been investigated. In MeCN–[NBu 4 ][PF 6 ] the first oxidation of [Mo 2 Cp 2 (μ-SMe) 2 (μ-PPh 2 )(μ-Cl)] ( 1 ) is quasi-reversible, contrasting with the essentially irreversible first oxidation of the thiolate analogue [Mo 2 Cp 2 (μ-SMe) 3 (μ-Cl)] ( 2 ) under similar conditions. The effects of lowering the temperature or increasing the scan rate on the oxidation of 1 were examined: the initial quasi-reversible oxidation at first became irreversible but at still higher scan rates or lower temperatures the oxidation was again quasi-reversible. This suggests that the oxidation of 1 in MeCN is followed by reversible coordination of acetonitrile to afford the species [Mo 2 Cp 2 (μ-SMe) 2 (μ-PPh 2 )(Cl)(MeCN)] + ( 7 + ). Cyclic voltammetry of [Mo 2 Cp 2 (μ-SMe) 3 (μ-Y)](Y = Br ( 3 ) or I ( 4 )) showed two quasi-reversible diffusion-controlled oxidation steps in CH 2 Cl 2 –[NBu 4 ][PF 6 ] or thf–[NBu 4 ][PF 6 ]. In acetonitrile, the fast loss of the halide ligands results in the formation of [Mo 2 Cp 2 (μ-SMe) 3 (MeCN) 2 ] + species.

cited By 10; International audience; A series of chemical and electrochemical transformations of systems in which a Mo2Cp2(μ-SMe)3 core is bridged by a μ-C2HnR ligand (n = 0-4) are described in this paper. The reaction of the alkylidene complex [Mo2Cp2(μ-SMe) 3(μ-η1:η2-CHCH2Tol)] (BF4) (1) with LiBun at 0°C produces the μ-σ,π-vinyl complex [Mo2Cp2(μ-SMe) 3(μ-η1:η2-CH=CHTol] (2) in good yield. The molecular structure of 2 has been confirmed by X-ray analysis. Upon treatment with NaBEL4 1 is readily converted into the semibridging alkyl species [Mo2Cp2(μ-SMe)3(μ-CH 2CH2Tol)] (3), which is also formed by electrochemical reduction of 1 in acidic medium. NMR and X-ray diffraction studies of 3 are consistent with, but do not definitively establish, the presence of a η1 α-agostic interaction. Density functional theory has been used to confirm the presence of agostic interactions in both 1 and 3 and also to explore the exchange pathways for these hydrocarbyl dimolybdenum systems. Electrochemical transformation of the μ-alkylidene complex 1 gives 3 as the major product when acid is present and a mixture of 2 and 3 when acid is absent, production of 2 being favored by low initial concentrations of 1. Theoretical, spectroscopic, and diffraction data are used to explain the formation and structures of closely related [Mo2Cp 2(μ-SMe)3(μ-C2HnR] z- complexes (n = 0-4 and z = 0, 1), including 1-3. © 2005 American Chemical Society.

Treatment of [Mo2(Cp)2(μ-SMe)3(MeCN)2]+ (1+) with NO3– or NO2– results in the conversion of one terminally bound acetonitrile ligand into an amidato bridge. The reaction produces [Mo2(Cp)2(μ-SMe)3{μ-η1,η1-OC(Me)NH}]0/+ (20/+) and involves the formation of an intermediate, which was detected by cyclic voltammetry but which could not be isolated, and which likely arises from the substitution of the NOx anion for one MeCN ligand. The electrochemical behaviour of 2+ was studied by cyclic voltammetry in THF and MeCN. The reduction of 2+ in the presence of acid (HBF4/H2O or HBF4/Et2O) in these solvents leads to the release of the amidate bridge. Controlled-potential electrolysis of 2+ in MeCN in the presence of acid produces 1+ quantitatively; the charge consumed (>1 F mol–1 of 1+) indicates that electrons are also used to reduce protons. This was confirmed by the formation of 2+ (in variable amounts depending on the conditions) on treating 2 with acid. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

The internalization of various cargo proteins and lipids from the mammalian cell surface occurs through the clathrin and lipid-raft endocytic pathways. Protein-lipid and protein-protein interactions control the targeting of signalling molecules and their partners to various specialized membrane compartments in these pathways. This functions to control the activity of signalling cascades and the termination of signalling events, and therefore has a key role in defining how a cell responds to its environment.

The reduction of [Mo2(Cp)2(µ-SMe)3(µ-O)]+ (1+) has been investigated by cyclic voltammetry and controlled-potential electrolysis in THF- and MeCN/NBu4PF6 without added acid or in the presence of various acids HX (HX: HTsO, CF3CO2H, HBF4). Reduction in the presence of acid follows an ECrevE mechanism in which the intermediate chemical step is an acid-base equilibrium between 1 and [Mo2(Cp)2(µ-SMe)3(µ-OH)]+ (2+). This electrochemical process is followed by protonation of the neutral µ-hydroxo complex 2 to afford different products which depend both on the solvent (THF or MeCN) and on the nature of the acid. Controlled-potential electrolysis of 1+ in the presence of HX (2 equiv.) leads to the generation of binding sites and finally gives products identical to those obtained from protonation of 2 by HX. The complex [Mo2(Cp)2(µ-SMe)3(µ-η1,η1-OCOCF3)] (3) which can be obtained either by protonation of 2 by CF3CO2H or by reduction of 1+ in the presence of CF3CO2H, has been characterized crystallographically. In the presence of HBF4 protonation of 1+ gives 22+. The reactivity of 2+ and of the complexes [Mo2(Cp)2(µ-SMe)3(H2O)(TsO)] (4) and [Mo2(cp)2(µ-SMe)3(H2O)L]+ (L = H2O or THF) (6+), all of which contain a terminal aqua ligand, has also been investigated. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

International audience; The design of the new FeMo heterobimetallic species [FeMo(CO)(5)(kappa(2)-dppe)(mu-pdt)] is reported. Mossbauer spectroscopy and density functional theory calculations give deep insight into the electronic and structural properties of this compound.

The purpose of this study was to evaluate the time-course effect of a 36-h treatment with ACTH (10−8m), transforming growth factor-β1 (TGFβ1; 10−10m), angiotensin II (AngII; 10−7m), and insulin-like growth factor I (IGF-I; 10−8m) on the steroidogenic capacity of bovine adrenocortical cells (BAC) and on messenger RNA (mRNA) levels of ACTH receptor, cytochrome P450c17, 3β-hydroxysteroid dehydrogenase (3βHSD), steroidogenic acute regulatory protein (StAR), and StAR protein. ACTH and IGF-I enhanced, in a time-dependent manner, the acute 2-h ACTH-induced cortisol production, whereas TGFβ1 and AngII markedly reduced it. ACTH, IGF-I, and AngII increased ACTH receptor mRNA, but the opposite was observed after TGFβ1 treatment. ACTH and IGF-I increased P450c17 and 3βHSD mRNAs, whereas AngII and TGFβ1 had the opposite effects. However, the effects of the four peptides on ACTH-induced cortisol production appeared before any significant alterations of the mRNA levels occurred. The most marked and rapid effect of the four peptides was on StAR mRNA. The stimulatory effect of ACTH was seen within 1.5 h, peaked at 4–6 h, and declined thereafter, but at the end of the 36-h pretreatment, the levels of StAR mRNA and protein were higher than those in control cells. IGF-I also enhanced StAR mRNA levels within 1.5 h, and these levels remained fairly constant. The effects of AngII on StAR mRNA expression were biphasic, with a peak within 1.5–3 h, followed by a rapid decline to almost undetectable levels of both mRNA and protein. TGFβ1 had no significant effect during the first 3 h, but thereafter StAR mRNA declined, and at the end of the experiment the StAR mRNA and protein were almost undetectable. Similar results were observed when cells were treated with ACTH plus TGFβ1. A 2-h acute ACTH stimulation at the end of the 36-h pretreatment caused a higher increase in StAR mRNA and protein in ACTH- or IGF-I-pretreated cells than in control cells, which, in turn, had higher levels than cells pretreated with TGFβ1, ACTH plus TGFβ1, or AngII.These results and the fact that the stimulatory (IGF-I) or inhibitory (AngII and TGFβ1) effects on ACTH-induced cortisol production were more pronounced than those on the ability of cells to transform pregnenolone into cortisol strongly suggest that regulation of StAR expression is one of the main factors, but not the only one, involved in the positive (IGF-I) or negative (TGFβ1 and AngII) regulation of BAC for ACTH steroidogenic responsiveness. A high correlation between steady state mRNA level and acute ACTH-induced cortisol production favors this conclusion.

