Master of Science in Biomedical Sciences / Department of Diagnostic Medicine/Pathobiology / Jodi L. McGill / The causative agent of tuberculosis (TB) in cattle is Mycobacterium bovis (M. bovis). γδ T cells are a unique subset of nonconventional T cells that play major roles in both the innate and adaptive arms of the immune system. Bovine γδ T cells have the capacity for multiple immune functions during infection with M. bovis. However, the alternative functions of γδ T cells as well as the responses of γδ T cells in vivo at the site of infection remain unclear. To identify novel functions for γδ T cells in response to M. bovis infections, RNA sequencing and transcriptomics analysis was completed on peripheral blood γδ T cells isolated from virulent M. bovis-infected cattle. Differentially expressed genes were confirmed with real-time PCR. In an attempt to model in vivo cell-to-cell interactions at the site of infection, γδ T cells were also isolated from naïve and M. bovis-infected calves and co-cultured with autologous, BCG-infected, monocyte-derived macrophages. γδ T cell chemokine and cytokine expression was analyzed via ELISA and real-time PCR. The characteristic lesions of bovine tuberculosis are well-organized pulmonary granulomas. To determine the relevance of the RNA-sequencing and in vitro co-culture results to in vivo infection, tissue samples from granulomatous lesions in the lungs and mediastinal lymph nodes of virulent M. bovis-infected cattle were collected 3 months after infection. mRNA transcripts for γδ T cells expression of-- IFN-γ, IL-17, IL-10, IL-22, and CCL2 were microscopically evaluated within the granulomas using an in situ hybridization system, RNAScope (Advanced Cell Diagnostics Inc.). Co-culture experiments and transcriptomics analysis revealed increased expression of chemokines and various cytokines by γδ T cells responding to M. bovis infection. The novel in situ hybridization assay revealed that cytokine expression by γδ T cells varied within the lesions, with significant levels of CCL2 and IFN-γ, and low expression of IL-10, IL-22, and IL-17 in situ at this time-point after infection. Co-culture experiments also revealed that γδ T cells from virulent M. bovis-infected cattle have the capacity to directly impact the viability of M. bovis in vitro. Our results suggest that γδ T cells accumulate within the granulomas, and influence host immunity to M. bovis by secretion of cytokines and chemokines, and direct cytotoxicity, in response to infected macrophages.

CD161 γδ T-cells have been implicated in the pathogenesis of Multiple Sclerosis however their role in health and chronic liver disease requires further exploration. In health, the majority of γδ T-cells expressed CD161 – a C-type lectin, and predominantly expressed the Vδ2 chain. The CD161+ γδ T-cells demonstrated a Th1-like pattern, expressing IFN-γ, TNF-α and Granzymes/Perforin when compared to the CD161- subset. The CD161+ γδ T-cells also expressed CCR6 and IL-18R thus also displaying a Th17-like pattern. These cells were also found in the lamina propria in the gut and rapidly expanded in the 1^st few weeks of life in the periphery. On gene array analysis, there were 409 genes expressed on the CD161+ γδ T-cells when compared to their CD161-ve counterparts including those coding for β2 receptors, CCL20, Acetycholinesterase, CCR1 and IL-18R. A potential clinical correlation to cardiac diseases was found when the upregulated genes were analysed. When the CD161+ γδ and CD161+ αβ T-cell populations were compared via gene-array, an association with a risk variant for coeliac disease was found. Thus in health, CD161+ γδ T-cells are not only a distinct subset of T-cells (confirmed by a FACS approach and gene array methods), but also the expression of CD161 may be linked to common genetic signals downstream in cell processes and disease pathogenesis, irrespective of T-cell subset population. In chronic liver disease there was a significant reduction in the periphery of CD161+ γδ T-cells in patients with chronic Hepatitis C (HCV) and an increase in patients with Primary Biliary Cirrhosis and Primary Sclerosing Cholangitis when compared with healthy individuals. The CD161+ γδ T-cells appeared to be of a different phenotype in HCV infection. There was no overall significant localisation into of CD161+ γδ T-cells patients with chronic liver disease or specifically in HCV infection. There was however a CD161+ γδ T-cell enrichment in the liver in patients with Non-Alcoholic Fatty Liver disease. The CD161+ γδ T-cells were also found in Hepatocellular Carcinoma tissue. Overall it appears the CD161+ γδ T-cells are indeed a unique subset, playing a distinct role in health, as part of an early innate response, but also potentially involved in disease pathogenesis.

