Zytotoxische T-Zellen (CD8+) spielen eine wichtige Rolle bei retroviralen Infektionen des Menschen, wie HIV und HTLV. Ihre molekulare Wirkweise war bisher aber nicht bekannt. Die Infektion von Mäusen mit dem retroviralen Friend Virus Komplex (FV) wurde daher als Modell verwendet, um die antiviralen Mechanismen gegen Retroviren aufzuklären. Als erstens wurde die Beteiligung von CD8+ T-Zellen bei der Kontrolle der FV Infektion in Depletion-Experimenten gezeigt. In akut FV infizierten Tieren konnten außerdem aktivierte Virus-spezifische CD8+ T-Effektorzellen nachgewiesen werden, welche zytotoxische Moleküle produzierten. Die Infektion von „knockout“ Mäusen ergab, dass die CD8+ T-Zellen hauptsächlich den Exozytoseweg benutzten, um FV-infizierte Zellen zu eliminieren. Die Anwesenheit eines der drei am Exozytoseweg beteiligten zytotoxischen Moleküle Perforin, Granzym A oder Granzym B war in der akuten Phase der Infektion ausreichend, um die Virusreplikation zu kontrollieren. Diese Ergebnisse weisen auf neue molekulare Mechanismen von Granzymen bei der Virusabwehr hin. In Gegensatz zur Abwehr des pathogenen FV Komplex benutzten CD8+ T-Zellen nicht den Exozytoseweg zur Abwehr von apathogenen Retroviren. Eine Infektion von Mäusen mit dem nicht-pathogenen F-MuLV induzierte keine Produktion von zytotoxischen Molekülen in CD8+ T-Zellen. Die Infektion von „knockout“ Mäusen zeigte, dass F-MuLV von CD8+ T-Zellen über den Fas/FasL Weg kontrolliert wurde. Das pathogene Potential von Retroviren scheint also einen Einfluß auf den jeweils verwendeten Abwehrmechanismus von zytotoxischen T-Zellen zu haben. Trotz der wichtigen Funktion von CD8+ T-Zellen bei der Kontrolle der akuten FV Infektion, spielten diese Zellen bei der persistierenden Infektion keine Rolle mehr. Die Untersuchungen von aktivierten CD8+ T-Zellen aus persistierend infizierten Mäusen zeigte, dass die Zellen agranulär waren und keine zytotoxischen Moleküle produzierten. Folglich hatten sie auch keine zytotoxische Aktivität in vitro. Der Exozytoseweg von Virus-spezifischen CD8+ T-Zellen war also in der persistierenden FV Infektion gestört. So entwickelten viele persistierend infizierte Tiere nach einer Reinfektion eine Splenomegalie, was zusätzlich beweist, dass die Funktion von Virus-spezifischen T-Zellen auch in vivo gestört war. Mäuse, die chronisch mit FV infiziert waren, wiesen also einen kompletten Funktionsverlust von CD8+ T-Zellen auf, der offensichtlich eine wichtige Voraussetzung für die Persistenz des Virus darstellt. Da die CD8+ T-Zellen nicht mehr funktionell waren, mussten vermutlich CD4+ T-Zellen über den Fas/FasL-Weg die Kontrolle über die chronische Infektion übernehmen. Das Verständnis der molekularen Mechanismen der CD8+ T-Zell Abwehr gegen Retroviren ermöglicht es neue Strategien der Immuntherapie gegen retroviralen Infektionen zu entwickeln. / Cytotoxic T-cells play an important role in the control of retroviral infections such as HIV and HTLV; however the molecular mechanisms used by these cells is not known. We used the infection of mice with the retroviral Friend Virus complex as model to investigate the antiviral mechanisms of CD8+ T-cells. The participation of CD8+ T-cells in the control FV infection was shown in depletion experiments. Activated virus-specific CD8+ T-cells were found in acutely FV infected animals and these cells produced cytotoxic molecules. The infection of knockout mice indicated that CD8+ T-cells eliminated infected cells via the exocytosis pathway. The presence of one of these three molecules involved in the exocytosis pathway (perforin, granzyme A or granzyme B) was sufficient to control virus-replication. These results suggest new molecular mechanisms of granzyme in the defence against retroviral infection. In contrast to the control of pathogenic FV complex, CD8+ T-cells do not need the exocytosis pathway to respond to non-pathogenic retroviruses. The infection of mice with non-pathogenic F-MuLV did not induce the production of cytotoxic molecules in CD8+ T-cells and knockout mice with defects in perforin, granzyme A and granzyme B were not susceptible to F-MuLV infection. In contrast, knockout mice with defect in the Fas/FasL pathway were unable to control virus-replication. These data indicate that CD8+ T-cells used the Fas/FasL pathway to control non-pathogenic retroviruses whereas the exocytosis pathway was crucial to keep pathogenic retroviruses in check. The establishment of chronic Friend virus infections is enabled by the induction of CD4+ regulatory T cells that impair CD8+ T-cell functions and allow the virus to escape (Dittmer et al., 2004). The current study characterizes this CD8+ T-cell dysfunction by analyzing the production and release of cytolytic molecules by virus-specific CD8+ T-cells. During acute infection, FV-specific CD8+ T cells produced the cytotoxic molecules perforin and granzyme B, and actively degranulated cytotxic granules. In contrast, CD8+ T cells of the same specificity from chronically infected mice neither produced any of these cytotoxic molecules nor showed evidence of degranulation. The defect in cytotoxine production in T cells from persistently infected mice did not occur at the level of transcription as both CD8+ T-cells from acutely and persistently infected animals had equal levels of mRNA for perforin and granzyme. CD8+ T-cells from persistently infected mice were unable to induce apoptosis in target cells. These results demonstrate a broad impairment of cytolytic CD8+ T-cell effector functions associated with chronic retroviral infection.

No description available.

Zytotoxische CD8+ T-Lymphozyten spielen in vielen inflammatorischen, aber auch primär neurodegenerativen Erkrankungen eine wichtige Rolle. Daher besitzt die Fragestellung inwiefern CD8+ ZTL Neurone direkt schädigen und ggf. welche mechanistischen Aspekte dieser Schädigung zugrunde liegen, eine hohe Relevanz. Um diese Fragestellung eingehender zu beleuchten, wurde mit dem OT-I-System gearbeitet. Dieses gut vorcharakterisierte CD8+ T-Zell-Modell besitzt den Vorteil, dass diese transgenen Zellen nur eine Peptidsequenz des Ovalbumin (OVA) Protein als spezifisches Antigen erkennen. Zunächst wurden in der vorliegenden Arbeit Co-Kultivierungs-Experimente durchgeführt. Hierzu wurden akut isolierte murine Hippokampus-Neurone unter verschiedenen Bedingungen mit OT-I Lymphozyten co-kultiviert. Hierbei konnte gezeigt werden, dass unter Antigenpräsentation der Neurone signifikant mehr Neurone in die Apoptose/Nekrose geführt werden, als unter Kontroll-Bedingungen, in denen entweder kein Antigen oder ein Antigen, das nicht von OT-I Lymphozyten erkannt wird, präsentiert wird. Nachdem die Antigen-abhängigen zytotoxischen Effekte auf Neurone gezeigt werden konnten, wurde mithilfe elektrophysiologischer Techniken die mechanistischen und funktionellen Konsequenzen des direkten neuronalen/OT-I-vermittelten Zellkontakts untersucht. Bei diesem experimentellen Ansatz wurde durch elektrisches Auslenken eines Neurons nach Kontakt mit einem OT-I Lymphozyt die passiven elektrischen Parameter der Neuronenmembran gemessen. In diesen Messungen konnte gezeigt werden, dass nach unmittelbarem Kontakt eines Neurons mit einem OT-I Lymphozyt der neuronale Membranwiderstand reduziert wird bzw. die Leitfähigkeit der Zellmembran erhöht wird. Diese Änderung der neuronalen Membran-Leitfähigkeit findet in einem Zeitraum von 10 min nach dem Zell-Zell-Kontakt statt. Auch hier konnte gezeigt werden, dass dieser Einfluss von OT-I Lymphozyten auf Neurone strikt Antigen-abhängig ist. Zur Untersuchung des Mechanismus der OT-I T-Lymphozyten auf Neurone wurde das Augenmerk auf verschiedene T-Zell-induzierte Apoptosewegegelegt. Es konnte gezeigt werden, dass durch Blockieren der Fas/FasL-Interaktion mittels eines Antikörpers kein Unterschied, weder in der neuronalen Apoptoserate nach Co-Kultivierung, noch eine Änderung der passiven neuronalen Membran-Leitfähigkeit auftritt. Weiterhin wurde die Rolle der von T-Zellen sezernierten Granula Perforin und Granzym B untersucht. Um den Einfluss dieser Granula aufzuklären, wurden OT-I Lymphozyten verwendet, die entweder defizient für Perforin oder Granzym B waren. In diesem experimentellen Ansatz wurde gezeigt, dass ausschließlich Perforin für die Erniedrigung des passiven neuronalen Membran-Widerstandes verantwortlich ist. Diese Erhöhung der neuronalen Membranleitfähigkeit führte aber nicht direkt zum neuronalen Zelltod. Vielmehr wurde durch die einhergehende Depolarisation des Neurons die elektrische Aktivität