Preferential Transfer of Certain Plasma Membrane Proteins onto T and B Cells by Trogocytosis

T and B cells capture antigens via membrane fragments of antigen presenting cells (APC) in a process termed trogocytosis. Whether (and how) a preferential transfer of some APC components occurs during trogocytosis is still largely unknown. We analyzed the transfer onto murine T and B cells of a large panel of fluorescent proteins with different intra-cellular localizations in the APC or various types of anchors in the plasma membrane (PM). Only the latter were transferred by trogocytosis, albeit with different efficiencies. Unexpectedly, proteins anchored to the PM's cytoplasmic face, or recruited to it via interaction with phosphinositides, were more efficiently transferred than those facing the outside of the cell. For proteins spanning the PM's whole width, transfer efficiency was found to vary quite substantially, with tetraspanins, CD4 and FcRγ found among the most efficiently transferred proteins. We exploited our findings to set immunodiagnostic assays based on the capture of preferentially transferred components onto T or B cells. The preferential transfer documented here should prove useful in deciphering the cellular structures involved in trogocytosis

Régulation de la gamma-glutamyltransférase humaine et effet de sa surexpression sur la cytotoxicité des anticancéreux dérivés du platine

Non disponible / Not availableLa y-glutamyl transferase (GG1) est une enzyme clé dans le métabolisme du giutathion (GSH),mais malgré son importance physiologique, les mécanismes de régulation de la GGT humaine sont très malconnus à cause de la complexité de son organisation génomique.Le premier objectif de notre travail était l'étude de la régulation du gène l (seul gène codant pour une protéine active) dans différentes lignées cellulaires. Nous avons montré qu'en réponse au TP A, aubutyrate de sodium, ou au TNFa, les modulations globales des 3 messagers issus du gène l (ARNm IA, IB eIc) reflètent la modulation de l'activité GGT. Cependant ces 3 ARNm sont régulés de manière indépendante et spécifique de la lignée considérée. Ces résultats montrent que les 3 ARNm issus du gène l sont probab1ement transcrits à partir de promoteurs indépendants, régulés différemment selon le modèlecellulaire. Nous avons ensuite étudié la régulation du promoteur B (dirigeant la transcription de l'ARNm IB)de la GGT humaine (seul promoteur décrit à l'heure actuelle) et nous avons montré que ce promoteur est induit par le TP A dans 2 lignées cellulaires. Cette induction passe par la fixation du facteur AP1 (composé d'une sous unité cJun au moins) à l'un des 3 sites AP1 proximaux étudiés. Nous avons également montré que 1es séquences situées en amont de ces sites AP1 sont impliquées dans la modulation de l'induction par le TP A et dans sa spécificité cellulaire.Enfin, nous avons localisé, cloné et partiellement caractérisé un nouveau promoteur sur le gène l humain de la GGT : celui qui dirige la transciption des ARNm Ic. Le second objectif de notre travail était de déterminer le rôle de la GGT dans la réponse cellulaire au cisplatine (CDDP) et au carboplatine, couramment utilisés en chimiothérapie. Nous avons montré qu'une des réponses rapides des cellules HeLa à un traitement par l'un de ces agents résulte en une induction du taux de GSH intracellulaire et de l'activité GGT ce qui laisse supposer un rôle important pour la GGT dans la réponse aux dérivés du platine. Nous avons utilisé une lignée HeLa isogénique, transfectée par l'ADNc de la GGT humaine (HeLa-GG1) qui expriment l'enzyme à un niveau 10 fois supérieur à celui de la lignée parentale HeLa. Cette lignée a permis de montrer que les cellules HeLa-GGT possèdent une résistance accrue aux CDDP et au carboplatine lorsque du GSH extracellulaire est fourni aux cellules. Dansle cas du carboplatine, la résistance est directement corrélée au taux de GSH intracellulaire. En revanche, le contenu intracellulaire en GSH ne semble pas être un facteur déterminant pour la réponse au CDDP dans notre modèle. En effet, les cellules HeLa cultivées dans un milieu complet possèdent un fort taux de GSHb intracellulaire et lorsque le milieu de culture est appauvri en cystéine elles perdent plus de 80 % de leur pool (indépendamment de leur activité GGT). Or, la cytotoxicité du CDDP est comparable dans ces deux conditions de culture. Afin d'expliquer la plus grande résistance des cellules HeLa-GGT au CDDP lorsque du GSH extracellulaire est fourni, nous avons étudié le catabolisme du GSH par la GGT au niveau extracellulaire. Nous avons alors montré que le CDDP est capable d'interagir avec le produit de dégradation du GSH par la GGT : la CysGly. Ce composé est d'ailleurs plus réactif que le GSH et il forme des adduits avec le CDDP 10 fois plus rapidement que le GSH. Nous avons également été capables de détecter cet adduit dans 1e milieu extracellulaire des cellules HeLa-GGT incubées en présence de CDDP et de GSH. Nous suggérons que ce mécanisme dépendant de la GGT pourrait être impliqué dans la résistance des ceUules au CDDP en diminuant la quantité de CDDP disponible et/ou sa toxicité

