Previous research has provided evidence that CD8 T cells mediate immunity against infection with Theileria parva. However, the immunity induced by one parasite strain doesn‟t give complete protection against other strains and this is associated with parasite strain specificity of the CD8 T cell responses. There is evidence that such strain specificity is a consequence of the CD8 T cell responses of individual animals being focused on a limited number of immunodominant polymorphic peptide-MHC determinants. Dominant responses to the Tp2 antigen have been demonstrated in animals homozygous for the A10 MHC haplotype. Three Tp2 epitopes recognised by A10+ animals (Tp249-59, Tp250-59 and Tp298-106) have been defined. This project set out to investigate the dominance of these epitopes and to examine the T cell receptor (TCR) repertoires of the responding T cells. The specific objectives were to: (i) Determine the dominance hierarchies of the three defined Tp2 epitopes in both A10-homozygous and -heterozygous cattle. (ii) Examine the clonal repertoires of epitope-specific responses by analysis of TCR gene expression. (iii) Isolate full-length cDNAs encoding TCR α and β chain pairs from T cell clones of defined epitope specificity and use them to generate cells expressing the functional TCRs. Using MHC class I tetramers the relative dominance of CD8 T cell responses were found to differ between A10-homozygous and heterozygous cattle. All A10-homozygous cattle examined had detectable responses to all 3 Tp2 epitopes, the Tp249-59 epitope consistently being the most dominant. By contrast, only some A10-heterozygous cattle had detectable responses to Tp2 and when present the response was specific only for the Tp298-106 epitope. Analyses of the sequences of expressed TCR β chains showed that the responses in individual animals were clonotypically diverse, but often contained a few large expanded clonotypes. The TCRs of Tp298-106–specific T cells showed preferential usage of the Vβ13.5 gene and the frequent presence of a “LGG” motif within the CDR3 of the B chain. A conserved (public) TCRβ clonotype shared by the Tp250-59-specific CD8 T cells from all A10-homozygous cattle was identified. The TCRα chains co-expressed with this public TCRβ clonotype were identified for a number of T cell clones. Lentivirus transduction of Jurkat cells with three full-length TCR α and β chain pairs resulted in successful expression of one of the α/β chain pairs as a functional TCR, thus providing the basis for future work to generate bovine T cells expressing defined TCRs in vitro.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13536号 / 論医博第2276号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 妹尾 浩, 教授 中川 一路 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Human Immunodeficiency Virus 1 (HIV-1) remains a significant public health concern due to the lack of a cure. In spite of anti-retroviral therapies, HIV-1 persists within infected cells as integrated transcriptionally silent proviruses. Re-activation after therapy interruption results in new HIV-1 replication. Attempts to clear this reservoir through the use of latency reversing agents by targeting cellular mechanisms that maintain HIV-1 in a latent state have been unsuccessful. In addition, subsets of latently infected cells exist within the reservoir that display differential capacities for provirus induction. In order to understand the nature of the reservoir and manipulate it therapeutically, more knowledge is needed regarding factors that bias a virus towards latency or replication at the time of infection. Because multiple mechanisms that regulate HIV-1 transcription, including chromatin remodeling, transcription factor activation and polymerase pausing, are regulated by the T cell receptor (TCR), I hypothesized that signaling at the time of infection determines proviral fate. I transduced Jurkat cell lines and primary CD4+ T cells with chimeric antigen receptors (CARs) that mimicked signaling from the TCR. These CARs spanned a 3-log range of binding affinities for their ligand, providing a tunable model. High levels of TCR stimulation during infection biased cells towards productive replication and the formation of an inducible latent reservoir. Examination of the mechanisms downstream from TCR signaling revealed that robust cellular activation led to a release of the repressor Negative Elongation Factor from the paused RNA Polymerase II, facilitating transcriptional elongation. Because signaling determined the presence of repressive factors, I sought to manipulate the balance between latency and expression through recruitment of repressors to the HIV-1 provirus using a nuclease-deficient CRISPR Associated Protein 9 fused to a Krüppel Associated Box Domain. I screened a pool of guide RNAs that mediated transcriptional repression of HIV-1. Our lab discovered that guides bound to the HIV-1 Long Terminal Repeat prevented viral re-activation in an integrated cell model of HIV-1 latency. The research presented here confirms my hypothesis that signals during infection have prolonged effects on latency reversal. I provide evidence that manipulation of these mechanisms represent therapeutic targets for cure efforts.

