Despite the remarkable success of adoptive T cell therapy in the treatment of melanoma and hematological malignancies, therapeutic capacity in a broad range of solid tumours is impaired due to immunosuppressive events that render tumour-specific T cells unable to persist and kill transformed cells. To address some of the limitations of ACT in solid tumours, our laboratory has developed a therapeutic modality utilizing oncolytic virus, which expresses a tumour-associated antigen, known as an oncolytic viral vaccine (OVV), in combination with tumour specific central memory T cells. With this therapeutic approach (ACT), we can achieve robust in vivo expansion of transferred cells resulting in the complete and durable tumour regression in multiple solid murine tumour models. However, we demonstrate that the curative potential is lost when the tumour stage and burden increase as expanded transferred cells differentiate to a dysfunctional state resulting in the progressive decline in the tumour-specific CD8+ T cell response. Thus, we believe that restoring the T cell response in late-stage tumours will lead to enhanced curative potential of ACT in late-stage tumours. We have previously shown that HDACi, MS-275, can enhance the therapeutic capacity of a T cell-based therapy in an aggressive brain tumour model. In addition, concomitant delivery of MS-275 with ACT ensures durable cures through immunomodulatory mechanisms. Strikingly, concomitant delivery of MS-275, a class 1 histone deacetylase inhibitor (HDACi), with ACT in late-stage tumours completely restores the transferred T cell response to similar levels observed in early-stage tumours resulting in the complete regression of advance-stage tumours. Furthermore, MS-275 enhanced the proliferative capacity and tumour-specific cytotoxic function of transferred cells, independently of tumour stage, type and mouse strain. Interestingly, we did not observe a complete reversal of T cell dysfunction, but rather observed that MS-275 conferred unique properties to T cells as the expression of some markers typically associated with T cell dysfunction was enhanced in addition to persistence and proliferation capacity. Moreover, concomitant delivery of MS-275 also restored the therapeutic capacity of endogenously primed tumour-specific CD8+ T cells expanded by an OVV in late-stage tumours, demonstrating the potential for general use for MS-275 in T cell-based therapies. Our data suggests the use of HDACi may potentiate T cell-based immunotherapies to overcome tumour-mediated T cell dysfunction in advanced stage solid tumours. / Thesis / Master of Science in Medical Sciences (MSMS)

Recombinant oncolytic virus (OV) vaccines that encode tumour-associated antigens are potent boosting agents for adoptive transfer of tumor-specific T cells (adoptive T cell therapy or ACT). Current strategies to exploit boosting vaccines for ACT rely on a priori knowledge of targetable tumour epitopes and isolation of matched epitope-specific T cells. Therefore, booster vaccines must be developed on a patient-by-patient basis, which severely limits clinical feasibility. To overcome the requirement for individualized pairing of vaccines and T cells, we propose a “universal” strategy for boosting tumor-specific T cells where the boost is provided through a synthetic receptor that can be engineered into any T cell and a matched vaccine. To this end, we are employing chimeric antigen receptors (CARs), which confer MHC-independent antigen specificity to engineered T cells, and a paired OV vaccine that encodes the CAR target. As proof-of-concept, we have developed and evaluated a model where murine TCR transgenic T cells are engineered with boosting CARs against a surrogate antigen for studies in immunocompetent hosts. In chapter 3, I optimized a murine CAR-T cell manufacturing protocol that allows for generation of highly-transduced T cells that maintain a predominantly central memory (Tcm) phenotype. This protocol leads to generation of highly functional CAR-T cell products that can be cryopreserved at the end of ex vivo culture for future use in adoptive transfer and vaccination studies. In chapter 4, I evaluated the in vivo boosting potential of our dual-specific CAR-T cells with paired OV vaccines. Adoptive transfer of these CAR-engineered tumor-specific T cells followed by