Réponse des Lymphocytes T Gamma-Delta à deux Complications de la transplantation rénale (le Cytomégalovirus et les anticorps spécifiques du donneur)

La transplantation rénale est la stratégie de suppléance rénale la plus performante. Le renforcement des thérapeutiques ciblant la réponse cellulaire T (i) a conduit à réévaluer la réponse allogénique humorale et (ii) a souligné deux complications majeures de la pression immunosuppressive : l infection à cytomégalovirus CMV et le risque de cancer. Dans le travail présenté ici, nous analysons d abord l impact histologique de deux de ces facteurs de détérioration de l allogreffon rénal : l infection à CMV avant une biopsie sur indication et la pathogénicité des anticorps anti-HLA dirigés contre le donneur, détectés par des techniques d identification en Single Antigen in situ dans le greffon. Nous montrons ensuite comment les lymphocytes T (LT) g Vd2neg font le lien entre CMV et DSA : induits par le CMV, les LT g Vd2neg participent aux lésions médiées par les DSA par leur capacité à réaliser une lyse dépendante de l anticorps (ADCC) impliquant le CD16. En plus de cette nouvelle fonction allogénique indirecte, les LT g Vd2neg possèdent une double réactivité anti-CMV et anti-tumoral. Nous présentons ici un modèle où les lymphocytes T g V 2neg s activent spécifiquement de façon TCR dépendante par la reconnaissance d un marqueur d intégrité épithéliale (EphA2) : ils détournent le mécanisme d interaction classique d EphA2 avec ses ligands naturels éphrines A1 et A4 pour s en faire un signal de costimulation, en s appuyant sur le contexte de stress pour renforcer son activation. Collectivement, nos résultats contribuent à mieux préciser la bioréactivité et le rôle des LT g V 2neg en transplantation rénale. Nos données suggèrent que chez l'homme, a fortiori lorsqu il est immunodéprimé, les LT g constituent un compartiment de surveillance lymphoide du stress, capable de censurer la dérégulation des cellules infectées ou transformées et de prendre part à la réponse allogénique par un mécanisme d ADCC.Kidney transplant is the most performant strategy for renal replacement therapy. Increasing treatment targetting T cell response has led (i) to reappraise the importance of humoral allogenic response, (ii) to underline two main complications subsequent to immunosuppressive pressure : cytomegalovirus infection and tumorigenesis. Here, we first report the pathological impact of two of these factors on kidney allograft deterioration : CMV infection prior a biopsy for cause and Donor Specific Alloantibodies (DSA) detected within the graft with single antigen flow bead assay. Then we showed that CMV-induced V 2neg g T cells are a new player and a potentially useful clinical biomarker in antibody-mediated lesions of kidney transplants. By engaging DSA on their Fcg-receptor CD16, g T cells participate in allograft lesions mediated by DSA through antibody-dependent cell cytotoxicity (ADCC). In addition to this new indirect allogenic function, CMV-induced V 2neg g T cells displayed a dual anti-CMV and anti-tumor reactivity. Finally, we here identified EphA2 as a new stress-regulated antigen targetting by a non V 2neg g T cell clone, which recognition implies the hijacking of the natural EphA2-ephrin interactions to activation. These data suggest that in humans, a fortiori when immunosuppressed, g T cells compose a lymphoid stress-surveillance compartment, capable of recognizing the dysregulated state of infected or transformed cells and to take part to allogenic response through an ADCC mechanism.BORDEAUX2-Bib. électronique (335229905) / SudocBORDEAUX1-Bib.electronique (335229901) / SudocSudocFranceF

