The generation of the cytotoxic CD8 T cell response is dependent on the functional outcomes imposed by the intrathymic constraints of differentiation and self-tolerance. Although thymic function can be partly replicated in vitro using OP9-DL1 cell cultures to yield CD8 αβ T cell receptor (TCR)-bearing cells from hematopoietic progenitor cells, a comprehensive and functional assessment of entirely in-vitro generated CD8 T cells derived from bone marrow hematopoietic stem cells (BM-HSCs) has not been established and remains controversial. Here we demonstrate that a phenotypic, molecular, and functional signature of in vitro-derived CD8 T cells is akin to that of ex vivo CD8 T cells. Transfer of in vitro-derived CD8 T cells into syngeneic and immunodeficient host mice showed no graft-versus-host response, while a robust homeostatic proliferation was observed, respectively. These findings, along with a diverse and broad TCR repertoire expressed by the in vitro-derived CD8 T cells, allowed for the successful generation of antigen (Ag)-specific T cells to be obtained from an entirely in vitro-generated CD8 T cell pool, which calls for further tailoring of their use against viral infections or malignancies. Furthermore, I demonstrate that Notch signaling regulates the expression of the cytolytic molecule Granzyme A in CD8+ T cells. This is supported by the inability of Notch-deprived TCR-signaled CD8 T cells to express Granzyme A, while CD8 T cells that received Notch signals readily expressed Granzyme A, suggesting that Notch signaling is a prerequisite for induction of this cytolytic molecule. We further demonstrate that Notch signaling by OP9 cells allows for efficient differentiation of conventional effector CD8 T cells from SAP-/- BM-derived HSCs and restricts differentiation of innate CD8 T cells while allowing for differentiation of IL17-producing CD8 T cells from BM-HSCs isolated from Itk-/-Rlk-/- (DKO) mice. Moreover, we reveal that the process of positive and negative selection in vitro is constrained by peptide-MHC (pMHC) class I expressed by the OP9 cells and disclose that the commitment of DP precursors to the CD8 T cell lineage is facilitated by Notch signaling. Our findings further establish the requirement for Notch receptor-ligand interactions throughout intrathymic T cell differentiation.

During viral infections, CD8+ T cells only respond to a select few epitopes derived from the respective foreign pathogen. These epitopes can be organized into a hierarchy, based on their ability to induce T cell priming. Such phenomenon is known as immunodominance. Cytotoxic T cells can be primed through the direct pathway, or the cross-priming pathway. The latter involves exogenously derived viral epitope presentation by uninfected professional antigen presenting cells. It has been previously reported that Lymphocytic Choriomeningitis nucleoprotein expressed in HEK cells (HEK-NP) could be cross presented to CD8+ T cells. In these studies we have used this same HEK-NP model to study the effects of LCMV-NP cross priming on the LCMV immunodominance hierarchy following viral challenge. Our results provide strong evidence that cross priming is an efficient route with which to induce cell-mediated immunity. We also highlight a regulatory role for cross priming in immunodominance by showing that a single dose of HEK-NP can completely shift the immunodominance hierarchy of a typical LCMV infection. Furthermore, we see that the induction of LCMV-NP cross priming boosts anti-viral immunity to subsequent LCMV infections. This work provides strong support for the physiological role that cross priming plays in normal cell-mediated immune responses. It may also provide relevant information to the realm of immunotherapy. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-07-10 14:33:18.115

No description available.

The long-term success of transplantation is limited by the need for immunosuppression; thus, tolerance induction is an important therapeutic goal. A 16-day treatment with rapamycin in mice led to indefinite graft survival of fully mismatched cardiac allografts, whereas untreated hearts were rejected after 8-10 days. Specific tolerance was confirmed through subsequent skin grafts and in vitro lymphocyte assays that showed recipient mice remained immunocompetent towards 3rd party antigens but were impaired in responding to donor antigens. Mechanisms that account for this tolerant state were then investigated. Splenic CD8+CD44+ memory T-cells were reduced in tolerant mice but had increased frequencies of the CD62LLO population. CD4+CD25+Foxp3+ regulatory T-cells were increased in tolerant mice. Through multiplex PCR, 4 regulatory T-cell related genes were found up-regulated and 2 proinflammatory genes were down-regulated in accepted hearts. This expression pattern may serve as a putative biomarker of tolerance in patients undergoing transplantation.

The long-term success of transplantation is limited by the need for immunosuppression; thus, tolerance induction is an important therapeutic goal. A 16-day treatment with rapamycin in mice led to indefinite graft survival of fully mismatched cardiac allografts, whereas untreated hearts were rejected after 8-10 days. Specific tolerance was confirmed through subsequent skin grafts and in vitro lymphocyte assays that showed recipient mice remained immunocompetent towards 3rd party antigens but were impaired in responding to donor antigens. Mechanisms that account for this tolerant state were then investigated. Splenic CD8+CD44+ memory T-cells were reduced in tolerant mice but had increased frequencies of the CD62LLO population. CD4+CD25+Foxp3+ regulatory T-cells were increased in tolerant mice. Through multiplex PCR, 4 regulatory T-cell related genes were found up-regulated and 2 proinflammatory genes were down-regulated in accepted hearts. This expression pattern may serve as a putative biomarker of tolerance in patients undergoing transplantation.

The generation of the cytotoxic CD8 T cell response is dependent on the functional outcomes imposed by the intrathymic constraints of differentiation and self-tolerance. Although thymic function can be partly replicated in vitro using OP9-DL1 cell cultures to yield CD8 αβ T cell receptor (TCR)-bearing cells from hematopoietic progenitor cells, a comprehensive and functional assessment of entirely in-vitro generated CD8 T cells derived from bone marrow hematopoietic stem cells (BM-HSCs) has not been established and remains controversial. Here we demonstrate that a phenotypic, molecular, and functional signature of in vitro-derived CD8 T cells is akin to that of ex vivo CD8 T cells. Transfer of in vitro-derived CD8 T cells into syngeneic and immunodeficient host mice showed no graft-versus-host response, while a robust homeostatic proliferation was observed, respectively. These findings, along with a diverse and broad TCR repertoire expressed by the in vitro-derived CD8 T cells, allowed for the successful generation of antigen (Ag)-specific T cells to be obtained from an entirely in vitro-generated CD8 T cell pool, which calls for further tailoring of their use against viral infections or malignancies. Furthermore, I demonstrate that Notch signaling regulates the expression of the cytolytic molecule Granzyme A in CD8+ T cells. This is supported by the inability of Notch-deprived TCR-signaled CD8 T cells to express Granzyme A, while CD8 T cells that received Notch signals readily expressed Granzyme A, suggesting that Notch signaling is a prerequisite for induction of this cytolytic molecule. We further demonstrate that Notch signaling by OP9 cells allows for efficient differentiation of conventional effector CD8 T cells from SAP-/- BM-derived HSCs and restricts differentiation of innate CD8 T cells while allowing for differentiation of IL17-producing CD8 T cells from BM-HSCs isolated from Itk-/-Rlk-/- (DKO) mice. Moreover, we reveal that the process of positive and negative selection in vitro is constrained by peptide-MHC (pMHC) class I expressed by the OP9 cells and disclose that the commitment of DP precursors to the CD8 T cell lineage is facilitated by Notch signaling. Our findings further establish the requirement for Notch receptor-ligand interactions throughout intrathymic T cell differentiation.

No description available.

No description available.

The limited success of HIV vaccine candidates to date highlights our need to better characterize protective cell-mediated immunity. Understanding correlates CD8+ T cell protection against HIV infection and progressive disease is essential for informing effective vaccine development, design and evaluation. CD8+ T cell responses with a robust polyfunctional and proliferative component are strongly linked to better disease outcomes. However, the specificity of polyfunctional and proliferative CD8+ T cell responses has not been thoroughly investigated. Additionally, the specificity of memory subsets and their connection to polyfunctionality and proliferation responses has not been adequately assessed. We address these gaps in knowledge and provide a better understanding of the fine specificity of HIV-specific CD8+ T cell responses. We hypothesize that the epitopes recognized by central memory (TCM) and effector memory (TEM) CD8+ T cells, defined by functional attributes, differ in chronic HIV-1 infection. Additionally, we hypothesize that polyfunctional and proliferative responses will better correlate with protection in HIV disease progression. The qualities of CD8+ T cell responses were evaluated using polyfunctional flow cytometry measuring both functional and phenotypic attributes of both TEM and TCM subsets in HIV infected individuals. We evaluated the quality and evolution of CD8+ T cell responses in HIV infected individuals shortly after seroconversion through to the chronic phase of infection, finding that early polyfunctional responses may result in better HIV disease outcomes. Additionally, we show that epitope-specificity differs between short-term cytokine/chemokine secretion and long-term proliferative assays. Importantly, we show that, at a cohort level, particular epitopes preferentially elicit specific qualities of CD8+ T cell responses in preference to others. This research improves our understanding of HIV pathogenesis and indicates that we can identify specific epitopes that can elicit protective responses and that early polyfunctional responses may slow HIV disease progression. Understanding the polyfunctional and proliferative capacities of HIV-specific effector and memory cells at various stages of HIV infection is of critical importance to the design of vaccines intended to elicit protective cell-mediated responses.

Ascorbic acid (AA), in popular speech vitamin C, is a commonly known nutrient. It is involved in several biological processes and deficiency can lead to scurvy. Recent publications have shown the impact of AA on epigenetic regulation in mice. Addition of AA, via enzymatic activity, enhances the generation of 5-hydroxymethylcytosine (5hmC), which is an intermediate in active demethylation of DNA. The role of AA on epigenetic changes in humans has to our knowledge never been studied. In this study, naïve CD4+ T cells from blood donors were used as a model system to investigate AAs possible role in methylation changes in the immune system. By using dot-blot assay, hydroxymethylated DNA immunoprecipitation (hmeDIP) and qPCR, changes in methylation executed by AA could be detected. A confirmation of AAs impact on epigenetic changes in mice was observed. AA enhanced the levels of 5hmC compared to untreated cells. The Jurkat cell line, a human T lymphocyte cell line, showed an opposite result. Treatment with AA decreased the levels of 5hmC compared to untreated cells. When comparing this result with the results obtained in human naïve T cells, the same observation was made. The difference between mouse and human in the ability of producing and metabolize AA could be a reason for this opposite result. Since AA had the ability to modify epigenetic changes in primary human CD4+ T cells, the results suggest that AA may have a function in the human immune system.