T cell receptors (TCR) bind to peptides from various sources on MHC (Major Histocompatibility Complex) molecules. A long-standing goal in the field is to understand the mechanisms of MHC-peptide exchange and MHC-TCR interactions. Here, I present work from three uniquely different systems that address the following: HLA-DR1 conformational stability, self-tolerant mechanisms of TCRs isolated from self-reactive TCR transgenic mice, and TCR cross-reactivity mechanisms between LCMV and VV. First, I present a crystal structure of HLA-DR1 in complex with A1L9 peptide, a peptide with two amino acid substitutions from the parental peptide. The singly substituted A1 peptide, which has a pocket 1 alanine substitution, decreases intrinsic half-life between MHC-peptide and increases susceptibility to HLA-DM mediated peptide exchange. This data agrees with previous models of HLA-DM-mediated peptide exchange in which the major determinant is located at the HLA-DR1 pocket 1. However, the L9 substituted peptide, which has a pocket 9 leucine substitution, displays the opposite phenotype: increased intrinsic half-life and decreased HLA-DM susceptibility. The crystal structure presented here shows that HLA-DR1 in complex with a doubly substituted peptide, A1L9, is in the same conformation as HLA-DR1 with the wild-type peptide, demonstrating that pocket 9 residues can rescue pocket 1 residue binding deficiencies and that HLA-DR1 stability is determined by amino acids along the peptide, not only at pocket 1. Next, I present crystal structures of two self-tolerant TCRs in complex with IAb-3K pMHC. To elucidate molecular mechanism for self-reactivity and self-tolerance, the TCRs J809.B5 and 14.C6 are compared to each other and its parental self-reactive TCR, YAe-62.8. In comparison to YAe-62.8, J809.B5 interacts with the same pMHC, but utilizes more peptide specific interactions, a mechanism that may distinguish self-reactive receptors from self-tolerant receptors. Additionally, the crystal structure of 14.C6 TCR, which bears a different CDR3α sequence from J809.B5, demonstrates that CDR3 sequences can modulate interactions of germline encoded CDR1 and CDR2 loops. Together, these results highlight that in addition to CDR3 VDJ recombination, diversity is generated in the mature TCR repertoire by differential chain pairing, either of which can affect the interactions of germline encoded CDR loops. Next, I present a detailed analysis of cross-reactive TCRs between Kb-GP34 and Kb-A11R. The mature LCMV-immune repertoire was analyzed by DNA deep sequencing of TCRβ CDR3 sequences, which led to the identification of new cross-reactive sequence motifs. Cross-reactive sequence motifs varied by each Vβ gene, suggesting a role of CDR1, CDR2, and CDR3 loop interplay in cross-reactivity. Lastly, I present the crystal structures of a GP34/A11R cross-reactive TCR in complex with both Kb-GP34 and Kb-A11R. Analysis of the crystal structures revealed that the two complexes are largely the same, despite differences in peptide sequences. Surprisingly, the TCR to peptide interactions were dominated by three out of eight peptide side-chains. Cross-reactivity between these two complexes is likely due to a large amount of interactions from TCR to MHC compared to interactions of TCR to peptide. We note two unique MHC-peptide interactions that may allow Kb to be an allele prone to cross-reactivity. The first is an interaction at the C-terminus of the A11R peptide which pulls A11R P7 asparagine away from TCR interactions. The second interaction is from an arginine at position 155, which sits at the interface between TCRα and TCRβ , and contributes the most buried surface area in the interaction interface. Because Kb’s arginine 155 is a long side chain that hydrogen bonds with the peptide backbone, and is also at the center of the TCR-peptide interface, GP34 and A11R peptide sequence differences may be occluded from TCR discrimination by Kb presentation. The data presented in this dissertation demonstrate that interactions between MHC-peptide and MHC-TCR act harmoniously and coopertively, whereby proximal interactions are affected by interactions elsewhere. While previous models of HLA-DR/HLA-DM interactions demonstrate the importance of interactions at HLA-DR1 pocket 1, I showed that pocket 9 also contributes to HLA-DR stability and therefore, HLA-DM susceptibility. I also showed that TCR CDR3 loop sequences affect germline CDR1/CDR2 loop interactions and vice versa. Lastly, I showed that allele specific MHC side chain interactions with the bound peptide influence TCR ligand binding and hence, TCR cross-reactivity.