vaccination with paired oncolytic vesicular stomatitis virus (VSV) vaccine leads to robust, but variable and transient, CAR-mediated expansion of tumour-specific CD8+ T-cells, resulting in delayed tumour progression in aggressive syngeneic tumour models. In chapter 5, I investigated the role of OV-induced type I interferon (IFN-I) responses on CAR-T cell boosting. I found that CAR-T cell expansion and anti-tumour function following OV vaccination is limited by the IFN-I response and can be further enhanced by blocking interferon alpha and beta receptor subunit 1 (IFNAR1). This IFN-I-mediated T cell suppression was found to be T cell-extrinsic and related to premature termination of OV infection and antigen expression in vivo. In chapter 6, I investigated the role of CD4+ T cell help in vaccine-mediated T cell boosting and evaluated different genetic engineering strategies to integrate pro-survival STAT5 signaling into the CAR-T cell product in an effort to improve persistence and long-term anti-tumour efficacy. The work presented herein describes a novel and clinically feasible approach to enhancing adoptive T cell therapies and contributes to the basic understanding of T cell biology in the context of CAR-engineering and cancer vaccination. / Thesis / Doctor of Philosophy (PhD) / Despite recent advances in cancer prevention, detection, and treatment, 2 in 5 Canadians are expected to be diagnosed with cancer in their lifetime and approximately 1 in 4 will succumb to their disease. New, more specific therapies are needed to improve responses to treatment and reduce therapy-related side effects. Cell therapy is a new way to treat cancer that uses the patient’s own immune cells as a living drug. The immune cells are taken from a patient’s blood or tumour, trained to attack cancer in the laboratory, and infused back into the patient where they will find and kill cancer cells. A major challenge with this strategy is that the trained immune cells do not always survive in the patient for long enough to get rid of the tumour. To “boost” the immune cells, we are developing a new strategy where the immune cells are genetically modified and combined with a vaccine to enhance their anti-tumor activity. Just like a vaccine against a bacteria or virus, this vaccine will tell the modified immune cells to turn on, make more of themselves, and to find and kill the cancer cells. By delivering this “go” signal through a vaccine, we think that the immune cells will be better able to survive and generate a stronger, longer-lasting immune response against the cancer. This thesis tests this approach in relevant mouse models of cancer and aims to understand how we can best design the immune cells and vaccine to work together in their tumour-killing activities.

Die Mehrzahl der im Thymus generierten CD4+CD25high regulatorischen T-Zellen (Treg) besitzt hohe Affinität gegenüber körpereigenen Antigenen. Es ist bekannt, dass T-Zell Rezeptoren (TCR) auf Treg Zellen in der Peripherie zusätzlich auch fremde Antigene verschiedener Pathogene wie Parasiten, Bakterien und Viren erkennen. Wenig ist bekannt über das klonale T-Zell Rezeptor Repertoire dieser Treg Populationen und ihre Beziehung zu CD4+CD25low effektor T-Zellen (Teff) im Menschen. In dieser Studie analysieren wir humane TCR auf expandierten Treg and Teff Zellen mit definierter Antigen Spezifität für Haupthistokompatibilitätskomplex (MHC) Klasse II restringierte „fremde“ Epitope des Cytomegalovirus (CMV). Bemerkenswerterweise fanden wir, dass der gleiche TCR Vb-CDR3 Klon in beiden funktionell unterschiedlichen Subpopulationen in vitro dominant expandiert ist. Im Unterschied zu ihren klonal-identischen Teff Gegenspielern, exprimieren die suppressiven Treg Zellen kaum CD127 und IL-2, aber hohe Mengen an IFNg und IL-10. Zusammen mit der signifikant erhöhten FOXP3 Expression, trotz unvollständiger foxp3-DNA Demethylierung, lassen sich die CMV-spezifischen CD4+CD25high Treg Zellen einem induzierten Treg (iTreg) Phänotyp zuordnen mit Ähnlichkeit zum beschriebenen Tr-1 Phänotyp. Darüber hinaus konnten wir die klonale TCR Identität auch in frisch isolierten CD4+CD25low und CD4+CD25high Subpopulationen bestätigen, was die Entstehung von CMV-spezifischen Treg Zellen bereits in vivo nahe legt. Periphere CD25high Treg Zellen supprimieren die anti-virale Immunantwort in Patienten