PLoS Pathog

Cytomegalovirus (CMV) is a leading infectious cause of morbidity in immune-compromised patients. γδ T cells have been involved in the response to CMV but their role in protection has not been firmly established and their dependency on other lymphocytes has not been addressed. Using C57BL/6 αβ and/or γδ T cell-deficient mice, we here show that γδ T cells are as competent as αβ T cells to protect mice from CMV-induced death. γδ T cell-mediated protection involved control of viral load and prevented organ damage. γδ T cell recovery by bone marrow transplant or adoptive transfer experiments rescued CD3ε-/- mice from CMV-induced death confirming the protective antiviral role of γδ T cells. As observed in humans, different γδ T cell subsets were induced upon CMV challenge, which differentiated into effector memory cells. This response was observed in the liver and lungs and implicated both CD27+ and CD27- γδ T cells. NK cells were the largely preponderant producers of IFNγ and cytotoxic granules throughout the infection, suggesting that the protective role of γδ T cells did not principally rely on either of these two functions. Finally, γδ T cells were strikingly sufficient to fully protect Rag-/-γc-/- mice from death, demonstrating that they can act in the absence of B and NK cells. Altogether our results uncover an autonomous protective antiviral function of γδ T cells, and open new perspectives for the characterization of a non classical mode of action which should foster the design of new γδ T cell based therapies, especially useful in αβ T cell compromised patients

Recent advances in γδ T cell biology: New ligands, new functions, and new translational perspectives

Gamma/delta (γδ) T-cells are a small subset of T-lymphocytes in the peripheral circulation but constitute a major T-cell population at other anatomical localizations such as the epithelial tissues. In contrast to conventional a/ß T-cells, the available number of germline genes coding for T-cell receptor (TCR) variable elements of γδ T-cells is very small. Moreover, there is a prefential localization of γδ T-cells expressing given Vgamma and Vdelta genes in certain tissues. In humans, γδ T-cells expressing the Vg9Vd2-encoded TCR account for anywhere between 50 and >95% of peripheral blood γδ T-cells, whereas cells expressing non-Vd2 genes dominate in mucosal tissues. In mice, there is an ordered appearance of γδ T-cell „waves“ during embryonic development, resulting in preferential localization of γδ T-cells expressing distinct VgammaVdelta genes in the skin, the reproductive organs, or gut epithelia. The major function of γδ T-cells resides in local immunosurveillance and immune defense against infection and malignancy. This is supported by the identification of ligands that are selectively recognized by the γδ TCR. As an example, human Vgamma9Vdelta2 T-cells recognize phosphorylated metabolites („phosphoantigens“) that are secreted by many pathogens but can also be overproduced by tumor cells, providing a basis for a role of these γδ T-cells in both anti-infective and anti-tumor immunity. Similarly, the recognition of endothelial protein C receptor by human non-Vdelta2 γδ T-cells has recently been identified to provide a link for the role for such γδ T-cells in immunity against epithelial tumor cells and cytomegalovirus-infected endothelial cells. In addition to „classical“ functions such as cytokine production and cytotoxicity, recent studies suggest that subsets of γδ T-cells can exert additional functions such as regulatory activity and – quite surpisingly – „professional“ antigen-presenting capacity. It is currently not well known how this tremendous extent of functional plasticity is regulated and what is the extent of γδ TCR ligand diversity. Due to their non-MHC-restricted recognition of unusual stress-associated ligands, γδ T-cells have raised great interest as to their potential translational application in cell-based immunotherapy. Topics of this Research Focus include: Molecular insights into the activation and differentiation requirements of γδ T-cells, role of pyrophosphates and butyrophilin molecules for the activation of human γδ T-cells, role of γδ T-cells in tumor immunity and in other infectious and non-infectious diseases, and many others. We are most grateful to all colleagues who agreed to write a manuscript. Thanks to their contributions, this E-book presents an up-to-date overview on many facets of the still exciting γδ T-cells

Caractérisation des fonctions effectrices des lymphocytes T [gamma delta V delta deux négatifs] impliqués dans la réponse immunitaire dirigée contre le cytomégalovirus (rôle de NKG2D, KIR2DS2 et de l'interaction avec les cellules dendritiques)

