Regulatorische T-Zellen (Treg) eröffnen neue immuntherapeutische Wege zur Kontrolle unerwünschter Immunreaktionen, jedoch wirft die Heterogenität dieser Zellen die Frage auf, welche Treg-Population für die klinische Anwendung. Darauf basierend werden in dieser Arbeit drei Fragestellungen bearbeitet: i) Bestimmung der Häufigkeit von Tregs und deren Subpopulationen in verschiedenen Altersgruppen bei Empfängern einer Organtransplantation (Tx) und einer gesunden Kontrollgruppe; ii) Vergleich der Suppressorkapazität verschiedener Treg-Populationen und in vitro-Expansion der Zellen unter Erhaltung ihrer Funktionalität; iii) Klärung der Differenzierungsmerkmale von Tregs und deren Verknüpfung mit konventionellen T-Zellen (Tconv) mittels Analyse des T-Zell-Rezeptor- (TCR) Repertoires. Sowohl bei gesunden Probanden als auch bei Tx-Empfänger konnte eine altersabhängige Verschiebung von naiven (TregN) hin zu dominant zentralen Gedächtnis-Zellen (TregCM) beobachtet werden, Treg von Tx-Empfängern hatten mehr Effektor-Memory-Zellen (EM) und sie waren mehr aktiviert. In Bezug auf die Kontrolle der frühen Tconv zeigen TregCM eine erhöhte Suppressorkapazität im Vergleich zu TregN. Außerdem sind im Gegensatz zu TregN nur TregCM dazu in der Lage, Apoptose bei Responderzellen zu induzieren. Der Grund hierfür könnte in der stärkeren Expression von CTLA-4 auf TregM liegen. Die Expansionskultur führte zur phänotypischen Veränderung der TregN, deren Umwandlung in TregCM mit einer verbesserten Suppressoraktivität verbunden ist. Die Daten legen nahe, dass das Expandieren mit gesamt Treg für die Adoptive-Treg-Therapie optimal sind, da sie der größte Anteil von ihnen die hochpotenten TregCM sind. TCR-Studien mittels Next Generation Sequencing zeigen weiter, dass TregM aus TregN entstehen, anstatt aus Tconv, in einem Antigen-gesteuerten Prozess. Diese Daten belegen erstmalig neue Erkenntnisse hinsichtlich der Unterschiede der TCR-Repertoires von TregM und Tconv beim Menschen. / Regulatory T cells (Treg) offer new immunotherapeutic options to control undesired immune reactions, but the heterogeinetiy of Treg raises the question which Treg population should be used for clinical translation Thus, this project involves three main parts: i) investigating Treg frequency and subsets distribution with age in healthy donors and transplant (Tx) patients; ii) comparing the suppressive capacity of Treg subsets and expanding them in vitro without losing functionality; iii) clarifyjing the differiation relationship of Treg subsets and their relation to conventional T cells (Tconv) by T cell receptor (TCR) repertoire analysis. From both healthy donors and Tx patients, an age-dependent shift from naïve Treg (TregN) to the dominant central-memory Treg (TregCM) was observed,; However,Treg in Tx patients contained more effector-memory EM cells, , and they were pre-activated due to the exposure to allo antigens,. Regarding control of early Tconv activation, TregCM showed enhanced suppressive capacity compared to TregN; furthermore, only TregCM could induce apoptosis of responder cells while TregN could not, which may result from thehigherexpression of cytotoxic T-lymphocyte antigen 4 (CTLA-4) on TregM. Following in vitro expansion of the Treg subsets, however, TregN converted mainly into TregCM phenotype with enhanced suppression activity. The poor proliferation capacity of TregEM might indicate EM as the terminal differential stage. These data suggest that expansion with total Treg is optimal for adoptive Treg therapy as the majority of them are the highly potent TregCM. Lastly, TCR repertoire study by next generation sequencing (NGS) indicate that TregM derived from TregN rather than Tconv in an antigen-driven process. The highest similarity of the TCR repertoires was observed between TregCM and TregEM. These data reveal new insights for the first time into the distinct TCR repertoires of Treg subsets and Tconv in human by NGS technology.