mit häufigen CMV-Reaktivierungen, was auf ihre Bildung als Reaktion chronischer Antigenexposition interpretiert werden kann. Unsere Ergebnisse beweisen erstmals direkt, dass aus dem gleichen humanen T-Zell Klon Teff und Treg Zellen mit identischer Spezifität entstehen können und lassen vermuten, dass die Treg Induktion in der Peripherie durch häufige Antigenexposition vorangetrieben wird. / The majority of thymically arised regulatory CD4+CD25high T cells (Treg) show high affinity to self-antigens. It has been proposed that T-cell receptors (TCR) on Treg cells in the periphery also recognize foreign-antigens from pathogens, such as bacteria and viruses. Studies in mice have shown that peripheral Treg cells can be generated not only from naïve T cells but also from effector T cells (Teff). However, in humans the clonal TCR-repertoire of these Treg populations and their relation to effector CD4+CD25low Teff is not sufficiently known up to date. Here, we analyzed human TCRs derived from expanded Treg and Teff cells with defined specificity to MHC class-II restricted “foreign” epitopes of Cytomegalovirus (CMV). Remarkably, we found that both functionally distinct subsets share the same dominant TCR-CDR3 clones in vitro. In contrast to their Teff counterparts, the Treg cells express low CD127 and IL-2, but high IL-10 upon antigen stimulation. Therefore, together with increased FOXP3 expression, but incomplete foxp3 DNA-demethylation, human CMV-antigen specific Treg cells exhibit an induced phenotype (iTreg) in vitro with similarity to recently described Tr-1 phenotype. Moreover, the clonal identity was confirmed in freshly isolated CD4+CD25low and CD4+CD25high subsets, suggesting their generation occurred already in vivo. Peripheral CD25high Treg cells suppress the anti-viral immune response in patients with frequent CMV-reactivations, implying their development as reaction on chronic antigen-exposure. Our results demonstrate directly for the first time, that the same human T-cell clone can possess the phenotype of Teff and Treg cells with specificity to identical foreign epitopes and suggest that Treg-induction in the periphery is supported by frequent antigen-exposure.

<p>The main focus of my thesis has been to develop protocols for generating antigen-specific cytotoxic T lymphocytes (CTLs) and T helper cells (T_H) for adoptive transfer to treat cytomegalovirus (CMV) disease and prostate cancer. CMV viremia is a severe complication in immunocompromised stem cell transplanted patients. Prostate cancer is a leading cause of death for men in Western countries. Although different in nature, CMV-infected cells and prostate cancer cells can both be eliminated through specific activation of the adaptive immune system. </p><p>To generate CMV pp65-specific T cells, I utilized dendritic cells (DCs) modified with an HLA-A*0201/pp65_495-503 peptide, a recombinant adenovirus coding for pp65, <i>in vitro</i> transcribed pp65 mRNA and a recombinant pp65 protein. Peptide stimulation yielded large numbers of peptide-specific CD8⁺ T cells with high lytic activity while adenovirus or mRNA stimulation resulted in the expansion of CTLs against multiple pp65 epitopes. The recombinant protein activated primarily CD4⁺ T_H cells. Stimulation with DCs co-modified with pp65 mRNA and pp65 protein simultaneously generated both pp65-specific CTLs and T_H cells. Such T cells would cover all pp65 epitopes while avoiding potential virus related biohazards. The mRNA/protein combinatory approach can be used to stimulate T cells <i>ex vivo</i> from virtually all stem cell donors for adoptive T cell transfer. </p><p>I have identified two immunogenic HLA-A*0201-restricted peptide epitopes from the prostate tissue antigen TARP. Repeated stimulations with TARP peptide-pulsed DCs yielded up to 20% TARP-directed CD8⁺ T cells even when starting from undetectable frequencies (<0.01%). The T cells could be sorted to 99% purity and expanded 1000-fold with retained specificity and activity. We also detected TARP-directed CD8⁺ T cells in the blood of prostate cancer patients. Therefore, TARP seems to have potential as antigen in DC vaccination or adoptive T cell therapy of prostate cancer. </p>