der Zelle vermindert, sodass es zu einem sogenannten „electrical silencing“ kommt. Dieser Umstand konnte auch in der Betrachtung der spontanen Netzwerkaktivität von Neuronenkulturen gezeigt werden. Hierfür wurden hoch dichte Neuronenkulturen auf MEA-Chips kultiviert. Mit Hilfe dieser MEA konnten die Summenfeldpotentiale der Neuronenkulturen detektiert werden. Hierbei wurde beobachtet, dass nach Beladung der Neuronen mit dem spezifischen OT-I-Antigen und OT-I Zellen eine Verringerung der spontanen Netzwerkaktivität einhergeht. Auch in diesem Effekt konnte eine Antigen-Spezifität nachgewiesen werden. Da der Prozess der zellulären Apoptose mit einem Anstieg der intrazellulären Ca2+-Konzentration einhergeht, und Perforin als Ca2+-durchlässiger unselektiver Porenbildner fungiert, wurden zur Überprüfung der Hypothese calcium imaging-Experimente durchgeführt. Analog zu den elektrophysiologischen Messungen wurde gezeigt, dass nach direktem Zell-Zell-Kontakt zwischen Neuron und OT-I Lymphozyt eine Erhöhung der intrazellulären Ca2+-Konzentration zu messen ist. Dass diese Änderung des neuronalen Ca2+-Einstroms durch Perforin-abhängige Membranporen hervorgerufen wird, konnte durch die Verwendung von Perforin-defizienten OT-I Lymphozyten bewiesen werden. Unter Verwendung von Perforin-defizienten OT-I Lymphozyten wurde keine Änderung der neuronalen Ca2+-Konzentration ermittelt. Weiterhin wurde in diesem experimentellen Ansatz gezeigt, dass auch der OT-I-vermittelte neuronale Ca2+-Anstieg strikt Antigen-abhängig ist.Zusammengefasst konnte in dieser Arbeit gezeigt werden, dass MHC-I/Antigen-vermittelte CD8+ Lymphozyten-Interaktion mit einem Neuron zu „electrical silencing“ des Neurons führt. Dieser Prozess ist klar Perforin-abhängig, führt jedoch nicht zum unmittelbaren Zelltod des Neurons. / Cytotoxic CD8+ T cells are considered as important effector cells contributing to neuronal damage in inflammatory and primary degenerative disorders in the CNS. Hence, it is highly relevant to know to what extent CD8+ T-lymphocytes can contribute to neuronal damage in these disorders. To challenge this question, we used the murine OT-I system. The advantage of this well-characterized transgenic model is that OT-I CD8+ T-lymphocytes are restricted to one single antigen – one peptide sequence of Ovalbumin (Ova). In a first set of experiments, OT-I lymphocytes were co-cultured with neurons that presented Ova in a MHC-I specific context on their surface. As control, neurons without any antigen or neurons that presented a scrambled peptide form (SIY) were used. These co-culture experiments indicates that neuronal killing by OT-I lymphocytes is a MHC-I and antigen-dependent mechanism. To clarify the underlying mechanism and the functionally consequences in this OT-I/neuron interaction, we performed electrophysiological patch-clamp analysis to measure the influence from one single OT-I T-cell on a single neuron. For this purpose, we established a special protocol to stimulate the neuronal membrane to measure the passive electrical parameters after a direct OT-I contact. These measurements revealed a significant antigen restricted reduction in neuronal membrane resistance. This effect could be detected within 10 min after the direct cell-cell contact. To challenge the underlying cellular mechanisms we analyzed several known apoptosis pathways. In a first set of experiments, we investigated the Fas/FasL interaction. To answer this question, we used a blocking FasL antibody, to interrupt this pathway. These experiments showed no changes in neuronal apoptosis, neither in co-cultivation experiments nor in the electrophysiological situation. As next step we investigate the role of CD8+ lymphocyte derived granula perforin and granzyme B. Therefore we used OT-I T-cells that are either deficient for perforin or granzyme B. Using these experimental conditions, we could show that only perforin is responsible for changing passive electrical parameters. However, these reductions in neuronal membrane resistance did not lead immediately in neuronal cell death, but rather led to a depolarization and therefore to an electrical silencing of the neuron. This electrical silencing was also shown to occur in the spontaneous network activity in a neuronal