T-Zellrezeptor (TZR)-Gentherapie zeigte erste Erfolge in klinischen Studien, jedoch wurden gleichzeitig Risikofaktoren deutlich. Ein Risikofaktor ist das falsche Paaren der transferierten TZR-Ketten mit den endogenen, was zu TZR-Molekülen von unbekannter Spezifität führt und die Oberflächenexpression und somit auch die Funktionalität des transgenen TZR reduziert. Dieser Aspekt wurde in generierten T-Zellklonen mit einer konstitutiven/endogenen TZR-Expression sowie einer zweiten induzierbaren/transgenen TZR-Expression untersucht. Es konnte gezeigt werden, dass nach Induktion der transgenen TZR-Expression der endogene TZR seine Funktionalität verlor, obwohl er noch auf der Oberfläche detektierbar war. Als Ursachen wurden neben einer reduzierten Oberflächenexpression des endogenen TZR auch falsch gepaarte TZR-Moleküle, die mit Hilfe der Fluoreszenz-Resonanz-Energie-Transfer-Methode detektiert wurden, gefunden. Die Modifikation des TZR durch den Einbau einer zweiten Cystein-Brücke, was das Paaren der korrespondierenden TZR-Ketten stabilisieren soll, führte in den T-Zellklonen zu keiner Reduktion der falsch-paarenden TZR-Moleküle. In primären Wildtyp-T-Zellen verbesserte sich das richtige Paaren des transgenen TZR leicht und konnte durch Codon-Optimierung der TZR-Gene weiter verbessert werden. Der zweite untersuchte Risikofaktor ist die Insertionsmutagenese durch den retroviralen Vektor. Die sichere Verwendbarkeit von differenzierten T-Zellen für die TZR-Gentherapie wurde in einem Tiermodel mit wiederholter T-Zellstimulierung, um weitere Mutationen während der Zellteilung zu provozieren, analysiert. Im Laufe der Zeit reicherten sich die transferierten T-Zellen in den Tieren dramatisch an, aber entwickelten sich nicht zu T-Zelllymphomen. Die Proliferationskapazität und die Funktionalität der transferierten T-Zellen wurden bestätigt. Die Polyklonalität der TZR-gen-modifizierten T-Zellen wurde mit Hilfe der linear-amplifizierten Polymerasekettenreaktion nachgewiesen. / T cell receptor (TCR) gene therapy is a new therapy for cancer which showed first clinical success but at the same time risk factors evolved. One risk factor is the mispairing of the TCR chains with the endogenous TCR chains which leads to TCRs with unknown specificities and to a reduced expression and functionality of the transferred TCR. This aspect was analyzed in dual TCR T cell clones which had one constitutive/endogenous TCR expression as well as a second inducible/transgenic TCR expression. It could be shown that the endogenous TCR lost its functionality after induction of the transgenic TCR expression although it was still detectable on the cell surface. The reason was found in the lower surface expression level of the endogenous TCR as well as in mispaired TCR dimers detected by fluorescence resonance energy transfer (FRET) technique. Modification of the TCR by insertion of a second cysteine bridge which should stabilize the pairing of the corresponding TCR chains did not reduce the TCR mispairing in the T cell clones. In primary wild-type cells, the pairing of the transgenic TCR improved slightly and could be further improved by codon-optimization of the TCR genes. The second analyzed possible side effect of TCR gene therapy is the insertional mutagenesis by the retroviral vector. The safety of differentiated T cells for TCR gene therapy was analyzed in an animal model with a repetitive T cell stimulation to provide the opportunity for mutations to occur during cell division. Over time, transferred T cells increased dramatically in the recipient mice, but did not lead to T cell lymphomas. The proliferative capacity and the functionality of transferred T cells were confirmed. The polyclonality of the TCR gene-modified T cells could be confirmed by linear amplification-mediated polymerase-chain reaction.

Die adoptive T-Zell-Therapie (ATT) ist eine sich schnell entwickelnde Immuntherapie, die bei Patienten, die an verschiedenen Krebsarten leiden, eine positive klinische Reaktion anzeigt. Eine Variante der ATT ist eine T-Zellen-Rezeptor (TCR)-Gentherapie, bei der Patienten-T-Zellen mit krebsspezifischen TCRs ausgestattet werden. Die Herstellung der TCR-erzeugten T-Zellen ist schnell und robust und erfordert eine geringe Anfangsmenge an Patienten-T-Zellen. Der Mangel an verfügbaren krebsspezifischen TCRs, die auf verschiedene Moleküle des menschlichen Leukozytenantigens (HLA) der Klasse I beschränkt sind, schließt jedoch viele Patienten von der Krebsbehandlung aus. Die Generierung einer krebsspezifischen TCR-Bibliothek, die aus gut definierten TCRs besteht, könnte die Zahl der Patienten, die an klinischen Studien teilnehmen, erhöhen. Das Ziel dieser Doktorarbeit war es, Epstein-Barr-Virus (EBV)-spezifische TCRs zu identifizieren und zu isolieren, um eine EBV-spezifische TCR-Bibliothek als ein nützliches Werkzeug der TCR-Gentherapie bei der Behandlung von EBV-bedingten Krebserkrankungen zu generieren. Insgesamt wurden neun EBV-spezifische TCRs von EBV-positiven Spendern isoliert und charakterisiert, die verschiedene pHLA-Komplexe von EBV-Latentmembranproteinen (LMP1, LMP2A) und Kernprotein (EBNA3C) erkannten. Zusätzlich wurde ein neuartiges immunogenes LMP1-Epitop (QQNWWTLLV) entdeckt, das auf HLA-C*15:02 beschränkt ist. Definierte EBV-spezifische TCRs können als Grundlage für die EBV-spezifische TCR-Bibliothek verwendet werden, die eine wertvolle Quelle von TCRs für die schnelle Generierung von EBV-spezifischen T-Zellen zur Behandlung von Krebspatienten mit verschiedenen HLA-Typen darstellt. / Adoptive T cell therapy (ATT) is a fast developing immunotherapy indicating positive clinical response in patients suffering from different type of cancers. One type of the ATT is a T cell receptor (TCR) gene therapy, which involves endowing patient T cells with cancer-specific TCRs. Manufacturing of the TCR-engineered T cells is fast and robust, requiring small initial amount of patient T cells. However, lack of available cancer-specific TCRs restricted to various human leukocyte antigen (HLA) class I molecules eliminates many patients from cancer treatment. Generation of a cancer-specific TCR library consisting of well-defined TCRs could increase the number of patients enrolled in clinical trials. The aim of this PhD thesis was to identify and isolate Epstein-Barr virus (EBV)-specific TCRs in order to generate the EBV-specific TCR library as a useful tool of the TCR gene therapy for treatment of EBV-related malignancies. In total, nine EBV-specific TCRs of EBV-positive donors that recognized various pHLA complexes of EBV latent membrane proteins (LMP1, LMP2A) and nuclear protein (EBNA3C) were isolated and characterized. Additionally, a novel immunogenic LMP1 epitope (QQNWWTLLV) restricted to a HLA-C*15:02 was discovered. Defined EBV-specific TCRs can be used as a basis for the EBV-specific TCR library, which provides a valuable source of TCRs for rapid generation of EBV-specific T cells to treat cancer patients with different HLA types.