T-lymphocytes (T-cell) therapy offers a treatment for cancers. Developing this technology in the future provides the opportunity to revolutionise treatment and to make cancer a chronic condition. T-cells in themselves are a type of lymphocytes (itself a type of white blood cell) that play a central role in cell mediated immunity. They can be distinguished from other lymphocytes, such as B-cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. T-cells have the capacity to destroy diseased cells, but tumours present a considerable challenge that reduces their impact. As cancer cells are frequently ‘invisible’ to the immune system, and they create an environment that suppresses T-cell activity., genetic engineering of T-cells can be used therapeutically to overcome these challenges. T-cells can be taken from the blood of cancer patients and then modified to recognise and destroy cancer-specific antigens.

No description available.

Les lymphomes T périphériques (PTCL) sont des néoplasmes rares et agressifs représentant environ 12% des lymphomes chez l’Homme. Nos travaux récents dans des souris p53-/- ont révélé une nouvelle entité de PTCL, émergeant de cellules Natural-Killer T-cell (NKT), un type particulier de lymphocyte T reconnaissant des antigènes lipidiques. Nous avons montré que ces lymphomes NKT (PTCL-NKT) présentent des caractéristiques de NKT stimulés chroniquement, et que la lymphomagenèse est initiée via l’activation chronique du TCR. Chez l’Homme, de nombreux PTCL sont suspectés pour être associés à des stimulations antigéniques chroniques, mais les mécanismes de transformation impliqués sont encore mal connus. Borrelia burgdorferi (Bb), l’agent responsable de la maladie de Lyme, provoque des infections chroniques dont l’implication dans certains lymphomes T cutanés (CTCL) a été suggérée. Cependant, cette observation manque de preuves cliniques et expérimentales. De manière intéressante, Bb est connue pour exprimer des glycolipides activateurs des NKT. Nous avons donc infecté des souris p53-/- avec des Bb vivantes, et montré que l’infection augmente significativement la fréquence des PTCL-NKT. Par traitement antibiotique précoce de souris infectées et par injections de Bb inactivées, nous avons également démontré que la chronicité de l’infection est nécessaire au développement de ces lymphomes. L’analyse phénotypique de ces PTCL-NKT a confirmé nos observations précédentes, montrant des caractéristiques de cellules NKT activées chroniquement, telles que l’expression de marqueurs d’activation et d’exhaustion (perte de NK1.1, surexpression de PD-1). Ces résultats suggèrent une implication de Borrelia dans la lymphomagenèse T. En se basant sur l’analyse de différents marqueurs phénotypiques et de leur production cytokinique, nous avons également montré que ces lymphomes présentent un profil dérégulé se rapprochant du sous-type NKT2. Une étude génomique par séquençage whole-exome sur 6 PTCL-NKT a révélé de larges pertes récurrentes du chromosome 13. Au sein de la zone minimale de délétion, nous avons identifié Jarid2, codant un facteur épigénétique impliqué dans le développement NKT par une activité histone-methytransférase. Ce gène est retrouvé altéré dans 20% des CTCL. De manière intéressante, les souris Jarid2-/- présentent une expansion périphérique de NKT au profil immature/NKT2, partageant donc des caractéristiques avec les PTCL-NKT. La perte de Jarid2 a été détectée dans presque tous les PTCL-NKT. Nous avons confirmé la perte de Jarid2 au niveau ARN et protéique. Nos résultats préliminaires montrent une hypométhylation de la lysine 9 de l’histone H3 (H3K9), la cible de Jarid2, soutenant un effet fonctionnel dans la physiopathologie des PTCL-NKT. Par conséquent, nous pensons que la perte de Jarid2 pourrait être un événement important de la lymphomagenèse NKT, puisque de plus en plus d’altérations de facteurs épigénétiques sont retrouvées dans les PTCL humains. Pour réponse à cette question, nous sommes notamment en train de générer des souris p53-/- x Jarid2-/-. En conclusion, nos données viennent renforcer le concept selon lequel certaines infections peuvent initier la transformation des cellules T par l’activation chronique du TCR. Nous avons également identifié un nouveau facteur épigénétique potentiellement impliqué dans la lymphomagenèse NKT / Peripheral T-cell lymphomas (PTCL) are aggressive and heterogeneous neoplasms that represent around 12% of Human lymphomas. Our recent work in p53-/- mice revealed a new PTCL entity, arising from Natural-Killer