Der DNA-Verpackungsmechanismus des humanen Cytomegalievirus (HCMV) ist charakteristisch für große DNA-Viren wie Herpesviren und ds-Bakteriophagen. Er beruht auf der Spaltung der konkatemeren DNA durch einen viralen, hetero-oligomeren Proteinkomplex, der Terminase. In der vorliegenden Arbeit konnten die funktionellen Domänen der Terminase-Untereinheit pUL89 in vitro identifiziert und charakterisiert werden. Neben einer Nuklease-Aktivität besitzt pUL89 auch die Fähigkeit dsDNA sequenz-unabhängig zu binden. Durch Nuklease-Untersuchungen konnte gezeigt werden, dass pUL89 sowohl dsDNA, als auch lineare DNA spaltet. PUL89 weist dabei eine größere Spezifität zu dsDNA auf. Des Weiteren konnte nachgewiesen werden, dass die Aminosäure D463 eine zentrale Funktion innerhalb der Nuklease-Aktivität besitzt. Durch kolorimetrische DNA-Bindungsuntersuchungen konnte die Aminosäure R544 als essenziell für die dsDNA-Bindungsfähigkeit von pUL89 identifiziert werden. Basierend auf den in vitro Ergebnissen wurden rekombinanten TB40/E-Virusmutanten mit Mutationen im ORF UL89 durch die En Passant Mutagenese generiert. Mit Hilfe dieser Viren sollte der Einfluss der Mutationen auf die Replikation des Virus charakterisiert werden. Es war möglich nachzuweisen, dass die Aminosäuren E534 und R544 eine essenzielle Aufgabe innerhalb von HCMV erfüllen, da die Mutation einer dieser Aminosäure zu nicht wachstums-fähigen BAC-Mutanten führte. Zur Charakterisierung dieser Konstrukte wurden die Zelllinien HELF Fi301-UL89 und HELF Fi301-vProm-UL89 verwendet. Durch Untersuchungen hinsichtlich der Wachstumseigenschaften, Proteinexpression, DNA-Spaltung, DNA-Bindung sowie elektronenmikroskopischen Aufnahmen, konnte gezeigt werden, dass die wachstums-kompetenten BAC-Mutanten keinen signifikanten Unterschied zum Wildtyp-Virus TB40/E zeigten. Sodass nachgewiesen werden konnte, dass die basischen Aminosäuren H565 und H571 keine essenzielle Funktion in pUL89 erfüllen. / The human cytomegalovirus DNA packaging mechanism is characteristic for large DNA viruses like Herpes viruses and ds bacteriophages. This mechanism is based on the cleavage of concatemeric DNA by the viral heterooligomeric protein complex terminase. This dissertation includes the identification and characterization of functional domains of the HCMV terminase subunit pUL89. PUL89 contain a nuclease activity and the ability to bind dsDNA. This protein shows the property to cut as well dsDNA as linear DNA. The amino acid D463 shows a significant role in this cleavage event. Colorimetric DNA binding experiments show the central role of R544 in DNA binding by pUL89. Based of the in vitro results recombinant TB40/E viruses with mutations in the ORF UL89 were generated. These viruses allow a characterization of the impact of virus replication. It was possible to show that the amino acids E534 and R544 have a functional role in HCMV. The mutation of one of these amino acids was enough to generate a growth deficient mutant. The stable cell lines HELF Fi301-UL89 and HELF Fi301-vProm UL89 were used for the characterization of the growth deficient mutants. The growth competent mutants H656A and H571A show no significant differences in comparison with the wild type TB40/E virus. This was verified by growth kinetics, protein expression characterizations, pulse field gel electrophoresis, DNA binding assays and electron microscopy.