No description available.

Neuroblastoma is an aggressive solid tumor that develops from immature cells of the nervous system and is almost exclusively diagnosed in infants and young children. Over the past decade a multitude of immune based therapies have been explored as therapeutic candidates for patients with neuroblastoma. The anti-GD2 monoclonal antibody, 3F8, and more recently, natural kill (NK) cell based therapies have been accepted as hopeful therapeutic options for patients with Neuroblastoma. These options however have many drawbacks including dose limiting pain, the development of tolerance, reliance on MHC mismatch and possible reliance on the invariant NK (iNK) cells population. Gamma Delta T cells, a subpopulation of T cells composed of a T cell receptor (TCR) with a gamma and a delta chain instead of an alpha and a beta; chain, have been shown to recruit a more robust immune response then both 3F8 and NK cells through their activation of antigen presenting cells (APCs) and non-reliance on MHC mismatch. Gamma Delta T cells are also able to recruit NK cells as well as other cytotoxic lymphocytes. For these reasons, it is believed that Gamma Delta T cell based treatment alone or in combination with an anti-GD2 monoclonal antibody may have a greater efficacy than either NK cells or an anti-GD2 monoclonal antibody alone. The intent of this thesis is to explore and evaluate the current state of Gamma Delta T cell based immunotherapy against the backdrop of NK cell based immunotherapy for neuroblastoma.

Le cytomégalovirus (CMV) est l’agent responsable de l’infection opportuniste la plus fréquemment rencontrée en transplantation d’organe. Chez les receveurs séronégatifs qui reçoivent un rein provenant d’un donneur séropositif, 50 % de ces patients peuvent développer une virémie, et 30 % une maladie. A court terme, malgré les traitements anti-viraux, elle est responsable d’une morbidité non négligeable. A long terme, le CMV est associé à une augmentation de la fréquence des sténoses artérielles, plus d’infections associées, plus de rejet aigu, plus de lésions de fibrose interstitielle et d’atrophie tubulaire, une moins bonne survie des greffons et des patients. La cohabitation et la coévolution du CMV avec l’homme depuis des milliers d’années ont aboutie à un état d’équilibre entre le virus et son hôte. Le virus s’est profondément adapté à son hôte afin d’échapper à la réponse immune. En réponse à cela, la réponse immunitaire anti-CMV occupe une part unique et majeure au sein de la réponse immune de l’hôte. Les lymphocytes T CD8+ spécifiques du CMV représentent par exemple 10.2% des lymphocytes T CD8+ mémoires. Avec l’âge, ils s’accumulent et peuvent représenter jusqu’à 30% du pool total de lymphocyte T CD8+. Le système immunitaire sous la contrainte du virus s’est donc refaçonné de façon à garder le contrôle du virus. Depuis 1999, un nouvel acteur de cette réponse immunitaire a été identifié : les lymphocytes T gamma delta Vdelta2-negative. Ces cellules sont impliquées habituellement dans la lutte contre les différents stress d’origine microbien et non microbien (tumeur). Elles interviennent plutôt localement (dans les épithéliums) par différents mécanismes et sont désormais considérées comme des effecteurs intermédiaires entre l’immunité innée et l’immunité adaptative. Leur expansion dans le sang est associée à la guérison de la maladie et à la résolution de l’infection à CMV. Elles ont par ailleurs in vitro une réactivité croisée contre des cellules infectées par le CMV et des cellules tumorales. Les lymphocytes T gamma delta Vdelta2-negative sont donc une représentation supplémentaire de l’énorme impact du CMV sur le système immunitaire de l’hôte. Dans ce travail, nous avons pu étendre et approfondir leur rôle en transplantation d’organe. Nous avons tout d’abord décrit que les lymphocytes T gamma delta Vdelta2-negative avaient un phénotype et une cinétique d’expansion exactement superposable aux lymphocytes T CD8+ spécifiques du CMV in vivo. Nous avons ensuite observé que l’expansion des lymphocytes T gamma delta Vdelta2-negative induits pas l’infection à CMV s’associait à une survenue moindre de cancer à long