Neuroendocrine cell populations were systematically studied and characterized in the thymus, an avian primary immune organ. The expression of the specific mRNAs for both Chromogranin A (CgA) and Carboxypeptidase E (CpE) in the thymus was first verified by RT-PCR. Additional evidence using immunofluorescent dual labeling, has demonstrated for the first time the co-existence of CgA and CpE in identical neuroendocrine cells at the protein level in a vetebrate primary lymphoid organ. These CpE- and CgA-positive cells were primarily found in the transition zone between the cortex and the medulla of the thymic lobules, an area known to contain numerous arterioles and to be heavily innervated by the autonomic nervous system, suggesting that these cell population can potentially receive input from each other, from the autonomous nervous system, from the circulation, or all of the above. (Neuro)endocrine messenger molecules produced by the thymic microenvironment, such as somatostatin (SST), seem to play a potentially important immunomodulatory role with regard to cell proliferation, differentiation, and migration, as well as cytokine production. The results showed that both SST and its receptor, SSTR2, are expressed locally within chicken thymus. The in vitro study showed that SST significantly inhibits IL-2 and concanavalin A (ConA) induced proliferation of thymocytes. In comparison with controls (medium containing IL-2 and ConA but without SST), addition of SST at 10-9 M and 10-6 M resulted in a nearly 20% decrease in proliferation (P < 0.01). The effects of somatostatin (SST) on the immune system, the role of SST on the gene expression of cytokines (IL-1, TGF, INF), chemokine receptors (CXCR4) as well as MHC-I components was assessed by real-time PCR. The question as to exactly which stimuli trigger the release of mediators such as somatostatin remains for future study. In addition, a complete inventory of all substances stored in the thymic LDCV and their effects on the developing T-cells when released in the microenvironment of the thymus are also questions that warrant further investigation.

A critical step in mounting an immune response is antigen recognition by T cells. This step proceeds by productive interactions between T cell receptors (TCR) on the surface of T cells and foreign antigen, in the form of peptide-major-histocompatibility-complexes (pMHC), on the surface of antigen-presenting-cells (APC). Antigen recognition is exceedingly difficult to understand because the vast majority of pMHC on APCs are derived from self-proteins. Nevertheless, T cells have been shown to be exquisitely sensitive, responding to as few as 10 antigenic pMHC in an ocean of tens of thousands of self pMHC. In addition, T cells are extremely specific and respond only to a small subset of pMHC by virtue of their specific TCR. To explain the sensitivity of T cells to pMHC it has been proposed that a single pMHC may serially bind multiple TCRs. Integrating present knowledge on the spatial-temporal dynamics of TCR/pMHC in the T cell-APC contact interface, we have constructed mathematical models to investigate the degree of TCR serial engagements by pMHC. In addition to reactions within clusters, the models capture the formation and mobility of TCR clusters. We find that a single pMHC serially binds a substantial number of TCRs in a TCR cluster only if the TCR/pMHC bond is stabilized by coreceptors and/or pMHC dimerization. In a separate study we propose that serial engagements can explain T cell specificity. Using Monte Carlo simulations, we show that the stochastic nature of TCR/pMHC interactions means that multiple binding events are needed for accurate detection of foreign pMHC. Critical to our studies are estimates of TCR/pMHC reaction rates and mobilities. In the second half of the thesis, we show that Fluorescence Recovery After Photobleaching (FRAP) experiments can reveal effective diffusion coefficients. We then show, using asymptotic analysis and model fitting, that FRAP experiments can be used to estimate reaction rates between cell surface proteins, like TCR/pMHC. Lastly, we use FRAP experiments to investigate how the actin cytoskeleton modulates TCR mobility and report effective reaction rates between TCR and the cytoskeleton.