Ziel der hier vorgelegten Untersuchung war es, die Bedeutung von S. aureus-Exotoxinen/Superantigenen als Pathogenitätsfaktor bei der AD zu analysieren, da kausalpathogenetisch ausgerichtete Experimentalansätze sowie systematische klinische Untersuchungen zu dieser Thematik bei Patienten mit AD ausstanden. In einer Querschnittsstudie war eine Assoziation zwischen dem Grad der S. aureus-Besiedlung und dem Schweregrad der AD nachzuweisen. Von 74 Kindern mit AD waren 60 (81%) mit S. aureus kolonisiert. S. aureus Exotoxin-sezernie-rende Stämme wurden bei 40 Patienten (53%) von der Haut isoliert. Am häufigsten wurden SEA- und SEC-sezernierende Stämme nachgewiesen, gefolgt von SEB, TSST-1 und SED. Der ausgeprägteste Schweregrad der AD wurde in der mit Exotoxin-sezernierenden S. aureus-kolonisierten Gruppe beobachtet. Für die Schwere der Erkrankung, gemessen am SCORAD-Score wurde eine Varianzaufklärung von 30% für die Exotoxine und 50% für die S. aureus-Infektion errechnet. In einer Subgruppe von Patienten wurde der Einfluß von S. aureus-Exotoxinen auf intradermale T-Zell-Rezeptor-Vß-Repertoir-Veränderungen untersucht. Bei den Patienten mit chronischer AD, die mit SEB-sezernierendem S. aureus besiedelt waren, war mittels immunhistologischer Untersuchung in der Haut nachzuweisen, dass zwischen 25% und 65% der intradermalen T-Zellen das zugehörige Superantigen-reaktive Vß-T-Zell-Repertoire gegenüber 5% bis 17% der T-Zellen im Blut exprimieren. Weder in der Haut noch im Blut war eine Akkumulation nicht-superantigenreaktiver T-Zell-Subpopulationen nachzuweisen. Auch fand sich keine selektive Akkumulation von Vß-T-Zell-Subpopulationen bei Kindern mit S. aureus-Kolonisierung ohne Exotoxinnachweis. Die Ergebnisse belegen, dass bei Kindern mit AD und positivem S. aureus-Exotoxinnachweis auf ekzematöser Haut ein Grossteil der dermal akkumulierten T-Zellen auf diese S. aureus-Exotoxine/Superantigene reagieren können und wesentlich an der Pathogenese der AD beteiligt sind. In einer Teilpopulation bei 58 Kindern mit AD wurden Prävalenz und Rolle von Serum-IgE-Antikörpern gegen die S. aureus-Exotoxine SEA und SEB untersucht. Bei 34% der Kinder mit AD (20/58) konnten wir spezifische IgE-Antikörper gegen SEA und/oder SEB nachweisen (45% zu SEB, 10% zu SEA und 45% zu SEA und SEB). Alle gegen SEA und SEB sensibilisierten Kindern waren mit S. aureus kolonisiert gegenüber 71% (27/38) der nicht-sensibilisierten Kinder. Der Grad der S. aureus-Besiedlung, die Prävalenz von SEB-sezernierendem S. aureus auf der Haut, sowie die Prävalenz von S. aureus-Hautinfektionen war in der sensibilisierten Gruppe höher. Die höchste Varianzaufklärung von 37% wurde zwischen dem Vorliegen von S. aureus-Hautinfektionen und dem Nachweis spezifischer SEA/SEB-IgE-Antikörper ermittelt; diese stellen somit einen Risikofaktor für eine Sensibilisierung gegen S. aureus-Exotoxine dar. Die SEA/SEB-sensibilisierte Gruppe zeigte einen höheren Schweregrad der AD, höhere Serum-Gesamt-Spiegel und eine polyvalente Sensibilisierung gegen Inhalations- und Nahrungsmittelallergene. Insgesamt belegen unsere klinischen, immunologischen und statistischen Ergebnisse, dass die S. aureus-Exotoxine einen wesentlichen Einfluss auf die Immunpathogenese der AD haben. Eine orale Therapie mit CyA kann bei S. aureus-kolonisierten Kindern die S. aureus-Besiedlungsdichte reduzieren. In der S. aureus-infizierten Gruppe war die Prävalenz von Exotoxin-produzierendem S. aureus und die Krankheitsaktivität höher, wobei eine Verminderung der Besiedlung nicht beobachtet wurde. Die Ergebnisse dieser Arbeit erlauben den Schluss, dass S. aureus-Exotoxine als Triggerfaktor die Exazerbation der AD im Kindesalter wesentlich unterstützen. Deswegen sollte in ein therapeutisches Konzept eine konsequente Prävention bzw. eine Behandlung von S. aureus-Infektionen einbezogen werden. Kinder mit bereits schwerer AD profitieren von einer immunmodulatorischen Therapie. / Background: The skin of patients suffering from atopic dermatitis (AD) exhibits a striking susceptibility to