The main focus of my thesis has been to develop protocols for generating antigen-specific cytotoxic T lymphocytes (CTLs) and T helper cells (TH) for adoptive transfer to treat cytomegalovirus (CMV) disease and prostate cancer. CMV viremia is a severe complication in immunocompromised stem cell transplanted patients. Prostate cancer is a leading cause of death for men in Western countries. Although different in nature, CMV-infected cells and prostate cancer cells can both be eliminated through specific activation of the adaptive immune system. To generate CMV pp65-specific T cells, I utilized dendritic cells (DCs) modified with an HLA-A*0201/pp65495-503 peptide, a recombinant adenovirus coding for pp65, in vitro transcribed pp65 mRNA and a recombinant pp65 protein. Peptide stimulation yielded large numbers of peptide-specific CD8+ T cells with high lytic activity while adenovirus or mRNA stimulation resulted in the expansion of CTLs against multiple pp65 epitopes. The recombinant protein activated primarily CD4+ TH cells. Stimulation with DCs co-modified with pp65 mRNA and pp65 protein simultaneously generated both pp65-specific CTLs and TH cells. Such T cells would cover all pp65 epitopes while avoiding potential virus related biohazards. The mRNA/protein combinatory approach can be used to stimulate T cells ex vivo from virtually all stem cell donors for adoptive T cell transfer. I have identified two immunogenic HLA-A*0201-restricted peptide epitopes from the prostate tissue antigen TARP. Repeated stimulations with TARP peptide-pulsed DCs yielded up to 20% TARP-directed CD8+ T cells even when starting from undetectable frequencies (&lt;0.01%). The T cells could be sorted to 99% purity and expanded 1000-fold with retained specificity and activity. We also detected TARP-directed CD8+ T cells in the blood of prostate cancer patients. Therefore, TARP seems to have potential as antigen in DC vaccination or adoptive T cell therapy of prostate cancer.

Les thérapies anti-tumorales se sont considérablement améliorées au cours de la dernière décennie. Toutefois, les traitements utilisés actuellement rencontrent d'importantes limitations, notamment dans le cas de cancers métastatiques, révélant l'urgence de développer de nouvelles approches. Ainsi, l'immunothérapie par transfert adoptif de cellules T représente une approche innovante particulièrement prometteuse. Son principe s'appuie sur l'injection de cellules T autologues spécifiques d'antigènes tumoraux, préalablement manipulées et amplifiées ex vivo, chez des patients rendus lymphopéniques par chimiothérapie et/ou radiothérapie. Toutefois, même si l'état lymphopénique est induit par ces 2 protocoles de conditionnements, leurs effets sur l'environnement de l'hôte ainsi que sur le devenir des cellules T greffées étaient, jusqu'à nos travaux, mal connus. Par le biais de modèles murins, nous avons pu démontrer que le devenir des cellules T diffère après transfert dans des souris irradiées ou traitées par chimiothérapie (Bu/Cy). Ainsi, après transfert dans des animaux irradiés, on observe une prolifération préférentielle des cellules T CD8, dépendante de l'IL-7, est observée alors qu'un transfert chez des souris traitées Bu/Cy se traduit par une prolifération rapide, indépendante de l'IL-7, des cellules T CD4. De plus, ces comportements sont associés à d'importantes modifications de l'environnement généré chez l'hôte. Plus spécifiquement, nous avons démontré, dans les organes lymphoïdes secondaires, que la localisation et la représentation des différentes sous-populations de cellules dendritiques présentes étaient différentiellement modulées par le type de conditionnement utilisé. Par ailleurs, l'élimination spécifique des cellules CD11c+ chez des souris traitées Bu/Cy était accompagnée d'une inhibition importante de la prolifération rapide des cellules T CD4 greffées. L'ensemble de nos travaux montrent que les traitements lymphopéniques génèrent des environnements distincts capables de moduler le devenir des cellules T greffées.Durant ma thèse, nous avons également abordé de façon originale un aspect novateur de l'environnement en étudiant le rôle potentiel des nutriments comme régulateurs métaboliques des fonctions effectrices des cellules T. La glutamine est l'acide aminé le plus abondant du plasma, pouvant contribuer aux besoins bionénergétiques et biosynthétiques des cellules T en prolifération. Nous avons démontré dans nos travaux qu'une carence en glutamine lors de l'activation de