Neuroendocrine cell populations were systematically studied and characterized in the thymus, an avian primary immune organ. The expression of the specific mRNAs for both Chromogranin A (CgA) and Carboxypeptidase E (CpE) in the thymus was first verified by RT-PCR. Additional evidence using immunofluorescent dual labeling, has demonstrated for the first time the co-existence of CgA and CpE in identical neuroendocrine cells at the protein level in a vetebrate primary lymphoid organ. These CpE- and CgA-positive cells were primarily found in the transition zone between the cortex and the medulla of the thymic lobules, an area known to contain numerous arterioles and to be heavily innervated by the autonomic nervous system, suggesting that these cell population can potentially receive input from each other, from the autonomous nervous system, from the circulation, or all of the above. (Neuro)endocrine messenger molecules produced by the thymic microenvironment, such as somatostatin (SST), seem to play a potentially important immunomodulatory role with regard to cell proliferation, differentiation, and migration, as well as cytokine production. The results showed that both SST and its receptor, SSTR2, are expressed locally within chicken thymus. The in vitro study showed that SST significantly inhibits IL-2 and concanavalin A (ConA) induced proliferation of thymocytes. In comparison with controls (medium containing IL-2 and ConA but without SST), addition of SST at 10-9 M and 10-6 M resulted in a nearly 20% decrease in proliferation (P < 0.01). The effects of somatostatin (SST) on the immune system, the role of SST on the gene expression of cytokines (IL-1, TGF, INF), chemokine receptors (CXCR4) as well as MHC-I components was assessed by real-time PCR. The question as to exactly which stimuli trigger the release of mediators such as somatostatin remains for future study. In addition, a complete inventory of all substances stored in the thymic LDCV and their effects on the developing T-cells when released in the microenvironment of the thymus are also questions that warrant further investigation.

Hydrogels, defined as three-dimensional, hydrophilic networks, offer extensive biomedical applications. The areas of application are heavily concentrated in drug delivery and tissue engineering because of the hydrogels’ ability to mimic extracellular matrixes of tissue while maintaining a high level of biocompatibility. Specifically, poly(ethylene glycol) (PEG) is a well-established biomaterial in hydrogel applications due to its high water-solubility, low toxicity, high biocompatibility, and stealth properties. This thesis discusses two applications of PEG-based degradable hydrogels. The first is the targeted, site-specific, controlled release of biologic drugs administered by inhalation. There are many challenges to designing a pulmonary delivery system for inhalation of biologic drugs such as low respirable fractions and short resident time in the lungs. In this report, the hydrogel microcarriers for encapsulated drugs were formed by cross-linked PEG and peptide sequences synthesized during a mild emulsion process. The microgels underwent freeze-drying in the presence of cryoprotectants and formulated for dry powder inhalation. The microgels displayed swelling properties to avoid local macrophage clearance in the lungs and exhibited triggered release and degradation in response to enzyme for disease specific release. Dry formulations were tested for aerosolization properties and indicated ability to be delivered to the deep lung by a dry powder inhaler. Lastly, microgels were successfully delivered to mice lungs via intratracheal aerosol delivery. This thesis also discusses the use of PEG-based hydrogel as a biomaterial microenvironment for encapsulated stem cells as a means of in vitro T cell differentiation. A 3D hydrogel system creates a biomimetic reconstruction of the cell’s natural microenvironment and allows us to adjust factors such as ligand density and mechanical properties of the hydrogel in order to promote cells differentiation. This report utilizes hydrogels of cross-linked hyaluronic acid and PEG to encapsulate mice bone marrow hematopoietic progenitor cells in the presence of notch ligands, displayed through stromal cells, magnetic microbeads, or immobilized within the hydrogel matrix. Mechanical properties of the hydrogels were tested and the release of encapsulated cells was performed by enzymatic degradation or dissolution. The differentiation data obtained indicated successful differentiation of stem cells into early T cells through the hydrogel system. / text

αβ-TCR+CD3+CD4-CD8-NK1.1- double negative (DN) T cells comprise 1-5% of T lymphocytes in mice and humans. Previous studies have demonstrated that DN T cells can suppress auto-, allo- and xeno-immune responses in an antigen-specific fashion. However, the mechanisms by which DN T cells regulate immune responses remain elusive. Whether DN T cells can regulate antigen presenting cells has not been investigated previously. The focus of this thesis is to determine the consequences of DN T cells interaction with antigen presenting cells (APCs) and the underlying mechanisms. In this thesis, using a murine skin transplantation model, we found that donor B cells, but not dendritic cells (DCs), are the major surviving donor APCs in recipients following donor lymphocyte infusion. Infusing donor B, but not non-B, cells resulted in significantly enhanced donor-specific skin allograft survival. Mice that had received donor B cells showed higher expression of activation markers on antigen-specific DN T cells. B cells could present alloantigen to DN T cells and prime DN T cell proliferation in an antigen-specific fashion. Activated DN T cells were not able to down regulate the expression of CD80 or CD86 on LPS-activated B cells, but they could kill activated allogeneic as well as syngeneic B cells via a perforin-dependent pathway in vitro. In addition, DN T cells expressed high levels of CTLA4 and were capable of down regulating CD80 and CD86 expressed on antigen-expressing mature DCs through CTLA4. DN T cells killed both immature and mature allogeneic DCs, as well as antigen-loaded syngeneic DCs, in an antigen-specific manner in vitro and in vivo, mainly through the Fas-FasL pathway. Taken together, the data presented in this thesis demonstrate, for the first time, that DN T cells are potent regulators of APCs and further clarify the mechanisms of DN T cell-mediated immune suppression. These findings provide novel insights for DN T cells to be developed as a potent immune suppression treatment for a variety of diseases.