network. The network activity was measured on a high density neuron network cultivated on a MEA. These MEA measurements revealed a decrease in the total spike activity after loading of OT-I lymphocytes on an antigen presenting neuronal network. Due to the increase of the intracellular Ca2+ level in the process of cell death and the Ca2+ selectivity of perforin membrane pores, we hypothesized that neuronal silencing and neuronal cell death elicited by perforin pores might lead to an intracellular Ca2+ increase. To proof this hypothesis we established a calcium imaging experiment in an OT-I/neuron contact situation. These measurements were done in the same manner as the electrophysiological measurements. Ca2+ imaging indicated increasing Ca2+ levels in neurons after application of perforin releasing OT-I lymphocytes. Furthermore, these experiments revealed a strictly antigen dependence for Ca2+ increase in target cells. In conclusion, we could show that MHC-I/antigen-mediated CD8+ lymphocyte interactions with neurons led to their electrical silencing. This process was perforin dependent. However this process was not causally linked to neuronal cell death.

While numerous experiments on NFAT were already performed with CD4+ T cells showing defective cytokine release and a reduced T helper cell development, no detailed studies existed for CD8+ T cells. From this point, we wanted to examine the impact of NFATc1 and c2 on the physiological functions of CD8+ T cells in vitro and in vivo. Therefore, we used a murine infection model with the bacteria Listeria monocytogenes and mice in which NFATc1 was specifically depleted in the T cell compartment. Our first in vitro studies showed a typical NFATc1 and c2 nuclear translocation and changes on mRNA levels upon T cell activation similarly in CD4+ as well as in CD8+ T cells extracted from wild type mice. NFAT nuclear translocation is important for target gene activation and generation of effector functions. Stimulated T cell populations lacking NFATc1 and/or NFATc2 showed a markedly decreased expression of Th1/Tc1 cytokines, as e.g. IL 2 and IFNγ being important for the clearance of intracellular pathogens. From our in vitro model for the generation of allogenically reactive cytotoxic CD8+ T cells, we revealed a decreased killing and lytic granule-release capacity in Nfatc1 inactivated CD8+ T cells whereas NFATc2-/- cytotoxic T cells did not show an altered cytotoxic response compared to wild type cells. Interestingly, we found lytic granules accumulated and mitochondria not getting translocated to the immunological synapse upon re-stimulation in NFATc1-deficient CD8+ T cells. Together with results showing the CsA insensitivity of the CTL killing/degranulation capacities, we assume that some major cellular processes are affected by NFATc1 which are not directly linked to the TCR-induced signal transduction cascade. We also showed the importance of NFATc1 in T cells during intracellular infections with the bacteria Listeria monocytogenes in an in vivo mouse model. After five days, only few bacteria were detected in wt mice whereas high amounts of Listeria particles were extracted from livers of Nfatc1fl/fl x Cd4 cre mice. Although the reactivity towards the pathogen was similar in both groups, a decreased cytokine expression in NFATc1-/- CD8+ T cells was observed together with an altered memory cell generation. Our results show the importance of NFATc1 in CD8+ T cells and give some clue for a possible connection to other basal cellular functions, as e.g. the formation of an immunological synapse. / Viele Experimente zur Rolle von NFAT wurden bereits anhand von CD4+ T Zellen durchgeführt und zeigten eine veränderte Zellphysiologie. Hingegen wurden CD8+ T Zellen diesbezüglich noch nicht intensiv studiert. Deshalb untersuchten wir den Einfluss von NFATc1 und NFATc2 auf die Funktion von CD8+ T Zellen in vitro und in vivo anhand des murinen Infektionsmodells mit dem Bakterium Listeria monocytogenes. Für die Versuche benutzen wir Mäuse, in denen das Protein NFATc1 spezifisch im T Zellkompartiment entfernt wurde. Erste Ergebnisse zeigten eine typische Translokation von NFATc1 und NFATc2 in den Zellkern. Eine Veränderung in der mRNA Expression nach Aktivierung, sowohl in CD4+ T Zellen als auch in CD8+ T Zellen, fand ebenfalls statt. NFATc defiziente CD4+ und CD8+ T Zellen wiesen eine verminderte Expression von Th1/Tc1 Zytokinen wie z.B. Interleukin-2 und Interferon γ auf, welche für die Bekämpfung intrazellulärer Pathogene wichtig sind. In unserem in vitro Modell fanden wir eine verminderte Abtötungsfähigkeit und eine Reduktion in der Freisetzung lytischer Granula in NFATc1-/- CD8+ T Zellen wohingegen eine NFATc2 Defizienz keine Auswirkungen auf die Zytotoxizität - verglichen mit wildtypischen Zellen - aufweist. Interessanterweise fanden wir eine Anhäufung von lytischen Granula und eine verminderte intrazelluläre Migration von Mitochondrien nach Ausbildung einer immunologischen Synapse in NFATc1-/- CD8+ T Zellen. Zusammen mit den Ergebnissen unserer CsA-Inhibierungsversuche nehmen wir an, dass einige allgemeine zelluläre Prozesse von NFATc1 beeinflusst werden, die nicht direkt von der T Zellrezeptor-induzierten Signalkaskade abhängen. Anhand eines in vivo Mausmodells zeigten wir auch die wichtige Rolle von NFATc1 in T Zellen während der Infektion mit Listeria monocytogenes. Fünf Tage nach Infektion konnten aus Nfatc1fl/fl x Cd4 cre Mäusen mehr Bakterienpartikel extrahiert werden als aus wt Mäusen. Wie in den in vitro Versuchen konnte auch hier eine geringere Zytokinproduktion der CD8+ T Zellen festgestellt werden allerdings wiesen die Mäuse auch eine geringere Bildung von Gedächniszellen auf. Unsere Ergebnisse zeigen, dass NFATc1 in CD8+ T Zellen eine wichtige Rolle spielt und auch Auswirkungen auf grundlegendere zelluläre Funktionen, wie die Ausbildung einer immunologischen Synapse, hat.

Cytotoxic T-lymphocytes are important for the control of viral replication during HIV infection, however the magnitude and breadth of HIV-specific CD8+ T-cell response does not correlate well. The purpose for this study was the examination of the HLA-B*2705-specific CD8+ T-cell response to the KRWIILGLNK (KK10) epitope as a definitive model of immune control over HIV replication. The breadth of the T-cell receptor (TCR) repertoire was determined for an association between the qualitative nature of this response and immune escape and therefore, disease progression. Methodology was developed and validated for TCR repertoire analysis in formaldehyde fixed antigen-specific CD8+ T-cells. The TCR repertoire for the KK10-specific CD8+ T-cell response was defined in cross-section and longitudinally for 6 HLA-B*2705+ patients. Comparison was made to cognate HLA-A*0201 CMV NV9 and HLA-B*2705 EBV RL9-specific CD8+ T-cell populations using the Simpson??s diversity index and the Morisita-Horn similarity index for standardized repertoire analysis. HLA-B*2705 KK10-specific TCR repertoire was not found to be a determinant of control. Greater clonotype variation was found within CMV-specific CD8+ T-cell populations, suggesting an association with reactivation of CMV and disease state. An association was found between KK10-specific population diversity and the prevalence of cognate KK10 epitope in vivo. Cross-reactivity observed for dominant KK10-specific clonotypes suggested that avidity of CD8+ T-cells was important for in vivo survival. Phenotype and function was tested through multiparameter analysis of HIV and CMV-specific CD8+ T-cells. Increased frequency of CD127 (IL-7R) and Bcl-2 expression within dominant populations was suggestive of selective advantage. Division of dominant and sub-dominant CMV-specific CD8+ T-cell populations into ??early?? and ??late?? differentiation phenotypes indicated virus-specific mechanisms of clonotype turn over. No simple association of TCR expression was found for HIV and CMV-specific CD8+ T-cells with published examples of definitive TCR bias. Over-represented TCR ??-chain families of patients were found in association with public clonotypes. Convergent recombination of TCR genes was demonstrated as a mechanism for the prevalence of shared clonotypes. Standardized assessment of T-cell repertoire successfully identified mechanisms of antigen-specific CD8+ T-cell recruitment. A substantial increase in sample numbers is required before this methodology can be used to accurately demonstrate the importance of TCR repertoire usage in the control of human viral infection.