cellules T CD4 par leur TCR entrainait un délai dans l'activation de la voie mTOR, une réduction de la production intracellulaire d'ATP aux temps précoces et se traduisait par une diminution de la prolifération. De plus, ces conditions étaient associées à une augmentation de la conversion de cellules CD4 T naïves, via TGFβ, en cellules régulatrices Foxp3+ , y compris en condition de polarization Th1. Par contre, la carence en glutamine n'a pas inhibé la différenciation Th2. Les cellules T Foxp3+ ainsi générées en condition limitante de glutamine présentaient in vivo des fonctions suppressives aussi efficaces que celles des cellules régulatrices nTregs. En effet, elles ont la capacité de bloquer l'induction de la colite provoquée par la greffe de cellules T effectrices dans des souris Rag2-/- . Nos travaux démontrent ainsi que l'environnement métabolique peut être un régulateur clé de la différenciation des cellules T CD4. L'ensemble de mes travaux de thèse ont mis en évidence de nouveaux paramètres capables de potentiellement modifier la survie et la réactivité des cellules T greffées. / Anti-tumor therapies have improved significantly over the decade. However, the currently used treatments have important limitations, notably for metastatic cancers, and the development of new approaches is therefore a high priority. Adoptive T cell therapy (ACT) represents an innovative strategy that has shown much promise. This therapy is based on the infusion of tumor-specific T cells, which have been manipulated and expanded ex vivo, into patients who have been rendered lymphopenic by chemotherapy and/or irradiation. It is interesting to note that while lymphodepletion is attained by the vast majority of conditioning regimens, the effects of these protocols on the host environment and potentially, on the destiny of adoptively-transferred T cells had not been elucidated prior to the studies which we initiated. Using a murine model, we found that the fate of adoptively-transferred T cells differs markedly in mice rendered lymphopenic by sub-lethal irradiation as compared to a busulfan/cyclophosphamide (Bu/Cy) chemotherapy regimen. Irradiation-mediated lymphopenia resulted in a skewed IL-7-dependent proliferation of donor CD8+ T cells, whereas Bu/Cy treatment led to an increased IL-7-independent, rapid CD4+ T cell proliferation. These alterations in T cell proliferation were associated with striking changes in the host microenvironment. More specifically, we demonstrated that the proportion and localization of different dendritic cell (DC) subsets in lymphoid organs were differentially affected by the type of conditioning. Furthermore, we found that these DC controlled the rapid donor CD4+ T cell division detected in Bu/Cy-treated mice as depletion of CD11c+ DC inhibited this proliferation. Altogether, our studies demonstrate that lymphopenic regimens generate distinct host environments which modulate the fate of adoptively-transferred T cells. Durind my PhD, we also investigated an original and novel aspect of the microenvironement by studying the potential role of nutrients as metabolic regulators of T cell effector function. Glutamine is the most abundant amino acid in the plasma and contributes to the bioenergetic and biosynthetic requirements of proliferating T cells. Here, we demonstrated that activation of CD4+ T cells under glutamine-deprived conditions results in a delayed mTOR activation with reduced early ATP production and decreased proliferation. Moreover, these conditions resulted in the conversion of naïve CD4+ T cells into Foxp3+ regulatory T cells (Tregs). This de novo Treg differentiation occurred even under Th1-polarizing conditions and was TGFβ-dependent. Interestingly, glutamine deprivation did not inhibit Th2 differentiation. Importantly, these converted Foxp3+ T cells showed enhanced in vivo persistence and were highly suppressive, completely protecting Rag-deficient mice from the development of autoimmune inflammatory bowel disease as efficiently as natural-occuring Tregs. Thus, our data reveal the external metabolic environment to be a key regulator of a CD4 T lymphocyte's differentiation. Altogether, the data generated during my PhD provide new insights into the identification of parameters that can potentially alter the survival and reactivity of adoptively-transferred T cells.