colonization with S. aureus. Some strains of S. aureus secrete exotoxins with T cell superantigen activity (toxigenic strains) and abnormal T cell functions are known to play a critical role in AD. Objective: The aim of this study was to determine the impact of exotoxin production by skin-colonizing S. aureus on disease severity and the presence of T-cell subsets in lesional skin. Furthermore, we investigated the effect of oral cyclosporin A in severe pediatric atopic dermatitis on disease severity and S. aureus colonization density. Methods: In a cross sectional study of 74 children with atopic dermatitis, the presence and density of toxigenic and non-toxigenic strains of S. aureus was correlated with disease severity. In a subgroup of patients the T cell receptor (TCR) Vß repertoire of peripheral blood and lesional T cells was investigated and correlated with individual superantigen activity of skin colonizing S. aureus. Furthermore, in a subgroup of patients, the presence of IgE antibodies to SEA and SEB was correlated with severity of the disease and the total and other unrelated allergen-specific IgE titers and density of colonization with S. aureus strains on atopic skin and episodes of superficial S. aureus skin infections. Eleven children with severe AD (SCORAD score > 50) were treated for eight weeks with 2.5 to 5 mg/kg CyA. In five children the skin was only colonized with S. aureus whereas the remaining six patients had clinically relevant skin infections with requirement for systemic antibiotic therapy. The isolates from the latter patients were sensitive for the selected antibiotics. Clinical and microbiological investigations were performed before and after CyA therapy. Results: 53% of children with AD were colonized with toxigenic strains of S. aureus producing SEC, SEA, TSST-1, SEB and SED in decreasing frequency. Children colonized with toxigenic S. aureus strains presented with higher disease severity as compared to the non-toxigenic and S. aureus negative groups. The influence of exotoxin production on the SCORAD score was determined as R2 = 0.3 (ie, 30% of the SCORAD score is explained by exotoxin production), whereas infection with S. aureus revealed R2 = 0.5. Patients colonized with toxigenic S. aureus exhibited shifts in the intradermal TCR Vß repertoire which correspond to the respective superantigen-responsive T cell subsets. In a subgroup of patients, twenty of 58 children (34%) were sensitized to superantigens (45% to SEB, 10% to SEA, 45% to SEA and SEB). In this group, severity of AD and levels of specific IgE to food and air allergens were higher. The degree of disease severity correlated to a higher extent with the presence of SEA/SEB-specific antibodies than with total serum IgE levels. Density of colonization with superantigen-secreting S. aureus strains was higher in the superantigen IgE-positive group. Sixty-three of these children experienced repeated episodes of superficial S aureus skin infections. The influence of S. aureus skin infection on the presence of SEA/SEB-specific antibodies was determined as R2 = 0.37 (ie, 37% of the the presence of SEA/SEB-specific antibodies is explained by S. aureus superficial skin infection). In the group of patients, who were treated with CyA, clinical signs and symptoms of AD improved in all patients (mean SCORAD score reduction from 74 to 29). However, disease severity was more supressed by CyA in the "colonized" patients compared with the patients with clinical S. aureus infections. Furthermore, there was a significant decrease in S. aureus density on atopic skin after CyA treatment in "colonized" patients but not in "infected" patients. The prevalence of exotoxin producing strains was higher in the "infected" group. Conclusion: The data demonstrate that S. aureus released exotoxins can modulate disease severity and dermal T cell infiltration. Patients, suffering from AD may take profit from both consequent prevention or treatment of S. aureus skin infection as well as immunmodulating approaches.