Bis heute sind die beiden Ratten-Cytomegalovirus (RCMV)-Isolate RCMV-England (RCMV-E) und RCMV-Berlin (RCMV-B) die einzig bekannten Viren, die ein Homolog von XCL1 kodieren, einem g-Chemokin, das vom Virus kopiert wurde um das Chemokin-Netzwerk des Wirts zu beeinträchtigen. Wie das Wirtshomolog lockt vXCL1 ausschließlich dendritische Zellen (DC) an, die den XC-Chemokinrezeptor 1 (XCR1) exprimieren. In dieser Arbeit wurde untersucht, inwieweit RCMV die XCL1-XCR1-Achse nutzt, um DC zu infizieren und sich im Wirt auszubreiten. In der Ratte konnten zwei DC-Hauptpopulationen identifiziert werden, XCR1+ CD4- und XCR1- CD4+ DC. Es konnte gezeigt werden, dass Überstände von RCMV-infizierten embryonalen Rattenfibroblasten ausschließlich die XCR1+ CD4- Population anlocken. Darüber hinaus konnte nachgewiesen werden, dass RCMV DC infiziert. Durch die Anreicherung wurden DC aktiviert. Während der Infektion inhibierte RCMV die Hochregulation von Reifungsmarkern, einschließlich CD40, CD86 und CCR7. Unabhängig von vXCL1 scheint RCMV die DC-Funktionalität durch das Herunterregulieren von Reifungsgenen zu lähmen. Um die Rolle von XCR1 und die Funktion von vXCL1 in vivo zu analysieren, wurden Xcr1+/+ und Xcr1-/- Ratten mit RCMV-B wt und RCMV-B D-vxcl1 infiziert. Während das XCR1- Expressionsniveau einen Einfluss auf die Geschwindigkeit der RCMV-Verbreitung in den Speicheldrüsen hatte, führte das Fehlen von vXCL1 zu einer starken Abnahme der Virusausbreitung. Die DC-Migration in die Speicheldrüsen war sowohl von vXCL1 als auch von XCR1 abhängig und war reduziert, wenn vXCL1 und XCR1 nicht vorhanden waren. Während der Infektion wurden CD8+ T-Zellen in die Speicheldrüsen rekrutiert. Diese Migration blieb jedoch aus, wenn Xcr1+/+ Ratten mit RCMV-B D-vxcl1 infiziert wurden. Zusammenfassend besitzt RCMV die Fähigkeit DC unabhängig von der vXCL1-Expression zu infizieren. RCMV verwendet vXCL1, um XCR1+ DC anzulocken, was entscheidend für die Virusausbreitung in die Speicheldrüsen zu sein scheint. / To date, the two Rat Cytomegalovirus (RCMV) isolates RCMV-England (RCMV-E) and RCMV-Berlin (RCMV-B) are the only known viruses that encode a homolog of XCL1, a gamma- chemokine adopted by viral piracy to interfere with the host’s chemokine network. Like its host homolog, vXCL1 exclusively attracts dendritic cells (DC) that express the XC chemokine receptor 1 (XCR1). In this work, it was investigated whether RCMV misuses the XCL1-XCR1 axis to infect DC in order to disseminate within the host. Initially, rat DC phenotyping revealed two major DC populations, XCR1+ CD4- DC and XCR1- CD4+. It could be shown that supernatants of RCMV-infected rat embryonic fibroblasts solely attracted the XCR1+ CD4- population. Moreover, RCMV was able to infect and replicate in DC. Due to digestion of the spleen and leukocyte enrichment DC became activated leading to full maturation 24 h after cell isolation. During infection, RCMV inhibited the upregulation of several maturation markers including CD40, CD86 and CCR7 and also led to reduced expression of MHCII, CD4 and XCR1. Regardless of vXCL1, RCMV appears to paralyze DC functionality by downregulating maturation genes. In order to analyze the role of XCR1 and vXCL1 function in vivo, Xcr1+/+ and Xcr1-/- rats were infected with RCMV-B wt and RCMV-B delta-vxcl1. Whereas the XCR1 expression level had an influence on the pace of RCMV-B wt dissemination to the salivary glands, the absence of vXCL1 led to a strong decrease in viral spread. DC migration to the salivary glands was dependent on vXCL1 as well as XCR1 and was markedly reduced when vXCL1 and XCR1 were not present. During infection, CD8+ T cells were recruited to the salivary glands, however, this migration was missing when Xcr1+/+ rats were infected with RCMV-B delta-vxcl1. In conclusion, RCMV has the ability to infect DC regardless of vXCL1 expression. RCMV uses vXCL1 to attract XCR1+ DC which appears to be important for viral dissemination to the salivary glands.