Cytotoxic CD8 T lymphocytes kill virus-infected cells and are critical for viral clearance from the body. Cytokines, particularly those sharing the common gamma receptor chain (gamma c), play a key role in this cytotoxic function as well as in the growth, differentiation and homeostasis of CD8 T lymphocytes. In order to exert these biological effects, cytokine-dependent signal transduction via the Janus kinase (Jak) / Signal Transducers and Activators of Transcription (STAT) pathway, the phosphoinositide 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK) pathways is required. In HIV infection however, the CD8 T lymphocytes become defective and are characterized by impaired cytotoxicity, altered differentiation patterns, and increased susceptibility to apoptosis. I hypothesized that impaired cytokine responsiveness resulting from defects in cytokine-dependent signal transduction contributes to the CD8 T cell impairment observed in HIV+ patients. I investigated the activation of the Jak/STAT signaling pathway to cytokines in CD8 T cells from HIV+ patients. Interestingly, these cells were responsive to IL-2, IL-4, IL-10, IL-15, and IL-21 at the level of their respective STAT activation. However, impairment of the IL-7 / IL-7Ralpha signaling axis was identified and characterized by a defect in STAT5 signaling. The impaired STAT5 activation correlated with a low IL-7Ralpha surface expression. The expanded population of IL- 7Ralphalow-expressing CD8 T cells, found particularly in viremic HIV+ patients, expressed higher levels of the transcriptional repressor Growth Factor Independent-1 (GFI1) compared to their IL-7Ralphahigh counterparts. This prompted further investigations into the role of GFI1 in IL-7Ralpha regulation in primary human CD8 T cells as a model. Though silencing of GFI1 did not modulate basal IL-7Ralpha expression, exogenous overexpression negatively regulated IL-7Ra surface levels. The gc cytokines, IL-2, IL-4, IL-7, and IL-15, but not IL-21, were found to efficiently suppress IL-7Ralpha expression however, only IL-4 simultaneously upregulated GFI1 expression. RNA interference studies targeting GFI1 in IL-4 stimulated CD8 T cells established a specific role for GFI1 in sustaining the suppression of IL-7Ralpha expression. Furthermore, transient downregulation of GFI1 in CD8 T cells subjected to IL- 4-dependent proliferation reduced their proliferative capacity. Other functions identified for GFI1 were in the suppression of CXCR4 and Bax expression in CD8 T cells. Studies aimed at identifying the signal transduction pathways responsible for regulating GFI1 and IL-7Ralpha expression revealed that IL-4-mediated downregulation of IL-7Ralpha expression required activation of the Jak/STAT and the PI3K pathways. On the other hand, IL-4-induced upregulation of GFI1 expression was mediated via the PI3K pathway. The JNK and P38 MAPK pathways appeared to be important as regulators of basal IL-7Ralpha expression levels, but had no statistically significant effects on GFI1 expression. To conclude, these studies have clarified the important biological effects of GFI1 in mature human CD8 T lymphocytes. Furthermore, exposure to IL-4 may generate CD8 T cell populations with an exhausted phenotype similar to those found in chronically-infected HIV+ patients, characterized by reduced cytotoxic activity and increased IL-4 production. Thus, the IL-4 study model may prove valuable for investigating the activity of human CD8 T cells in such chronic diseases and those characterized by a type 2 cytokine profile.