Sleeping Beauty (SB) Transposon-basierte Vektoren werden als Alternative zu viralen Vektoren für T-Zell-Gentherapie erforscht und ermöglichen eine schnelle und kostengünstige Genmanipulation von T-Zellen. Die Verwendung von Transposon-Vektoren erfordert jedoch die DNA-Elektroporation von T-Zellen, die sich schädlich auf T-Zellen auswirkt. DNA-elektroporierte T-Zellen weisen eine verringerte Lebensfähigkeit und eine verzögerte Aktivierung nach Stimulation des T-Zell-Rezeptors (TCR) auf. Um die Nachteile der Transposon-basierten T-Zell-Genmanipulation zu überwinden, haben wir neuartige SB-Vektoren entwickelt. Durch die Kombination von SB Transposon-basierten Minicircle-Vektoren mit SB100X Transposase-mRNA konnten T-Zellen effizient genmodifiziert werden. Unser Ansatz reduzierte die T-Zell-Mortalität und steigerte gleichzeitig die Transfektionseffizienz. Mit diesen neuartigen Vektoren wurde die stabile Expression verschiedener TCRs und CARs in über 50% der eingesetzten T-Zellen erreicht. Gentechnisch manipulierte T-Zellen konnten Antigen-spezifisch stimuliert werden und zeigten effiziente Zytokin-Sekretion und Tumorzell-Lyse. Weiterhin haben wir miRNAs entwickelt, die die Expression der endogenen TCR-Ketten unterdrücken. Der Einbau dieser miRNAs in die TCR-Expressionskassette erhöhte die Oberflächenexpression des therapeutischen TCRs, verringerte die Fehlpaarung mit endogenen TCR-Ketten und erhöhte die T-Zell-Funktionalität. Ein direkter Vergleich von SB- und Virus-modifizierten T-Zellen zeigte sowohl in vitro als auch in vivo eine vergleichbare Wirksamkeit der modifizierten T-Zellen hinsichtlich Zytokin-Sekretion, Tumorzell-Lyse und Tumorkontrolle. In dieser Arbeit konnte gezeigt werden, dass SB Minicircle-Vektoren die Herstellung von genetisch modifizierten T-Zellen ermöglichen und diese Tumor-spezifische Wirksamkeit aufweisen. Dieser Ansatz könnte die Herstellung therapeutischer T-Zellen für die personalisierte T-Zell-Gentherapie vereinfachen und beschleunigen. / Sleeping Beauty (SB) transposon-based vectors have entered clinical trials as an alternative to viral vectors for T cell gene therapy, offering time- and cost-efficient engineering of therapeutic T cells. However, transposon vectors require DNA electroporation into T cells, which we found to cause adverse effects. T cell viability was decreased, and DNA-transfected T cells showed delayed activation upon T cell receptor (TCR) stimulation regarding blast formation and proliferation. To overcome the limitations of transposon-based T cell engineering, we investigated the effect of DNA electroporation on T cells and developed novel SB vectors. T cells could efficiently be engineered with Sleeping Beauty vectors by combining SB transposon minicircles and SB100X transposase mRNA. Our approach reduced T cell mortality and substantially enhanced transfection efficiency. We achieved stable expression of several TCRs and CARs in more than 50% of the transfected T cells compared to 15% when conventional plasmids were used. T cells engineered to express a tumor-specific TCR mediated effective tumor cell lysis and cytokine secretion upon antigen-specific stimulation. Furthermore, we developed miRNAs to silence the expression of the endogenous TCR chains. Incorporation of these miRNAs into the TCR expression cassette increased surface expression of the therapeutic TCR, diminished mispairing with endogenous TCR chains, and enhanced T cell functionality. Importantly, a direct comparison of SB minicircle- and RV-engineered T cells in vitro as well as in vivo demonstrated equal T cell efficacy with regards to cytokine release, tumor cell lysis and tumor control. We demonstrated that SB minicircles enable the generation of gene-modified T cells with tumor-specific reactivity. Our approach facilitates the manufacturing of therapeutic T cells with superior biosafety and accelerates the generation of patient-specific T cell products for personalized T cell gene therapy.

Adoptiver Transfer EBV-spezifischer, polyklonaler T-Zelllinien findet Anwendung bei Prophylaxe und Therapie EBV-assoziierter Erkrankungen. Der Ansatz hat den Nachteil der aufwändigen Herstellung der T-Zelllinien, welche aufgrund der Stimulation mit EBV transformierten B-Zelllinien oft nicht die gewünschten EBV Antigene, sondern immundominante EBV Antigene erkennen. Eine Alternative zum polyklonalen T Zelltransfer stellt die Übertragung EBV-spezifischer T-Zellrezeptoren (TCRs) dar. Dadurch können subdominante EBV Antigene angegangen werden, die von Tumorzellen tatsächlich exprimiert werden. In den hier beschriebenen Arbeiten, verwendeten wir peptidbeladene dendritische Zellen (DCs), um selektiv CD4+ T-Zellen gegen ein Epitop aus dem EBV Protein EBNA2 anzureichern. Es gibt Hinweise darauf, dass DCs sich besonders zur Stimulation von T-Zellen eignen, die subdominante EBV Antigene erkennen. Die TCR Gene eines solchen CD4+ T-Zellklons, sowie zweier CD8+ Klone, wurden in Vektoren kloniert, mit denen die EBV Spezifität der Klone auf andere T-Zellen übertragen werden sollte. Wie bereits zuvor in anderen Laboren beobachtet, waren auch unsere TCR modifizierten T-Zellen zunächst nicht in der Lage, EBV infizierte Zielzellen effektiv zu attackieren. Erst durch Modifikation der Vektorstrategie (2A Peptidlinker als Ersatz für das IRES Element (internal ribosomal entry site)) sowie der TCR Gene (Codon-Optimierung) konnte eine deutlich verbesserte Expression und Funktion der modifizierten T-Zellen erreicht werden. Außerdem hing die Effektivität der modifizierten T-Zellen essentiell