T-cell (NKT), a particular type of T cell recognizing lipidic antigens. We found that NKT lymphomas (NKTL) present features of chronically stimulated NKT-cells and that lymphomagenesis is driven through chronic TCR activation by microbial glycolipids. In human, many PTCL are suspected to be associated with chronic antigenic stimulation, but this transformation mechanism is still poorly understood.Borrelia burgdorferi (Bb), the causative agent of Lyme disease, induces chronic infection and has recently been suggested to be involved in cutaneous T-cell lymphomas (CTCL). However, this observation lacks clinical and experimental proofs. Interestingly, Bb is known to express NKT-activating glycolipids. We therefore infected p53-/- mice by live intradermal Bb injection and showed that Bb infection significantly increased NKTL rate. Phenotypic characterization of these NKTL confirmed our previously described features of chronically stimulated NKT-cells, with expression of activation and exhaustion markers (loss of NK1.1, upregulation of PD-1). Based on surface markers, transcription factors and cytokine production analysis, we also found that our lymphomas mostly present a NKT2 subtype profile, sometimes surprisingly mixed with NKT17 or NKT1. Genomic study by whole-exome sequencing on few of these lymphomas revealed recurrent large losses in the chromosome 13. Within the minimal deletion region, we identified Jarid2, a gene involved in NKT development by epigenetic regulation and which is found altered in 20% of CTCL. Jarid2 loss was detected in almost all NKTL. Interestingly, Jarid2-/- mice show increased NKT number in the periphery with an immature/NKT2 phenotype, sharing features with our NKTL.Thus, we believe that Jarid2 loss may be an important event in NKT lymphomagenesis, as more and more epigenetic factors are found mutated in several human PTCL. To answer this question we are currently breeding p53-/- x Jarid2-/- mice. In conclusion, our data reinforced the concept that chronic bacterial activation of T-cells through their TCR can effectively drive T-cell transformation. We also identified a new potential epigenetic factor that may be involved in lymphomagenesis

Mucosal associated invariant T-cells (MAIT) are a phylogenetically conserved subset of alpha/beta T-cells with natural killer-like (NK) activity. MAIT are defined by the expression of an invariant T-cell receptor alpha (TCRα) chain; in mice and humans this chain uses the orthologous mVα19/hVα7.2-Jα33 genes respectively. Available evidence indicates that MAIT are restricted by MR1, a highly conserved MHC class I-related molecule, and that their development is dependent on B lymphocytes. They appear to constitute part of the innate immune response, but their precise functional role is poorly understood. This study aimed to characterise MAIT and MR1 in ruminants, and to further the knowledge and understanding of these unique cells. Using PCR primers based on partial database sequences, orthologous full-length TCRα chains were identified in circulating bovine and ovine T cells. The germline elements of the respective α chains were identified and their overall frequency of expression within the bovine TCRα repertoire determined. Experiments using the orthologous TCRα chain as a marker for MAIT cells to examine expression in bovine and ovine blood and various tissues showed that spleen and mesenteric lymph nodes contained the highest frequency of MAIT cells. Use of the same technique to study levels of this marker in cattle of different ages revealed very low numbers of MAIT cells in neonatal animals, followed by a marked increase in the first 3 weeks of life. Analyses of MAIT TCRα expression in different T cell subsets showed that, unlike mice and humans in which MAIT cells are predominantly within the CD4-/CD8- T-cell population, MAIT cells in bovine blood are predominantly CD8+. Full-length cDNAs were isolated for bovine and sheep MR1 and their sequences were found to display marked cross-species conservation. Using a specific PCR, MR1 was shown to be expressed in peripheral blood and by different lineages of Theileria-transformed cells. Alternatively-spliced transcripts of MR1 were detected in both cattle and sheep and several of these retained an intact open-reading frame. Constructs of bovine MR1 and an MR1/MHC chimera were prepared in a eukaryotic expression vector but these failed to give detectable cell surface expression following transfection into Cos-7, despite positive intracellular expression.