Background: Although infectious diseases are less common in high-income countries compared to low-income countries, they should still be seriously considered as a relevant public health issue. Some professions, such as healthcare workers, laboratory workers, and care providers may be at a particularly high risk of acquiring infections. In Germany, work-related infectious diseases are after skin diseases, the most common cause of occupational diseases reported to the Institution for Statutory Social Accident Insurance and Prevention in the Health Care and Welfare Services (BGW). An occupational disease, as defined by the WHO, is “any disease contracted primarily as a result of an exposure to risk factors arising from work activity”, although this definition varies between countries. In order for infections to be recognized as an occupational disease, either the identification of an index case is needed or it must be shown that the likelihood in which a particular case of illness was attributable to the occupation: the probability of causation must be greater than 50% (the “more-likely-than-not” rule). A general “rule-of-thumb” is to equate the probability of causation of 50% with a relative risk of disease equal to two (the “doubling of the risk”). This principle is used by many countries for the recognition of an occupational disease. Few studies have concentrated on the risk of infectious disease in daycare workers, who may be at higher risk than the general population due to their frequent and close contact to young children. Research questions: The primary aim of this review was to summarize the evidence on the relationship between being a daycare worker working with children and the possible increased risk for infections not preventable by vaccines. Furthermore, research gaps were to be identified. Finally, the implications for practice and health policy based on the evidence were to be described. Methods: For the systematic reviews with meta-analysis, the Medline and Embase databases were searched using search strings defined according to the Population, Exposure, Comparison, and Outcomes (PECO) applicable to the research questions in order to find studies on vaccine non-preventable infections in daycare workers published since 2000. The search hits were evaluated using predefined inclusion and exclusion criteria by two independent reviewers. A separate manual search was performed by reviewing the reference lists of key articles and systematic reviews. The “citation tracking factor” by Google scholar was used to find additional relevant studies. The resulting studies were extracted and were assessed in eight risk of bias domains for the judgement of study quality. With a meta-analysis, the pooled risk of infections for daycare workers compared to the general or a reference population was calculated. The quality of evidence was assessed by the Grading of Recommendations Assessment, Development, and Evaluation (GRADE). Results: After evaluating the 6879 records, ten methodologically adequate studies were identified regarding parvovirus B19 infection (four studies) and cytomegalovirus (CMV) infection (six studies). No adequate studies on other infections were found. For parvovirus B19 infection, three cross-sectional studies and one retrospective cohort study were identified. The pooled parvovirus B19 seroprevalence in daycare workers was 70.3% (95% CI 59.5-80.4). Of three studies investigating the relative risk (RR) of parvovirus B19 infection on daycare workers, only one study evaluated seroconversion rates. There was an indication for an increased risk of parvovirus B19 infection for daycare workers compared to the unexposed population (RR = 1.12, 95% CI 0.98–1.27) using prevalence estimators. Furthermore, daycare workers had a higher parvovirus B19 seroconversion rate compared to the unexposed population (RR = 2.63, 95% CI 1.27–5.45) in the low risk of bias study. For CMV infection, five cross-sectional studies and one cohort study were included. The pooled CMV seroprevalence of daycare workers was 59.3% (95% CI 47.6-70.9). The four studies investigating risk of infection indicated an increased seroprevalence for daycare workers compared to a reference population (prevalence ratio, RR=1.54, 95% CI 1.33-1.77). No study evaluated CMV seroconversions for daycare workers. Conclusions: The findings suggest higher parvovirus B19 and cytomegalovirus seroprevalence for daycare workers compared to the general population. There is a need for longitudinal and higher-quality studies regarding infections not preventable by vaccines in daycare workers, as well as a need to study other infections for which daycare workers may be at higher risk. Nonetheless, when the actual occupational seroconversion risk is considered by taking into account the pre-occupational seroprevalences, the pooled relative risks for both parvovirus B19 and CMV infection are compatible with a doubled seroconversion risk corresponding to a probability of causation due to the occupation of at least 50%. Preventative efforts in the workplace are needed based on the legally required risk assessment at the workplace. Moreover, it is important to raise awareness of the potential risk of infection in women trying to conceive or during pregnancy. Recommendations to prevent infections in the day care center include using gloves and frequent handwashing after exposure to young children’s bodily fluids, cleaning surfaces, and avoiding intimate contact with young children if pregnant, although these measures alone may not completely protect the daycare worker from infection. Currently, in Germany, an employment ban for pregnant daycare workers depends on the federal state. To avoid occupational risks for pregnant daycare workers, scientific-based guidelines should be developed and applied consistently.