Transforming growth factor beta (TGFbeta) has been reported to be a potent growth inhibitor of epithelial cells. The purpose of the present work was to study in vitro and in vivo the effects of overexpression of a dominant-negative type II TGFbeta receptor on the proliferation and differentiation of Y-1 cells. Stable transfections were performed with a mutant TbetaRII (TbetaRII-KR) fused with the Enhanced Fluorescent Green Protein (EGFP). The expression of this fusion protein and its overexpression were demonstrated by northern blot and immunoblot with EGFP and TbetaRII probes and antibodies respectively. The membrane localization of this fusion protein was confirmed by confocal microscopy. The functionality of this fusion protein was demonstrated by its blocking effects on TGFbeta action on DNA synthesis and on Y-1 expression of steroidogenic acute regulatory protein (StAR) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Moreover, in nude mice the tumorigenicity of cells stably transfected with the fusion protein was higher than that of cells stably transfected with EGFP alone. Taken together, the present results show that TbetaRII-KR/EGFP blocks the effects of TGFbeta1 on Y-1 cells and acts as a potent dominant-negative receptor preventing TGFbeta signaling.

Transforming growth factor beta 1 (TGF beta 1) is a potent inhibitor of several differentiated functions in bovine adrenal fasciculata cells (BAC). In addition, these cells express and secrete this factor. To determine whether this peptide plays an autocrine role in BAC, cells were transfected with 10 microM unmodified sense (SON) or antisense (AON) oligonucleotide complementary to the translation initiation region of the TGF beta 1 mRNA in an attempt to inhibit TGF beta 1 protein synthesis. We investigated first, the cellular uptake, the stability, and the intracellular distribution of 32P-labeled TGF beta 1 AON and SON; and second, the effects of both oligonucleotides on BAC specific functions. We have demonstrated that in BAC, the TGF beta 1 AON uptake reached a plateau after 8 h of transfection (16% of the radioactivity added) and remained fairly constant for at least 24 h. In contrast, the uptake of TGF beta 1 SON reached a plateau after 2 h of transfection (8% of the radioactivity added), remained stable for only 3 h, and then declined. After 8 h of transfection, followed by 44 h of culture without oligonucleotides, the intracellular level of TGF beta 1 AON was still high with about 8% of the radioactivity added, whereas that of TGF beta 1 SON represented only 1.2%. Moreover, AON was present in the cytoplasmic and nuclear fractions, and it was hybridized in both compartments. However, TGF beta 1 SON was present mainly in the cytoplasmic fraction where it was not hybridized. Neither TGF beta 1 AON nor SON modified TGF beta 1 mRNA levels; however, TGF beta 1 AON, but not SON, caused the disappearance of TGF beta 1 immunoreactivity inside the cells. Finally, the steroidogenic responsiveness of BAC transfected with TGF beta 1 AON increased about 2-fold, and this was associated with a 2-fold increase of the mRNA levels of both cytochrome P450 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase. Neither TGF beta 1 SON nor a scrambled oligonucleotide containing the same number of G nucleotides as TGF beta 1 AON had any effect on these parameters. Thus, these studies demonstrate that TGF beta 1 has an autocrine inhibitory effect on BAC differentiated functions, an effect that can be overcome by TGF beta 1 AON.

Technological developments of multiparametric flow cytometry come along with the generation of new dyes. The APC-tandem dyes, which combine the fluorophores APC and Cy7/H7, allow the detection of a specific signal in the APC-Cy7/H7 channel along with an unexpected nonspecific signal in the APC channel. Depending on the magnitude of the latter, it may be a handicap for interpreting the data of multicolor labeling experiments. We investigated the alteration of the APC-tandem dyes by labeling peripheral blood cells with antibodies directed toward leukocyte surface proteins and by analyzing cells by flow cytometry. Our results show that the APC-Cy7/H7 tandem fluorochromes degraded over time. Nonspecific APC signal was observed with the various antibodies tested only upon cell attachment but not under bead linkage. Moreover, the percentage of degradation of the APC-Cy7/H7 dyes was dependent on the cell type analyzed. Interestingly, nonspecific APC signal strongly decreased when the metabolic activity of immunolabeled cells was inhibited or when cells were incubated with vitamin C. This study demonstrates that the APC-tandem dyes are the target of cell-dependent degradation, which may be antagonized. These findings will allow cytometer users to optimize their multicolor panels.

Cell-surface TGFbeta (transforming growth factor beta) receptors partition into membrane rafts and the caveolin-positive endocytic compartment by an unknown mechanism. In the present study, we investigated the determinant in the TGFbeta type II receptor (TbetaRII) that is necessary for membrane raft/caveolar targeting. Using subcellular fractionation and immunofluorescence microscopy techniques, we demonstrated that the extracellular domain of TbetaRII mediates receptor partitioning into raft and caveolin-positive membrane domains. Pharmacological perturbation of glycosylation using tunicamycin or the mutation of Mgat5 [mannosyl(alpha-1,6)-glycoprotein beta-1,6-N-acetylglucosaminyltransferase V] activity interfered with the raft partitioning of TbetaRII. However, this was not due to the glycosylation state of TbetaRII, as a non-glycosylated TbetaRII mutant remained enriched in membrane rafts. This suggested that other cell-surface glycoproteins associate with the extracellular domain of TbetaRII and direct their partitioning in membrane raft domains. To test this we analysed a GMCSF (granulocyte/macrophage colony-stimulating factor)-TbetaRII chimaeric receptor, which contains a glycosylated GMCSF extracellular domain fused to the transmembrane and intracellular domains of TbetaRII. This chimaeric receptor was found to be largely excluded from membrane rafts and caveolin-positive structures. Our results indicate that the extracellular domain of TbetaRII mediates receptor partitioning into membrane rafts and efficient entrance into caveolin-positive endosomes.

International audience; The μ-alkylidyne complex [Mo2Cp2(μ-SMe)3(μ-CCH2Ph)] (1) reacts with HBF4 in acetonitrile to give the unstable bis-nitrile species [Mo2Cp2(μ-SMe)2(μ-CCH2Ph)](NCCH3)2](BF4) (2). Treatment with either borohydride or chloride converts 2 into [Mo2Cp2(μ-SMe)2(μ-CCH2Ph)(μ-κ1:κ1-BH4)] (3) or [Mo2Cp2(μ-SMe)2(μ-CCH2Ph)(μ-Cl)] (4), respectively. Clean evolution of 4 in non-degassed solvent affords the novel μ-thioalkylidene derivative [Mo2(O)(Cl)Cp2(μ-SMe)(μ-MeSCCH2Ph)](5).