von der als Zielzelle verwendeten LCL ab. Die hier beschriebenen Arbeiten zeigen die erfolgreiche Übertragung von TCRs gegen EBV Antigene auf T-Zellen. Die so modifizierten T-Zellen erlangten anti-EBV Aktivität und sprechen daher für die prinzipielle Anwendbarkeit TCR-modifizierter T-Zellen zur Behandlung EBV-assoziierter Erkrankungen. / Adoptive transfer of polyclonal Epstein-Barr-virus (EBV)-specific T cell lines has been used as prophylaxis and therapy in patients with EBV-associated malignancies. This approach, however, is limited by the difficult expansion of polyclonal T cells directed mainly against dominant EBV antigens presented on EBV-transformed B cell lines (LCLs). Isolating EBV-specific T cell receptors (TCRs) for transduction of T cells is an alternative strategy to confer T cell immunity against EBV antigens including subdominant EBV antigens. In this study, we have used peptide-pulsed DCs to selectively expand EBV-specific CD4+ T cell clones against an EBNA2-derived epitope. Data suggested that peptide-pulsed DCs are particularly effective in stimulating T cells specific for subdominant EBV antigens. TCR genes from one of these clones as well as from two CD8+ T cell clones were identified by RACE PCR. TCR alpha and beta chains where then cloned into retroviral vectors for transduction of T cells to equip them with anti-EBV specificity. The TCR-modified T cells where then tested for their function towards LCLs to assess the chances for the use of EBV-redirected T cells in adoptive immunotherapy of EBV-associated disease. Like in previous reports, our EBV-specific TCRs at first did not confer effective activity against LCLs. Instead, we had to apply modifications to the TCR vectors to improve expression and function of the introduced TCRs. Codon optimization as well as replacement of the IRES site by a 2A peptide linker was required to significantly increase expression and function of transduced TCRs. Also, we found that the effectiveness of TCR transduced T cells is dependent on the target cell chosen. Our data show successful transfer of functionally active EBV-specific TCRs into T cells to render them effective against LCLs, representing the basis for the development of TCR-transgenic T cells for adoptive T cell transfer in EBV-associated disease.

Thesis summary The process of B cell lymphogenesis in swine remains uncertain. Some reports indicate that pigs belong to a group of animal that use ileal Peyers's patches (IPP) for the generation of B cells while others point to the possibility that the bone marrow is functional throughout life. The functional subpopulations of B cells in swine are also unknown. Together with other ruminants, and also birds, γδ T cells in swine may account for >70% of all T cells which is in apparent contrast with humans and mice. The purpose of this thesis was to address these discrepancies and unresolved issues. The results disprove the existing paradigm that the IPP is primary lymphoid tissue and that B cells develop in IPP in an antigen-independent manner. On the other hand, it shows that bone marrow is fully capable of B cell lymphogenesis and remains active at least for the same period of time as it had been speculated for the IPP. This thesis also identified functionally different subsets of porcine peripheral B cells, and shows that CD21 molecules can be expressed in differential forms. Finally, this thesis identifies two lineages of γδ T cells that differ in many functional and phenotype features. This finding may explain why γδ T cells constitute of minority of lymphocytes in circulation of humans and mice.

O timo é um órgão linfoide primário, localizado em região mediastinal, cuja importância funcional é a diferenciação e maturação de todas as subpopulações de linfócitos T provenientes da medula óssea assim como a seleção de células autorreativas. Sua hipoplasia ou aplasia resultam em síndromes de imunodeficiência. Embora de vital importância, o estudo clínico de sua função não é rotineiro na prática clínica, o que pode ser atribuído a sua dificuldade de avaliação em razão de sua localização, necessidade de uso de métodos de imagem não inócuos ao paciente (tomografia computadorizada (TC), PET-SCAN) e complexidade das análises em sangue periférico de subpopulações de células T por citometria de fluxo e, mais recentemente, medição de T cell receptor excision circles (TRECs), por PCR. Um possível método da avaliação do timo sem radiação ionizante ou dor ao paciente seria a elastografia de timo por ultrassom e seu uso na prática clínica poderia substituir a TC, como ocorre na avaliação de lesões hepáticas ou mamárias. Objetivo - Este estudo se propõe a 1. Implantar este método na avaliação da função tímica, 2. Estabelecer valores de referência de TRECs na faixa etária estudada, 3. Investigar se há correlação entre os dois parâmetros. Métodos - Foram incluídas sessenta e quatro crianças de 0-5 anos em acompanhamento no ambulatório de cirurgia infantil sem doença sistêmica ou infecção aguda, e que iriam coletar amostra de sangue para exames pré-operatórios. Quarenta e oito destas coletaram amostra de sangue para avaliação de TRECs, vinte e nove realizaram elastografia num mesmo momento, porém apenas 13 destas apresentaram resultado confiável. A média da idade foi de 36 ± 16meses, predomínio do grupo foi masculino (75%), nascidos a termo (72%) e a principal intervenção cirúrgica foi do tipo urológica de pequeno porte. A elastografia mostrou média de 1,21 ± 0,24m/s, sem diferença significativa quando comparada ano a