Régulation de la gamma-glutamyltransférase humaine et effet de sa surexpression sur la cytotoxicité des anticancéreux dérivés du platine

La y-glutamyl transferase (GG1) est une enzyme clé dans le métabolisme du giutathion (GSH),mais malgré son importance physiologique, les mécanismes de régulation de la GGT humaine sont très malconnus à cause de la complexité de son organisation génomique.Le premier objectif de notre travail était l'étude de la régulation du gène l (seul gène codant pour une protéine active) dans différentes lignées cellulaires. Nous avons montré qu'en réponse au TP A, aubutyrate de sodium, ou au TNFa, les modulations globales des 3 messagers issus du gène l (ARNm IA, IB eIc) reflètent la modulation de l'activité GGT. Cependant ces 3 ARNm sont régulés de manière indépendante et spécifique de la lignée considérée. Ces résultats montrent que les 3 ARNm issus du gène l sont probab1ement transcrits à partir de promoteurs indépendants, régulés différemment selon le modèlecellulaire. Nous avons ensuite étudié la régulation du promoteur B (dirigeant la transcription de l'ARNm IB)de la GGT humaine (seul promoteur décrit à l'heure actuelle) et nous avons montré que ce promoteur est induit par le TP A dans 2 lignées cellulaires. Cette induction passe par la fixation du facteur AP1 (composé d'une sous unité cJun au moins) à l'un des 3 sites AP1 proximaux étudiés. Nous avons également montré que 1es séquences situées en amont de ces sites AP1 sont impliquées dans la modulation de l'induction par le TP A et dans sa spécificité cellulaire.Enfin, nous avons localisé, cloné et partiellement caractérisé un nouveau promoteur sur le gène l humain de la GGT : celui qui dirige la transciption des ARNm Ic. Le second objectif de notre travail était de déterminer le rôle de la GGT dans la réponse cellulaire au cisplatine (CDDP) et au carboplatine, couramment utilisés en chimiothérapie. Nous avons montré qu'une des réponses rapides des cellules HeLa à un traitement par l'un de ces agents résulte en une induction du taux de GSH intracellulaire et de l'activité GGT ce qui laisse supposer un rôle important pour la GGT dans la réponse aux dérivés du platine. Nous avons utilisé une lignée HeLa isogénique, transfectée par l'ADNc de la GGT humaine (HeLa-GG1) qui expriment l'enzyme à un niveau 10 fois supérieur à celui de la lignée parentale HeLa. Cette lignée a permis de montrer que les cellules HeLa-GGT possèdent une résistance accrue aux CDDP et au carboplatine lorsque du GSH extracellulaire est fourni aux cellules. Dansle cas du carboplatine, la résistance est directement corrélée au taux de GSH intracellulaire. En revanche, le contenu intracellulaire en GSH ne semble pas être un facteur déterminant pour la réponse au CDDP dans notre modèle. En effet, les cellules HeLa cultivées dans un milieu complet possèdent un fort taux de GSHb intracellulaire et lorsque le milieu de culture est appauvri en cystéine elles perdent plus de 80 % de leur pool (indépendamment de leur activité GGT). Or, la cytotoxicité du CDDP est comparable dans ces deux conditions de culture. Afin d'expliquer la plus grande résistance des cellules HeLa-GGT au CDDP lorsque du GSH extracellulaire est fourni, nous avons étudié le catabolisme du GSH par la GGT au niveau extracellulaire. Nous avons alors montré que le CDDP est capable d'interagir avec le produit de dégradation du GSH par la GGT : la CysGly. Ce composé est d'ailleurs plus réactif que le GSH et il forme des adduits avec le CDDP 10 fois plus rapidement que le GSH. Nous avons également été capables de détecter cet adduit dans 1e milieu extracellulaire des cellules HeLa-GGT incubées en présence de CDDP et de GSH. Nous suggérons que ce mécanisme dépendant de la GGT pourrait être impliqué dans la résistance des ceUules au CDDP en diminuant la quantité de CDDP disponible et/ou sa toxicité.NANCY1-SCD Pharmacie-Odontologie (543952101) / SudocSudocFranceF