The rubella virus (RV) was the first virus shown to be teratogenic in humans. The wealth of data on the clinical symptoms associated with congenital rubella syndrome is in stark contrast to an incomplete understanding of the forces leading to the teratogenic alterations in humans. This applies not only to RV, but also to congenital viral infections in general and includes (1) the mode of vertical transmission, even at early gestation, (2) the possible involvement of inflammation as a consequence of an activated innate immune response, and (3) the underlying molecular and cellular alterations. With the progress made in the development of pluripotent stem cell-based models including organoids and embryoids, it is now possible to assess congenital virus infections on a mechanistic level. Moreover, antiviral treatment options can be validated, and newly emerging viruses with a potential impact on human embryonal development, such as that recently reflected by the Zika virus (ZIKV), can be characterized. Here, we discuss human cytomegalovirus (HCMV) and ZIKV in comparison to RV as viruses with well-known congenital pathologies and highlight their analysis on current models for the early phase of human development. This includes the implications of their genetic variability and, as such, virus strain-specific properties for their use as archetype models for congenital virus infections. In this review, we will discuss the use of induced pluripotent stem cells (iPSC) and derived organoid systems for the study of congenital virus infections with a focus on their prominent aetiologies, HCMV, ZIKV, and RV. Their assessment on these models will provide valuable information on how human development is impaired by virus infections; it will also add new insights into the normal progression of human development through the analysis of developmental pathways in the context of virus-induced alterations. These are exciting perspectives for both developmental biology and congenital virology.

O citomegalovírus (CMV) é uma séria ameaça aos receptores de transplante de medula óssea. A reativação está associada com uma imunidade mediada por células TCD8+ defeituosa. Nosso objetivo foi correlacionar as diferentes subpopulações de células TCD8+ com a reconstituição imunológica dos pacientes, especificamente a imunidade anti-CMV, analisando as subpopulações de células T infundidas nas diferentes modalidades de transplante de medula óssea. Receptores de transplante alogênico de células tronco mobilizadas para o sangue periférico (n=16) ou coletadas diretamente da medula óssea (n=28) e receptores de transplante autólogo de células tronco mobilizadas para o sangue periférico (n=22) foram avaliados. Verificamos que as transferências de células mobilizadas para o sangue periférico dos doadores, tanto nos transplantes alogênicos como autólogos, são proporcionalmente enriquecidas por subpopulações de células memória efetora e efetora, comparadas às transferências de células procedentes diretamente da medula óssea. Este enriquecimento por subpopulações de células TCD8+ mais diferenciadas foi também correlacionado com maior número de células contendo altos níveis de granzima B, considerado um marcador para linfócitos citotóxicos, sendo também encontrado em maior número nas transferências de células do sangue periférico. Entretanto, no pós-transplante, observou-se que somente os receptores de transplante autólogo de células tronco mobilizadas para o sangue periférico, e não os das outras modalidades de transplante, exibiam números elevados de células T CD8+ de memória-efetora e efetora. Ao mesmo tempo, estes receptores apresentaram menos freqüentemente episódios de reativação pelo CMV, e mais freqüentemente produziram IFN-gama em resposta ao CMV. Portanto, a transferência de células do sangue periférico, desde que em ambiente autólogo, está associada não só com a transferência de células TCD8+ com um fenótipo mais maduro, mas também com uma persistência mais prolongada das mesmas, podendo proporcionar uma resposta imunológica antiviral mais rápida e eficiente, como esperado para as células de memória versus naïve. / Cytomegalovirus (CMV) is a serious threat to the recipients of bone marrow transplantation. Reactivation is associated with defective CD8+ T cell-mediated immunity. We aimed to correlate the different subsets of CD8+ T cells with the patients\' immune reconstitution, specifically anti CMV immunity, by analyzing the CD8+ T cell subsets infused in the different types of bone marrow transplantation. Recipients of allogeneic transplant of peripheral blood stem cells (n=16) or bone marrow (n=28) and recipients of autologous transplant of peripheral blood stem cells (n=22) were evaluated. We show that infusions of stem cells derived from donor\'s peripheral blood, either allogeneic or autologous, are proportionally enriched for the memory-effector and effector phenotypes, compared to the infusions of stem cells of bone marrow origin. This increased number of more differentiated subsets of CD8+ T cells was also correlated with an increased number of cells containing high levels of granzyme B, which is another reliable marker of cytotoxic lymphocyte, and which was also more evident in autologous recipients. However, post-transplant, we observed that only the recipients of autologous peripheral blood cells, and not the recipients of the other transplant modalities, exhibited very high numbers of memory-effector and effector TCD8+ cells. At the same time, they less frequently presented CMV reactivation, and more frequently produced IFN-gama in response to CMV antigens. Thus, transfer of stem cells from peripheral blood, provided in an autologous setting, is associated with transfer and prolonged survival of CD8+ T cells with a more mature phenotype, which may provide a more rapid and efficient anti-viral immune response, as expected for memory versus naïve cells.

