The normal physiological activation of naive T cells requires the engagement of both the T cell receptor (TCR) and the co-stimulatory molecule, CD28. However, a group of monoclonal antibodies (mAbs) have been identified that are able to activate T cells in vitro and in vivo via CD28 engagement alone. Two defining characteristics found in all CD28 superagonist mAbs are their membrane proximal CD28 epitopes and the requirement for mAb immobilisation. To investigate whether agonistic mAbs to similar cell molecules could be identified based on epitope position alone, mAbs to the inhibitory receptor PD-1 were generated and characterised. Using a drastic mutation-based epitope mapping technique, one mAb was identified with a membrane proximal epitope along with two other mAbs with membrane distal epitopes. These mAbs were tested for triggering activity in a hybridoma stimulation assay. mAb stimulation was observed with all three mAbs but only in cells expressing a PD-1 chimera that associated with the TCR and the strength of activation was dependent on epitope location. Cross-linking of a monomeric PD-1/CD28 chimera with a pair of anti-PD-1 mAbs resulted in signalling in this system, however, suggesting a role for ligand aggregation in addition to epitope position in mAb signalling. To further investigate the role of epitope position in CD28 superagonism, a cell line expressing a chimeric form of CD28 was created wherein the superagonistic mAb epitope was moved to a membrane distal position. When stimulated with a CD28 superagonist mAb signalling was no longer observed. However stimulation with another mAb that had an epitope to a membrane proximal location on the chimera resulted in superagonistic effect. These results show that epitope location is the dominant cause of T cell stimulation observed by CD28 superagonist mAbs and that epitope dependent mAb signalling is possible in other T cell surface molecules. The work described in this thesis has implications for both the development of immune modulating mAb therapeutics and for the general mechanism of triggering of cell surface receptors dependent on extrinsic tyrosine kinases.

T-cell activation is a complex process crucial for a proper function of immune system. It has been extensively studied and its main features are well understood. However, some of the events involved in T-cell signalling are still unclear. After T-cell receptor stimulation, Src-family kinase Lck drives the initiation of signalling by tyrosine phosphorylation. Phosphorylation of several downstream targets is dependent on the redistribution of Lck to the different compartment of the plasma membrane, called lipid rafts. In lipid rafts, active Lck is juxtaposed and activates raft-resident substrates which then trigger downstream signalling. The critical in this process is the mechanism of Lck translocation to lipid rafts which has not been studied so far and represents the topic of great academic and clinical interests. Previously, we identified the adaptor protein RACK1 as a candidate protein mediating the redistribution of Lck to lipid rafts by linking it to the microtubular network. In this thesis, we analysed the structural features and functional role of RACK1 in its interaction with Lck. We show here, using the SYF cell lines expressing the wild type and various mutated forms of Lck, that intact SH3 or SH2 domains of Lck are required for an effective RACK1-Lck complex formation. We also documented...

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 100-117).

T cell activation and thymic selection are thought to be determined by the binding propensity (avidity or affinity) of the T cell receptor (TCR) to its ligands. However, binding propensity quantified by previous 3D TCR–pMHC kinetics such as using tetramer staining or surface plasmon resonance (SPR) under estimate TCR–pMHC interaction due to neglecting physiological conditions. Recent studies considering membrane contribution in TCR–pMHC interaction reported 2D kinetics and force regulated bond dissociation kinetics have better prediction to biological responses in CD8+ T cells. In this study, we further tested the findings in CD4+ T cells and CD4+ CD8+ (double-positive, DP) thymocytes. We analyzed TCR–pMHC interaction for a well-characterized panel of altered peptide ligands (APLs) on multiple transgenic mouse TCR systems. Using ultrasensitive 2D mechanical assays, in situ 2D kinetic measurements show better sensitivity than the SPR 3D kinetic measurements in gauging the ligand potency and thymic selection. Furthermore, force-regulated bond lifetime of TCR–pMHC interaction amplifies the discrimination in recognition of APLs and thymic selection. When force was applied to TCR–pMHC–CD4/8 bonds, two distinct patterns emerged: agonist/negative selecting ligands formed CD4/8-dependent catch-slip bonds where lifetime first increased, reached a maximum, then decreased with increasing force, whereas antagonist/positive selecting ligands formed slip-only bonds where lifetime monotonically decreases with increasing force. Our results highlight an important role of mechanical force in ligand discrimination and suggest a new mechanism for T cell activation and thymic selection that is distinct from previous models based on 3D measurements.

