The direction of plasma membrane exchange between lymphocytes and accessory cells by trogocytosis is influenced by the nature of the accessory cell.

Exchange of plasma membrane fragments, including cell-surface proteins and lipids, in conjugates formed between lymphocytes and their cellular partners is a field of intense investigation. Apart from its natural occurrence during Ag recognition, the process of membrane transfer can be triggered in experimental or therapeutic settings when lymphocytes targeted by Abs are conjugated to FcgammaR-expressing accessory cells. The direction of membrane capture (i.e., which of the two cells is going to donate or accept plasma membrane fragments) can have important functional consequences, such as insensitivity of tumor cells to treatment by therapeutic mAbs. This effect, called antigenic modulation or shaving, occurs as a result of a process in which the FcgammaR-expressing cells remove the mAb and its target protein from the tumor cells. We therefore analyzed this process in conjugates formed between various FcgammaR-expressing cells and a series of normal or tumor T and B cells opsonized with different Abs capable of triggering membrane exchange (including the therapeutic Ab rituximab). Our results show that the direction of membrane capture is dictated by the identity of the FcgammaR-expressing cell, much more so than the type of lymphocyte or the Ab used. We found that monocytes and macrophages are prone to be involved in bidirectional trogocytosis with opsonized target cells, a process they can perform in parallel to phagocytosis. Our observations open new perspectives to understand the mechanisms involved in trogocytosis and may contribute to optimization of Ab-based immunotherapeutic approaches.

HSV ICP0 recruits USP7 to modulate TLR-mediated innate response.

Pattern recognition receptors represent the first line of defense against invading pathogens. Herpes simplex virus (HSV) encodes multiple ligands detected by these receptors, yet persists in the majority of infected individuals indicating a breakdown in host defense against the virus. Here we identify a novel mechanism through which HSV immediate-early protein ICP0 inhibits TLR-dependent inflammatory response by blocking NF-kappaB and JNK activation downstream of TLR signal activation. This process depends on ICP0-mediated translocation of USP7 (HAUSP) from the nucleus to cytoplasm. We show that nuclear USP7 migrates to the cytoplasm in response to TLR engagement, a process that contributes to termination of TLR response. Cytoplasmic USP7 binds to and deubiquitinates TRAF6 and IKKgamma, thus terminating TLR-mediated NF-kappaB and JNK activation. These findings suggest that USP7 is part of a negative feedback loop regulating TLR signaling and that ICP0 exploits this physiologic process to attenuate innate response to HSV. ICP0 inhibition of the TLR response serves to uncouple the innate and adaptive immune response, thereby playing a key role in HSV pathogenesis and persistence.

Ligand binding but undetected functional response of FcR after their capture by T cells via trogocytosis.

Intercellular 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.

Suitability of various membrane lipophilic probes for the detection of trogocytosis by flow cytometry.

Trogocytosis is a recently discovered phenomenon whereby lymphocytes capture fragments of the plasma membrane from antigen presenting cells (APCs). Using APCs labeled with widely used fluorescent lipophilic probes, we previously described a trogocytosis analysis protocol (TRAP) useful to understand the mechanisms and biological consequences of this process and to identify lymphocytes reacting specifically with antigen-bearing APCs. We have compared the suitability of 22 different fluorescent lipophilic probes for use in TRAP assays with cytotoxic T lymphocytes (CTL). The criteria we used were: simple and efficient incorporation in APC membranes, minimal passive diffusion among cells but efficient transfer onto T cells during trogocytosis. Sphingosin-based probes were found to incorporate inefficiently into cells. For others with unsaturated lipid chains, we found a tendency for extensive passive diffusion. In the end, about a third of the probes tested were found to be suitable in TRAP assays, which all carry either C16 or C18 saturated carbon chains, including some that can be excited with a red laser. Moreover, we found it possible to combine TRAP assays based on lipophilic probes with intracellular cytokine detection. We have identified a set of new lipophilic fluorescent probes suitable for TRAP assays in combination with intracellular staining.

Chemical labels metabolically installed into the glycoconjugates of the target cell surface can be used to track lymphocyte/target cell interplay via trogocytosis: comparisons with lipophilic dyes and biotin.

Trogocytosis, the process whereby lymphocytes capture membrane components from the cells they interact with, is classically evidenced by the transfer of fluorescent lipophilic compounds or biotinylated proteins from target cells to T or B cells. A particular class of molecules, not studied explicitly so far in the context of trogocytosis is glycoconjugates. Here, we used a method to metabolically install chemical labels in target cell glycoconjugates. Working with those target cells, we describe the conditions allowing CTL to be detected based on glycoconjugate trogocytosis triggered by antigen or stimulatory antibodies. Accordingly, we used this method to monitor the CTL response triggered in mice after vaccination. In addition, we documented the applicability of this approach to the detection of CD4(+) T and B cells. Overall, glycoconjugates were transferred between target cells and lymphocytes during trogocytosis with efficiencies comparable or higher than measured for biotinylated proteins or lipophilic dyes incorporated into general membrane lipids. From a technological point of view, our approach can be employed to detect reactive lymphocytes via glycoconjugate trogocytosis. More generally, we believe that the ever-growing ability to employ chemistry in living systems to label particular compounds will be powerful in unraveling the contributions of glycosylation to various aspects of T and B cells biology.

Different mechanisms for gamma-glutamyltransferase-dependent resistance to carboplatin and cisplatin.