Human cytomegalovirus (HCMV) is a widespread human herpesvirus pathogen and prototypical member of the β-herpesvirus subfamily. Like all herpesviruses, the virus establishes a lifelong latent infection following host exposure, which has the potential to reactivate periodically and contribute to recurrent disease processes. In individuals with weak or compromised immune systems, such reactivation can lead to profound pathology. Understanding how latent infections are maintained is important for uncovering how HCMV causes disease. The study of viral genes that are expressed during latent infection grants insight into how latency is regulated and how it could be therapeutically targeted. To that end, this project has sought to evaluate the functional significance of one such viral gene termed LUNA in the context of latency. In models of experimental latent infection based on primary myeloid cells, levels of viral gene transcription were found to be significantly reduced following infection with LUNA deletion mutant viruses, consistent with corresponding observable changes in post-translational histone modifications over the viral promoters of latency-associated genes. Additionally, using luciferase reporter systems, latency-associated viral gene promoters became activated in response to the expression of wild-type LUNA. Together, these findings argue for a role of LUNA in regulating viral gene expression during latent HCMV infection. One possible mechanism by which LUNA may fulfil its role is by targeting cellular ND10 structures, known intrinsic inhibitors of herpesvirus gene expression, for disruption. In support of this, latently infected cells were found to be devoid of ND10, a phenotype that was recapitulated by the direct expression of wild-type LUNA. Furthermore, mutation studies confirmed the identification of a novel deSUMOylase activity encoded by LUNA that was responsible for mediating ND10 disruption. Use of a catalytically inactive LUNA mutant in transcriptional analyses of latent infection also generated similar results as with the LUNA deletion viruses. Overall, these data support the hypothesis that LUNA serves as an important regulator of viral gene expression during latency, which is likely linked to its ability to target ND10 structures for disruption, thus raising the possibility that inhibition of deSUMOylation may serve as a novel therapeutic strategy to target latent HCMV infection.

O citomegalovírus (CMV) é uma séria ameaça aos receptores de transplante de medula óssea. A reativação está associada com uma imunidade mediada por células TCD8+ defeituosa. Nosso objetivo foi correlacionar as diferentes subpopulações de células TCD8+ com a reconstituição imunológica dos pacientes, especificamente a imunidade anti-CMV, analisando as subpopulações de células T infundidas nas diferentes modalidades de transplante de medula óssea. Receptores de transplante alogênico de células tronco mobilizadas para o sangue periférico (n=16) ou coletadas diretamente da medula óssea (n=28) e receptores de transplante autólogo de células tronco mobilizadas para o sangue periférico (n=22) foram avaliados. Verificamos que as transferências de células mobilizadas para o sangue periférico dos doadores, tanto nos transplantes alogênicos como autólogos, são proporcionalmente enriquecidas por subpopulações de células memória efetora e efetora, comparadas às transferências de células procedentes diretamente da medula óssea. Este enriquecimento por subpopulações de células TCD8+ mais diferenciadas foi também correlacionado com maior número de células contendo altos níveis de granzima B, considerado um marcador para linfócitos citotóxicos, sendo também encontrado em maior número nas transferências de células do sangue periférico. Entretanto, no pós-transplante, observou-se que somente os receptores de transplante autólogo de células tronco mobilizadas para o sangue periférico, e não os das outras modalidades de transplante, exibiam números elevados de células T CD8+ de memória-efetora e efetora. Ao mesmo tempo, estes receptores apresentaram menos freqüentemente episódios de reativação pelo CMV, e mais freqüentemente produziram IFN-gama em resposta ao CMV. Portanto, a transferência de células do sangue periférico, desde que em ambiente autólogo, está associada não só com a transferência de células TCD8+ com um fenótipo mais maduro, mas também com uma persistência mais prolongada das