<p><p>LAT is a transmembrane adaptor protein that is critical for the emanation of signals downstream of the TCR. Following TCR engagement, LAT is phosphorylated on multiple tyrosine residues, allowing it to serve as a scaffold for a multi-protein signaling complex. Mutation of tyrosine 136 on LAT abrogates binding of PLC-&#947;1. The disruption of this interaction has severe consequences on TCR-mediated calcium signaling and MAPK activation. Mice harboring a mutation at this tyrosine, LATY136F (LAT<super>m/m</super>) mice, have drastically impaired thymocyte development; however, CD4<super>+</super> T cells in the periphery rapidly expand and instigate a fatal lymphoproliferative syndrome. In order to bypass the severe developmental defects exhibited in LAT<super>m/m</super> mice, our laboratory previously developed a conditional knock-in mouse line in which the mutated LAT allele is expressed in mature T cells following deletion of a floxed wildtype LAT allele (ERCre<super>+</super>LAT<super>f/m</super> mice). LAT<super>f/m</super> mice develop a similar lymphoproliferative syndrome as LAT<super>m/m</super> mice. We used both of these mouse models to analyze the contribution of two other proteins that are essential for TCR-mediated signaling, RasGRP1 and Gads, in LAT-mediated autoimmunity. </p><p><p>Analysis of LAT<super>m/m</super>RasGRP1<super>-/-</super> mice demonstrated that the additional deletion of RasGRP1 increased the thymocyte development block and, as a result, young mice contained markedly reduced T cell populations. However, by four months of age, a lymphoproliferative disease had developed in these mice. To bypass the severe developmental block, we analyzed LAT<super>f/m</super>RasGRP1<super>-/-</super> mice and observed that they developed disease similarly to LAT<super>f/m</super> mice. We also assessed the effect of Gads deletion in both mouse models of LAT disease. LAT<super>m/m</super>Gads<super>-/-</super> mice had an even more dramatic block in the DN stage of thymocyte development compared to LAT<super>m/m</super> controls, although by four months of age CD4<super>+</super> T cells had expanded. Following deletion of the wildtype LAT allele, LAT<super>f/m</super>Gads<super>-/-</super> mice also developed disease. Our results indicated that LAT-mediated autoimmunity can occur independently of the critical T cell signaling components RasGRP1 and Gads. </p><p><p>In addition, we more closely examined RasGRP1-mediated Erk activation in T cells. RasGRP1 is a Ras-guanyl nucleotide exchange factor that is required for positive selection of thymocytes, activation of T cells, and control of T cell mediated-autoimmunity. While the importance of various RasGRP1 structural domains has previously been explored, RasGRP1 also contains a tail domain of unknown function. To elucidate the physiological role of this domain, we generated knock-in mice expressing RasGRP1 without the tail domain, RasGRP1<super>d/d</super> mice. Analysis of these mice demonstrated that deletion of the tail domain led to impaired T cell development but, with age, CD4<super>+</super> T cells expanded and auto-antibodies were produced. RasGRP1<super>d/d</super> thymocytes were unable to activate Erk and underwent aberrant thymic selection processes. Mechanistically, the tail-deleted form of RasGRP1 was not able to traffic to the cell membrane following stimulation, indicating a potential reason for its inability to activate Erk. While the DAG-binding C1 domain of RasGRP1 has long been recognized as an important factor mediating Erk activation, our data revealed the physiological relevance of the tail domain of RasGRP1 in the control of Erk signaling.</p> / Dissertation