Helper T cells (Th) are a vital component of the immune system responsible for directing other immune cells to eliminate pathogens and cancer. Specifically, Th cells facilitate B cell and cytotoxic T cell (Tc) activation and recruitment and enhance their function against cancer and infectious diseases. Th cells are a valuable resource for improving Tc responses in cancer treatment and have become a focus of immunotherapeutic research. While it is increasingly clear that helper T cells serve an important role, the details about which entities produce an effective Th cell response remain unclear. CD5 is a T cell co-receptor that negatively regulates T cell activation and helps fine-tune the TCR repertoire by altering TCR signaling during the selection process in the thymus. This work discusses the role of the co-receptor CD5 in influencing Th cell metabolism, as well as the study of two T cells called LLO118 and LLO56 that have different CD5 expression levels, and their functional response to altered peptides. Antibodies have revolutionized the world of cancer research and accelerated the development of therapies that trigger the immune system to target disease. In recent years, many antibody-based immunotherapies have emerged as effective candidates for combating cancer due to their refined specificity and ability to target a variety of epitopes. However, many therapies, such as those that target CD19 on B cell cancers, are also present on healthy cells, destroying both cancerous and healthy cells alike. Thymidine kinase 1 (TK1) is an enzyme involved in the DNA salvage pathway that converts thymidine into the nucleotide thymine. Recently, TK1 has been shown to be overexpressed on the surface of many cancers such as acute lymphoblastic leukemia. Importantly, TK1 is not expressed on the surface of healthy cells, making it an ideal cancer-specific antigen that can be targeted for cancer treatment. This work discusses our efforts to characterize TK1-specific single-chain antibodies from a yeast display library. According to the World Health Organization, genomics is defined as the study of all genes and their related functions. In contrast to genetics, genomics analyzes the entire DNA makeup of an organism rather than a single gene. In the past 20 years, the cost of genomic sequencing has decreased dramatically, making it affordable and accessible. A key area that must be addressed with genomic testing involves education about their promise, challenges, potential consequences, and ethical considerations. Genomic testing provides a powerful opportunity to educate everyone on scientific and ethical issues to increase understanding on the subject. This work discusses the influence of personal genomics in society and focuses on the importance, benefits, and consequences of genomics education in the classroom, clinic, and the public.

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.

According to the centers for disease control and prevention (CDC) and the world healthorganization (WHO), heart disease and immune related diseases such as diabetes and cancer areamong the leading causes of death around the world. Thus, the regulation of the function ofimmune cell plays a key role in health and disease. Calcium (Ca2+) ions play a critical role inimmune cell activation, function and in a robust immune response. Defects in Ca2+ signalinginfluences the development of cardiac disease, Alzheimer disease, immune cell metabolism,muscle dysfunction, and cancer. Each immune cell is unique in its activation and function,making it relevant to understand how activation of each type of immune cell is regulated. Herewe describe the role of the nBMP2 protein in macrophage activation and function and the role ofthe CD5 co-receptor in helper T cell activation and function.The nuclear bone morphogenetic protein 2 (nBMP2) is the nuclear variant of the bonemorphogenetic protein 2 (BMP2), a growth factor important in heart development, neurogenesis,bone, cartilage and muscle development. To better understand the function of nBMP2, transgenicnBMP2 mutant mice were generated. These mice have a slow muscle relaxation and cognitivedeficit caused in part by abnormal Ca2+ mobilization. Mutant nBMP2 mice also have an impairedsecondary immune response to systemic bacterial challenge. Here we have further characterizedmacrophage activation and function from mutant nBMP2 mice before and after bacterialinfection. We describe how nBMP2 influences the Ca2+ mobilization response and phagocytosisin macrophages, revealing a novel role of the nBMP2 protein in immune cell regulation.CD5 is a surface marker on T cells, thymocytes, and the B1 subset of B cells. CD5 isknown to play an important role during thymic development of T cells. CD5 functions as anegative regulator of T cell receptor (TCR) signaling and fine tunes the TCR signaling response.Here we describe our characterization of CD5 regulation of Ca2+ signaling in naïve helper Tcells. We also outline our findings examining how CD5-induced changes in helper T cellactivation influence other biological processes such as immune cell metabolism, the diversity ofthe gut microbiome, and cognitive function and behavior. Thus, this work elucidates theinfluence of the CD5 co-receptor on the functional outcomes in multiple systems when CD5 isaltered.