International audience; The electrochemical reduction of isomeric complexes, [Mo2Cp2(μ-SMe)3(μ-η1:η1-HCCPh)]+ (1+) and [Mo2Cp2(μ-SMe)3(μ-η1:η2-C[double bond, length as m-dash]CHPh)]+ (3+), where the hydrocarbyl bridges in a η1:η1- or a η1:η2 mode, has been studied by cyclic voltammetry and controlled-potential electrolysis in thf–[NBu4][PF6] and CH2Cl2–[NBu4][PF6], in the absence and in the presence of acid. The binding mode of the CC fragment induces different electrochemical behaviour of the complexes in acid-free solutions since 1+ reduces in two diffusion-controlled one-electron steps while the first reduction of 3+ is characterized by slow electron transfer kinetics. Controlled-potential reduction of both 1+ and 3+ produces a mixture of the acetylide [Mo2Cp2(μ-SMe)3(μ-η1:η2-CCPh)] (2) and alkylidyne complexes [Mo2Cp2(μ-SMe)3(μ-η1-CCH2Ph)] (4). In the presence of acid, the electrochemical reduction of 1+ and of 3+ occurs according to ECE processes. The nature of the products formed by controlled-potential reduction of 1+ depends on the nature of the acid and of the solvent. The transient formation of a complex with a μ-alkenyl ligand, either [Mo2Cp2(μ-SMe)3(μ-η1:η2-CH[double bond, length as m-dash]CHPh)] (7) or an isomer, is suggested by the oxidative electrochemistry of 7 and by its reaction with acids. In thf–[NBu4][PF6] in the presence of an excess of acid (HBF4/Et2O) and of phenylacetylene, electrolysis of 1+ gives rise to catalytic reduction of phenylacetylene to styrene. However, unidentified reactions limit the efficiency of this process. The reduction of 3+ in acidic medium produces the alkyl complex [Mo2Cp2(μ-SMe)3(μ-CH2CH2Ph)] (6) through alkylidyne [Mo2Cp2(μ-SMe)3(μ-η1-CCH2Ph)] (4) and alkylidene [Mo2Cp2(μ-SMe)3(μ-η1-CHCH2Ph)]+ (5+) intermediates. Some ethylbenzene was formed after reduction of 5+ in the presence of acid. These results show an effect of the binding mode of the hydrocarbyl bridge on the mechanism and products of the reduction of the corresponding complexes.

The electrochemical oxidation of [Mo2(cp)2(μ-SMe)3(μ-N2Ph)] and [Mo2(cp)2(μ-SMe)3(μ-N2HPh)]+ complexes where the diazo bridge adopts either an η1 or an η1:η1 coordination mode has been studied by cyclic voltammetry and controlled-potential electrolysis in THF– and CH2Cl2–[NBu4][PF6]. The electrochemical oxidation of [Mo2(cp)2(μ-SMe)3(μ-η1-N2Ph)] 1 and of [Mo2(cp)2(μ-SMe)3(μ-η1-N2HPh)]+1-H+++ triggers the isomerization of the diazo bridge to the η1:η1 mode found in 2+++ and 2-H2+2+2+ respectively. The electrochemical oxidation of [Mo2(cp)2(μ-SMe)3(μ-η1:η1-N2Ph)] 3 and of [Mo2(cp)2(μ-SMe)3(μ-η1:η1-HN2Ph)]+3-H+++ with a syn (“up–up”) arrangement of the Me substituents of the equatorial sulfur bridges is also followed by an isomerization to 2+++ and 2-H2+2+2+, respectively, with an anti (“up–down”) configuration of the equatorial Me groups. The rates of the isomerization 1++ → 2+++, 1-H2+2+2+ → 2-H2+2+2+, and 3-H2+2+ → 2-H2+2+2+ were studied by cyclic voltammetry at different scan rates and at different temperatures. The isomerization of the protonated complexes with either a hydrazido(2−) or a diazene bridge (respectively 1-H2+2+2+ and 3-H2+2+) is faster than that of the diazenido precursors (respectively 1++ and 3++). The diazenido complex 2+++ protonates readily, affording 2-H2+2+2+, while 2-H3+3+ undergoes proton loss.

Signaling by the transforming growth factor-β (TGF-β) family of growth factors involves cell surface receptor serine/threonine kinases and downstream components such as SARA and Smurfs, which bind the receptor substrates called the Smad proteins. These components partition between two endocytic pathways, which lead to two separate functions in TGF-β signaling. On the one hand, clathrin-dependent endocytosis promotes signaling by leading the receptor to the early endosome where SARA is localized. On the other, non-clathrin pathways, which are enriched for the Smurf ubiquitin ligases, lead the receptor to degradation. However, in polarized cells, ligand addition induces TGF-β receptor trafficking into junctional regions of the cell where activation of Smad-dependent and -independent pathways mediates the process of epithelial-to-mesenchymal transition, which is critical during development and tumorigenesis. In this chapter, we will focus on how compartmentalization of TGF-β receptors and their downstream components controls TGF-β signaling and its biological functions

The conjugation of ubiquitin to proteins involves a cascade of activating (E1), conjugating (E2), and ubiquitin-ligating (E3) type enzymes that commonly signal protein destruction. In TGFbeta signaling the inhibitory protein Smad7 recruits Smurf2, an E3 of the C2-WW-HECT domain class, to the TGFbeta receptor complex to facilitate receptor degradation. Here, we demonstrate that the amino-terminal domain (NTD) of Smad7 stimulates Smurf activity by recruiting the E2, UbcH7, to the HECT domain. A 2.1 A resolution X-ray crystal structure of the Smurf2 HECT domain reveals that it has a suboptimal E2 binding pocket that could be optimized by mutagenesis to generate a HECT domain that functions independently of Smad7 and potently inhibits TGFbeta signaling. Thus, E2 enzyme recognition by an E3 HECT enzyme is not constitutively competent and provides a point of control for regulating the ubiquitin ligase activity through the action of auxiliary proteins.

The Golgi enzyme β1,6 N-acetylglucosaminyltransferase V (Mgat5) is up-regulated in carcinomas and promotes the substitution of N-glycan with poly N-acetyllactosamine, the preferred ligand for galectin-3 (Gal-3). Here, we report that expression of Mgat5 sensitized mouse cells to multiple cytokines. Gal-3 cross-linked Mgat5-modified N-glycans on epidermal growth factor and transforming growth factor–β receptors at the cell surface and delayed their removal by constitutive endocytosis. Mgat5 expression in mammary carcinoma was rate limiting for cytokine signaling and consequently for epithelial-mesenchymal transition, cell motility, and tumor metastasis. Mgat5 also promoted cytokine-mediated leukocyte signaling, phagocytosis, and extravasation in vivo. Thus, conditional regulation of N-glycan processing drives synchronous modification of cytokine receptors, which balances their surface retention against loss via endocytosis.

Endocytosis of cell surface receptors is an important regulatory event in signal transduction. The transforming growth factor beta (TGF-beta) superfamily signals to the Smad pathway through heteromeric Ser-Thr kinase receptors that are rapidly internalized and then downregulated in a ubiquitin-dependent manner. Here we demonstrate that TGF-beta receptors internalize into both caveolin- and EEA1-positive vesicles and reside in both lipid raft and non-raft membrane domains. Clathrin-dependent internalization into the EEA1-positive endosome, where the Smad2 anchor SARA is enriched, promotes TGF-beta signalling. In contrast, the lipid raft-caveolar internalization pathway contains the Smad7-Smurf2 bound receptor and is required for rapid receptor turnover. Thus, segregation of TGF-beta receptors into distinct endocytic compartments regulates Smad activation and receptor turnover.

Introduction and hypothesis : B-acute lymphoblastic leukemia (B-ALL) expressing the fusion transcript ETV6/RUNX1 is one of the most frequent subtype of childhood leukemia. This disease is associated with a good survival rate but 5 to 10% of patients relapse more than five years after treatment most notably in extra-hematopoietic sanctuary sites like testis. CD9, a protein member of the tetraspanin family, has been shown to be under-expressed in ETV6/RUNX1 B-ALL. This protein is involved in hematopoietic stem cell engraftment. Its expression has also been correlated with the risk of metastasis and is associated with a poor clinical outcome in various types of cancer. Our hypothesis is that CD9, through its functional properties on migration and homing, could be a key actor of ETV6/RUNX1 leukemia relapse. The purpose of our study was to investigate the effect of CD9 expression on motility and engrafment of pre-B lymphocytes. Methods and results : We used the human pre-B lymphoblastic REH cell line which express CD9. By lentiviral transduction of small hairpin RNAs targeting CD9 mRNA, we generated REH cells depleted in CD9 protein. We performed engraftment assays in vivo using Nod Scid Gamma immunodeficient mice. Mice injected with REH cells depleted in CD9 had a longer survival rate than mice injected with REH control cells. In vitro analysis of adhesion on fibronectin demonstrated that cellular adhesion is dependent of membrane expression of CD9. As well, the more CD9 is expressed, the higher migration rate in response to CXCL12 chemokine is. F‐actin labelling after CXCL12 stimulation showed also an increased F‐actin polymerization in CD9‐positive cells and the formation of actin extensions where CD9 and actin are colocalized. No modifications in CXCR4 (receptor of CXCL12) internalization has been found in CD9 depleted cell lines, which suggests that CD9 does not affect the receptor itself but the signalling pathway downstream the receptor. We investigated the activation of RAC1 protein, a Rho GTPase known to induce the formation of actin-rich lamellipodia in diverse cellular functions including migration. We demonstrated that cells depleted in CD9 have lost their ability to activate RAC1 in response to CXCL12. The inhibition and the depletion of RAC1 respectively have the same effect as CD9 depletion on the migration in response to CXCL12 in vitro and on the survival in vivo. Finally, we tested the ability of REH cells and REH depleted in CD9 cells to migrate to testis in vivo after xenograft in mouse model. We showed that only CD9 positive-REH cells were found in testis tissue after xenograft. Moreover we tested in vitro, the ability of testis tissue to induce the migration of REH cells in a Boyden chamber model. We showed that testis tissue induced the migration of REH cells and this migration was strongly decreased using either an inhibitor of CXCR4, an inhibitor of RAC1 or an antibody targeting specifically CD9. Conclusion : Our data show that CD9 is a key actor in pre‐B lymphoblasts adhesion, CXCL12 dependant migration and actin remodelling. We show for the first time in pre-B cells a link between CD9, RAC1 pathway and homing in testis which is a preferential site of late relapse. This work is supported by CNRS, University of Rennes 1, University Hospital of Rennes, Ligue Régionale contre le Cancer (sections 22, 35 and 56) (MPA, VG, MBT), Région Bretagne (MPA, MBT, VG), SFR Biosit UMS 3480 (VG, MBT), Association Laurette Fugain (VG). Disclosures No relevant conflicts of interest to declare.

International audience; The electrochemical reduction of the imide complex [Mo2(cp)2(μ-SMe)3(μ-NH)]+ (1+) has been investigated in THF and MeCN electrolytes by cyclic voltammetry, controlled-potential electrolysis and coulometry. In the absence of free protons, the electrochemical reduction produces the amide derivative [Mo2(cp)2(μ-SMe)3(μ-NH2)] (2) after consumption of 1 F mol-1 of 1+. In THF in the presence of acid, the reduction of 1+ occurs through a two-electron process. The presence of acid also results in the shift of the equilibrium between 1+ and amide dication 22+ (MeCN electrolyte) or induces an isomerisation of the imide ligand (THF electrolyte). This allows the electrolysis to be conducted at a potential 600 mV less negative than the reduction potential of 1+. Controlled-potential electrolyses in the presence of acid (2 equiv HTsO) produce the ammine derivative. Ammonia is released from these compounds either by coordination of the solvent (MeCN electrolyte) or by the binding of chloride to the ammine-tosylate complex (electrolyses in THF in the presence of acid and chloride). The final products, isolated almost quantitatively (>95%), are [Mo2(Cp)2(μ-SMe)3(MeCN)2] + and [Mo2(cp)2(μ-SMe)3(μ-Cl)], respectively.

The expression and the maintenance of specific differentiated function of Leydig cells are regulated not only by gonadotropin but by locally produced factors, which may act as autocrine regulators. Many factors, in particular growth factors, have been postulated to have such a type of effect on testicular cells, but very few fulfilled the three criteria required to establish a paracrine/autocrine role: (a) presence of receptors and biological action on local cells; (b) local secretion regulated by physiological signals; and (c) blockade of the factor or its receptors must modify the function of local cells. In the present work we demonstrate that two factors, insulin-like growth factor 1 (IGF-I) and transforming growth factor beta1 (TGFbeta1) fulfilled the three criteria: (a) IGF-I stimulates the transcription of the genes encoding Leydig cell differentiated function, leading to an enhanced steroidogenic responsiveness to LH/hCG; (b) Leydig cells (LC) express and secrete IGF-I and this secretion is enhanced by hCG; and (c) incubation of LC with IgG anti-IGF-I, but not with IgG-control, markedly reduced the steroidogenic responsiveness to LH/hCG. In contrast to IGF-I, TGFbeta is a potent inhibitor of LC differentiated function. Moreover, LC express TGFbeta1 mRNA and secrete this peptide. To prove that the locally produced TGFbeta has an autocrine role, LC were transfected with 10 microM of an antisense oligonucleotide (AON) complementary to the translation initiation region of TGFbeta1 mRNA. Transfection with AON but not with sense deoxynucleotide induces a complete disappearance of TGFbeta immunoreactivity in LC and an enhanced hCG-induced testosterone production by LC. This increased steroidogenic responsiveness was associated with a significant enhancement of both LH/hCG receptor mRNA and P450c17 mRNA. Taken together, the above results show that both factors play an autocrine role, although opposite, on Leydig cell function.

Cellular movement from one place to another is regulated by various guidance cues. The precise perception of these signals in the three-dimensional environment of a multicellular organism is remarkably complex. Recent work is now revealing that guided cell movement also requires spatial control of signaling events by endocytic dynamics.

Autoimmune cytopenias represent a well-known complication of chronic lymphocytic leukemia (CLL). The most frequent one is autoimmune haemolytic anemia (AIHA), occurring in about 5% of the patients during the course of the disease. The prognostic significance of autoimmune cytopenias remains uncertain and controversial. In previous studies, a positive direct antiglobulin test (DAT) was found in up to 35% of CLL cases, however only a minority of the patients would develop AIHA at any time of the disease. The aim of this monocentric study was to explore the clinical and biological characteristics and the outcome of CLL patients showing a positive direct antiglobulin test at any time during the course of the disease. We reviewed all CLL patients seen at our institution who had at least one positive DAT between January 2007 and May 2013. Fifty-four patients were found, representing about 10% of our CLL cohort. The sex ratio was 1,8 (35M/19F). Median age at diagnosis was 66,2 years (range 44,7 to 87,3). According to the Binet classification, 41 (76 %) patients were in stage A, 7 in stage B, and 6 in stage C. Study of usual prognostic parameters evidenced that these patients represented a very high risk group: 82% of cases harbored unmutated IGHV, and CD38 was expressed in 60% cases. Cytogenetic alterations were studied by FISH analysis: 11q deletion, trisomy 12 and 17p deletion were found in 12,5%, 22,9% and 14,3% cases respectively. In this cohort, with a median follow up of 85 months, 41 (76 %) patients required treatment. The first line of treatment was initiated for CLL progression in 33 cases, for symptomatic AIHA along with CLL progression in 5, and for symptomatic AIHA (without CLL progression) in 3. The median time to first treatment was very short (16 months), and the median overall survival of this cohort was 84 months. In 22 cases, the DAT was positive from the time of diagnosis, while in 34 patients it became positive later during the course of CLL. Only 19 patients (35%) developed symptomatic AIHA. DAT specificity was IgG, IgG+ C or C for respectively 36, 10 and 8 patients. Interestingly, there was no impact of the time of first positive DAT during the course of the disease (at diagnosis or later). Moreover, occurrence of a symptomatic AIHA had no impact on treatment free survival or overall survival when compared with cases with positive DAT only. Among these cases, 41 were in stage A at diagnosis and up to 78% harbored unmutated IGHV (as compared with 30 % in our institutional stage cohort). In order to evaluate the impact of a positive DAT on a homogenous population, we focused on the 31 IGHV unmutated stage A cases and compared them with our cohort of IGHV unmutated stage A with consistently negative DAT. We found a significant prevalence of VH1-69 and VH 3-21 use (43 %) and a high percentage belonging to a stereotyped subset (34%). The majority of the cases (19/28 tested) were responders to in vitro B-cell receptor stimulation by anti IgM. In this population, time to first treatment (26 months) was slightly shorter but median overall survival (55 months) was significantly reduced highlighting the poor response to treatment. In conclusion, occurrence of a positive DAT at any time of the course of CLL is associated with poor outcome and has the same adverse prognostic impact as the onset of a symptomatic AIHA. Moreover, study of B-Cell receptor (BCR stimulation by anti-IgM, CDR3 stereotypy) show that these patients may represent a pathophysiological distinct subgroup in which antigen stimulation is likely to play a major role. Disclosures: No relevant conflicts of interest to declare.

Introduction CD9 is a membrane protein, member of the tetraspanin family. Recent publications have reported the role of CD9 on engraftment of hematopoietic stem cells, and on cancer stem cell potential. The expression of CD9 has been correlated to the risk of metastases and to a poor clinical outcome in various types of cancer. Surprisingly, CD9 protein is downregulated in ETV6/RUNX1 pre-B acute lymphoblastic leukemia. The purpose of our study is to investigate the effect of CD9 expression on migration and engraftment abilities of pre-B lymphocytes. Materials and Methods The CD9-positive Nalm6 and REH (ETV6/RUNX1) pre-B cells were used. By lentiviral transduction of shRNA targeting mRNA, we generated Nalm6 and REH cell lines depleted in CD9 protein. Engraftment tests were performed in vivo using Nod Scid Gamma immunodeficient mice. REH and Nalm6 cells were detected in bone marrow by CD10 and respectively CD19 or HLA-DR labelling. Ability of the different cell lines to adhere on fibronectin and to migrate through double chambers system in response to CXCL12 were measured in vitro. We also investigated the presence of membrane villosities on REH and REH shCD9 cell surface by scanning electron microscopy. Finally, F-actin polymerization after CXCL12 stimulation was measured by rhodamin-phalloidin labelling. Results In vivo engrafments tests showed that the human cells detected in bone marrow is strongly enriched in CD9 positive cells compared to the initially injected population. This result suggests that CD9 facilitates pre-B lymphobasts homing. An in vitro analysis of adhesion on fibronectin demonstrated that cellular adhesion is dependent on membrane expression of CD9. As well, the more CD9 is expressed, the higher the migration rate in response to CXCL12 chemokine is. The analysis of membrane villosities on REH cell surface revealed that cells over expressing CD9 had longer villosity than shCD9 transducted cell lines. Moreover, F-actin labelling after CXCL12 stimulation showed an increased F-actin polymerization in CD9-positive cells and the formation of actin extensions. Conclusion We provide novel evidence that CD9 is a key player of pre-B lymphoblasts engraftment, adhesion and CXCL12 dependant migration. CD9 expression is related to actin remodelling. We are now investigating a potential link between CD9 and RAC1 activation in response to CXC12. Therefore, the expression level of CD9 could impact leukemic blasts abilities to spread and be responsible of relapses. This work is supported by CNRS, University of Rennes 1, University Hospital of Rennes, la Ligue Régionale contre le Cancer (committee 22, 35 and 56) (MPA, VG, MBT), SFR Biosit UMS 3480 (VG, MBT), Association Laurette Fugain (VG) and Europe Career Integration Grant (MBT). Disclosures: No relevant conflicts of interest to declare.

Cells communicate through signals that must be propagated from cell surface to nucleus. Tracking the signals generated by the transforming growth factor-β protein reveals a surprising partner in this process.

Antigen-driven signals are involved in the pathogenesis and progression of CLL with a particular occurrence within the lymph node microenvironment. We have previously shown that ex-vivo BCR engagement promoted simultaneous down-regulation of CXCR4 and CD62L membrane expressions in CLL cells from patients at risk of disease progression only. Functionally, cells down-regulating CXCR4 and CD62L in response to BCR triggering displayed both a reduction in migration toward CXCL12 and in adhesion to lymphatic endothelial cells. We studied a cohort of 73 previously untreated patients, 36 of whom were IGHV mutated and 37 were IGHV unmutated. The distribution of the extent of CXCR4 down-regulation was identical among CXCR4 responders, whether IGHV mutated (median 49% [Q1:28-Q3:62]) or unmutated (median 43% [Q1:31-Q3:66]). We also studied the expression of another chemokine receptor, CXCR5, which is also expressed at the membrane of CLL cells and plays an important role in the homing and trafficking of lymphocytes to the lymphoid follicles. Sustained antigenic stimulation of CLL cell samples (n=25) promoted down-regulation of CXCR4 and CXCR5 in a strictly correlated manner (r= 0,9) i.e. in the same subsets of cells. The extent of BCR-dependent decrease of the two proteins was strikingly representative of the heterogeneous capacity of the B-CLL cells to respond to BCR engagement in a given patient. Moreover, treatment with the specific PKD inhibitor CID755673 blocked significantly both CXCR4 and CXCR5 BCR-mediated decrease (n=11), demonstrating that PKDs specifically target these 2 molecules, whereas CD62L down-regulation was not significantly affected by the PKD inhibitor. At the functional level, treatment with the PKD inhibitor restored CLL cell migration capacity in response to CXCL12. PKD phosphorylation/activation in response to BCR stimulation, which involvesPI3K-d, was required for CXCR4-phosphorylation and its down-regulation. We then studied the clinical relevance of CXCR4 down-regulation after BCR engagement. The capacity to down-regulate CXCR4 was significantly related to shorter PFS (p=0.043). This cohort allowed exploring the link with IGHV mutational status. 36/37 unmutated IGHV cases had a significant CXCR4 down-regulation after in vitro BCR stimulation, in concordance with the ultimately constant progression of the disease. Conversely, IGHV mutated cases fell into two groups: a) one group of 14/36 patients, with very low ( In conclusion, the capacity of cells to down-regulate cell surface chemokine receptors and L-selectin is reflecting the size of the cell subset responding to BCR signaling. Interestingly, BCR signaling capacity to down-regulate CXCR4 is variable among IGHV mutated samples and strongly linked to the presence of lymph nodes as well as disease progression with time. It is tempting to speculate that depending on the BCR-mediated CXCR4/CXCR5 down-regulation capacity, some IGHV mutated patients might not benefit from Btk or PI3K-d inhibitors. Given the crucial importance of down-regulation of these two chemokine receptors after BCR engagement, we suggest that PKD could be promising new therapeutic target in CLL. Disclosures Levy: Roche: Honoraria; Janssen: Honoraria; Gilead: Honoraria. Cymbalista:Janssen: Honoraria, Research Funding; Gilead: Honoraria; Roche: Honoraria; Karyopharm: Honoraria.

Chronic lymphocytic leukemia (CLL) is a chronic lymphoproliferative disorder characterized by a heterogeneous clinical evolution, with indolent and progressive forms, as well as by analtered immune system; the latter being consistently found during disease progression. Our laboratory has previously demonstrated the existence of clonal CD5⁺/CD19⁺ subpopulations that secrete soluble cytokines, such as IL10 and TGFβ1, and express the transcription factor FOXP3. Based on these results, my work aimed to further characterize phenotypically and functionally these subpopulations, and notably the IL10 secreting one and to by follow its evolution during therapy with ibrutinib, a Btk inhibitor. Functionally, my results demonstrate that CD5⁺ CD19⁺ B lymphocytes have the ability to inhibit CD4⁺ T cells proliferation, to induce regulatory T cells differentiation and to reduce Th1 differentiation. Moreover, we also evidenced that IL10 producing subpopulation shares common phenotypic and activation features with the CLL cells that are part of the proliferative fraction. Furthermore, the higher frequency of the IL-10⁺ found in progressive patients compared to the indolent ones reveals that IL10 secretion by CLL B cells is linked to disease progression. Finally, Ibrutinib therapy induced a rapid decrease of the absolute number of IL10⁺ cells, which behave like the cells expressing low levels of the CXCR4, a marker of the proliferative fraction. Altogether, our results highlight the crucial role of the immunoregulatory CD5⁺/CD19⁺ subpopulations in CLL progression, and provide new clues on the mechanisms underlying the therapeutic effects of ibrutinib.; La leucémie lymphoïde chronique (LLC) est une hémopathie B qui se caractérise par une évolution clinique hétérogène et un dysfonctionnement du système immunitaire. Des travaux antérieurs du laboratoire ont mis en évidence l’existence de sous-populations lymphocytaires B CD5⁺/CD19⁺ qui sécrètent des facteurs solubles immuno-régulateurs, tels que l’IL10 et le TGFβ1 et, qui expriment le facteur de transcription FOXP3. Mon travail de thèse a consisté à poursuivre la caractérisation phénotypique et fonctionnelle de ces sous populations,et notamment celle sécrétant de l’IL10, tout en évaluant son évolution chez des patients en cours de traitement par ibrutinib, un inhibiteur de Btk. Sur le plan fonctionnel,mes travaux démontrent que les lymphocytes B de LLC ont la capacité d’inhiber la prolifération lymphocytaire T CD4⁺, d’induire une différenciation lymphocytaire T régulatrice et d’inhiber la différenciation Th1. Mes résultats démontrent également que la sous population IL10⁺ présente des caractéristiques phénotypiques et d’activation communes aux cellules de la fraction proliférative. De plus, la fréquence de la population IL10⁺, plus élevée chez les patients évolutifs, révèle que la capacité de sécrétion de l’IL10 par les cellules B de LLC est corrélée à la progression. Enfin, le traitement par ibrutinib induit une diminution rapide de la sous-population IL10+ qui se comporte comme celle de la sous-population CXCR4ˡᴼᵂ, un marqueur de la fraction proliférative. L’ensemble de ces données souligne l’importance des sous-populations immuno-régulatrices dans l’évolutivité de la LLC et apporte de nouveaux éléments sur les mécanismes d’action de l’ibrutinib.

Chronic Lymphocytic Leukemia (CLL) is a clonal B cell malignancy of the elderly. This neoplasm is characterized by a heterogeneous clinical course from indolent chronic disease to progressive lymphadenopathy that remains incurable. The aim of my PhD was to undertake a comprehensive phenotypic and functional analysis of the B cell subpopulations responsible for their survival advantage. CLL B cell purification from 30 patients indicate the presence in various extents of leukemic B cell subpopulations producing immune-regulatory cytokines, notably IL-10 and TGFβ1. Remarkably, these CLL subpopulations express the FOXP3 transcription factor, a marker of regulatory T cells. These subpopulations present a phenotypic signature, distinguishable from regulatory B cells populations, with specific markers of activated memory B cells. Functional studies prove their regulatory capacities on T cell differentiation, proliferation and secretion, contributing to the T cell dysfunction observed in CLL. Finally, statistical analysis combining IL-10, TGFβ1 and FOXP3 expressions for these subpopulations allows generating a poly-functional index correlated with two major risk factors of CLL progression. Our data characterize novel CLL B cell subpopulations that are involved in disease progression and give a rational to CLL B cell survival in secondary lymphoid organs., La Leucémie Lymphoïde Chronique (LLC) se caractérise par une hétérogénéité d’évolution avec des formes indolente et progressive. Cette dernière reste incurable avec les options thérapeutiques classiques. Mon objectif de thèse a consisté à définir phénotypiquement et fonctionnellement le potentiel régulateur des lymphocytes B de LLC induisant un contexte favorable à leur survie. Après purification des cellules B leucémiques de 30 patients LLC, nos résultats montrent que les sous-populations B sécrètent des cytokines immuno-régulatrices dont l’IL-10 et le TGFβ1. De façon intéressante, ces sous-populations lymphocytaires expriment également le facteur de transcription FOXP3, caractéristique des cellules T régulatrices. La signature phénotypique de ces sous-populations est spécifique au néoplasme avec des marqueurs de lymphocytes B mémoires activés. Nos approches fonctionnelles in vitro démontrent que ces sous-populations B modulent la prolifération et orientent la différenciation et les sécrétions des cellules T, contribuant à l’absence d’immuno-surveillance chez les patients. Enfin, une analyse statistique combinant les expressions de l’IL-10, du TGFβ1 et de FOXP3 dans ces sous-populations B permet de définir un indice polyfonctionnel qui corrèle avec deux facteurs clés du risque de progression de la LLC. L’ensemble de mes travaux de thèse a permis de caractériser de nouvelles sous-populations B impliquées dans la progression de la maladie et donne un rationnel à la survie des cellules leucémiques dans l’environnement ganglionnaire.

Based on extrinsic and intrinsic factors, skin complexion of an individual evolves in time. The Joss of its homogeneity is linked to the appearance of hyperpigmented les ions. The latters are induced by chronic sun exposure and appear with the age, as in the solar lentigo disorder. Despite its well-known characterization at the macroscopic levels, only few studies explore the skin fonctions of the solar lentigo with non-invasive tools. At the cellular and molecular levels, this lesion results from an altered process of pigmentation that is involved in the regulation of the cutaneous photo-protection. Despite the changes of the functional dialogue between the epiderrnic and derrnic layers, no study describes the functional characteristics of primary ce lis isolated from the solar lentigo. The first aim of my project consisted on a functional exploration of the solar lentigo by the use of diverse biometrological parameters. The study was carried out on a cohort of 80 women, some of them had few (grade l) and others many solar lentiges (grade 2) on their faces. Thanks to photographie measurements to determine the grade, various biometrological approaches had quantified the rates of sebum, melanin, hemoglobin, moisturizing, light reflection and the colour (L *, a*, b* and 1T A). Results of the statistical analyses revealed that the quantity of sebumdiscriminates the skin territories of the cheek and forehead, 2) the rates of melanin, hemoglobin, light reflection and the colour were differential between affected (solar lentigo) and not affected zones, within the volunteers' cheek territory, and 3) decreased rates of light reflection and hemoglobin, as well as, increased rate moisturizing, were observed within the lesional zone between both grades. Altogether, our data highlighted some of the biometrological parameters, as indicators of the skin territory, the lesional vs non lesional areas and the progression (grade 2 vs 1) of solar lentigo The second aim covered morphological and functional analyses of the solar lentigo's primary fibroblasts. This study was carried out on a cohort of 10 volunteers and two biopsies, containing the peri-lesional and lesional areas, were taken from each person. From these biopsies, human primary fibroblasts were isolated and grown. An immunofluorescence approach revealed that the fibroblasts of solar lentigo (FL) and those from adjacent healthy areas (FS) did not depict similar morphological characteristics with a differential organization of their actin cytoskeleton. Functional approaches demonstrated that FL displayed decrease of their metabolic activity, theirproliferation rates and their migration capacity, compared to FS. On the contrary, FL showed increased secretion capacity in terms of soluble factors. Our in vitro mode! of primary fibroblasts(FL/FS), which showed similarities with in vivo fibroblast's characteristics, might be considered as an appropriate cellular mode! to test active principles targeting skin complexion heterogeneity in women with mature skin. Using clinicat and translational research approaches, both objectives highlighted indicators and biomarkers of the solar lentigo. This work contributed to better understand the impact of the intrinsic and extrinsic factors in the Joss of complexion homogeneitv., La couleur de la peau chez un individu, appelée teint ou complexion, évolue au cours du temps et dépend de facteurs extrinsèques et intrinsèques. Une perte de son homogénéité est liée à l'apparition de lésions cutanées hyperpigmentées. Elles sont notamment provoquées par une exposition chronique au soleil et apparaissent avec l'âge, comme dans le cas du lentigo actinique. Si cette lésion hyperpigmentée bénigne est bien caractérisée à l'échelle macroscopique, peu d'études explorent ses fonctions cutanées grâce à des outils non invasifs. À l'échelle cellulaire et moléculaire, cette lésion hyperpigmentée bénigne résulte d'une altération du processus de pigmentation lors de la régulation du phénomène de photo-protection cutané. Si le modèle actuel d'une perte de cette régulation prend en compte l'altération du dialogue fonctionnel entre les couches épidermique et dermique, aucune étude ne décrit les caractéristiques fonctionnelles des cellules primaires extraites du lentigo actinique. Le premier objectif de mon projet a consisté en une exploration fonctionnelle du lentigo actinique par l'utilisation de divers paramètres biométrologiques. L'étude a été réalisée sur une cohorte de 80 femmes dont certaines présentent peu (grade 1) et, d'autres plusieurs, lentigosactiniques (grade 2) sur le visage. Après illustration du grade par une mesure photographique, différentes approches biométrologiques ont quantifié les taux de sébum, de mélanine, d'hémoglobine, d'hydratation, de réflexion de la lumière et de couleur (L *, a*, b* et lT A). Les résultats des analyses statistiques montrent que 1) la quantité de sébum discrimine les territoires cutanés de la joue et du front, 2) les taux de mélanine, d'hémoglobine, de réflexion de la lumière et de couleur sont différentiels entre les zones lésées (lentigo actinique) et non lésées, adjacentes au sein du territoire de la joue chez un volontaire, 3)que la diminution des taux de réflexion de la lumière et d'hémoglobine, ainsi que l'augmentation du taux d'hydratation, est observée au sein de la zone lésée entre les grades I et 2. L'ensemble de ces données ont mis en évidence certains paramètres biométrologiques comme indicateurs du territoire cutané, de la zone lésée vs non lésée et de l'évolution (grade 2 vs I) du lentigo actinique.Le second objectif a porté sur une analyse morphologique et fonctionnelle des fibroblastes primaires du lentigo actinique. L'étude a été réalisée sur une cohorte de 10 volontaires sur lesquels deux biopsies contenant les zones lésées et non lésées, adjacentes ont été prélevées. À partir de ces biopsies, les fibroblastes primaires humains ont été mis en culture. Une approche d'immunotluorescence révèle que les fibroblastes de lentigo actinique (FL) et ceux de la zone saine adjacente (FS) n'ont pas les mêmes caractéristiques morphologiques avec une organisation différentielle de leur cytosquelette d'actine. Une approche fonctionnelle montre que les FL ont une diminution de leur activité métabolique, de leur taux de prolifération et de leur capacité migratoire. À l'inverse, les FL sont dotés d'une augmentation de leur capacité sécrétoire en terme de facteurs solubles. Notre modèle in vitro de fibroblastes primaires (couples FL/FS), qui présentent des similitudes avec les caractéristiques décrites in vivo, représenterait un modèle cellulaire adéquat pour tester des principes actifs dont l'efficacité permettrait de réduire l'hétérogénéité du teint chez les femmes à peaux matures. Ces deux objectifs, qui ont été réalisés par des approches en recherche clinique et translationnelle, ont permis de mettre en évidence des indicateurs et des biomarqueurs du lentigoactinique qui permettront de mieux comprendre l'impact des facteurs intrinsèques et extrinsèques dans la perte d'homogénéité du teint liée au lentigo actinique.