T cells are an essential part of the adaptive immune system and protect the body from intracellular infections. The specificity with which αßTCR-bearing T cells recognize cognate antigen presented on MHC molecules is paramount to maintaining the balance between mounting effector functions against pathogens and establishing peripheral tolerance to self. The mechanism by which T cells translate qualitative differences in TCR:pMHC binding to sensitive proximal signalling events which ultimately result in specific Tcell effector responses to infected cells but not to self is mostly unknown. To address how T cell signalling responds to qualitative differences in TCR triggering by pMHC, I established a system of stimulating T cells bearing the 1G4 TCR specifically in vitro with a panel of four NY-ESO-1_156-165 peptide variant MHC tetramers. Single amino acid substitutions to the NY-ESO-1_156-165 peptide conferred a maximum 35-fold difference in the monomeric affinity for the 1G4 TCR. The system allows the highly controlled investigation of very rapid TCR proximal signalling events simultaneously and quantitatively using flow cytometry. Stimulations with pMHC tetramers showed rapid sensitive sequential signalling responses which were able to confer ligand discrimination. Very early signalling events such as CD3ζ phosphorylation showed analogue responses to the different affinity pMHC tetramers. Later signalling events including phospho-ERK showed a distinct on/off switch-like response. The amplitude of the very early analogue signalling responses determined the extent of later digital ERK signals. This indicates that a certain analogue signalling threshold must be passed to result in T cell activation. The thymocyte protein Themis has been shown proximal TCR signalling to modulate thymocyte selection thresholds. Its deletion results in profound defects in positive thymocyte selection. Themis locates to the LAT signalosome of the TCR signalling cascade via Grb2, yet its molecular function is unknown. Employing the system I established, I demonstrate that Themis-k/d cells show increased levels of CD3z-chain phosphorylation, phospho-ERK signalling and signal-induced apoptosis which was independent of the ERK signal. This shows that Themis is a global attenuator of proximal TCR signalling. We are currently investigating possible associations of Themis to proteins phosphastases such as SHP-1 which could attenuate TCR proximal protein tyrosine signalling events.

Protein-protein recognition plays a central role in the surveillance of self and non-self in the mammalian immune system and ultimately in cellular survival within the organism. Two systems of fundamental importance to the immune system are the Tumour Necrosis Factor (TNF) and the T cell receptor (TCR) families. High-throughput methods developed within the Oxford Protein Production Facility have been successfully applied to the production of members of the TNF receptor and ligand superfamilies for structural characterisation. The TNF receptor DR6 was successfully refolded from E.coli inclusion bodies using a rapid-dilution technique and yielded diffraction quality crystals. Data collected from these crystals will be used to obtain an x-ray crystallographic model of DR6. Vascular Endothelial Growth Inhibitor (VEGI) was produced as a soluble recombinant protein in E.coli, and formed a number of poorly diffracting crystals, it is hoped that further trials and optimization of conditions will lead to improved data quality. Lymphotoxin β receptor was produced in a Eukaryotic system. This has shed light on the complications posed by signal peptide cleavage and glycosylation on the production of protein for crystallization trials. TNF superfamily proteins are ideal targets for the design of novel therapeutic agents due to their involvement in a number of disease pathologies. Various methods of molecular docking and small molecule design were applied to the search for potential inhibitors of receptor binding for the TNF ligand proteins TRAIL and BAFF. A number of potential drug leads were identified from the National Cancer Institute drug database. The Natural Killer (NK) T cell restricted TCRs recognise CD1d-presented glycolipid. Determination of the crystal structures of the invariant NK TCR and the NK restricted TCRs 5E and 5B shows that these proteins adopt the canonical structures of class I MHC restricted TCRs. This suggests that the binding of CD1d-glycolipid by these receptors will conform to the same model of binding seen for the class I MHC restricted TCRs.