Chez les individus sains, les lymphocytes T gdelta représentent entre 0,5 et 10 % des lymphocytes T totaux.Des études antérieures menées au laboratoire ont montré que ce pourcentage est augmenté chez des transplantés rénaux développant une infection à cytomégalovirus (CMV). De plus, des clones T gdeltaVdelta2neg isolés chez ces patients présentent une forte réactivité in vitro contre des fibroblastes infectés par le CMV. Dans ce travail de thèse , nous nous sommes intéressés aux mécanismes d'activation des lymphocytes T gdelta Vdelta2neg. Nous avons montré que deux molécules peuvent être impliquées dans cette activation : NKG2D et KIR2DS2. La molécule KIR2DS2 peut potentialiser le signal transmis par le TCR pour induire la production des cytokines IFN-g et TNF-a. Si l'engagement par la molécule NKG2D ne va pas induire la sécrétion de cytokines, cette molécule peut agir comme molécule stimulatrice per se ou comme molécule de co-stimulation sur la cytotoxicité de lignées ou de clones Tgdelta V2neg. Dans la deuxième partie, nous nous sommes intéressés aux interactions entre les lymphocytes T gdelta Vdelta2neg et les cellules dendritiques (DC) infectées par le CMV. Nos résultats montrent que, même dans des conditions où le pourcentage de DC infectées est faible, les clones T gdelta Vdelta2neg vont sécréter du TNF-a et de l'INF-g au contact des DC préalablement mises au contact avec le CMV. De plus, nos résultats suggèrent que lors de l'infection, les DC vont être protégées de la lyse par ces clones. L'ensemble de ces résultats permettent d'approfondir nos connaissances sur les mécanismes d'activation des lymphocytes T gdelta V delta2neg, ouvrant de nouvelles perspectives pour l'utilisation thérapeutique de ces cellules.In healthy individuals, gdelta T lymphocytes represent 0,5 to 10 % of total T lymphocytes. Previous studies in our laboratory have shown that this percentage can be increased in renal transplant recipients who have developed cytomegalovirus (CMV) infections. Moreover, V delta2neg gdelta T cell clones isolated from these recipients show a strong reactivity in vitro against CMV infected fibroblasts. In this work, we have focused our attention on the mechanisms of V delta2neg gdelta T lymphocyte activation. Two molecules are involved in this activation : NKG2D and KIR2DS2. We observed that KIR2DS2 was able to increase the TCR signal involved in IFN-g and TNF-a production. Although the engagement of the NKG2D molecule did not induce cytokine secretion, this molecule could act as a stimulatory molecule per se, or as a co-stimulatory molecule on V delta2neg gdelta T cell clones or lines cytotoxicity. In the second part, we analyzed the interaction between Vdelta2neg gdelta T cells and CMV-infected dendritic cells (DC). Our results show that, even when the percentage of CMV-infected DC was low, TNF-a and INF-g production by Vdelta2neg gdelta T cell clones was enhanced when they were co-cultured with DC previously incubated with CMV. Moreover, our results suggest that when the infection occurs, the DC will be protected from the Vdelta2neg gdelta T cell clone cytotoxicity. Taken together, these results increase our knowledge on the activation mechanisms of Vdelta2neg gdelta T lymphocytes by opening new approaches for the therapeutic use of these cells.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Etude de l'implication des lymphocytes Tgð humains dans la réponse immunitaire dirigée contre le Cytomegalovirus