αβ-TCR+CD3+CD4-CD8-NK1.1- double negative (DN) T cells comprise 1-5% of T lymphocytes in mice and humans. Previous studies have demonstrated that DN T cells can suppress auto-, allo- and xeno-immune responses in an antigen-specific fashion. However, the mechanisms by which DN T cells regulate immune responses remain elusive. Whether DN T cells can regulate antigen presenting cells has not been investigated previously. The focus of this thesis is to determine the consequences of DN T cells interaction with antigen presenting cells (APCs) and the underlying mechanisms. In this thesis, using a murine skin transplantation model, we found that donor B cells, but not dendritic cells (DCs), are the major surviving donor APCs in recipients following donor lymphocyte infusion. Infusing donor B, but not non-B, cells resulted in significantly enhanced donor-specific skin allograft survival. Mice that had received donor B cells showed higher expression of activation markers on antigen-specific DN T cells. B cells could present alloantigen to DN T cells and prime DN T cell proliferation in an antigen-specific fashion. Activated DN T cells were not able to down regulate the expression of CD80 or CD86 on LPS-activated B cells, but they could kill activated allogeneic as well as syngeneic B cells via a perforin-dependent pathway in vitro. In addition, DN T cells expressed high levels of CTLA4 and were capable of down regulating CD80 and CD86 expressed on antigen-expressing mature DCs through CTLA4. DN T cells killed both immature and mature allogeneic DCs, as well as antigen-loaded syngeneic DCs, in an antigen-specific manner in vitro and in vivo, mainly through the Fas-FasL pathway. Taken together, the data presented in this thesis demonstrate, for the first time, that DN T cells are potent regulators of APCs and further clarify the mechanisms of DN T cell-mediated immune suppression. These findings provide novel insights for DN T cells to be developed as a potent immune suppression treatment for a variety of diseases.

A critical step in mounting an immune response is antigen recognition by T cells. This step proceeds by productive interactions between T cell receptors (TCR) on the surface of T cells and foreign antigen, in the form of peptide-major-histocompatibility-complexes (pMHC), on the surface of antigen-presenting-cells (APC). Antigen recognition is exceedingly difficult to understand because the vast majority of pMHC on APCs are derived from self-proteins. Nevertheless, T cells have been shown to be exquisitely sensitive, responding to as few as 10 antigenic pMHC in an ocean of tens of thousands of self pMHC. In addition, T cells are extremely specific and respond only to a small subset of pMHC by virtue of their specific TCR. To explain the sensitivity of T cells to pMHC it has been proposed that a single pMHC may serially bind multiple TCRs. Integrating present knowledge on the spatial-temporal dynamics of TCR/pMHC in the T cell-APC contact interface, we have constructed mathematical models to investigate the degree of TCR serial engagements by pMHC. In addition to reactions within clusters, the models capture the formation and mobility of TCR clusters. We find that a single pMHC serially binds a substantial number of TCRs in a TCR cluster only if the TCR/pMHC bond is stabilized by coreceptors and/or pMHC dimerization. In a separate study we propose that serial engagements can explain T cell specificity. Using Monte Carlo simulations, we show that the stochastic nature of TCR/pMHC interactions means that multiple binding events are needed for accurate detection of foreign pMHC. Critical to our studies are estimates of TCR/pMHC reaction rates and mobilities. In the second half of the thesis, we show that Fluorescence Recovery After Photobleaching (FRAP) experiments can reveal effective diffusion coefficients. We then show, using asymptotic analysis and model fitting, that FRAP experiments can be used to estimate reaction rates between cell surface proteins, like TCR/pMHC. Lastly, we use FRAP experiments to investigate how the actin cytoskeleton modulates TCR mobility and report effective reaction rates between TCR and the cytoskeleton.