Thesis (Ph.D. in Immunology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 138-153). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (M.Sc.)--Memorial University of Newfoundland, Faculty of Medicine, 1998. / Typescript. Bibliography: leaves 193-209.

T cells are a cornerstone of the mammalian adaptive immune system. A range of T-cell subsets exist that can orchestrate the overall immune response to pathogens or cancers, either by directly killing infected cells or licensing other cells to do so. Dysregulation of this important process can result in immunodeficiency or autoimmunity. Although T cells have been studied extensively over many decades, the detailed mechanisms underlying T-cell activation remain to be fully resolved. This thesis describes the development of new optogenetic approaches for the modulation of T-cell signalling dynamics and the interrogation of key events in T-cell activation to help investigate this question. Optogenetics is a rapidly emerging technique whereby light can be used to control the spatial and temporal activation, or inactivation of signalling pathways at unprecedented resolution. The methods described in this work utilise the blue light-responsive LOV2 photo-domain from the common oat A. Sativa, which is the foundation of the both the ‘LOVTRAP’ and ‘TULIPs’ optogenetic toolkits. T-cell antigen receptor (TCR) microclusters arise early during the interaction between T cells and antigen presenting cells (APCs). These TCR signalling platforms contain the proteins necessary for sustained T-cell activation, yet the processes underlying their formation and dissociation are still not fully characterised as they have been difficult to investigate with current chemical and genetic manipulations of T cells. Using two optogenetics systems combining either LOVTRAP or TULIPs and the microcluster- scaffolding protein LAT (Linker for the Activation of T cells), it was possible to modulate early T-cell signalling events and measure functional outputs in real-time. Unfortunately, the biological limitations of these LAT-based systems meant that they could not be used to quantitatively investigate microcluster formation. However, in an alternative approach, a drug-inducible, light-controllable chimeric antigen receptor was successfully developed that yielded important new insights into the rapid rate of signal decay within the TCR signalling pathway and the temporal dynamics of T-cell activation over several timescales. T cell-dependent bispecific antibodies (TDBs) are a new class of immuno-therapeutics that can specifically direct a T-cell response towards tumours, by crosslinking the TCR complex to a surface- expressed target on the cancerous cells. However, their mechanism of action has not been studied in detail. The close apposition of the T cell and target cells driven by the TDB interaction can result in the steric exclusion of phosphatases, such as CD45, away from the TCR at the TDB-generated cell-cell interface due to their large, rigid extracellular domains. Using the myeloma-expressed antigen, FcRH5, it was found that membrane-proximal epitopes of FcRH5 drive more robust TCR clustering and increased CD45 exclusion than membrane distal epitopes, which strongly correlated with effective killing of the target cell. These findings have important implications for therapeutic design and implementation of TDBs.

abstract: CD8+ T-lymphocytes (CTLs) are central to the immunologic control of infections and are currently at the forefront of strategies that enhance immune based treatment of a variety of tumors. Effective T-cell based vaccines and immunotherapies fundamentally rely on the interaction of CTLs with peptide-human leukocyte antigen class I (HLA-I) complexes on the infected/malignant cell surface. However, how CTLs are able to respond to antigenic peptides with high specificity is largely unknown. Also unknown, are the different mechanisms underlying tumor immune evasion from CTL-mediated cytotoxicity. In this dissertation, I investigate the immunogenicity and dysfunction of CTLs for the development of novel T-cell therapies. Project 1 explores the biochemical hallmarks associated with HLA-I binding peptides that result in a CTL-immune response. The results reveal amino acid hydrophobicity of T-cell receptor (TCR) contact residues within immunogenic CTL-epitopes as a critical parameter for CTL-self/nonself discrimination. Project 2 develops a bioinformatic and experimental methodology for the identification of CTL-epitopes from low frequency T-cells against tumor antigens and chronic viruses. This methodology is employed in Project 3 to identify novel immunogenic CTL-epitopes from human papillomavirus (HPV)-associated head and neck cancer patients. In Project 3, I further study the mechanisms of HPV-specific T-cell dysfunction, and I demonstrate that combination inhibition of Indoleamine 2, 3-dioxygenase (IDO-1) and programmed cell death protein (PD-1) can be a potential immunotherapy against HPV+ head and neck cancers. Lastly, in Project 4, I develop a single-cell assay for high-throughput identification of antigens targeted by CTLs from whole pathogenome libraries. Thus, this dissertation contributes to fundamental T-cell immunobiology by identifying rules of T-cell immunogenicity and dysfunction, as well as to translational immunology by identifying novel CTL-epitopes, and therapeutic targets for T-cell immunotherapy. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2017