Der adoptive Transfer von T-Zell-Rezeptor (TZR) modifizierten T-Zellen gegen krebsspezifische Antigene ist ein vielversprechender Ansatz in der Immuntherapie. Geeignete Zielmoleküle für diese Therapie sollten wichtig für das Überleben von Krebszellen sein und zudem in ausreichenden Mengen auf der Zelloberfläche exprimiert werden, um von T-Zellen erkannt zu werden. Die Identifizierung dieser Zielmoleküle ist jedoch eine Herausforderung und erfordert eine intensive Charakterisierung, um eine ausreichende Prozessierung und Präsentation auf den Tumorzellen zu validieren. Ziel dieser Arbeit war, HLA-A2-spezifische Neoepitope als Zielmoleküle für adoptive T-Zell-Therapie zu validieren. Dafür wurden erfolgreich Immunantworten in einem humanen transgenen Mausmodell nach Peptidimmunisierung induziert und TZRs mit hoher Affinität isoliert. Trotz einer hohen funktionellen Avidität von H3.3K27M-spezifischen T-Zellen wurde keine Erkennung von Tumorzellen erreicht. Zweitens wurden TZR-transduzierte T-Zellen gegen die häufige Melanommutation Rac1P29S isoliert, welche zytotoxisch gegen Melanomzelllinien waren. Letztlich wurde beobachtetet, dass TZRs mit hoher Affinität gegen gespleißte Kras und Rac2 Epitope, welche durch Proteasom-katalysiertes Peptidspleißen erzeugt wurden, keine Immunantwort gegen endogen exprimierte Mutationen hervorrufen konnten. Daraus lässt sich schließen, dass gespleißte Epitope wahrscheinlich seltener vorkommen als zuvor angenommen und daher möglicherweise irrelevant für die adoptive T-Zelltherapie sind. Diese Daten deuten darauf hin, dass die Auswahl von Zielmolekülen für die adoptive T-Zell-Therapie mit Hilfe reverser Immunologie auf der Grundlage von Bindungsalgorithmen und der Häufigkeit von Mutationen allein nicht ausreicht. Daher sind vor der Isolierung und Charakterisierung von TZRs zusätzliche Strategien wie z.B. die Analyse des MHC-Immunopeptidoms erforderlich, um die Auswahl geeigneter Zielmoleküle für die T-Zelltherapie zu verbessern. / Adoptive transfer of T cell receptor (TCR)-engineered T cells against tumour-specific neoantigens is a promising approach in cancer immunotherapy. Ideally, targeted antigens are crucial for cancer cell survival and are generated in sufficient amounts to be recognised by T cells. However, the identification of ideal targets remains challenging and requires intensive characterisation to validate sufficient antigen processing and presentation by the tumour cells. This thesis focused on the validation of HLA-A2 binding neoepitopes carrying the recurrent cancer mutations H3.3K27M, Rac1P29S, Rac2P29L or KrasG12V as targets for adoptive T cell therapy. After peptide immunisation, immune responses in a human transgenic mouse model were elicited and high-affinity TCRs successfully isolated. Although H3.3K27M-specific T cells showed high functional avidity, no recognition of cells endogenously expressing mutant H3.3 was achieved. Furthermore, a mechanism to target the common melanoma mutation Rac1P29S with a TCR raised against a heterologous mutation with higher peptide-MHC affinity was described. TCR-transduced T cells induced cytotoxicity against Rac1P29S expressing melanoma cell lines. Lastly, high-affinity TCRs specific for mutant Kras and Rac2 spliced epitopes generated by proteasome-catalysed peptide splicing were successfully isolated, however, TCR-transduced T cells did not induce an immune response against endogenously expressed mutant transgenes. The results indicate that spliced epitopes are probably less abundant than previously estimated and therefore may play a minor role in the generation of targets for adoptive T cell therapy. These data suggest that target selection using a reverse immunology approach based on binding algorithms and frequency of mutations alone is not sufficient. Thus, additional strategies to improve the selection of suitable targets such as the analysis of the MHC immunopeptidome are required prior to TCR isolation and characterisation.