terme chez les patients transplantés rénaux. Nous avons pu montrer que leur activation était sous la dépendance d’une interaction entre leur TCR et un ligand. Enfin, une autre voie d’activation dépendante du CD16, faisant intervenir les complexes immuns CMV-IgG anti-CMV a aussi été identifiée. Nos travaux depuis 10 ans ont donc démontré que les lymphocytes T gamma delta Vdelta2-negative occupaient une place majeure dans la réponse immune anti-CMV au même titre que les lymphocytes T CD8+. L’intégration de ces cellules à l’immunologie anti-CMV devrait permettre de mieux comprendre certains effets indirects induits par le virus, et pourrait être utile dans le suivi de la réponse immune anti-CMV en transplantation d’organe. L’identification de leur ligand pourrait permettre enfin de tester assez rapidement de nouveaux protocoles d’immunothérapie anti-virale ou anti-tumorale. / Cytomegalovirus (CMV) infection is the most frequent opportunistic infection encountered in solid-organ transplantation. Fifty percent of seronegative kidney transplant recipients (KTR) who receive a kidney from a seropositive donor may develop a CMV infection which causes a disease in 30% of cases. In the long term, CMV is associated with an increased incidence of arterial stenosis, more opportunistic infections, more acute rejection episodes, more interstitial fibrosis and tubular atrophy, and a poorer graft and patient survivals. For thousands years, the co-evolution between the CMV and the immune system allowed to a state of equilibrium between the virus and the host. The virus has deeply adapted to its host in order to escape the immune response. In response, the anti-CMV immune reaction takes up an important and unique place. For example, the CMV-specific CD8+ T cells represent an average 10.2% of the memory CD8+ T cell compartment in CMV-seropositive healthy individuals. With age, these cells accumulate and can represent around 30% of CD8+ T lymphocytes. Therefore under the long-lasting pressure of the virus, the immune system has redesigned in order to keep the control of the virus. Since 1999, a new actor was identified within the immune system: the Vdelta2-negative gamma delta T cells. These cells are involved against various microbial and non microbial stresses. They act locally in epithelia by different mechanisms and are now considered as intermediate effectors between innate and adaptive immunity. In vivo in KTR, their blood expansion is associated with the resolution of CMV infection. In vitro, they share a cross-reactivity against CMV-infected cells and tumor cells. Therefore, the Vdelta2-negative gamma delta T cells are new representatives of the huge impact of CMV on the host immune system. In this work, we were able to extend and get further insight into their role in organ transplantation. In vivo, we first described that Vdelta2-negative gamma delta T cells displayed a phenotype and an expansion kinetic similar to that of CMV-specific CD8+ T cells. Next, we observed that the CMV-induced Vdelta2-negative gamma delta T cells expansion was associated with a lower occurrence of cancer in long-term KTR. In vitro, experiments of transfer of gamma delta TCR allowed us to show that their activation against tumor ligands was TCR-dependent and that different tumor ligands could be recognized by each Vdelta2-negative gamma delta TCR studied. In addition, we observed that the recognition of CMV-infected cells was not only TCR-dependent, but under the dependence of a multi molecular complex involving co stimulatory signals. Finally, we also identified a new CD16-dependent pathway of activation in gamma-delta T cells, involving IgG-opsonised CMV. In summary, Vdelta2-negative gamma delta T cells take up a major place within the anti-CMV immune response in addition to CD8+ T lymphocytes. The integration of these cells to the anti-CMV immunology should provide a better understanding of some indirect effects of the virus and could be useful to monitor the immune response against CMV in solid-organ transplant recipients. Moreover, identification of their ligands could provide interesting tools for new protocols of anti-CMV and anti-tumor immunotherapy.