In this work, we investigated the effect of gamma-glutamyltransferase (GGT) overexpression on cell viability after carboplatin treatment and compared with cisplatin. Carboplatin challenge of HeLa cells induced GGT and glutamate-cystine ligase (GCL) activities by 2- and 1.4-fold, respectively and concomitantly increased the intracellular reduced glutathione (GSH) level (1.5-fold). To study the role of GGT, HeLa-GGT cells, a stably transfected cell line overexpressing GGT (120-150 mU/mg protein) and the parental HeLa cells (10-15 mU/mg protein) were used. Both cell lines exhibited comparable viability (IC(50) approximately 150 microM) after carboplatin treatment when cultured in standard (250 microM cystine) medium. Culture in low (50 microM) cystine medium resulted in a dramatic decrease (approximately 90%) of the intracellular GSH level and to a 2.5-fold increase of carboplatin cytotoxicity (IC(50) approximately 60 microM). When GSH (50 microM) was included in the culture medium, only HeLa-GGT cells exhibited increased resistance to carboplatin. Using partially purified GGT from HeLa-GGT cells, we show that cisplatin forms adducts with cysteinylglycine, depending only on GGT activity whereas carboplatin did not efficiently react with cysteinylglycine. Thus, in this model system, GGT activity can affect platinum drugs cytotoxocity by two different ways: cisplatin can be detoxified extracellularly after reaction with the -SH group of cysteinylglycine; in the case of carboplatin, the supply of GSH precursors, initiated by GGT, increases the intracellular level of the tripeptide and provides enhanced defensive mechanisms to the cell.

Enhanced resistance of HeLa cells to cisplatin by overexpression of gamma-glutamyltransferase.

Gamma-glutamyltransferase (GGT), which is a key enzyme for the cellular glutathione (GSH) homeostasis, was shown to be overexpressed in human tumor cells selected for resistance to cisplatin and to influence the resistance of experimental tumors in vivo. We first established that cisplatin treatment of HeLa cells was accompanied by an early 3-fold induction of GGT synthesis, enhancing the possibility that this enzyme plays an important role in the cell defenses against this anticancer drug. This role was then studied using a GGT-transfected HeLa cell line (HeLa-GGT) exhibiting 10 times the activity of the parental HeLa cells (120-150 and 10-14 mU/mg protein, respectively). Both cell lines showed comparable intracellular GSH levels and cisplatin resistance when cultured in high (250 microM) or low (50 microM) cysteine-containing medium. When 50 microM of GSH were included in the low-cysteine culture medium only HeLa-GGT cells partially recovered their intracellular GSH and exhibited an increased resistance to cisplatin. Cisplatin treatment also inhibited GGT-dependent production of reactive oxygen species, a process depending on the availability of cysteinylglycine produced during GSH catabolism. Furthermore, we showed that cisplatin forms adducts with cysteinylglycine 10 times more rapidly than with GSH, and that these adducts were formed only in the extracellular medium of HeLa GGT cells. This extracellular mechanism could at least partially account for the increased resistance of GGT-rich cells to cisplatin.

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 FcRgamma 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.

Improving administration regimens of CyaA-based vaccines using TRAP assays to detect antigen-specific CD8(+) T cells directly ex vivo.

Vaccination with recombinant adenylate cyclase of Bordetella pertussis (CyaA) carrying antigen is a promising approach to target antigen-presenting cells. We have used Trogocytosis Analysis Protocol (TRAP) assays to monitor immune responses raised by different vaccination regimens with recombinant CyaA carrying the ovalbumin antigen. We find that the intradermal, intramuscular or subcutaneous routes are all superior to intravenous injections, and actually lead to a sufficiently high frequency of reactive CTL to be detected and characterized directly ex vivo by TRAP assay or other standard assays. Finally, for all routes, we find a clear boosting effect upon re-injection of the vaccine.

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.

A simple trogocytosis-based method to detect, quantify, characterize and purify antigen-specific live lymphocytes by flow cytometry, via their capture of membrane fragments from antigen-presenting cells.

We have developed a method exploiting the phenomenon of trogocytosis to detect lymphocytes reacting specifically with target cells by flow cytometry. Trogocytosis is a process by which lymphocytes capture fragments of the plasma membrane from the antigen-presenting cells (APCs) expressing their cognate antigen. For this method, a label (such as a fluorescent lipid or biotin) is first incorporated in the membrane of APCs. These labeled cells are then co-cultured for a few hours with a population of cells containing the lymphocytes to be detected. After this period of stimulation, lymphocytes that have performed trogocytosis are identified by their acquisition of the label initially present on the APC membrane using flow cytometry. A major advantage of this method is its compatibility with the simultaneous detection of phenotypic and/or functional markers on the lymphocytes. Furthermore, cells can be recovered alive and active after detection of trogocytosis, and are therefore available for further characterization or even conceivably for therapeutic purposes.

Phorbol ester regulation of the human gamma-glutamyltransferase gene promoter.

In the present study the molecular mechanisms underlying tetradecanoylphorbol-13-acetate (TPA) mediated regulation of the human gamma-glutamyltransferase (GGT) gene were examined. TPA challenge of HeLa cells resulted in an increase of GGT mRNA and enzyme activity. Deletion analysis of the promoter revealed that the -348 to +60 fragment was able to mediate TPA induced expression. Gel shift and supershift analyses showed that TPA treatment increased nuclear protein binding to a putative AP-1 site (-225 to -214) and that c-Jun was part of the complex. This AP-1 element, when cloned either in its native arrangement or as tandem repeat 5' of the minimal thymidine kinase promoter, mediated a significant increase of luciferase activity after TPA treatment of transfected HeLa cells, while its mutated counterpart abolished the induction. The same AP-1 element was able to mediate TPA induced expression in HepG2 cells. Collectively these results indicate that like other GSH metabolising enzymes, GGT too is a target for AP-1 mediated regulation.