Thesis (Ph. D.)--Case Western Reserve University, 2010. / [School of Medicine] Department of Molecular Virology. Includes bibliographical references.

Our immune system must be able to rapidly fight against pathogens, but at the same time be tightly regulated to prevent harmful autoimmune and inflammatory responses. This intricate balance is controlled in part by T lymphocytes. Therapies targeting T cells have the potential to revolutionise the ways in which inflammation and autoimmune diseases are treated. However, before this can be achieved, a better quantitative understanding of the molecular processes controlling the functions of these cells is required. T cell signalling is tightly regulated by a series of complex molecular networks, which converge on key transcription factors, including Nuclear Factor-κB (NF-κB), Nuclear Factor of Activated T cells (NFAT), and Activator Protein 1 (AP-1). Using a combination of single-cell time-lapse imaging, and genome-wide assays probing for chromatin accessibility and gene expression, this study provides a better understanding of the mechanisms underpinning T cell activation and signalling. One central tenet of T cell activation is that activation-associated gene expression is triggered by the binding of the cognate antigen to the T cell receptor (TCR), and enhanced by co-stimulatory receptors, including CD28, which act to augment TCR signalling. This study shows that activation- associated gene expression programmes (induced by calcium ionophore ionomycin and phorbol 12-myristate 13-acetate (PMA) in Jurkat T cells) are closely associated with specific chromatin landscapes. Further to this, data shown here indicate that the integration between TCR and co- stimulatory receptor signalling occurs at the chromatin level, and plays a pivotal role in regulating T cell activation. Using live-cell imaging, this study also shows that information about the diverse external signals received by T cells could be encoded within the dynamic nuclear translocations of key transcription factors. In particular, TCR signals appear to be processed by the duration of NFAT nuclear occupancy. TCR stimulation in the presence of a co-stimulatory signal resulted in the rapid nuclear import and export of NFAT proteins. In contrast, when TCR stimulation was applied without a co-stimulatory signal, prolonged nuclear occupancy of NFAT was observed. Further investigation suggested that the sustained activity of NFAT could confer a ‘signal memory’ within the TCR signalling network, thus providing a potential mechanism for preventing premature T cell turn-off during transient T cell-Antigen presenting cell interactions. This new detailed picture of T cell biology moves the field towards better therapeutic strategies for numerous diseases.

A critical step in mounting an immune response is antigen recognition by T cells. This step proceeds by productive interactions between T cell receptors (TCR) on the surface of T cells and foreign antigen, in the form of peptide-major-histocompatibility-complexes (pMHC), on the surface of antigen-presenting-cells (APC). Antigen recognition is exceedingly difficult to understand because the vast majority of pMHC on APCs are derived from self-proteins. Nevertheless, T cells have been shown to be exquisitely sensitive, responding to as few as 10 antigenic pMHC in an ocean of tens of thousands of self pMHC. In addition, T cells are extremely specific and respond only to a small subset of pMHC by virtue of their specific TCR. To explain the sensitivity of T cells to pMHC it has been proposed that a single pMHC may serially bind multiple TCRs. Integrating present knowledge on the spatial-temporal dynamics of TCR/pMHC in the T cell-APC contact interface, we have constructed mathematical models to investigate the degree of TCR serial engagements by pMHC. In addition to reactions within clusters, the models capture the formation and mobility of TCR clusters. We find that a single pMHC serially binds a substantial number of TCRs in a TCR cluster only if the TCR/pMHC bond is stabilized by coreceptors and/or pMHC dimerization. In a separate study we propose that serial engagements can explain T cell specificity. Using Monte Carlo simulations, we show that the stochastic nature of TCR/pMHC interactions means that multiple binding events are needed for accurate detection of foreign pMHC. Critical to our studies are estimates of TCR/pMHC reaction rates and mobilities. In the second half of the thesis, we show that Fluorescence Recovery After Photobleaching (FRAP) experiments can reveal effective diffusion coefficients. We then show, using asymptotic analysis and model fitting, that FRAP experiments can be used to estimate reaction rates between cell surface proteins, like TCR/pMHC. Lastly, we use FRAP experiments to investigate how the actin cytoskeleton modulates TCR mobility and report effective reaction rates between TCR and the cytoskeleton. / Science, Faculty of / Mathematics, Department of / Graduate