Thesis--Wisconsin. / Vita. Includes bibliographical references (leaves 127-146).

No description available.

Immune reconstitution following T-cell depletion consists of expansion of circulating T-cells or de novo synthesis of T-cells from the thymus. The IL-7/IL-7 receptor signalling pathway is critical for the maturation and differentiation of thymocytes before they leave the thymus as mature T-cells. Viral infections including HIV have been shown to decrease IL-7Ralpha (CD127) expression on circulating CD4+ and CD8+ T-cells. However little is known about the effects of HIV infection on CD127 expression and activity in thymocytes despite existing evidence of HIV infection of the thymus. Thymic function is altered in HIV infection leading to a dysregulation of the thymic epithelial network and reduced thymic output which may contribute, in part, to impaired immune reconstitution in progressive HIV disease. In vitro studies demonstrate that HIV infection interrupts thymopoiesis resulting in a developmental block in thymopoiesis similar to that seen in models of IL-7/IL-7R deficiencies suggesting a role for altered IL-7 signalling in HIV associated thymic dysfunction. Therefore we hypothesize that thymic dysfunction which occurs in HIV infection is due to reduced IL-7R and/or altered IL-7 signalling in thymocytes resulting in impaired de novo T-cell synthesis. In order to address this hypothesis an in vitro system for the functional study of human thymocytes has been optimized. The research conducted as part of this thesis assessed if in vitro HIV infection or if cytokines that are upregulated in the course of HIV infection altered CD127 expression on maturing thymocytes. It also evaluated if in vitro HIV infection disrupts thymocyte function at different stages of maturation and whether this disruption in function is due to impaired IL-7/IL-7R signalling. The host factors IL-7, TNF- and IL-4, which are upregulated in HIV infection, are found to downregulate CD127 expression on thymocytes. IL-4 pre-treatment of thymocytes reduced the ability of IL-7 to induce STAT-5 phosphorylation. Furthermore following in vitro HIV infection of thymocytes, CD127 expression of single positive CD8 thymocytes was decreased. In vitro HIV infection altered IL-7 activity as demonstrated by lower levels of Bcl-2 and phospho-STAT-5 expression in thymocytes following IL-7 stimulation. These accumulated results suggests that HIV may play a role in impaired thymic function by altering IL-7 responsiveness. Understanding the mechanisms of thymic dysfunction in HIV infection may provide some insight into therapies leading to immune reconstitution through increased thymic output.

Immune reconstitution following T-cell depletion consists of expansion of circulating T-cells or de novo synthesis of T-cells from the thymus. The IL-7/IL-7 receptor signalling pathway is critical for the maturation and differentiation of thymocytes before they leave the thymus as mature T-cells. Viral infections including HIV have been shown to decrease IL-7Ralpha (CD127) expression on circulating CD4+ and CD8+ T-cells. However little is known about the effects of HIV infection on CD127 expression and activity in thymocytes despite existing evidence of HIV infection of the thymus. Thymic function is altered in HIV infection leading to a dysregulation of the thymic epithelial network and reduced thymic output which may contribute, in part, to impaired immune reconstitution in progressive HIV disease. In vitro studies demonstrate that HIV infection interrupts thymopoiesis resulting in a developmental block in thymopoiesis similar to that seen in models of IL-7/IL-7R deficiencies suggesting a role for altered IL-7 signalling in HIV associated thymic dysfunction. Therefore we hypothesize that thymic dysfunction which occurs in HIV infection is due to reduced IL-7R and/or altered IL-7 signalling in thymocytes resulting in impaired de novo T-cell synthesis. In order to address this hypothesis an in vitro system for the functional study of human thymocytes has been optimized. The research conducted as part of this thesis assessed if in vitro HIV infection or if cytokines that are upregulated in the course of HIV infection altered CD127 expression on maturing thymocytes. It also evaluated if in vitro HIV infection disrupts thymocyte function at different stages of maturation and whether this disruption in function is due to impaired IL-7/IL-7R signalling. The host factors IL-7, TNF- and IL-4, which are upregulated in HIV infection, are found to downregulate CD127 expression on thymocytes. IL-4 pre-treatment of thymocytes reduced the ability of IL-7 to induce STAT-5 phosphorylation. Furthermore following in vitro HIV infection of thymocytes, CD127 expression of single positive CD8 thymocytes was decreased. In vitro HIV infection altered IL-7 activity as demonstrated by lower levels of Bcl-2 and phospho-STAT-5 expression in thymocytes following IL-7 stimulation. These accumulated results suggests that HIV may play a role in impaired thymic function by altering IL-7 responsiveness. Understanding the mechanisms of thymic dysfunction in HIV infection may provide some insight into therapies leading to immune reconstitution through increased thymic output.