Chez le sujet sain, les cellules T(gamma delta) circulantes ne représentent que 0,5-6 % des lymphocytes T. Une étude dans notre laboratoire a montré que ces cellules subissent une expansion chez les transplantés rénaux développant une infection à Cytomégalovirus (CMV). Nous montrons ici qu'elle est concomittante de la résolution de l'infection, indiquant que la détermination du pourcentage de cellules T(gamma delta) est un facteur fiable de bon pronostic chez ces patients. Ce phénomène survient également chez les transplantés hépatiques, cardiaques, pulmonaires et cardio-pulmonaires. L'expansion de ces cellules s'accompagne de leur activation et ne concerne que les sous-populations V(delta)1+, V(delta)3+ et V(delta)5+, suggérant que toutes les cellules T(gamma delta) hormis les cellules T V(delta)2+ peuvent subir une expansion après infection à CMV. L'analyse de la diversité jonctionnelle du TCR gamma delta révèle une restriction marquée de leur répertoire, indiquant une sélection induite par un antigène. Les cellules T V(delta)1+ et V(delta)3+ des transplantés rénaux infectés par le CMV sont capables de proliférer spécifiquement in vitro en présence de lysats de fibroblastes infectés par le CMV. Chez le sujet sain, les cellules T V(delta)2 présentent également un répertoire restreint après infection à CMV et s'expandent in vivo mais plus faiblement, suggérant que l'immunosuppression amplifie un phénomène physiologique. Les cellules T V(delta)2 amplifiées chez les transplantés rénaux expriment le récepteur natural killer (NKR) CD94, mais pas le NKG2A ni le NKG2B. La diversité combinatoire limitée des cellules T(gamma delta) et la restriction de leur répertoire chez les patients infectés par le CMV nous a permis de prouver qu'une population T monoclonale peut exprimer un phénotype NKR hétérogène. Ces résultats indiquent que le CMV est un facteur environnemental majeur qui forge le répertoire des lymphocytes T(gamma delta) circulants chez l'homme, et que ces cellules peuvent jouer un rôle dans la réponse immunitaire dirigée contre le CMV, en particulier chez les immunodéprimés.In healthy individuals, circulating (gamma delta) T cells only represent 0,5-6 % of total T lymphocytes. Previous study in our laboratory demonstrated that an expansion of circulating (gamma delta)T cells occurs in renal transplanted patients when they develop an infection with cytomegalovirus (CMV). Here, we show that this expansion is concomitant with the resolution of CMV infection in renal allograft recipients, pointing to (gamma delta)T cell percentage determination as a reliable prognosis factor to predict the resolution of CMV infection. Moreover, CMV-associated (gamma delta)T cell expansion also occurs in liver, heart, lung and heart+lung transplanted patients. This increase is associated with an activation of (gamma delta)T cells, and concerned only V(delta)3+ and V(delta)5+ sub-populations suggesting that all (gamma delta)T cells but V(delta)2+ cells expand after CMV infection. Analysis of (gamma delta)TCR junctionnal diversity revealed a marked restriction in the repertoire of expanded cells, suggesting their antigen-driven selection. V(delta)1 and V(delta)3 T cells from CMV-infected kidney recipients were able to specifically proliferate in vitro in the presence of CMV-infected fibroblast lysates.In healthy subjects, V(delta)2neg (gamma delta)T cells similarly present a restricted repertoire after CMV infection and expand in vivo but to a much lower level, suggesting that immunosuppression amplify a physiologic phenomenon. Expanded V(delta)2neg (gamma delta)T cells in transplanted patients express natural killer cell receptors (NKR), notably CD94 but not NKG2A or B. Limited combinatorial diversity of (gamma delta)T cells and their very restricted repertoire in CMV-infected patients permitted to prove that a monoclonal T cell population can express a heterogenous NKR-phenotype. Altogether, these finding indicate that CMV is a major environmental factor forging the circulating (gamma delta)T cell repertoire in human, and that these cells might play an important role in the immune response to CMV infection, particularly in immunosuppressed patients.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Am J Transplant

Universal prophylaxis for cytomegalovirus (CMV) prevention is viable but, compared with a preemptive strategy, leads to higher incidence of late-onset disease (LOD) associated with poor patient and graft survival. The purpose of this study was to compare LOD with early onset disease (EOD), with a focus on the highest risk kidney transplant recipients (KTRs): CMV seronegative recipients transplanted from seropositive donors (D+R-). Since CMV control depends on both antiviral treatment and specific immune response, we also compared Vδ2-negative (Vδ2(neg) ) γδ T cell expansion involved in CMV infection resolution. EOD was defined as occurring 3 mo after transplantation. Depending on the period, universal prophylaxis or preemptive treatment was used. Overall, 168 D+R- KTRs were included between 2003 and 2011. LOD was associated with a lower peak DNAemia (p = 0.04), fewer recurrences (odds ratio 0.16; 95% confidence interval 0.05-0.55; p = 0.01) and shorter anti-CMV curative treatment (40 vs. 60 days, p < 0.0001). As a corollary, we found that Vδ2(neg) γδ T cell expansion was faster in LOD than in EOD (31 vs. 168 days after the beginning of CMV disease, p < 0.0001). In D+R- KTRs, universal prophylaxis is associated with more LOD, which had better infection management and a faster immune response. These results support the use of universal prophylaxis over a preemptive strategy and reappraise outcomes of LOD