HSV Encoded ICP-0 Inhibits TLR Signaling in CLL Cells by Targeting TRAF-6

Abstract Innate immunity to viral vectors has largely been overlooked in development of gene therapy strategies and may play a key role in shaping immune responses and potentially clinical outcomes. In the setting of cancer immune therapy, the innate response may influence subsequent evolution of an anti-tumor adaptive immune response. We have studied two forms of HSV based amplicon vectors packaged in the presence or absence of HSV helper virus for use in immune therapy of chronic lymphocytic leukemia (CLL). Our previous work showed that the presence of helper-virus in HSV amplicon stocks impedes the development of tumor-specific adaptive immunity, and inhibits CTL development. We asked whether treatment outcomes could be explained by differences in the innate immune response elicited by the two vectors. We characterized innate immune response in CLL B cells transduced by helper-virus free (HF-HSV) vs. helper-virus containing (H+-HSV) amplicon vectors. Transduction of CLL B cells with HF-HSV induced robust innate cytokine/chemokine response that was not seen when transduction at similar efficiency was performed with H+-HSV vector. We profiled mRNA expression of TLR 1–10 in CLL B cells by quantitative PCR and confirmed the expression of TLR 2 and 9, which play a key role in mediating HSV innate response. Using 293 stably expressing TLR2/6 and TLR9, we show that the discrepancy in innate response of CLL B cells to transduction with HF-HSV and H+-HSV corresponded to differences seen in these viruses’ capacity to activate TLR-2 and TLR-9. Pre-incubation with cyclohexamide restored H+-HSV capacity to activate TLR2 and 9 suggesting that H+-HSV encodes an inhibitory protein that interferes with TLR signaling. A screen of the four HSV immediate early (IE) proteins (ICP-0, ICP-22, ICP-27 and ICP-47) encoded by HSV Helper-virus identified ICP-0 as a likely candidate for inhibition of TLR signaling. Ectopic expression of ICP-0 inhibited innate response to HF-HSV in TLR2/6. In addition, ICP-0 inhibited other TLRs (TLR3, 4, 7, and 8) and IL-1 receptor mediated NF-κB activation, suggesting that ICP-0 targets a common signaling protein downstream of these receptors. Over-expression of various TLR signaling components identified TRAF-6 as a target for ICP-0 mediated inhibition of the NF-κB response. Finally, we correlated HSV vector-mediated innate response to capacity to prime an adaptive anti-CLL T cell response. A comparison of immune function of CD40L and B7.1 encoded by either hf-HSV or H+-HSV to prime autologous T cell response in vitro showed a clear advantage for HF-HSV amplicons encoding CD40L and B7.1 compared to H+-HSV amplicons. Our results highlight a novel mechanism through which HSV suppresses host innate immunity through a hitherto un-described ICP-0 inhibition of TLR/IL-1R signaling. The work also establishes a potential link between vector mediated innate immunity and ability to prime an adaptive anti-tumor immune response against CLL cells, which has important implications in relation to development of gene therapy vectors for clinical testing

HSV ICP0 Inhibits TLR-Mediated NF-κB Response to TLR Signaling

Abstract HSV infection activates a robust innate response through engagement of multiple pattern recognition receptors (PRR) including both TLR (TLR2 and TLR9) as well as non-TLR. Signaling events downstream of these receptors activate NF-κB and IRF3 responsive genes and initiate an innate inflammatory response aimed at controlling viral replication and spread. In this work, we studied immune suppressive activity of a replication-defective HSV virus and identified the immediate early protein ICP0 as a negative regulator of both NF-κB and IRF3 signaling. ICP0 possesses an ubiquitin E3 ligase function through its NH2 RING domain, as well as de-ubiquitinating activity through its association with the cellular de-ubiquitinating enzyme USP7 (HAUSP). We show that these two domains of ICP0 function independently to suppress IRF3 and NF-κB signaling, respectively and, in the process, effectively shut down host innate immunity to HSV infection. Although ICP0 inhibition of IRF3 has been reported, inhibition of TLR-mediated NF-κB response has not been previously described. We show that ICP0 globally inhibits NF-κB response to all TLR receptors as well as IL-1R. ICP0 exerts this activity by associating with USP7 and altering its cellular localization from a nuclear to cytoplasmic protein. In the cytosol, USP7 associates with and de-ubiquitinates TRAF6 and IKK-γ (NEMO), two signaling components of the TLR-mediated NF-κB pathway that are poly-ubiquitinated upon TLR activation. ICP-0 expression vectors harboring point mutations/deletions that target the RING domain E3 ligase function or compromise ICP-0 ability to bind USP7 would selectively inhibit its ability to interfere with either IRF3 or NF-kB signaling, respectively. In support of this, knockdown of endogenous USP7 by RNAi severely impaired ICP0-mediated inhibition of NF-κB response while leaving its capacity to inhibit IRF3 intact. In contrast, over-expression of USP-7 was sufficient to inhibit TLR-mediated NF-κB response. Ability of ICP-0 to inhibit both IRF3 and NF-κB signaling pathways, the former through its E3 ligase function and the latter through its association with USP-7, affords HSV comprehensive protection from host immunity during repeated cycles of lytic infection and reactivation from latency. The work also identifies a rare example of how two seemingly contradictory biologic functions resident within ICP0, namely ubiquitin E3 ligase activity at the NH2 terminus RING domain and de-ubiquitinating activity through association with USP-7, could cooperate to inhibit multiple signaling pathways necessary for efficient silencing of innate immunity. Finally, the work also identifies a previously unknown function for USP7 in regulating innate signaling beyond its known function as a regulator of p53

Ligand Binding but Undetected Functional Response of FcR after Their Capture by T Cells via Trogocytosis

International audienceIntercellular transfer of cell surface proteins by trogocytosis is common and could affect T cell responses. Yet, the role of trogocytosis in T cell function is still elusive, and it is unknown whether a molecule, once captured by T cells, harbors the same biological properties as in donor APC. In this study, we showed that FcgammaR as well as the associated FcRgamma subunit could be detected at high levels on murine and human T cells after their intercellular transfer from FcgammaR-expressing APC. Capture of FcgammaR occurred during coculture of T cells with FcgammaR-expressing APC upon Ab- or Ag-mediated T cell stimulation. Once captured by T cells, FcgammaR were expressed in a conformation compatible with physiological function and conferred upon T cells the ability to bind immune complexes and to provision B cells with this source of Ag. However, we were unable to detect downstream signal or signaling-dependent function following the stimulation of FcgammaR captured by T cells, and biochemical studies suggested the improper integration of FcgammaR in the recipient T cell membrane. Thus, our study demonstrates that T cells capture FcgammaR that can efficiently exert ligand-binding activity, which, per se, could have functional consequences in T cell-B cell cooperation

HSV Amplicon Transduction Activates an Innate Immune Response in CLL B Cells;Implications for Immune Therapy and Vaccine Development