Based on extrinsic and intrinsic factors, skin complexion of an individual evolves in time. The Joss of its homogeneity is linked to the appearance of hyperpigmented les ions. The latters are induced by chronic sun exposure and appear with the age, as in the solar lentigo disorder. Despite its well-known characterization at the macroscopic levels, only few studies explore the skin fonctions of the solar lentigo with non-invasive tools. At the cellular and molecular levels, this lesion results from an altered process of pigmentation that is involved in the regulation of the cutaneous photo-protection. Despite the changes of the functional dialogue between the epiderrnic and derrnic layers, no study describes the functional characteristics of primary ce lis isolated from the solar lentigo. The first aim of my project consisted on a functional exploration of the solar lentigo by the use of diverse biometrological parameters. The study was carried out on a cohort of 80 women, some of them had few (grade l) and others many solar lentiges (grade 2) on their faces. Thanks to photographie measurements to determine the grade, various biometrological approaches had quantified the rates of sebum, melanin, hemoglobin, moisturizing, light reflection and the colour (L *, a*, b* and 1T A). Results of the statistical analyses revealed that the quantity of sebumdiscriminates the skin territories of the cheek and forehead, 2) the rates of melanin, hemoglobin, light reflection and the colour were differential between affected (solar lentigo) and not affected zones, within the volunteers' cheek territory, and 3) decreased rates of light reflection and hemoglobin, as well as, increased rate moisturizing, were observed within the lesional zone between both grades. Altogether, our data highlighted some of the biometrological parameters, as indicators of the skin territory, the lesional vs non lesional areas and the progression (grade 2 vs 1) of solar lentigo The second aim covered morphological and functional analyses of the solar lentigo's primary fibroblasts. This study was carried out on a cohort of 10 volunteers and two biopsies, containing the peri-lesional and lesional areas, were taken from each person. From these biopsies, human primary fibroblasts were isolated and grown. An immunofluorescence approach revealed that the fibroblasts of solar lentigo (FL) and those from adjacent healthy areas (FS) did not depict similar morphological characteristics with a differential organization of their actin cytoskeleton. Functional approaches demonstrated that FL displayed decrease of their metabolic activity, theirproliferation rates and their migration capacity, compared to FS. On the contrary, FL showed increased secretion capacity in terms of soluble factors. Our in vitro mode! of primary fibroblasts(FL/FS), which showed similarities with in vivo fibroblast's characteristics, might be considered as an appropriate cellular mode! to test active principles targeting skin complexion heterogeneity in women with mature skin. Using clinicat and translational research approaches, both objectives highlighted indicators and biomarkers of the solar lentigo. This work contributed to better understand the impact of the intrinsic and extrinsic factors in the Joss of complexion homogeneitv.; La couleur de la peau chez un individu, appelée teint ou complexion, évolue au cours du temps et dépend de facteurs extrinsèques et intrinsèques. Une perte de son homogénéité est liée à l'apparition de lésions cutanées hyperpigmentées. Elles sont notamment provoquées par une exposition chronique au soleil et apparaissent avec l'âge, comme dans le cas du lentigo actinique. Si cette lésion hyperpigmentée bénigne est bien caractérisée à l'échelle macroscopique, peu d'études explorent ses fonctions cutanées grâce à des outils non invasifs. À l'échelle cellulaire et moléculaire, cette lésion hyperpigmentée bénigne résulte d'une altération du processus de pigmentation lors de la régulation du phénomène de photo-protection cutané. Si le modèle actuel d'une perte de cette régulation prend en compte l'altération du dialogue fonctionnel entre les couches épidermique et dermique, aucune étude ne décrit les caractéristiques fonctionnelles des cellules primaires extraites du lentigo actinique. Le premier objectif de mon projet a consisté en une exploration fonctionnelle du lentigo actinique par l'utilisation de divers paramètres biométrologiques. L'étude a été réalisée sur une cohorte de 80 femmes dont certaines présentent peu (grade 1) et, d'autres plusieurs, lentigosactiniques (grade 2) sur le visage. Après illustration du grade par une mesure photographique, différentes approches biométrologiques ont quantifié les taux de sébum, de mélanine, d'hémoglobine, d'hydratation, de réflexion de la lumière et de couleur (L *, a*, b* et lT A). Les résultats des analyses statistiques montrent que 1) la quantité de sébum discrimine les territoires cutanés de la joue et du front, 2) les taux de mélanine, d'hémoglobine, de réflexion de la lumière et de couleur sont différentiels entre les zones lésées (lentigo actinique) et non lésées, adjacentes au sein du territoire de la joue chez un volontaire, 3)que la diminution des taux de réflexion de la lumière et d'hémoglobine, ainsi que l'augmentation du taux d'hydratation, est observée au sein de la zone lésée entre les grades I et 2. L'ensemble de ces données ont mis en évidence certains paramètres biométrologiques comme indicateurs du territoire cutané, de la zone lésée vs non lésée et de l'évolution (grade 2 vs I) du lentigo actinique.Le second objectif a porté sur une analyse morphologique et fonctionnelle des fibroblastes primaires du lentigo actinique. L'étude a été réalisée sur une cohorte de 10 volontaires sur lesquels deux biopsies contenant les zones lésées et non lésées, adjacentes ont été prélevées. À partir de ces biopsies, les fibroblastes primaires humains ont été mis en culture. Une approche d'immunotluorescence révèle que les fibroblastes de lentigo actinique (FL) et ceux de la zone saine adjacente (FS) n'ont pas les mêmes caractéristiques morphologiques avec une organisation différentielle de leur cytosquelette d'actine. Une approche fonctionnelle montre que les FL ont une diminution de leur activité métabolique, de leur taux de prolifération et de leur capacité migratoire. À l'inverse, les FL sont dotés d'une augmentation de leur capacité sécrétoire en terme de facteurs solubles. Notre modèle in vitro de fibroblastes primaires (couples FL/FS), qui présentent des similitudes avec les caractéristiques décrites in vivo, représenterait un modèle cellulaire adéquat pour tester des principes actifs dont l'efficacité permettrait de réduire l'hétérogénéité du teint chez les femmes à peaux matures. Ces deux objectifs, qui ont été réalisés par des approches en recherche clinique et translationnelle, ont permis de mettre en évidence des indicateurs et des biomarqueurs du lentigoactinique qui permettront de mieux comprendre l'impact des facteurs intrinsèques et extrinsèques dans la perte d'homogénéité du teint liée au lentigo actinique.

Proteins play an important role in cells via their enzymatic activity and the irinteractions. Their functions are mainly based on the protein structure. In order to detect their presence and to characterize their structure, we used optical properties of nanostructures. The localized surface plasmon resonance (LSPR), as well as the surface enhanced Raman scattering (SERS), allowed us to detect various proteins. We also optimized nanostructures to build a sensitive, reproducible and specific biosensor based on SERS. Indeed, specific detection of one pathological biomarker, the Manganese Super Oxide Dismutase (MnSOD) protein, was investigated by using optically optimized and aptamer-functionalized nanostructures. Using this system, we were able to detect the MnSOD at physiological concentration in body fluids, such as serum and saliva. Finally, the structural study of the Spleen Tyrosine kinase (Syk) protein by SERS, allowed us to demonstrate that its structure varied with its phosphorylation levels. A complementary Western Blot analysis showed that the Syk kinase activity depended also on its phosphorylation state, meaning that the structure and the activity of Syk were linked. Altogether, these data contributed to a better understanding of the interface between physics and biology., Les protéines jouent un rôle important dans les cellules, via leur activité enzymatique et les interactions qu’elles mettent en jeu. Ces fonctions sont principalement basées sur la structure des protéines. Afin de détecter leur présence, et de caractériser leur structure, nous nous sommes appuyés sur les propriétés optiques des nanostructures. La résonance des plasmons de surface localisés (RPSL), ainsi que la diffusion Raman exaltée de surface(DRES), nous ont permis de détecter différentes protéines. Une optimisation des nanostructures nous a également permis de concevoir un biocapteur basé sur la DRES, qui soit sensible, reproductible et spécifique. En effet, la détection spécifique d’un biomarqueur pathologique, la protéine Manganèse Super Oxide Dismutase (MnSOD), a été réalisée grâce à l’utilisation de nanostructures optimisées et fonctionnalisées avec un aptamère (séquence ADN). Avec ce système, nous avons démontré la détection de la MnSOD à des concentrations physiologiques dans des fluides corporels comme le sérum et la salive. Enfin, l’étude de la structure de la protéine Spleen Tyrosine kinase (Syk), par DRES, nous a permis de mettre en évidence un réarrangement structurale de Syk lors de sa phosphorylation. Une étude complémentaire par Western Blot montre que son activité kinase est dépendante de son état de phosphorylation indiquant que la structure et l’activité de Syk sont liées. L’ensemble de ces travaux contribue à une meilleure connaissance de l’interface entre la physique et la biologie.