Cytotoxic CD8 T lymphocytes kill virus-infected cells and are critical for viral clearance from the body. Cytokines, particularly those sharing the common gamma receptor chain (gamma c), play a key role in this cytotoxic function as well as in the growth, differentiation and homeostasis of CD8 T lymphocytes. In order to exert these biological effects, cytokine-dependent signal transduction via the Janus kinase (Jak) / Signal Transducers and Activators of Transcription (STAT) pathway, the phosphoinositide 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK) pathways is required. In HIV infection however, the CD8 T lymphocytes become defective and are characterized by impaired cytotoxicity, altered differentiation patterns, and increased susceptibility to apoptosis. I hypothesized that impaired cytokine responsiveness resulting from defects in cytokine-dependent signal transduction contributes to the CD8 T cell impairment observed in HIV+ patients. I investigated the activation of the Jak/STAT signaling pathway to cytokines in CD8 T cells from HIV+ patients. Interestingly, these cells were responsive to IL-2, IL-4, IL-10, IL-15, and IL-21 at the level of their respective STAT activation. However, impairment of the IL-7 / IL-7Ralpha signaling axis was identified and characterized by a defect in STAT5 signaling. The impaired STAT5 activation correlated with a low IL-7Ralpha surface expression. The expanded population of IL- 7Ralphalow-expressing CD8 T cells, found particularly in viremic HIV+ patients, expressed higher levels of the transcriptional repressor Growth Factor Independent-1 (GFI1) compared to their IL-7Ralphahigh counterparts. This prompted further investigations into the role of GFI1 in IL-7Ralpha regulation in primary human CD8 T cells as a model. Though silencing of GFI1 did not modulate basal IL-7Ralpha expression, exogenous overexpression negatively regulated IL-7Ra surface levels. The gc cytokines, IL-2, IL-4, IL-7, and IL-15, but not IL-21, were found to efficiently suppress IL-7Ralpha expression however, only IL-4 simultaneously upregulated GFI1 expression. RNA interference studies targeting GFI1 in IL-4 stimulated CD8 T cells established a specific role for GFI1 in sustaining the suppression of IL-7Ralpha expression. Furthermore, transient downregulation of GFI1 in CD8 T cells subjected to IL- 4-dependent proliferation reduced their proliferative capacity. Other functions identified for GFI1 were in the suppression of CXCR4 and Bax expression in CD8 T cells. Studies aimed at identifying the signal transduction pathways responsible for regulating GFI1 and IL-7Ralpha expression revealed that IL-4-mediated downregulation of IL-7Ralpha expression required activation of the Jak/STAT and the PI3K pathways. On the other hand, IL-4-induced upregulation of GFI1 expression was mediated via the PI3K pathway. The JNK and P38 MAPK pathways appeared to be important as regulators of basal IL-7Ralpha expression levels, but had no statistically significant effects on GFI1 expression. To conclude, these studies have clarified the important biological effects of GFI1 in mature human CD8 T lymphocytes. Furthermore, exposure to IL-4 may generate CD8 T cell populations with an exhausted phenotype similar to those found in chronically-infected HIV+ patients, characterized by reduced cytotoxic activity and increased IL-4 production. Thus, the IL-4 study model may prove valuable for investigating the activity of human CD8 T cells in such chronic diseases and those characterized by a type 2 cytokine profile.

Infectious diseases remain amongst leading causes of death in people aged 65 years and older; therefore, much research is focused on determining the immune components that contribute to age-dependent increased susceptibility to, and increased mortality from, infections. CD8⁺ T cells are critical for clearing intracellular pathogens through production of cytokines and direct killing of infected cells. Age-dependent CD8⁺ T cell alterations have been described, including decreased numbers of naïve CD8⁺ T cell precursors and decreased numbers and function during infection. This dissertation explores the mechanisms contributing to these changes. First, we demonstrated that multiple mechanisms contribute to changes in the CD8⁺ T cell pool with age. CD8⁺ T cells from unimmunized T cell receptor transgenic (TCRTg) old mice undergo massive virtual memory (VM) conversion with age; both homeostatic proliferation and cross-reactivity may contribute to the generation and accumulation of VM cells with age. These VM cells exhibit an age-dependent replicative impairment to cognate antigen, which points to possible detrimental functional consequences due to changes in the overall T cell pool. Second, we evaluated the cell intrinsic contribution to the decreased old CD8⁺ T cell response. With in vitro stimulation, old CD8⁺ T cells exhibit decreased ability to enter into late cell divisions and decreased production of effector molecules. In addition, we found that old CD8⁺ T cells have decreased expression of the master transcription factor T-bet, which correlates to decreased effector function and terminal differentiation in vivo. Collectively, these results identify possible cell-intrinsic targets for improving CD8⁺ T cell immunity. Finally, we measured whether a Listera monocytogenes live vaccine model induces protective immune responses in old mice. We found that vaccination conferred little protection in old mice upon pathogen challenge. These results contrast with other vaccine models, which may allow for pinpointing both the vaccine and immune components required for generating strong protective immunity in the elderly. Collectively, this dissertation demonstrates that CD8⁺ T cell precursors, effector cells, and memory cells exhibit profound changes with, age and identifies both possible mechanisms contributing to these alterations as well as possible therapeutic/vaccine targets for improving immunity in the elderly.