Das Ziel der vorliegenden Arbeit bestand darin, ein Vektorsystem zu entwickeln, dass den simultanen Transfer verschiedener Transgene in CD8+ und CD4+ T-Zellen und dadurch die Herstellung eines immunotherapeutischen T-Zell-Produkts ermöglicht, welches aus zwei unterschiedlich modifizierten T-Zell-Subtypen besteht. Im ersten Teil der Arbeit wurde die Targeting-Technologie von lentiviralen auf γ-retrovirale Vektoren übertragen. Anschließend wird die Herstellung von Vektoren beschrieben, die spezifisch für murines CD4 oder CD8 sind. Deren Spezifität wurde zum einen durch die exklusive Expression von GFP in CD4+ oder CD8+ Zellen und zum anderen durch den Dosis-abhängigen Verlust des GFP-Signals nach Inkubation dieser Zellen mit CD4- und CD8-blockierenden Antikörpern nachgewiesen. Im dritten Teil der Arbeit wird gezeigt, dass MVm8 und MVm4 primäre T-Zellen spezifisch transduzieren. MVm8-vermittelter Transfer des Ovalbumin (OVA)-reaktiven TZRs OT-I führte zu T-Zellen, die OVA+ Tumor-Zelllinien erkannten und Interferon-γ sezernierten. Der vierte Teil dieser Arbeit beschäftigt sich mit der in vivo Transduktion primärer T-Zellen mithilfe von MVm8, welches den OT-I-TZR und eine Luciferase transferiert (MVm8/OT-I-luc). Durch systemische Applikation von MVm8/OT-I-luc wurden T-Zellen in vivo transduziert. Durch Immunisierungen konnten antigen-spezifisches Homing, Expansion und eine anschließende Kontraktion in vivo transduzierter T-Zellen gezeigt werden. Mäuse mit starker OT-I-luc-Expression waren gegenüber einer Infektion durch OVA-transgene listeria monocytogenes geschützt. Zusammenfassend lässt sich sagen, dass das in dieser Arbeit entwickelte Vektorsystem in der Lage ist zwischen Subtypen von T-Zellen zu unterscheiden und sie simultan mit unterschiedlichen Transgenen auszustatten. Für MVm8 konnte gezeigt werden, dass es T-Zellen direkt in vivo transduzieren kann. / The aim of this thesis was to generate a vector system that allows the simultaneous transfer of different transgenes into CD8+ and CD4+ T cells, allowing the generation of a immunotherapeutic T cell product comprised of two differently engineered T cell subsets. The first part of the thesis describes the transfer of the measles virus (MV) envelope-based targeting technology from lentiviral (LV) to γ-retroviral (gRV) vectors. The second part reports the generation of two targeting vectors specific for murine CD4 or CD8. The exclusive specificity of MVm4 and MVm8 was proven by expression of GFP in CD4+ and CD8+ reporter cells, respectively, but not in CD4-CD8- cells after transduction, and by a dose-dependent loss of GFP signal after incubation of reporter cells with CD4 or CD8 blocking antibodies before transduction. The third part shows that MVm8 but not MVm4 transduced primary T cells. MVm8-mediated transfer of the ovalbumin (OVA)-reactive TCR OT-I resulted in T cells secreting interferon-γ (IFNγ) upon recognition of OVA+ tumor cell lines. The final part of this thesis describes the in vivo transduction of primary T cells using MVm8 transferring OT-I and a luciferase (MVm8/OT-I-luc). To this end, B6 mice deficient for Rag2 have been repopulated with either polyclonal (B6) or monoclonal T cells derived from P14-TCR transgenic mice (P14). One day later the transferred T cells were transduced in vivo by systemic application of MVm8/OT-I-luc. Upon immunization in vivo-transduced T cells homed, expanded and contracted repeatedly in an antigen-dependent manner. Finally, mice exhibiting strong luc-signals showed improved protection against infections by OVA-transgenic listeria monocytogenes (LM-OVA). In conclusion, the viral vector system developed within this thesis is able to discriminate between the two main T cell subsets and to equip them with distinct transgenes simultaneously.