Le développement d'un vaccin anti-paludique est limité par notre connaissance incomplète des effecteurs agissant contre P.falciparum. Nous avons mis en évidence que les cellules T Vγ9Vδ2 sont activées par la forme intra-érythrocytaire (schizonte) et par les phosphoantigènes de P.falciparum, et peuvent inhiber la croissance du parasite in vitro par un mécanisme dépendant de la granulysine ciblant la forme invasive du parasite (mérozoïte). Ces résultats suggérent que les lymphocytes T Vγ9Vδ2 jouent un rôle dans le contrôle précoce de la charge parasitaire. Cependant, le mécanisme médiant l’interaction entre les schizontes, les mérozoïtes les cellules T Vγ9Vδ2 et reste élusif. L'objectif de cette thèse est d’étudier les interactions entre les stades sanguins de P. falciparum et les cellules T Vγ9Vδ2, afin de mieux comprendre leurs activités anti-parasitaires, dans le but à long terme d’une utilisation clinique. Dans ce travail, nous étudions l'importance du contact direct avec les parasites dans l’activation et les activités anti-parasitaires des cellules T Vγ9Vδ2, par des approches de microscopie confocale et de cytométrie en flux. Nous suggérons que les cellules T Vγ9Vδ2 forment peu ou pas de contacts avec les mérozoïtes, et très peu de contacts avec le schizonte. De plus, nous montrons que, contrairement à une lignée cellulaire tumorale cible (Daudi), le contact avec les schizontes n'affecte pas l'activation des cellules T Vγ9Vδ2, suggérant que les phosphoantigenes du parasite sont libérés dans le milieu. Nous démontrons que les phosphoantigènes produits par la voie DOXP sont probablement libérés par un processus actif, dépendant des new permeation pathways (NPP). Ensembles, ces résultats suggèrent que l'activation et l'activité antiparasitaire des cellules T Vγ9Vδ2 n’est pas dépendante du contact, mais est médié par des facteurs solubles. / The limited knowledge of immune effector against Plasmodium falciparum precludes the development of a malaria efficient vaccine. We have recently evidenced that Vγ9Vδ2 T cells act as a new immune effector early in malaria infection. These cells are activated by the mature intraerythrocytic form (schizont) and by parasite-derived antigens (phosphoantigens). After activation, they inhibit in vitro parasite growth by targeting the extraerythrocytic invasive form (merozoite), by a granulysin-dependent mechanism. However, the mechanism by which Vγ9Vδ2 T cells are activated by schizonts and target merozoites remains elusive. The aim of this PhD project is to describe the interactions between P.falciparum blood stages and γδ T cells, in order to better understand their anti-parasitic activities and in the long term goal to manipulate these cells to prevent malaria. In the work, we investigate the importance of cell to parasite contact in Vγ9Vδ2 T cell activation and anti-parasitic activity by time-lapse and fixed confocal imaging, and cytometry. We suggest that Vγ9Vδ2 T cells form little or no contacts with merozoites, and very few contacts with the mature intraerythrocytic (schizont) form of the parasite. Moreover, we show that cytotoxic activities are elicited by schizonts, but that contrary to a known tumor cell line (Daudi cells), contact has no effect on the level of activation, suggesting that parasite-derived phosphoantigens are secreted in the microenvironment. We pursue the characterization of the parasite-derived phosphoantigens and demonstrate that they are produced by the DOXP pathway. Lastly, we show that phosphoantigens are most likely released by an active process, dependent on the new permeation pathways. Altogether, these results shed light on an unconventional mode of activation by P.falciparum blood stages and antiparasitic activity of Vγ9Vδ2 T cells, which is not contact-dependent, but rather is mediated by soluble factors.

Antigen presentation and recognition are key processes of the immune system necessary to initiate the adaptive immune response. Longstanding goals of these fields have been to understand the molecular mechanism of MHC II-peptide binding, the way in which dysregulation of this process can lead to disease, and determining how γδTCRs recognize their ligands. To examine some of these outstanding questions, I designed photocleavable peptides that could bind HLA-DR1 and could be used to facilitate peptide exchange. I also performed studies to understand whether peptide exchange on HLA-DR1 can be affected by glycation modifications, which occur in hyperglycemic conditions such as diabetes. I observed that while glycation modifications on HLA-DR1 did not affect peptide exchange, these modifications decreased the catalytic effect of HLA-DM on this reaction, which could affect antigen presentation in diabetic patients. For studies on antigen recognition by γδTCRs, I focused on γδNKT cells, a subset of γδT cells known to play a role during Listeria infection. I used four different variants of the γδNKT TCR to study the restrictions on Vγ junctional region usage by this TCR for ligand recognition. I found that all the TCR variants I examined could recognize cells infected with Listeria, indicating that this TCR is not restricted by γ-chain usage in order to recognize ligand. My research generated reagents that could serve in future studies of HLA-DR1 peptide binding and contributed to understanding the effect of hyperglycemic conditions on antigen presentation, as well as provided greater understanding of γδTCR restriction for ligand recognition.