Abstract Since gene therapy vectors are designed to shuttle therapeutic genes into various tissues, efficiency transduction is often viewed as the most critical measure of a vector’s clinical usefulness. However, other variables such as activation of the host’s innate immunity may play an equally important role shaping the clinical outcome. We have studied the application of helper-free HSV (hf-HSV) amplicon vectors to deliver costimulatory ligands into chronic lymphocytic leukemia (CLL) B cells for the purpose of enhancing their antigen presenting capacity. In this work, we examine the innate immune response of CLL B cells to transduction with helper-virus free HSV (hf-HSV) amplicon vectors. We screened culture supernatant of CLL B cells transduced with helper-free HSV amplicon for secretion of IL-6, TNF-α and interferon β by ELISA and observed significant induction of these cytokines upon hf-HSV transduction as compared to non-transduced and CLL cells transduced with helper virus-containing (H+-HSV) HSV amplicons. We also compared the effects of hf-HSV and H+-HSV amplicon vector transduction on surface expression of NKG2D ligand. CLL B cells transduced with hf-HSV but not H+-HSV amplicons upregulated the costimulatory NKG2D ligands MICA and MICB. To investigate mechanisms of HSV activation of innate immunity, we screened a panel of human TLR receptors for recognition of HSV amplicons using 293T cells transfected with individual human TLR receptors and assaying NF-κB driven luciferase activity as a read-out. We found that HSV amplicon activates TLR3 (receptor for double stranded RNA), TLR8 (receptor for single stranded RNA) and TLR9 (receptor for unmethylated CpG motifs). CLL B cells express predominantly TLR9 and to a lesser extent TLR8. TLR3, on the other hand is not expressed on resting B cells and is upregulated upon exposure to interferon β. In 293T cells lacking TLR receptors, viable hf-HSV but not UV-inactivated amplicons up-regulated surface expression of both MICA and MICB and activated interferon-β promoter driven luciferase, indicating the presence of a TLR-independent HSV activated innate response. To establish the presence of TLR-dependent NKG2D ligand induction, CLL B cells (expressing TLR7, 8 and 9) were incubated with their specific ligands (Loxaribine and CpG ODN motifs) for 24 hours and MICA and MICB expression confirmed by flow-cytometry. In summary, our data shows that hf-HSV amplicon vectors activate multiple aspects of innate immunity involving both TLR dependent and independent pathways, which translates into expression of cytokines and costimulatory ligands by transduced cells. The presence of helper virus on the other hand completely abolishes this response. In the setting of a vaccine development, the evoked innate response may prime the adaptive T cell response leading to improved clinical outcome

Tumor necrosis factor alpha induces gamma-glutamyltransferase expression via nuclear factor-kappaB in cooperation with Sp1

Gamma-glutamyltransferase (GGT) cleaves the gamma-glutamyl moiety of glutathione (GSH), an endogenous antioxidant, and is involved in mercapturic acid metabolism and in cancer drug resistance when overexpressed. Moreover, GGT converts leukotriene (LT) C4 into LTD4 implicated in various inflammatory pathologies. So far the effect of inflammatory stimuli on regulation of GGT expression and activity remained to be addressed. We found that the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) induced GGT promoter transactivation, mRNA and protein synthesis, as well as enzymatic activity. Remicade, a clinically used anti-TNFalpha antibody, small interfering RNA (siRNA) against p50 and p65 nuclear factor-kappaB (NF-kappaB) isoforms, curcumin, a well characterized natural NF-kappaB inhibitor, as well as a dominant negative inhibitor of kappaB alpha (IkappaBalpha), prevented GGT activation at various levels, illustrating the involvement of this signaling pathway in TNFalpha-induced stimulation. Over-expression of receptor of TNFalpha-1 (TNFR1), TNFR-associated factor-2 (TRAF2), TNFR-1 associated death domain (TRADD), dominant negative (DN) IkappaBalpha or NF-kappaB p65 further confirmed GGT promoter activation via NF-kappaB. Linker insertion mutagenesis of 536 bp of the proximal GGT promoter revealed NF-kappaB and Sp1 binding sites at -110 and -78 relative to the transcription start site, responsible for basal GGT transcription. Mutation of the NF-kappaB site located at -110 additionally inhibited TNFalpha-induced promoter induction. Chromatin immunoprecipitation (ChIP) assays confirmed mutagenesis results and further demonstrated that TNFalpha treatment induced in vivo binding of both NF-kappaB and Sp1, explaining increased GGT expression, and led to RNA polymerase II recruitment under inflammatory conditions

Tumor necrosis factor α induces γ-glutamyltransferase expression via nuclear factor-κB in cooperation with Sp1