Immune reconstitution following T-cell depletion consists of expansion of circulating T-cells or de novo synthesis of T-cells from the thymus. The IL-7/IL-7 receptor signalling pathway is critical for the maturation and differentiation of thymocytes before they leave the thymus as mature T-cells. Viral infections including HIV have been shown to decrease IL-7Ralpha (CD127) expression on circulating CD4+ and CD8+ T-cells. However little is known about the effects of HIV infection on CD127 expression and activity in thymocytes despite existing evidence of HIV infection of the thymus. Thymic function is altered in HIV infection leading to a dysregulation of the thymic epithelial network and reduced thymic output which may contribute, in part, to impaired immune reconstitution in progressive HIV disease. In vitro studies demonstrate that HIV infection interrupts thymopoiesis resulting in a developmental block in thymopoiesis similar to that seen in models of IL-7/IL-7R deficiencies suggesting a role for altered IL-7 signalling in HIV associated thymic dysfunction. Therefore we hypothesize that thymic dysfunction which occurs in HIV infection is due to reduced IL-7R and/or altered IL-7 signalling in thymocytes resulting in impaired de novo T-cell synthesis. In order to address this hypothesis an in vitro system for the functional study of human thymocytes has been optimized. The research conducted as part of this thesis assessed if in vitro HIV infection or if cytokines that are upregulated in the course of HIV infection altered CD127 expression on maturing thymocytes. It also evaluated if in vitro HIV infection disrupts thymocyte function at different stages of maturation and whether this disruption in function is due to impaired IL-7/IL-7R signalling. The host factors IL-7, TNF- and IL-4, which are upregulated in HIV infection, are found to downregulate CD127 expression on thymocytes. IL-4 pre-treatment of thymocytes reduced the ability of IL-7 to induce STAT-5 phosphorylation. Furthermore following in vitro HIV infection of thymocytes, CD127 expression of single positive CD8 thymocytes was decreased. In vitro HIV infection altered IL-7 activity as demonstrated by lower levels of Bcl-2 and phospho-STAT-5 expression in thymocytes following IL-7 stimulation. These accumulated results suggests that HIV may play a role in impaired thymic function by altering IL-7 responsiveness. Understanding the mechanisms of thymic dysfunction in HIV infection may provide some insight into therapies leading to immune reconstitution through increased thymic output.