ano. Observamos uma média de TRECs de 195,6 ± 120,5 cópias/µL, mostrando valores significativamente mais altos quando comparados a adolescentes hígidos da base de dados do laboratório. Os valores de TRECs observados mostram uma ampla variabilidade na faixa etária estudada, sem diferença significativa quando separados por idade ano a ano. Não se encontrou correlação significativa entre a dureza do timo analisada à elastografia e valores de TRECs em sangue periférico. Concluímos que a elastografia é um método que possibilita a avaliação das dimensões e função do timo em crianças a partir de 2 anos de idade, entretanto estudos adicionais são necessários para que se possa recomendar a larga implantação deste método com essa finalidade / The thymus is a primary lymphoid gland responsible for the maturation of T cells as well as the immunological central tolerance. It has been a neglected organ by physicians, despite its relevance in early immunity. Thymic function can be indirectly measured by Computerized Tomography imaging and PET SCAN, T cell subpopulation flow cytometry. More recently, in the beginning of this century, a direct measurement represented by TRECs (T cell receptors excision circles) was developed. Classical thymic imaging has used ionized radiation, which poses a major risk for the pediatric patient and new techniques are needed. Objectives and methods - In this work, we tested the use of elastography ultrasound for the evaluation of the thymus in a group of < 5-year- old healthy children. In parallel, we measured TRECs in peripheral blood and compared the values obtained from both methods. We have reached sixty-four children at the pediatric surgery outpatients ambulatory, scheduled for minor surgeries. A sample of blood was taken during pre operatory and then patients were sent to the imaging service for elastography. Of all, sixty-four had undertaken TRECs and seventeen, elastography. The median age was 36 ±16 months and we had 75% of boys for surgical correction of urologic minor defects. The elastography results showed a median of 1.2 ± 0.24 m/s in all ages, the same stiffness as the liver, as shown in other works. Our median TREC/µL value was 195.6 ± 120.5 copies/µL showing a trend of reduction in older ages, and with statistical significance when compared with healthy teenagers\' values from the lab database. We concluded that elastography may be a good diagnostic tool for thymus evaluation, and additional works are needed for its recommendation in clinical practice. Our TRECs values showed a large variability, as also demonstrated in previous works, and a trend of reduction over age. We could not observe any significant correlation between elastography and TRECs values

O timo é um órgão linfoide primário, localizado em região mediastinal, cuja importância funcional é a diferenciação e maturação de todas as subpopulações de linfócitos T provenientes da medula óssea assim como a seleção de células autorreativas. Sua hipoplasia ou aplasia resultam em síndromes de imunodeficiência. Embora de vital importância, o estudo clínico de sua função não é rotineiro na prática clínica, o que pode ser atribuído a sua dificuldade de avaliação em razão de sua localização, necessidade de uso de métodos de imagem não inócuos ao paciente (tomografia computadorizada (TC), PET-SCAN) e complexidade das análises em sangue periférico de subpopulações de células T por citometria de fluxo e, mais recentemente, medição de T cell receptor excision circles (TRECs), por PCR. Um possível método da avaliação do timo sem radiação ionizante ou dor ao paciente seria a elastografia de timo por ultrassom e seu uso na prática clínica poderia substituir a TC, como ocorre na avaliação de lesões hepáticas ou mamárias. Objetivo - Este estudo se propõe a 1. Implantar este método na avaliação da função tímica, 2. Estabelecer valores de referência de TRECs na faixa etária estudada, 3. Investigar se há correlação entre os dois parâmetros. Métodos - Foram incluídas sessenta e quatro crianças de 0-5 anos em acompanhamento no ambulatório de cirurgia infantil sem doença sistêmica ou infecção aguda, e que iriam coletar amostra de sangue para exames pré-operatórios. Quarenta e oito destas coletaram amostra de sangue para avaliação de TRECs, vinte e nove realizaram elastografia num mesmo momento, porém apenas 13 destas apresentaram resultado confiável. A média da idade foi de 36 ± 16meses, predomínio do grupo foi masculino (75%), nascidos a termo (72%) e a principal intervenção cirúrgica foi do tipo urológica de pequeno porte. A elastografia mostrou média de 1,21 ± 0,24m/s, sem diferença significativa quando comparada ano a ano. Observamos uma média de TRECs de 195,6 ± 120,5 cópias/µL, mostrando valores significativamente mais altos quando comparados a adolescentes hígidos da base de dados do laboratório. Os valores de TRECs observados mostram uma ampla variabilidade na faixa etária estudada, sem diferença significativa quando separados por idade ano a ano. Não se encontrou correlação significativa entre a dureza do timo analisada à elastografia e valores de TRECs em sangue periférico. Concluímos que a elastografia é um método que possibilita a avaliação das dimensões e função do timo em crianças a partir de 2 anos de idade, entretanto estudos adicionais são necessários para que se possa recomendar a larga implantação deste método com essa finalidade / The thymus is a primary lymphoid gland responsible for the maturation of T cells as well as the immunological central tolerance. It has been a neglected organ by physicians, despite its relevance in early immunity. Thymic function can be indirectly measured by Computerized Tomography imaging and PET SCAN, T cell subpopulation flow cytometry. More recently, in the beginning of this century, a direct measurement represented by TRECs (T cell receptors excision circles) was developed. Classical thymic imaging has used ionized radiation, which poses a major risk for the pediatric patient and new techniques are needed. Objectives and methods - In this work, we tested the use of elastography ultrasound for the evaluation of the thymus in a group of < 5-year- old healthy children. In parallel, we measured TRECs in peripheral blood and compared the values obtained from both methods. We have reached sixty-four children at the pediatric surgery outpatients ambulatory, scheduled for minor surgeries. A sample of blood was taken during pre operatory and then patients were sent to the imaging service for elastography. Of all, sixty-four had undertaken TRECs and seventeen, elastography. The median age was 36 ±16 months and we had 75% of boys for surgical correction of urologic minor defects. The elastography results showed a median of 1.2 ± 0.24 m/s in all ages, the same stiffness as the liver, as shown in other works. Our median TREC/µL value was 195.6 ± 120.5 copies/µL showing a trend of reduction in older ages, and with statistical significance when compared with healthy teenagers\' values from the lab database. We concluded that elastography may be a good diagnostic tool for thymus evaluation, and additional works are needed for its recommendation in clinical practice. Our TRECs values showed a large variability, as also demonstrated in previous works, and a trend of reduction over age. We could not observe any significant correlation between elastography and TRECs values

Há um amplo espectro de doenças causadas por estreptococos do grupo A (GAS), e são consideradas um problema de saúde pública em vários países, principalmente os em desenvolvimento, com aproximadamente 600 milhões de casos/ano. As infecções causadas por GAS podem ocasionar doenças invasivas como faringite e pioderma com seqüelas auto-imunes graves como a febre reumática (FR) e glomerulonefrite. A FR acomete principalmente crianças e jovens adultos. A FR apresenta diversas manifestações, sendo a doença reumática cardíaca (DRC) a seqüela mais grave, caracterizada por lesões cardíacas valvares progressivas e permanentes. O tratamento, frequentemente envolve cirurgia cardíaca para a correção de lesões valvulares, o que acarreta alto custo para o Sistema Único de Saúde no Brasil e em vários países. Em trabalhos anteriores sobre os mecanismos desencadeadores das lesões reumáticas no coração, foi possível identificar o papel do linfócito T como mediador principal da autoimunidade, através da análise do receptor de células T infiltrantes de lesão cardíaca de indivíduos com DRC. Várias expansões oligoclonais com diferentes tamanhos da região que reconhece o antígeno, CDR3 foram encontradas. No presente trabalho, analisou-se a atividade tímica através da quantificação de fragmentos circulares excisados pelo rearranjo do gene do receptor do linfócito T (TREC) em linfócitos T de sangue periférico de indivíduos com FR e DRC. Também foi avaliada a presença de células T naïve e de memória através de citometria de fluxo. Os resultados do presente trabalho mostraram que a quantidade de TREC em amostras de sangue periférico do grupo de pacientes com FR/DRC foi significantemente menor quando comparada a observada em indivíduos saudáveis. Interessantemente, em ambos os grupos a quantidade de TREC apresentou correlação negativa com a idade dos indivíduos estudados. Os resultados indicaram diferenças na atividade tímica em pacientes com FR/DRC, provavelmente decorrente do processo autoimune que envolve linfócitos T / There is a wide spectrum of diseases caused by group A streptococci (GAS), that still being considered a public health problem in developing countries, with about 600 million cases per year. Infections by GAS can cause invasive diseases such as pharyngitis and pyoderma leading to serious autoimmune complications such as rheumatic fever (RF) and glomerulonephritis. RF mainly affects children and young adults, and presents different manifestations. Rheumatic heart disease (RHD) is considered the most serious complication leading to valvular lesions that are characterized by progressive and permanent heart damage, which entails high cost to the Public Health System in Brazil and worldwide. In previous works that focused on the mechanisms leading to rheumatic heart lesions, we identified the role of T lymphocytes as principal mediator of autoimmune reactions. Through the in deep analysis of infiltrating T-cell receptor repertoire of patients with RHD, we identified oligoclonal expansions with different sizes of CDR3 that is the region of antigen recognition. In the present study we analyzed the thymic activity through T cell receptor excision circles (TREC) quantification in T cells from peripheral blood of RF/RHD patients. We also evaluated naïve and memory T cells from peripheral blood by flow cytometry. Our results showed that the amount of TREC in the peripheral blood of patients was significantly lower when compared to the healthy individuals. In addition, both groups showed that the amount of TREC is negatively correlated with age. These results indicated that the thymic activity in RF/RHD patients is altered probably due to the autoimmune process that involves T lymphocytes