International audienceGamma-glutamyltransferase (GGT) cleaves the gamma-glutamyl moiety of glutathione (GSH), an endogenous antioxidant, and is involved in mercapturic acid metabolism and in cancer drug resistance when overexpressed. Moreover, GGT converts leukotriene (LT) C4 into LTD4 implicated in various inflammatory pathologies. So far the effect of inflammatory stimuli on regulation of GGT expression and activity remained to be addressed. We found that the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) induced GGT promoter transactivation, mRNA and protein synthesis, as well as enzymatic activity. Remicade, a clinically used anti-TNFalpha antibody, small interfering RNA (siRNA) against p50 and p65 nuclear factor-kappaB (NF-kappaB) isoforms, curcumin, a well characterized natural NF-kappaB inhibitor, as well as a dominant negative inhibitor of kappaB alpha (IkappaBalpha), prevented GGT activation at various levels, illustrating the involvement of this signaling pathway in TNFalpha-induced stimulation. Over-expression of receptor of TNFalpha-1 (TNFR1), TNFR-associated factor-2 (TRAF2), TNFR-1 associated death domain (TRADD), dominant negative (DN) IkappaBalpha or NF-kappaB p65 further confirmed GGT promoter activation via NF-kappaB. Linker insertion mutagenesis of 536 bp of the proximal GGT promoter revealed NF-kappaB and Sp1 binding sites at -110 and -78 relative to the transcription start site, responsible for basal GGT transcription. Mutation of the NF-kappaB site located at -110 additionally inhibited TNFalpha-induced promoter induction. Chromatin immunoprecipitation (ChIP) assays confirmed mutagenesis results and further demonstrated that TNFalpha treatment induced in vivo binding of both NF-kappaB and Sp1, explaining increased GGT expression, and led to RNA polymerase II recruitment under inflammatory conditions

gamma-Glutamyl transpeptidase catalyses the extracellular detoxification of cisplatin in a human cell line derived from the proximal convoluted tubule of the kidney.

Nephrotoxicity is a side-effect and the main factor limiting the clinical use of cisplatin. In vivo, the administration of the cysteinecontaining tripeptide glutathione (GSH) has been found to reduce nephrotoxicity, but the biochemical mechanism of this protective action is not fully understood. The present study was designed to gain insights into the mechanism by which GSH prevents cisplatin nephrotoxicity. We also wanted to verify the hypothesis of whether the protective action of GSH is mediated by products of the extracellular breakdown of GSH catalysed by g-glutamyl transpeptidase (GGT), an enzyme that is highly expressed in kidney tubular cells. The study was performed in HK-2 cells, derived from the immortalisation of human kidney proximal tubule cells. We investigated the influence of modulators of GGT activity and/or thiols on the antiproliferative activity of cisplatin and on the intracellular GSH content. We determined the antiproliferative activity of cisplatin, platinum cellular accumulation and DNA platination following precomplexing of the drug with thiols. The antiproliferative effect of cisplatin was minimally affected by the addition of GSH. However, when the antiproliferative assay was performed in the presence of glycyl-glycine (GlyGly), to serve as a transpeptidation acceptor and thus to stimulate GGT-mediated GSH catabolism, cisplatin-induced growth inhibition was largely prevented. This effect was not mediated through an increase of intracellular GSH levels, which were not affected by the GlyGly supplementation. The thiol dipeptide cysteinyl-glycine, i.e. the GSH catabolite generated by GGT activity, showed a higher reactivity against cisplatin in vitro than GSH, as was shown by the more rapid oxidation of its –SH groups. The cisplatin/GSH or cisplatin/cysteinyl-glycine adducts did not display an antiproliferative effect. However, 2 h precomplexing with GSH in the presence of GGT, or directly with the GSH catabolite cysteinyl-glycine, decreased the antiproliferative effect of cisplatin and drug-induced DNA platination to a greater extent than precomplexing with GSH alone. The results of the present study show that, in HK-2 cells, extracellular GSH decreases the antiproliferative effects of cisplatin only upon its hydrolysis by GGT, thereby supporting the hypothesis that the extracellular metabolism of GSH by GGT plays a role in modulating cisplatin nephrotoxicity. A primary role in the protection of HK-2 cells appears to be played by cysteinyl-glycine, the proximal product of the GGT-mediated hydrolysis of GSH, which shows a high reactivity against CDDP resulting in the rapid inactivation of the drug