Toxoplasma gondii, an obligate intracellular protozoan parasite, establishes a latent, chronic infection by forming cysts preferentially in the brain after replication of tachyzoites in various organs during the acute stage of infection. Chronic infection with T. gondii is one of the most common parasitic diseases in humans. The immune system is required for maintaining the latency of chronic infection. Reactivation of infection can occur in immunocompromised individuals, such as AIDS patients, which results in the development of life-threatening toxoplasmic encephalitis (TE). IFN-gamma-dependent, cell mediated immune responses play an essential role in preventing the reactivation of chronic infection of T. gondii in the brain. In my dissertation study, we examined the mechanisms of IFN-gamma-mediated prevention of TE by using models of reactivation of chronic infection in BALB/c mice. This strain of mouse is genetically resistant to T. gondii infection and establishes a latent chronic infection as do immunocompetent humans, and therefore provides an excellet model for this purpose. Our laboratory previously demonstrated that both T cells and IFN-gamma-producing non-T cells are required for genetic resistance of BALB/c mice against development of TE. However, the function of T cells required for the resistance is still unclear. Therefore, in the present study, we examined whether IFN-gamma production or perforin-mediated cytotoxicity of T cells play an important role in their protective activity against TE. Immune T cells were obtained from infected IFN-gamma-knockout (IFN-g-/-), perforin-knockout (PO), and wild-type (WT) BALB/c mice, and transferred into infected, sulfadiazine-treated athymic nude mice which lack T cells but have IFN-gamma-producing non-T cells. Control nude mice that had not received any T cells developed severe TE due to reactivation of infection and died after discontinuation of sulfadiazine treatment. Animals that had received immune T cells from either PO or WT mice did not develop TE and survived. In contrast, nude mice that had received immune T cells from IFN-gamma-/- mice developed severe TE and died as early as control nude mice. T cells obtained from spleens of the animals that had received either PO or WT T cells both produced large amounts of IFN-gamma following stimulation with T. gondii antigens in vitro. In addition, the amounts of IFN-gamma mRNA expressed in the brains of PO T-cell recipients did not differ from those of WT T-cell recipients. These results indicate that IFN-gamma production, but not perforin-mediated cytotoxic activity, by T cells is required for prevention of TE in genetically resistant BALB/c mice. In our attempt to identify a T cell population(s) that produces IFN-gamma in the brain and plays an important role for prevention of TE, we analyzed T cell receptor (TCR) Vb chain usage in T cells expressing IFN-gamma in the brains of infected BALB/c mice. We found T cells bearing TCR V beta8 chain to be the most frequent IFN-g-producing population in the brains of infected animals. To examine the role of IFN-gamma production by this T cell population for prevention of TE, V beta8+ immune T cells purified from spleens of infected BALB/c and IFN-g-/- mice were transferred into infected, sulfadiazine-treated athymic nude mice. After discontinuation of sulfadiazine treatment, control nude mice that had not received any T cells and animals that had received Vb8+ T cells from IFN-g-/- mice all died due to reactivation of infection (TE). In contrast, animals that had received the cells from WT mice survived. These results indicate that IFN-gamma production by Vb8+ T cells in the absence of any other T cell population can prevent reactivation of infection. Thus, V beta8+ T cells play a crucial role in genetic resistance of BALB/c mice to TE through their production of IFN-gamma. When V beta8+ immune T cells were divided into CD4+ and CD8+ subsets, a potent protective activity was observed only in the CD8+ subset whereas a combination