mesmas, podendo proporcionar uma resposta imunológica antiviral mais rápida e eficiente, como esperado para as células de memória versus naïve. / Cytomegalovirus (CMV) is a serious threat to the recipients of bone marrow transplantation. Reactivation is associated with defective CD8+ T cell-mediated immunity. We aimed to correlate the different subsets of CD8+ T cells with the patients\' immune reconstitution, specifically anti CMV immunity, by analyzing the CD8+ T cell subsets infused in the different types of bone marrow transplantation. Recipients of allogeneic transplant of peripheral blood stem cells (n=16) or bone marrow (n=28) and recipients of autologous transplant of peripheral blood stem cells (n=22) were evaluated. We show that infusions of stem cells derived from donor\'s peripheral blood, either allogeneic or autologous, are proportionally enriched for the memory-effector and effector phenotypes, compared to the infusions of stem cells of bone marrow origin. This increased number of more differentiated subsets of CD8+ T cells was also correlated with an increased number of cells containing high levels of granzyme B, which is another reliable marker of cytotoxic lymphocyte, and which was also more evident in autologous recipients. However, post-transplant, we observed that only the recipients of autologous peripheral blood cells, and not the recipients of the other transplant modalities, exhibited very high numbers of memory-effector and effector TCD8+ cells. At the same time, they less frequently presented CMV reactivation, and more frequently produced IFN-gama in response to CMV antigens. Thus, transfer of stem cells from peripheral blood, provided in an autologous setting, is associated with transfer and prolonged survival of CD8+ T cells with a more mature phenotype, which may provide a more rapid and efficient anti-viral immune response, as expected for memory versus naïve cells.

Computationally driven drug design/discovery efforts generally rely on accurate assessment of the forces that guide the molecular recognition process. HINT (Hydropathic INTeraction) is a natural force field, derived from experimentally determined partition coefficients that quantifies all non-bonded interactions in the biological environment, including hydrogen bonding, electrostatic and hydrophobic interactions, and the energy of desolvation. The overall goal of this work is to apply the HINT-based atomic level description of molecular systems to biologically important proteins, to better understand their biochemistry – a key step in exploiting them for therapeutic purposes. This dissertation discusses the results of three diverse projects: i) structural modeling of human sphingosine kinase 2 (SphK2, a novel anticancer target) and binding mode determination of an isoform selective thiazolidine-2,4-dione (TZD) analog; ii) structural modeling of human cytomegalorvirus (HCMV) alkaline nuclease (AN) UL98 (a novel antiviral target) and subsequent virtual screening of its active site; and iii) explicit treatment of interfacial waters during protein-protein docking process using HINT-based computational tools. SphK2 is a key regulator of the sphingosine-rheostat, and its upregulation /overexpression has been associated with cancer development. We report structural modeling studies of a novel TZD-analog that selectively inhibits SphK2, in a HINT analysis that identifies the key structural features of ligand and protein binding site responsible for isoform selectivity. The second aim was to build a three-dimensional structure of a novel HCMV target – AN UL98, to identify its catalytically important residues. HINT analysis of the interaction of 5’ DNA end at its active site is reported. A parallel aim to perform in silico screening with a site-based pharmacophore model, identified several novel hits with potentially desirable chemical features for interaction with UL98 AN. The majority of current protein-protein docking algorithms fail to account for water molecules involved in bridging interactions between partners, mediating and stabilizing their association. HINT is capable of reproducing the physical and chemical properties of such waters, while accounting for their energetic stabilizing contributions. We have designed a solvated protein-protein docking protocol that explicitly models the Relevant bridging waters, and demonstrate that more accurate results are obtained when water is not ignored.