Das Zytokin Interleukin-7 (IL-7) ist für die Entstehung und das Überleben reifer T Zellen von zentraler Bedeutung. Die Gabe von IL-7 führt sowohl in der Maus als auch im Menschen zu erhöhten T Zellzahlen und einem veränderten T Zellphänotyp. Folglich könnte sich die therapeutische Gabe von IL-7 bei Patienten mit geschwächtem Immunsystem positiv auswirken. Diese Hypothese wird derzeit in mehreren klinischen Studien untersucht. Bisher wurde allerdings nur die Wirkung von IL-7 auf T-Zellen studiert. Zu dessen Wirkung auf andere Immun- oder Stromazellen sowie deren IL-7-abhängigen Beitrag zur Regulation der T-Zellhomöostase ist nur wenig bekannt. Daher war es Ziel der Arbeit, den Einfluss einer therapeutischen Gabe von IL-7 auf adoptiv-transferierte T-Zellen in IL-7-Rezeptor (IL-7R)-kompetenten und defizienten lymphopenischen Mäusen zu studieren. Die Untersuchungen bestätigen, dass die Gabe von IL-7 T-Zellantworten unterstützt, zeigen jedoch auch, daß viele dieser Effekte von IL-7R-exprimierenden Wirtszellen abhängig sind. Dies weist darauf hin, dass IL-7R-vermittelte Signale in Wirtszellen indirekt T-Zellantworten beeinflussen. Zudem zeigte sich, dass effiziente anti-Tumor-T Zellantworten von IL 7R-vermittelten Signalen in Wirtszellen abhängen. Vor allem nicht-hämatopoetische Wirtszellen fungieren hier als Regulatoren der IL-7-Therapie-vermittelten T Zelldifferenzierung. Unsere Ergebnisse bestätigen außerdem, dass Stromazellen in verschiedenen Organen il-7 exprimieren und zeigen darüber hinaus, dass diese Zellen durch die Gabe von IL-7 beeinflusst werden. Wir folgern daraus, dass die Effekte der IL-7-Therapie auf T Zellhomöostase teilweise indirekt über il-7-exprimierende Stromazellen vermittelt werden. Um diese Zellen genauer identifizieren und untersuchen zu können, haben wir ein neues transgenes Mausmodell charakterisiert, was es erleichtern wird, die beteiligten molekularen Signalwege zu analysieren und den Erfolg der adoptiven T Zelltherapie zu verbessern. / Interleukin-7 (IL-7) is an essential cytokine required for the development and maintenance of mature T cell. Its availability is limited under normal conditions, but rises during lymphopenia, leading to increased T cell proliferation. The administration of recombinant IL-7 to normal or lymphopenic mice and humans results in increased T cell numbers and altered T cell phenotype. Hence, IL-7 administration could mediate therapeutic benefits in immunocompromised patients and is currently tested in several clinical trials. However, besides its well-studied effects on T cells little is known about the effect of IL-7 on other immune and non-immune cells and their influence on T cell homeostasis. Therefore, we evaluated the effect of IL-7 therapy on adoptively transferred T cells in IL-7 receptor (IL-7R)-competent and IL-7R-deficient lymphopenic mice. We confirm the benefits of IL-7 therapy on T cell responses but additionally show that many of these effects are dependent on IL-7R expression by host cells, indicating that IL-7R signaling in host cells modulates T cell responses. We show that efficient T cell responses against cancer are dependent on host IL-7R signaling. Based on studies in bone-marrow chimeric mice, we identify non-hematopoietic host cells as main regulators of IL-7 therapy-modulated T cell differentiation. We conclude from these data that IL-7 therapy affects non-hematopoietic stromal cells that modulate the success of adoptive T cell therapy. Our results confirm that stromal cells in various organs express il-7 and show that these cells are targeted by IL-7 therapy in vivo. Hence, we propose that il-7-expressing cells regulate IL-7 therapy-modulated T cell homeostasis. To identify and study these il-7 expressing stromal cells in more detail, we characterized a new transgenic mouse model that will facilitate determining the molecular pathways to improve the success of adoptive T cell therapy.

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.