of both subsets provided greater protection than did the CD8+Vb8+ population alone. These results indicate that CD8+ subset of V beta8+ T cells is a major afferent limb of IFN-gamma-mediated resistance of BALB/c mice against TE, although the CD4+ subset of the T cell population works additively or synergistically with the CD8+V beta8+ population. T cells need to enter into the brains of infected mice to demonstrate their protective activity against TE. This migration is mediated, in part, by endothelial adhesion molecules. Since IFN-gamma is essential for preventing reactivation of chronic infection with this parasite in the brain, we examined whether this cytokine plays an important role in expression of lymphocyte and endothelial adhesion molecules and recruitment of T cells into the brain during chronic infection with T. gondii using IFN-g-/- and WT BALB/c mice. Although the number of cerebral vessels expressing intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) increased in both WT and IFN-g-/- mice following infection, there were more VCAM-1+ vessels in brains of infected WT than infected IFN-g-/- mice; in contrast, numbers of ICAM-1+ vessels did not differ between strains. We did not detect endothelial E-selectin, P-selectin, MAdCAM-1 or PNAd in any of the brains. Significantly fewer CD8+ T cells were recruited into brains of infected IFN-g-/- than WT mice. Treatment of infected IFN-g-/- mice with recombinant IFN-gamma restored the expression of VCAM-1 on their cerebral vessels and recruitment of CD8+ T cells into their brains, confirming an importance of this cytokine for up-regulation of VCAM-1 expression and CD8+ T cell trafficking. In infected WT and IFN-g-/- animals, almost all cerebral CD8+ T cells had an effector/memory phenotype (LFA-1high, CD44high and CD62Lneg) and approximately 38% were positive for a4b1 integrin (the ligand for VCAM-1). In adoptive transfer of immune spleen cells, pre-treatment of the cells with a monoclonal antibody against a4 integrin markedly inhibited recruitment of CD8+ T cells into the brain of chronically infected wild-type mice. These results indicate that IFN-g-induced expression of endothelial VCAM-1 and its binding to a4b1 integrin on CD8+ T cells is important for recruitment of the T cells into the brain during the chronic stage of T. gondii infection. Since we found strong expression of ICAM-1 on endothelia and LFA-1 on T cells in the brains of infected mice, LFA-1/ICAM-1 interaction, in addition to a4b1 integrin/VCAM-1 interaction, may also be involved in this process. As mentioned earlier, CD8+ T cells are crucial for prevention of TE in BALB/c mice. Therefore, IFN-gamma-mediated expression of VCAM-1 and its binding to a4b1 integrin for recruitment of CD8+ T cells may play a critical role in genetic resistance of BALB/c mice to development of TE. / Ph. D.

Klasické schéma vývoje hematopoetických buněk předpokládá časné oddělení lymfoidního a myeloidního prekurzoru. V poslední době jsou navrhovány složitější modely, které předpokládají větší flexibilitu hematopoezy a navrhují existenci progenitorů s lymfoidním i myeloidním potenciálem. Akutní hybridní leukémie jsou malignity, které podle různých kritérií nelze jednoznačně zařadit k lymfoidní nebo k myeloidní linii a jejichž chování spíše dává za pravdu novým modelům hematopoezy. Předkládaná práce se zabývala především výzkumem dětských leukémií s přesmykem z lymfoidní do myeloidní linie během indukční léčby. Jedná se o rozsáhlý projekt, v jehož rámci si diplomová práce si kladla za úkol určit liniové zařazení leukemických blastů pomocí detekce přestaveb genů pro imunoglobuliny a T-buněčné receptory (TCR). Potvrdili jsme, že myeloidní buňky derivované v průběhu léčby pochází u všech pacientů z původního lymfoidního klonu. Dále jsme u těchto případů zkoumali expresi vytipovaných genů ve srovnání s běžnými druhy leukémií. Třetí částí práce byl výzkum prognostického významu přítomnosti přestaveb TCR (a tedy příslušnosti k lymfoidní linii) u leukémií z T-lymfoidní řady.