Innate (and Innate-like) Lymphoid Cells: Emerging Immune Subsets With Multiple Roles Along Transplant Life.

International audienceTransplant immunology is currently largely focused on conventional adaptive immunity, particularly T and B lymphocytes, which have long been considered as the only cells capable of allorecognition. In this vision, except for the initial phase of ischemia/reperfusion, during which the role of innate immune effectors is well established, the latter are largely considered as "passive" players, recruited secondarily to amplify graft destruction processes during rejection. Challenging this prevalent dogma, the recent progresses in basic immunology have unraveled the complexity of the innate immune system and identified different subsets of innate (and innate-like) lymphoid cells. As most of these cells are tissue-resident, they are overrepresented among passenger leukocytes. Beyond their role in ischemia/reperfusion, some of these subsets have been shown to be capable of allorecognition and/or of regulating alloreactive adaptive responses, suggesting that these emerging immune players are actively involved in most of the life phases of the grafts and their recipients. Drawing upon the inventory of the literature, this review synthesizes the current state of knowledge of the role of the different innate (and innate-like) lymphoid cell subsets during ischemia/reperfusion, allorecognition, and graft rejection. How these subsets also contribute to graft tolerance and the protection of chronically immunosuppressed patients against infectious and cancerous complications is also examined

Immunol Rev

Cytomegalovirus (CMV) infection is responsible for significant morbidity and mortality in immunocompromised patients, namely solid organ and hematopoietic cell transplant recipients, and can induce congenital infection in neonates. There is currently an unmet need for new management and treatment strategies. Establishment of an anti-CMV immune response is critical in order to control CMV infection. The two main human T cells involved in HCMV-specific response are αβ and non-Vγ9Vδ2 T cells that belong to γδ T cell compartment. CMV-induced non-Vγ9Vδ2 T cells harbor a specific clonal expansion and a phenotypic signature, and display effector functions against CMV. So far, only two main molecular mechanisms underlying CMV sensing have been identified. Non-Vγ9Vδ2 T cells can be activated either by stress-induced surface expression of the γδT cell receptor (TCR) ligand annexin A2, or by a multimolecular stress signature composed of the γδTCR ligand endothelial protein C receptor and co-stimulatory signals such as the ICAM-1-LFA-1 axis. All this basic knowledge can be harnessed to improve the clinical management of CMV infection in at-risk patients. In particular, non-Vγ9Vδ2 T cell monitoring could help better stratify the risk of infection and move forward a personalized medicine. Moreover, recent advances in cell therapy protocols open the way for a non-Vγ9Vδ2 T cell therapy in immunocompromised patients

Harnessing γδ T cells in anticancer immunotherapy

International audienceγδ T lymphocytes are involved in the stress response to injured epithelia and in tissue homeostasis by limiting the dissemination of malignant or infected cells and by regulating the nature of the subsequent adaptive immune response. γδ T cells have potent MHC-unrestricted cytotoxicity, a high potential for cytokine release and broad-spectrum recognition of cancer cells, and as such, are attractive effectors for cancer immunotherapy. Current expectations are going beyond ex vivo manipulation of the Vγ9Vδ2 T subset, and target novel γδ T cell subsets, properties or receptors, to harness these unconventional T lymphocytes against cancer. This Opinion article discusses novel aspects of γδ T cell function during the course of anticancer therapies, as well as new avenues for their clinical implementation