La transplantation de sang de cordon ombilical (TSCO) constitue un traitement de choix pour une multitude de pathologies hématologiques malignes et non malignes chez l’enfant et dans certains cas l’adulte. La TSCO est associée à certaines complications, dont une reconstitution immunitaire plus lente et une incidence élevée d’infections opportunistes, notamment celles reliées au cytomégalovirus (CMV) et au virus varicella-zoster (VZV). Dans le cadre de ce travail, nous nous sommes intéressés dans un premier temps à la caractérisation de la reconstitution immunitaire spécifique au CMV et au VZV. Nos résultats ont démontré que la reconstitution de l’immunité cellulaire ne requiert ni un statut séropositif pré-transplantation ni le développement de la maladie. De plus, des reconstitutions spontanées ont été détectées chez certains patients séronégatifs vis-à-vis du CMV ou du VZV. Outre le fait qu’elle se manifeste surtout à partir de 6 mois post-transplantation, ladite reconstitution mérite le qualificatif de « protectrice » en termes de réactivations virales et du développement de signes cliniques lorsqu’une fréquence de 150 cellules produisant l’IFN-γ/million est dépassée. Toutefois, moins de 5% des patients développent une réponse T anti-VZV et anti-CMV au cours 100 premiers jours suivant la TSCO. Il est donc possible que les lymphocytes CD8+ T provenant du SCO, comparativement à leurs homologues provenant de la moelle osseuse (MO), présentent un défaut de fonctionnalité, communément appelé « épuisement clonal ». La caractérisation du répertoire de récepteurs inhibiteurs exprimés par les cellules T CD8+ suivant la TSCO ou la transplantation de moelle osseuse (TMO) a révélé une augmentation significative de la fréquence des cellules exprimant PD-1 tôt suivant la transplantation. Cette population, caractérisée majoritairement par un phénotype effecteur-mémoire (EM), démontre une perte significative de la capacité proliférative et exprime moins d'IFN-γ, d'IL-2, de TNF-α et de CD107a. Une meilleure caractérisation de la reconstitution immunitaire après TSCO permettrait, d'une part de sélectionner des biomarqueurs en vue d’une meilleure gestion des patients à risques de développer des infections virales et/ou de rechuter, et d'autre part d'améliorer leur pronostic. / Umbilical cord blood transplantation (UCBT) is a treatment of choice for a variety of hematological malignancies and non-malignant diseases in children and, in some cases, in adults. UCBT is associated with a slower immune reconstitution and a high incidence of viral infections, especially related to cytomegalovirus (CMV) and the varicella-zoster virus (VZV). As part of this work, we aimed to assess the reconstitution of CMV and VZV-specific T cell responses. Neither pre-transplant serostatus nor disease development is required for development of T cell mediated immunity. Moreover, spontaneous reconstitution detected in some patients who were seronegative for CMV or VZV. Detected especially after 6 months post-transplant, antiviral responses are protective in terms of viral reactivation and development of clinical signs, when a frequency of 150 cells producing d'IFN-γ / million is achieved. However, less than 5% of patients develop an antiviral response during the first 100 following UCBT. Compared to their bone marrow (BM) counterparts, UCB CD8+ T lymphocytes may be functionally impaired, a state commonly called « clonal exhaustion ». Characterization of the inhibitory receptors repertoire expressed by CD8+ T cells following UCBT and BMT showed a significant increase in the frequency of cells expressing PD-1 early after transplantation. This population, mainly characterized by effector phenotype, showed a significant loss of proliferative capacity and produced less IFN-γ, IL-2, TNF-α and CD107a. An improved understanding of the CD8+ T cell compartment following UCBT, as well as biomarkers related to T cell exhaustion will decrease infection, transplant related mortality and correlate with better prognosis

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) γδ Vd2neg font le lien entre CMV et DSA : induits par le CMV, les LT γδ 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 γδ Vd2neg possèdent une double réactivité anti-CMV et anti-tumoral. Nous présentons ici un modèle où les lymphocytes T γδ Vδ2neg 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 γδ Vδ2neg en transplantation rénale. Nos données suggèrent que chez l'homme, a fortiori lorsqu’il est immunodéprimé, les LT γδ 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 Vδ2neg γδ T cells are a new player and a potentially useful clinical biomarker in antibody-mediated lesions of kidney transplants. By engaging DSA on their Fcγ-receptor CD16, γδ 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 Vδ2neg γδ 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 Vδ2neg γδ 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, γδ 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.