O diabetes mellitus tipo 1 (DM-1) é uma doença autoimune órgão-específica caracterizada pela destruição seletiva das células pancreáticas produtoras de insulina. A imunossupressão em altas doses seguida do transplante autólogo de células-tronco hematopoéticas (TACTH) constitui uma alternativa terapêutica recente e promissora para o DM-1 recém-diagnosticado, impedindo a progressão da destruição das células pancreáticas produtoras de insulina e induzindo independência insulínica por um período prolongado na maioria dos pacientes. O princípio dessa terapia baseia-se na eliminação das células autorreativas pela imunossupressão intensa e na reconstituição de um sistema imunológico novo e tolerante após o transplante. Com o objetivo de avaliar a função do timo e sua contribuição na geração do repertório de células T nos pacientes com DM-1 após o TACTH, nesse trabalho foram avaliados os níveis de T cell receptor excision circles (TRECs) em células T do sangue periférico e a diversidade do repertório de células T dos pacientes com DM-1 (n=23) antes e em diversos períodos após o transplante. A quantificação absoluta dos níveis de TRECs (número de moléculas de TRECs/100g de DNA) foi realizada pela técnica de PCR em tempo real e a avaliação do repertório de células T foi realizada pela técnica de TCRBV CDR3 Spectratyping. Dentre os vinte e três pacientes, vinte alcançaram a independência insulínica por períodos variáveis de tempo e três não responderam ao tratamento. Não foi observada a restrição do repertório de células T nos pacientes com DM-1 no período pré-transplante, ou seja, quando recém-diagnosticados. Foram identificadas cinco famílias V (7, 18, 19, 20 e 22) em expansão clonal nos pacientes com DM-1. As famílias V 7, 18, 19, 20 apresentaram-se em expansão clonal antes do transplante e se mantiveram com frequência elevada após o transplante, enquanto a família V 22 apresentou aumento da frequência somente nos períodos mais tardios após o transplante. Nos primeiros meses após o transplante, houve redução do número de moléculas de TRECs e restrição do repertório de células T. Contudo, um ano após o transplante, o número de moléculas de TRECs atingiram valores normais e o repertório de células T apresentou-se com ampla diversidade. Nossos resultados mostraram que o TACTH foi capaz de induzir mudanças na composição do repertório de células T dos pacientes com DM-1 após a terapia de IAD/TACTH, evidenciadas por alterações qualitativas e quantitativas dos picos de CDR3 do TCR, sugerindo a reconstituição de um repertório de células T diverso até dois anos pós-transplante. Embora tenha ocorrido reativação da função tímica após o transplante, evidenciada pelo aumento dos níveis de TRECs de um ano e meio a cinco anos pós-transplante, a diversidade do repertório das células T diminuiu a partir de dois anos e meio pós-transplante, sugerindo uma reconstituição tímica de novo de células T naive que expressam preferencialmente algumas cadeias V. Estas evidências imunológicas poderiam explicar a melhora clínica (independência insulínica) temporária observada na maioria dos pacientes após a terapia de IAD/TACTH. / Type 1 diabetes mellitus (T1D) is an organ-specific autoimmune disease characterized by insulin-producing pancreatic cell destruction. High-dose immunosuppression followed by autologous hematopoietic stem cell transplantation (AHSCT) is a recent and promising therapeutic approach for treatment of T1D, preventing the progress of destruction of pancreatic cells and inducing insulin independence for a prolonged period in most patients. The rationale of the AHSCT is based on the elimination of autoreactive cells by the intense immunosuppression and on the reconstitution of a new and tolerant immune system after transplantation. Aiming at assessing the thymic role in the production of new T cell repertoire in T1D patients after AHSCT, in this study was evaluated the levels of T cell receptor excision circles (TRECs) in T cells of peripheral blood as well as the clonality and diversity of T cell repertoire in T1D patients (n=23) before and several periods after transplantation. The absolute quantification of TRECs levels (number of molecules of TRECs/100ng of DNA) was performed by real-time PCR and the analysis of T cell repertoire was performed by TCRBV CDR3 Spectratyping. Among the twenty-three patients, twenty achieved insulin independence for variable periods and three did not respond to the treatment. The T cell repertoire in T1D patients was not restricted in pre-transplantation, i.e., when newly diagnosed. It was identified five V families (7, 18, 19, 20 e 22) in the clonal expansion in T1D patients. The V families 7, 18, 19, 20 were in clonal expansion before transplantation and maintained with high frequency after transplantation, whereas the V 22 family increased its frequency only in the later periods after transplantation. It was observed that the numbers of molecules of TRECs decreased and the T cell repertoire was restricted in the early months after transplantation. However, the levels of TRECs were normalized and the T cell repertoire showed diversity one year after transplantation. Our results indicate that AHSCT was able to induce changes in the composition of the T cell repertoire of patients after AHSCT, evidenced by qualitative and quantitative changes in the composition of T-cell receptor -chain CDR3 peaks, suggesting the reconstitution of diverse T cell repertoire up to two years after transplantation. Although there was reactivation of thymic function after transplantation, as evidenced by increased levels of TRECs from one and a half year to five years after transplantation, the diversity of the T cells repertoire decreased from two and a half years after transplantation, suggesting a reconstruction of new naive T cells that preferentially express some V chains. These immunological evidences could explain the temporary clinical improvement (insulin independence) observed in most patients after IAD / AHSCT therapy.

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.

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.