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapeutic option for many malignant diseases. However, the efficacy of allo-HSCT is limited by the occurrence of destructive graft-versus-host disease (GVHD). Since allogeneic T cells are the driving force in the development of GVHD, their activation, proliferation, and differentiation are key factors to understanding GVHD pathogenesis. On the other hand, antigen-presenting cells (APCs) are essential for allogeneic T-cell priming and the development of GVHD. The T-box transcription factor T-bet is a master regulator for IFN-γ production and Th1 differentiation. T-bet also regulates the functions of APCs including dendritic cells (DCs) and B cells. Therefore, we investigated the role of T-bet in T cell responses, as well as on APC functions, in acute GVHD (aGVHD) using murine models of allogenic bone marrow transplantation (allo-BMT). In Chapter 2, we evaluated the roles of T-bet and IFN-γ in T-cell responses. T-bet-/- T cells induced significantly less GVHD compared with either wild-type (WT) or IFN-γ-/- counterparts in CD4-driven major histocompatibility complex (MHC)- or minor histocompatibility antigen (miHA)-mismatched models. We defined several T-bet-dependent but IFN-γ-independent molecules that may account for this distinct outcome. Further study indicates that T-bet also controls the optimal activity of Th17 cells to induce GVHD. Moreover, the compromised graft-versus-leukemia (GVL) effect of T-bet-/- T cells could be essentially reversed by IL-17 neutralization. Thus, targeting T-bet or regulating its downstream effectors independent of IFN-γ may be a promising strategy to control GVHD in the clinic. In Chapter 3, we evaluated the role of T-bet on APCs and found that T-bet-/- recipients developed much milder GVHD than their WT counterparts in MHC-mismatched or CD4-depedent miHA-mismatched models. As the functional readout of APCs, allogeneic donor T cells, particular CD4 subpopulation, significantly reduced IFN-γ production, proliferation and migration, and caused less damage in liver and gut in T-bet-/- recipients. We further observed that T-bet on recipient hematopoietic APCs, particular DCs, was primarily responsible for donor T-cell response and pathogenicity in GVHD. In fact, Trail/DR5 interaction served as a major signaling pathway responsible for donor T-cell apoptosis and impaired GVHD development in T-bet-/- recipients. Furthermore, T-bet expression on the host is largely dispensable for the GVL effect. Taken together, we propose that T-bet is a potential therapeutic target for the control of GVHD through regulating T cells as well as APCs. We believe further exploration and understanding of the immunobiology of T-bet in controlling the activities of T cells and APCs in GVHD will expand therapeutic options for the continuing success of allo-HSCT.