Une résidence à long terme de lymphocytes T (LTs) au sein de la plupart des tissus non lymphoïdes a été récemment décrite, notamment à la suite d’infections. Ces cellules confèreraient à l’hôte une meilleure protection en cas de réinfection. À l'aide de deux approches expérimentales différentes, l'injection d'anticorps bloquant l’entrée des LTs dans les ganglions lymphatiques (LNs) et la génération de parabioses par chirurgie, nous avons pu mettre en évidence, à l’état basal, la résidence d’une proportion significative des LTs αβ mémoires CD4+, des LTs αβ régulateurs CD4+ et d’une sous-population des LTs γδ dans les organes lymphoïdes secondaires. Les LTs CD4+ régulateurs et mémoires résidents ont en commun de nombreuses caractéristiques phénotypiques et fonctionnelles, et partagent avec leurs homologues issus de tissus non lymphoïdes une signature transcriptionnelle commune de résidence. Les LTs γδ résidents, quant à eux, arborent des caractéristiques phénotypiques et fonctionnelles proches de celles des cellules du système immunitaire inné. Si le microbiote semble jouer un rôle important dans la résidence des LTs αβ CD4+ des plaques de Peyer (PPs), son rôle ne semble pas être prépondérant dans la résidence de ces cellules au sein des LNs. Comme dans de nombreux tissus non lymphoïdes, la sous-expression de S1PR1 pourrait en partie expliquer la résidence des LTs αβ CD4+. Par contre, les LTs γδ seraient, eux, retenus dans les tissus lymphoïdes de par des interactions étroites avec les macrophages. Enfin, la résidence des LTs αβ augmente avec l'âge au point que la majorité des LTs CD4+ régulateurs et mémoires des LNs et des PPs sont en fait résidents chez des souris âgées. Nos résultats montrent que la résidence des cellules T n'est pas seulement une caractéristique des tissus non lymphoïdes mais qu’elle peut être étendue aux organes lymphoïdes secondaires. Le rôle respectif de ces différentes populations de LTs devra être exploré. / In the last decade, numerous data have demonstrated the existence of T cells residing in non-lymphoid tissues, mostly after infectious diseases. These resident memory T cells may represent a first line of defense against pathogens at front-line sites of microbial exposure upon reinfection. Using two different experimental approaches such as the injection of integrin-neutralizing antibodies that inhibits the entry of circulating lymphocytes into lymph nodes and long-term parabiosis experiments, we have highlighted the long-term residence of a substantial proportion of regulatory and memory CD4 αβ T cells and γδ T cells within the secondary lymphoid organs of specific pathogen free mice. Resident γδ T cells display innate-like characteristics. Lymph node-resident regulatory and memory CD4 αβ T cells share many phenotypic and functional characteristics, including a core transcriptional profile, with their cell-counterparts from non-lymphoid tissues. Microbiota plays an important role in αβ T-cell residence in Peyer’s patches but only a small one if any in lymph nodes. Like in many non-lymphoid tissues, S1PR1 down-regulation may account forαβ T-cell residency within secondary lymphoid organs although other mechanisms may account for this especially in the case of lymph node memory CD4 T cells. Specific in vivo cell-depletion strategies have allowed us to demonstrate that macrophages are the main actors involved in the long-term retention of γδ T cells in secondary lymphoid organs. Strikingly, T-cell residence increases with age to the point that the majority of regulatory and memory CD4 αβ T cells from LNs and Peyer’s patches are in fact resident T cells in old mice. Altogether, our results show that T-cell residence is not only a hallmark of non-lymphoid tissues but can be extended to secondary lymphoid organs.

No description available.

Das human pathogene Varicella-Zoster Virus (VZV) gehört zur Familie der Herpesviren und ist weltweit verbreitet. Die Primärinfektion verursacht Varicellen, welche durch einen bläschenartigen Hautausschlag charakterisiert ist. Im Anschluss daran etabliert VZV eine lebenslange Latenz und verursacht nach Reaktivierung Herpes Zoster. Seit 2004 ist der Lebendimpfstoff aus attenuierten Virionen des VZV-Stammes V-Oka in Deutschland empfohlen. Im Gegensatz zur Infektion mit zirkulierenden virulenten VZV Stämmen tritt nach Verimpfung des Vakzin-Stammes V-Oka kein Exanthem auf. Die Haut ist der Hauptreplikationsort von VZV und immunologische Unterschiede zwischen virulentem VZV und dem Vakzin-Stamm treten hier am deutlichsten auf. In der vorliegenden Arbeit konnte eine neue Immunevasionsstrategie virulenter VZV Stämme aufgedeckt werden, welche erklären könnte, wie virulente VZV Stämme frühe antivirale Immunantworten umgehen. In Hautläsionen von Herpes Zoster Patienten konnte eine massive Infiltration von myeloiden inflammatorischen Dendritischen Zellen beobachtet werden. In vitro Studien mit Monozyten abgeleiteten Dendritischen Zellen (DC), welche inflammatorische DC repräsentieren, zeigten, eine signifikant erhöhte Expression von CD1c Molekülen nach Infektion mit dem Vakzin-Stamm, sowie virulentem VZV. Funktionelle Untersuchungen mit intraepithelialen CD1c-restringierten gamma delta T Zellen zeigten, dass DC nach Infektion mit dem Vakzin-Stamm phänotypisch und funktionell reiften und somit die T Zellen zur IFN-gamma Sekretion stimulierten. Im Gegensatz dazu wurde die funktionelle Reifung von DC, die mit virulentem VZV infiziert waren, geblockt. Folglich wurde kein bioaktives IL-12 sezerniert, welches als entscheidendes Cytokin zum Aufbau einer antiviralen T-Helfer 1 Immunantwort beiträgt. Darüber hinaus konnte gezeigt werden, dass virulentes VZV die Signalkaskade des Toll-like Rezeptors 2 (TLR2) in DC inhibiert und somit die IL-12 Produktion verhindert. / Varicella-zoster virus (VZV) which belongs to the family of herpesviruses is restricted to humans and distributed worldwide. Primary infection of VZV causes chickenpox characterized by a disseminated rash. Thereafter, VZV establishes a lifelong latency and can be reactivated to cause herpes zoster. Since 2004 the attenuated strain V-Oka of VZV was licensed for Germany to immunize children against VZV infection. In contrast to infection by circulating virulent VZV strains, vaccination with V-Oka remains asymptomatic. The skin is the major replication site of VZV and immunological differences between virulent VZV and the vaccine should become most apparent within this immune organ. In summary, this study discovered a new immune evasion strategy of virulent VZV strains which might explain how virulent VZV strains overcome innate antiviral responses. A strong infiltration of myeloid-derived inflammatory DCs has been detected in skin lesions of herpes zoster patients. In vitro studies with monocyte-derived dendritic cells (DCs), reflecting inflammatory DCs, showed that they were efficiently infected by both, the vaccine and a virulent VZV strain. Intriguingly, a significant upregulation of CD1c molecules on VZV-infected DCs was observed. Functional investigations using intraepithelial CD1c-restricted gamma delta T cells revealed that DCs infected with the vaccine virus were fully instructed to mature, thereby promoting IFN-gamma secretion of gamma-delta T cells. In striking contrast, DCs infected with virulent VZV strains were efficiently blocked to mature functionally. In detail, they did not secrete bioactive IL-12 which is an instrumental cytokine for generation of antiviral T helper 1 responses. Moreover, virulent VZV blocked Toll-like receptor 2 (TLR2) signaling in DCs thereby preventing production of bioactive IL-12 which in turn inhibited IFN-gamma secretion by gamma-delta T cells.