Immune reconstitution following T-cell depletion consists of expansion of circulating T-cells or de novo synthesis of T-cells from the thymus. The IL-7/IL-7 receptor signalling pathway is critical for the maturation and differentiation of thymocytes before they leave the thymus as mature T-cells. Viral infections including HIV have been shown to decrease IL-7Ralpha (CD127) expression on circulating CD4+ and CD8+ T-cells. However little is known about the effects of HIV infection on CD127 expression and activity in thymocytes despite existing evidence of HIV infection of the thymus. Thymic function is altered in HIV infection leading to a dysregulation of the thymic epithelial network and reduced thymic output which may contribute, in part, to impaired immune reconstitution in progressive HIV disease. In vitro studies demonstrate that HIV infection interrupts thymopoiesis resulting in a developmental block in thymopoiesis similar to that seen in models of IL-7/IL-7R deficiencies suggesting a role for altered IL-7 signalling in HIV associated thymic dysfunction. Therefore we hypothesize that thymic dysfunction which occurs in HIV infection is due to reduced IL-7R and/or altered IL-7 signalling in thymocytes resulting in impaired de novo T-cell synthesis. In order to address this hypothesis an in vitro system for the functional study of human thymocytes has been optimized. The research conducted as part of this thesis assessed if in vitro HIV infection or if cytokines that are upregulated in the course of HIV infection altered CD127 expression on maturing thymocytes. It also evaluated if in vitro HIV infection disrupts thymocyte function at different stages of maturation and whether this disruption in function is due to impaired IL-7/IL-7R signalling. The host factors IL-7, TNF- and IL-4, which are upregulated in HIV infection, are found to downregulate CD127 expression on thymocytes. IL-4 pre-treatment of thymocytes reduced the ability of IL-7 to induce STAT-5 phosphorylation. Furthermore following in vitro HIV infection of thymocytes, CD127 expression of single positive CD8 thymocytes was decreased. In vitro HIV infection altered IL-7 activity as demonstrated by lower levels of Bcl-2 and phospho-STAT-5 expression in thymocytes following IL-7 stimulation. These accumulated results suggests that HIV may play a role in impaired thymic function by altering IL-7 responsiveness. Understanding the mechanisms of thymic dysfunction in HIV infection may provide some insight into therapies leading to immune reconstitution through increased thymic output.

T lymphocytes (T cells) represent one of the major cell populations of the immune system. Named by the place of their development, the thymus, several types can be distinguished as the αβ T cells, the γδ T cells, the mucosa-associated invariant T cells (MAIT), and the natural killer T (NKT) cells. The αβ lineages of CD4+ THelper and the CD8+ T cytotoxic cells with the T cell receptor (TCR) composed of α- and β-chain are major players of the adaptive immune system. In the thymus, CD4+ and CD8+ single positive (SP) αβ cells represent the ultimate result of positive and negative selection of CD4+CD8+ double positive (DP) thymocytes. The DP population derives from the double negative (DN) thymocytes that develop from bone marrow-derived progenitors through different stages (DN1-DN4) that are characterized by CD25 and CD44 surface expression. NFATc1, a member of the Nuclear Factor of Activated T cells (NFAT) transcription factors family, is critically involved in the differentiation and function of T cells. During thymocyte development, the nuclear expression of NFATc1 reaches the highest level at the DN3 (CD44-CD25+) stage. The hematopoietic cell-specific ablation of NFATc1 activity results in an arrest of thymocyte differentiation at the DN1 (CD44+CD25-) stage. On the other hand, over-expression of a constitutively active version of NFATc1 results in an impaired transition of DN3 cells to the DN4 (CD44-CD25-) stage, suggesting that a certain threshold level of NFATc1 activity is critical at this point. ChIP-seq and RNA-seq analysis allowed us the identification of NFATc1/A target genes involved in lineage development as the Tcra and Tcrb gene loci. Furthermore, we identified multiple NFATc1-regulated genes that are involved in γδ T cell development. In the mouse models, Rag1Cre-Nfatc1fl/fl and Rag1Cre-E2fl/fl, in which the activity of NFATc1 or inducible NFATc1 in the latter is impaired during the early stages of thymocyte development, we observed increased numbers of γδ T cells. These γδ T cells showed an unusual overexpression of CD4, a lack of CD24 expression, and overexpression of the anti-apoptotic gene Bcl2a1a. We hypothesize that during the DN stages NFATc1 plays an important role in regulating crucial steps of αβ thymocyte development and when NFATc1 activity is missing this may disturb αβ development resulting in alternative cell fates like γδ T cells. / T-Lymphozyten (T-Zellen) sind eine wichtige Säule des Immunsystems. Benannt nach dem Ort ihrer Entstehung, dem Thymus, werden sie verschiedenen Linien zugeordnet, den αβ-T-Zellen, den γδ-T-Zellen, den Mukosa assoziierten invarianten T-Zellen (MAIT) und den natürlichen Killer T-Zellen (NKT). Bei den αβ-T-Zellen mit einem T-Zell-Rezeptor (TZR) aus α- und β-Kette unterscheidet man die CD4+ Helfer T- von den CD8+ zytotoxischen T-Zellen. Beide sind zentrale Bestandteile des adaptiven Immunsystems. Einfach positive (SP) CD4+ und CD8+ αβ-T-Zellen gehen aus der positiven und negativen Selektion doppelt-positiver (DP) CD4+CD8+ Zellen hervor. Zuvor durchlaufen sie als doppelt-negative (DN) Thymozyten, die von Vorläufern aus dem Knochenmark abstammen, verschiedene Stadien (DN1-DN4), die durch unterschiedliche Expression der Adhäsionsmoleküle CD25 und CD44 gekennzeichnet sind. Der Transkriptionsfaktor NFATc1, ein Mitglied der Familie Nukleärer Faktoren Aktivierter T-Zellen (NFAT), ist maßgeblich an der Differenzierung und der Funktion von T-Zellen beteiligt. Während der Thymozytenenreifung kann eine zunehmende nukleäre Expression von NFATc1 beobachtet werden, die im DN3-Stadium (CD44-CD25+) den höchsten Wert erreicht. Eine Deletion der NFATc1-Aktivität in frühen Vorläuferzellen führte zu einem Arrest der Thymozytendifferenzierung im DN1-Stadium (CD44+CD25-). Andererseits hemmte die Überexpression einer konstitutiv aktiven Form von NFATc1 den Übergang von DN3-Zellen in das DN4-Stadium (CD44-CD25-), was vermuten lässt, dass ein bestimmter Schwellenwert der NFATc1-Aktivität zu diesem Zeitpunkt von entscheidender Bedeutung ist. Mittels ChIP- und RNA-Sequenzierung gelang es, NFATc1-Zielgene im Thymus zu identifizieren. Hierzu zählten unter anderen die Tcra und Tcrb Gene. Transkriptions-analysen an isolierten DN-Thymozyten offenbarten daneben mehrere NFATc1-regulierte Gene, die an der Entwicklung von γδ-T-Zellen beteiligt sind. In den Mausmodellen, Rag1Cre-Nfatc1fl/fl und Rag1Cre-E2fl/fl, bei denen die Aktivität von NFATc1 bzw. der induzierbaren NFATc1-Aktivität zu frühen Stadien der Thymozytenentwicklung beeinträchtigt ist, stieg die Zahl von γδ-T-Zellen an. Diese γδ-T-Zellen zeigen eine unübliche Überexpression des CD4 Markers, eine fehlende Expression des Oberflächenmarkers CD24 und eine Überexpression des antiapoptotischen Gens Bcl2a1a. Wir vermuten daher, dass NFATc1 an der erfolgreichen Reifung von αβ-T-Zellen direkt beteiligt ist und fehlende NFATc1-Aktivität die Entwicklung alternativer Linien wie von γδ-T-Zellen befördert.

No description available.

<p>An immunological mechanism to account for the regulation of peripheral self-reactive T cells, which escape central tolerance in the thymus, during the primary activation of naïve, foreign antigen-specific T cells remains to be established. Contemporary models of primary T cell activation that attempt to describe how this occurs differ significantly in the cellular interactions necessary for naïve CD4+ T helper cell activation. It is generally accepted that most CD8+ T cells are dependent upon CD4+ T helper cells for their activation. </p><p>The Infectious Non-Self and Danger Models of CD4+ T cell activation propose that interaction of a naïve T cell with an appropriately armed dendritic cell is sufficient, whereas the Two-step, Two-signal Model proposes additional cellular interactions are necessary. The major goal of this thesis was to establish and utilize an in vitro experimental system that would allow one to begin to delineate which model most validly describes the cellular interactions required for generation of primary immune responses from naïve T cells. Employing a population of naïve T cells uncontaminated with any partially or fully activated cells is essential for such a study.</p><p>The results presented in this thesis show, that when thymocytes are employed as a source of responding naïve T cells, cellular interactions, in addition to interaction with bone marrow derived dendritic cells, are required for the activation of naïve thymic T cells. The primary activation of thymic T cells to generate CD4+ IL-2 producing cells, and CD8+ IFN-g producing cells and cytotoxic T cells upon stimulation with splenic allogeneic stimulator cells is critically dependent upon the presence of a syngeneic population of radiation resistant, CD4+ T cells found in the spleen of normal mice. Additionally, when such cells are present as a source of help for thymocytes, allogeneic bone marrow derived dendritic cells fail to stimulate the generation of optimal cytotoxic and cytokine responses from naïve thymic T cells. However, they do stimulate thymocytes to cycle and up regulate the ligand for the costimulatory molecule CD40, CD40L.</p><p>The results presented within also show that the optimal activation of naïve thymic T cells to generate CD4+ IL-2 producing cells, and CD8+ IFN-g producing cells and cytotoxic T cells, requires the presence of allo-MHC bearing Ig+ B220+ B cells. The removal of B220+ cells by magnetic cell sorting from the allogeneic spleen reveals that the generation of CD8+ cytotoxic T cells and IFN-g producing cells from thymocytes is markedly reduced compared to unsorted allogeneic spleen cells. However, IL-2 and IL-4 cytokine producing cells are still detectable. Potential reasons for the generation of the latter cytokine producing cells are discussed. The results presented in this thesis have revealed insights into the cellular interactions involved in the activation of naïve thymic T cells.

<p>An immunological mechanism to account for the regulation of peripheral self-reactive T cells, which escape central tolerance in the thymus, during the primary activation of naïve, foreign antigen-specific T cells remains to be established. Contemporary models of primary T cell activation that attempt to describe how this occurs differ significantly in the cellular interactions necessary for naïve CD4+ T helper cell activation. It is generally accepted that most CD8+ T cells are dependent upon CD4+ T helper cells for their activation. </p><p>The Infectious Non-Self and Danger Models of CD4+ T cell activation propose that interaction of a naïve T cell with an appropriately armed dendritic cell is sufficient, whereas the Two-step, Two-signal Model proposes additional cellular interactions are necessary. The major goal of this thesis was to establish and utilize an in vitro experimental system that would allow one to begin to delineate which model most validly describes the cellular interactions required for generation of primary immune responses from naïve T cells. Employing a population of naïve T cells uncontaminated with any partially or fully activated cells is essential for such a study.</p><p>The results presented in this thesis show, that when thymocytes are employed as a source of responding naïve T cells, cellular interactions, in addition to interaction with bone marrow derived dendritic cells, are required for the activation of naïve thymic T cells. The primary activation of thymic T cells to generate CD4+ IL-2 producing cells, and CD8+ IFN-g producing cells and cytotoxic T cells upon stimulation with splenic allogeneic stimulator cells is critically dependent upon the presence of a syngeneic population of radiation resistant, CD4+ T cells found in the spleen of normal mice. Additionally, when such cells are present as a source of help for thymocytes, allogeneic bone marrow derived dendritic cells fail to stimulate the generation of optimal cytotoxic and cytokine responses from naïve thymic T cells. However, they do stimulate thymocytes to cycle and up regulate the ligand for the costimulatory molecule CD40, CD40L.</p><p>The results presented within also show that the optimal activation of naïve thymic T cells to generate CD4+ IL-2 producing cells, and CD8+ IFN-g producing cells and cytotoxic T cells, requires the presence of allo-MHC bearing Ig+ B220+ B cells. The removal of B220+ cells by magnetic cell sorting from the allogeneic spleen reveals that the generation of CD8+ cytotoxic T cells and IFN-g producing cells from thymocytes is markedly reduced compared to unsorted allogeneic spleen cells. However, IL-2 and IL-4 cytokine producing cells are still detectable. Potential reasons for the generation of the latter cytokine producing cells are discussed. The results presented in this thesis have revealed insights into the cellular interactions involved in the activation of naïve thymic T cells.