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Cytotoxic CD8 T lymphocytes kill virus-infected cells and are critical for viral clearance from the body. Cytokines, particularly those sharing the common gamma receptor chain (gamma c), play a key role in this cytotoxic function as well as in the growth, differentiation and homeostasis of CD8 T lymphocytes. In order to exert these biological effects, cytokine-dependent signal transduction via the Janus kinase (Jak) / Signal Transducers and Activators of Transcription (STAT) pathway, the phosphoinositide 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK) pathways is required. In HIV infection however, the CD8 T lymphocytes become defective and are characterized by impaired cytotoxicity, altered differentiation patterns, and increased susceptibility to apoptosis. I hypothesized that impaired cytokine responsiveness resulting from defects in cytokine-dependent signal transduction contributes to the CD8 T cell impairment observed in HIV+ patients. I investigated the activation of the Jak/STAT signaling pathway to cytokines in CD8 T cells from HIV+ patients. Interestingly, these cells were responsive to IL-2, IL-4, IL-10, IL-15, and IL-21 at the level of their respective STAT activation. However, impairment of the IL-7 / IL-7Ralpha signaling axis was identified and characterized by a defect in STAT5 signaling. The impaired STAT5 activation correlated with a low IL-7Ralpha surface expression. The expanded population of IL- 7Ralphalow-expressing CD8 T cells, found particularly in viremic HIV+ patients, expressed higher levels of the transcriptional repressor Growth Factor Independent-1 (GFI1) compared to their IL-7Ralphahigh counterparts. This prompted further investigations into the role of GFI1 in IL-7Ralpha regulation in primary human CD8 T cells as a model. Though silencing of GFI1 did not modulate basal IL-7Ralpha expression, exogenous overexpression negatively regulated IL-7Ra surface levels. The gc cytokines, IL-2, IL-4, IL-7, and IL-15, but not IL-21, were found to efficiently suppress IL-7Ralpha expression however, only IL-4 simultaneously upregulated GFI1 expression. RNA interference studies targeting GFI1 in IL-4 stimulated CD8 T cells established a specific role for GFI1 in sustaining the suppression of IL-7Ralpha expression. Furthermore, transient downregulation of GFI1 in CD8 T cells subjected to IL- 4-dependent proliferation reduced their proliferative capacity. Other functions identified for GFI1 were in the suppression of CXCR4 and Bax expression in CD8 T cells. Studies aimed at identifying the signal transduction pathways responsible for regulating GFI1 and IL-7Ralpha expression revealed that IL-4-mediated downregulation of IL-7Ralpha expression required activation of the Jak/STAT and the PI3K pathways. On the other hand, IL-4-induced upregulation of GFI1 expression was mediated via the PI3K pathway. The JNK and P38 MAPK pathways appeared to be important as regulators of basal IL-7Ralpha expression levels, but had no statistically significant effects on GFI1 expression. To conclude, these studies have clarified the important biological effects of GFI1 in mature human CD8 T lymphocytes. Furthermore, exposure to IL-4 may generate CD8 T cell populations with an exhausted phenotype similar to those found in chronically-infected HIV+ patients, characterized by reduced cytotoxic activity and increased IL-4 production. Thus, the IL-4 study model may prove valuable for investigating the activity of human CD8 T cells in such chronic diseases and those characterized by a type 2 cytokine profile.