[EN]It is well illustrated that the generation of Tregs is the main mechanism responsible for maintaining immune tolerance during developmental exposure to non-inherited maternal antigen (NIMA). Moreover, the presence of NIMA-specific Tregs in the uterus of pregnant mice promote reproductive fitness by enforcing maternal tolerance to overlapping paternal antigens expressed by the fetus during next-generation pregnancies. However, the reason why perinatal T cell lineage is biased towards immune tolerance is poorly understood. Due to the fact that terminal deoxynucleotidyl transferase (TdT) is not expressed in neonatal T cells in the mouse, neonatal T cells have a less diverse TCR repertoire. This is known to limit their specificity and to increase their affinity for MHC/peptide complexes. At the start of the present work, we postulated that expressing high affinity TCR might be the reason that forces the development of antigen-specific Tregs in neonates. We undertook our study with the aim to investigate the mechanisms underlying mouse NIMA-specific Treg development in the perinatal period. Using 2W1S-OVA+ heterozygous mouse model in which 2W1S antigen was transformed into surrogate NIMA for half of the offspring, we observed an increased frequency of 2W1S-specific Tregs in NIMA-2W1S-exposed animals. Moreover, we also observed that periphery-derived NIMA-2W1S Tregs had a less diverse TCR repertoire and were phenotypically distinct from thymus-derived SELF-2W1S-specific Tregs. In order to investigate whether the lack of diversity was responsible for the development of neonatal NIMA-specific Tregs, we generated transgenic mice where TdT expression was enforced in T cells before birth. We found that transgenic TdT added clonal TCR diversity but did not prevent the development of T cell clones with neonatal type TCR repertoire and did not modify the frequency of neonatal NIMA-specific Tregs. On the contrary, TdT expression increased significantly generation of SELF-specific Tregs to levels similar to that of NIMA-specific Tregs. Taken together, our data indicate that the developmental pathways of NIMA- and SELF-specific Treg repertoire are different in terms of inducing and maintaining neonatal tolerance. / [FR]Il est bien illustré que la génération périnatal de Treg est le principal mécanisme responsable du maintien de la tolérance immunitaire fœtale qui se développe suite à l'exposition aux antigènes maternels non-hérités (NIMA). De plus, la présence de Tregs spécifiques des NIMA dans l'utérus des femmes enceintes favorise la capacité de reproduction en renforçant la tolérance maternelle aux mêmes antigènes paternels exprimés par le fœtus pendant les grossesses de prochaine génération. Cependant, la raison pour laquelle la lignée des cellules T fœtales est biaisée en faveur de la tolérance immunitaire est mal comprise. Chez la souris, en raison du manque d'expression de la désoxynucléotidyl transférase terminale (TdT), les cellules T néonatales ont un répertoire de TCR moins diversifié. Ceci est connu pour limiter leur spécificité et augmenter leur affinité pour les complexes CMH / peptide. Au début du présent travail, nous avons émis l'hypothèse que l'expression de TCRs de haute affinité pourrait être la raison qui force le développement de Treg spécifiques chez les nouveau-nés. Nous avons plus particulièrement entrepris notre étude dans le but d'étudier les mécanismes sous-jacents au développement de Tregs spécifiques des NIMA chez la souris pendant la période périnatale. En utilisant le modèle de souris hétérozygotes pour 2W1S-OVA+ dans lequel l'antigène 2W1S a été transformé en NIMA pour la moitié de la progéniture, nous avons observé une fréquence accrue de Tregs spécifiques de 2W1S chez les animaux exposés au NIMA. De plus, nous avons également observé que les Treg NIMA-2W1S dérivés de la périphérie avaient un répertoire de TCRs moins diversifié et étaient phénotypiquement distincts des Tregs spécifiques de SELF-2W1S dérivés du thymus. Afin de déterminer si le manque de diversité était responsable du développement de Tregs néonataux spécifiques de NIMA, nous avons généré des souris transgéniques où l'expression de TdT était appliquée dans les cellules T avant la naissance. Nous avons constaté que le TdT transgénique ajoutait une diversité de TCR clonale, mais n'empêchait pas le développement de clones de cellules T avec un répertoire TCR de type néonatal et ne modifiait pas la fréquence des Treg néonataux spécifiques du NIMA. Au contraire, l'expression de TdT a augmenté de manière significative la génération de Tregs spécifiques de SELF-2W1S à des niveaux similaires à ceux des Treg spécifiques de NIMA-2W1S. Prises ensembles, nos données indiquent que les voies de développement du répertoire des Tregs néonataux spécifiques de NIMA et SELF sont différentes en termes d'induction et de maintien de la tolérance néonatale. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished