Studies on the role of the tyrosine kinase Itk in T cell development and function

Aus bisher publizierten Studien war bekannt, dass der Guanosinnukleotid-Austauschfaktor Vav1 und die Tyrosin-Kinase Itk, ein Mitglied der Familie der Tec Kinasen, in ähnlichen T-Zell Aktivierungs-Signalwegen agieren. Beide Proteine interagieren mit Cbl-b, einer E3-Ubiquitin-Ligase. Während meiner Doktorarbeit untersuchte ich das genetische Zusammenspiel dieser drei Proteine mit Hilfe von Itk/Vav1 sowie Itk/Cbl-b Doppel-knockout Mäusen. Die Abwesenheit von Cbl-b in Itk–/– CD4+ T-Zellen führte wieder zu einer normalen T-Zell-Proliferation und IL-2 Produktion nach T-Zell-Rezeptor-Aktivierung, während die Produktion von IL-4 nur teilweise wieder erlangt werden konnte. Diese „Rettung“ des Itk-Defekts in Abwesenheit von Cbl-b ist sehr ähnlich der Beobachtung, dass der Defekt von Vav1-defizienten T-Zellen ebenfalls durch die Entfernung von Cbl-b aufgehoben werden konnte. Unsere Studien zeigen daher auf genetischer Ebene, dass Itk- und Vav1-abhängige Signalwege in T-Zellen mechanistisch ähnlich agieren bzw. durch Cbl-b ähnlich reguliert werden. Die Analyse von Vav1–/–Itk–/– Mäusen hat ergeben, dass die Entstehung von T-Zellen in Abwesenheit von Itk und Vav1 stark beeinträchtig war und nur sehr wenige CD4+ und CD8+ T-Zellen vorhanden waren. Wir konnten zeigen, dass diese starke Reduktion durch einen erhöhten Zelltod von Thymozyten und einer Blockade der positiven Selektion während der Thymozytenreifung hervorgerufen worden ist. Unsere Daten zeigen daher zum ersten Mal, dass die kombinierte Aktivität von Vav1 und Itk für die Entstehung von T-Zellen und die Generierung des peripheren T-Zell-Pools notwendig ist. Studien haben gezeigt, dass Itk für die Entstehung der sogenannten „konventionellen“ T-Zellen wichtig ist. In Abwesenheit dieser Tyrosin-Kinase ist deshalb eine spezielle Population von CD44hi Memory-Phänotyp (MP) T-Zellen relativ erhöht. Mittels einer Affymetrix gene chip Analyse konnten wir zeigen, dass der Transkriptionsfaktor PLZF in Itk knockout T-Zellen hochreguliert ist. Die erhöhte Expression dieses Faktors wurde auf die CD44hi CD4+ T-Zellen zurückgeführt. T-Zellspezifische transgene Überexpression von PLZF in Mäusen induzierte ein T-Zell-intrinsisches Programm, welches zur Entstehung dieser CD44hi T-Zellen führte. Die entstandenen MP CD4+ und CD8+ T-Zellen produzierten IFNγ nach Stimulierung mit PMA und Ionomycin, was eine charakteristische Eigenschaft von den sogenannten „innate-like“ T-Zellen ist. Die Veränderung der Verteilung von naiven versus MP T-Zellen in den PLZF transgenen Mäusen konnte schon während der T-Zell-Entwicklung beobachtet werden. Weiter zeigten die transgenen Mäuse, aufgrund eines Entwicklungsdefektes, eine stark verringerte Anzahl von CD1d-abhängigen NKT-Zellen in Thymus und Milz. Zudem konnten wir zeigen, dass die transge-nen CD4+ T-Zellen nach T-Zell-Rezeptor-Stimulierung reduzierte Mengen an IL-2 und IFNγ, jedoch erhöhte Mengen von IL-4 produzierten. Die Daten des zweiten Teils meiner Doktorarbeit weisen darauf hin, dass PLZF ein wichtiger Transkriptionsfaktor für die Reifung und Funktion von CD44hi MP T-Zellen mit „innate-like“ Eigenschaften ist.Vav1 and the Tec family kinase Itk act in similar T cell activation pathways. Both molecules inte-ract with members of the Cbl family of E3 ubiquitin ligases, and signaling defects in Vav1–/– T cells are rescued upon deletion of Cbl-b. During my PhD study I investigated the relation between Itk and Cbl-b or Vav1 by generating Itk/Cbl-b and Itk/Vav1 double-deficient mice. Deletion of Cbl-b in Itk–/– CD4+ T cells restored proliferation and partially IL-2 production, and led also to a variable rescue of IL-4 production. Thus, Itk and Vav1 act mechanistically similar in peripheral T cells, since the defects in Itk–/– T cells, like in Vav1–/– T cells, are rescued if cells are released from the negative regulation mediated by Cbl-b. In addition, only few peripheral CD4+ and CD8+ T cells were present in Vav1–/–Itk–/–mice due to severely impaired thymocyte differentiation. Vav1–/–Itk–/– thymocyte numbers were strongly reduced compared to wildtype, Itk–/–or Vav1–/– mice, and DP thymocytes displayed increased cell death and impaired positive selection. Therefore, our data also reveal that the combined activity of Vav1 and Itk is required for proper T cell development and the generation of the peripheral T cell pool. Itk has also been shown to be essential for the development of conventional T cells. As a consequence, Itk–/– mice have a relative increase in Memory phenotype (MP) T cells with innate characteristics. We could show in an Affymetrix gene chip approach that the transcription factor PLZF is up-regulated in the Itk–/– T cell population. Immunoblot analysis revealed that PLZF was primarily expressed in the CD4+CD44hi population both in wildtype and Itk–/– T cells. This observation suggested a link between PLZF expression and MP CD4+ T cells. Transgenic expression of PLZF during T cell development and in CD4+ and CD8+ T cells induced a T cell intrinsic program leading to an increase in peripheral CD44hi MP CD4+ and CD8+ T cells and a corresponding decrease of naïve CD44lo T cells. The MP CD4+ and CD8+ T cells produced IFNγ upon PMA/ionomycin stimulation, thus showing innate-like function. Changes in the naïve versus memory-like subset distribution were already evident in SP thymocytes, indicating PLZF-induced T cell developmental alterations. In addition, CD1d-restricted NKT cells in PLZF transgenic mice showed impaired development and were severely reduced in the periphery. Finally, after anti-CD3/CD28 stimulation, CD4+ transgenic T cells showed reduced IL-2 and IFNγ production but increased IL-4 secretion due to enhanced IL-4 production of the CD44hiCD62L+ subset. Our data indicate that PLZF is a novel transcriptional regulator of the development of CD44hi MP T cells with a characteristic partial innate-like phenotype

Indoleamine 2,3-Dioxygenase in Human Hematopoietic Stem Cell Transplantation

In recent years tryptophan metabolism and its rate limiting enzyme indoleamine 2,3-dioxygenase (IDO) have attracted increasing attention for their potential to modulate immune responses including the regulation of transplantation tolerance. The focus of this review is to discuss some features of IDO activity which particularly relate to hematopoietic stem cell transplantation (HSCT). HSCT invariably involves the establishment of some degree of a donor-derived immune system in the recipient. Thus, the outstanding feature of tolerance in HSCT is that in this type of transplantation it is not rejection, which causes the most severe problems to HSCT recipients, but the reverse, graft-versus-host (GvH) directed immune responses. We will discuss the peculiar role of IDO activity and accelerated tryptophan metabolism at the interface between immune activation and immune suppression and delineate from theoretical and experimental evidence the potential significance of IDO in mediating tolerance in HSCT. Finally, we will examine therapeutic options for exploitation of IDO activity in the generation of allo-antigen-specific tolerance, i.e. avoiding allo-reactivity while maintaining immunocompetence, in HSCT

Transcriptional signatures of Itk-deficient CD3+, CD4+ and CD8+ T-cells

<p>Abstract</p> <p>Background</p> <p>The Tec-family kinase Itk plays an important role during T-cell activation and function, and controls also conventional versus innate-like T-cell development. We have characterized the transcriptome of Itk-deficient CD3⁺T-cells, including CD4⁺and CD8⁺subsets, using Affymetrix microarrays.</p> <p>Results</p> <p>The largest difference between Itk^-/-and Wt CD3⁺T-cells was found in unstimulated cells, e.g. for killer cell lectin-like receptors. Compared to anti-CD3-stimulation, anti-CD3/CD28 significantly decreased the number of transcripts suggesting that the CD28 co-stimulatory pathway is mainly independent of Itk. The signatures of CD4⁺and CD8⁺T-cell subsets identified a greater differential expression than in total CD3⁺cells. Cyclosporin A (CsA)-treatment had a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Bioinformatic analysis of NFAT-sites of the group of transcripts similarly regulated by Itk-deficiency and CsA-treatment, followed by chromatin-immunoprecipitation, revealed NFATc1-binding to the <it>Bub1</it>, <it>IL7R, Ctla2a</it>, <it>Ctla2b</it>, and <it>Schlafen1 </it>genes. Finally, to identify transcripts that are regulated by Tec-family kinases in general, we compared the expression profile of Itk-deficient T-cells with that of Btk-deficient B-cells and a common set of transcripts was found.</p> <p>Conclusion</p> <p>Taken together, our study provides a general overview about the global transcriptional changes in the absence of Itk.</p

The protein tyrosine kinase Tec regulates a CD44highCD62L- Th17 subset

The generation of Th17 cells has to be tightly controlled during an immune response. In this study, we report an increase in a CD44 2 effector/memory Th17 populations

Einführung in die partiellen Differentialgleichungen anhand der Methode der Charakteristiken

Der Inhalt der folgenden Arbeit ist das genaue Ausarbeiten der Methode der Charakteristiken an verschiedenen partiellen Differentialgleichungen, sowie die Verwendung von klassischen und schwachen Lösungen für partielle Differentialgleichungen. In der ersten Herangehensweise wird die Methode an verschiedenen Fällen einer linearen Transportgleichung entwickelt. Dabei wird der klassische Lösungsbegriff für partielle Differentialgleichungen erläutert und verwendet. Im zweiten Zugang wird die Methode der Charakteristiken an nichtlinearen hyperbolischen Differentialgleichungen wieder aufgegriffen. Bei den verwendeten Gleichungen kann weder Existenz noch Eindeutigkeit einer Lösung für beliebig lange Zeiten vorausgesetzt werden, weshalb ein neues Lösungskonzept benötigt wird, das Konzept einer schwachen Lösung. Im letzten Kapitel finden die erarbeiteten Themengebiete ihre Anwendung und werden mit geeigneter Interpretation in einem Verkehrsflussmodell betrachtet

Stress-induced recruitment of epiplakin to keratin networks increases their resistance to hyperphosphorylation-induced disruption

Epiplakin is a large (>725 kDa) cytoskeletal protein exclusively expressed in epithelial tissues. It has a unique structure, consisting entirely of plakin repeat domains (PRDs), one of the hallmarks of spectraplakin protein family members. Previous studies, including the phenotypic analyses of knockout mice, failed to reveal the biological function of epiplakin. Using in vitro binding assays, we show here that all but one of the 16 PRDs of mouse epiplakin bind to keratins of basal keratinocytes. Nevertheless, in primary keratinocyte cell cultures, epiplakin only partially colocalized with keratin intermediate filament networks. However, upon application of cellular stress in the form of keratin hyperphosphorylation, osmotic shock or UV irradiation, the entire cytoplasmic epiplakin pool became associated with keratin. In response to such types of stress, epiplakin initially translocated to the still-intact keratin filament network and remained associated with keratin after its disruption and transformation into granular aggregates. Time-course experiments revealed that serine/threonine (okadaic acid) and tyrosine (orthovanadate) phosphatase inhibitor-induced filament disruption in differentiated keratinocytes proceeded faster in epiplakin-deficient cells compared with wild-type cells. Our data suggest that epiplakin plays a role in keratin filament reorganization in response to stress, probably by protecting keratin filaments against disruption in a chaperone-like fashion

The transcriptional regulator PLZF induces the development of CD44 high memory phenotype T cells

Transcriptional pathways controlling the development of CD44hi memory phenotype (MP) T cells with “innate-like” functions are not well understood. Here we show that the BTB (bric-a-brac, tramtrack, broad complex) domain-containing protein promyelocytic leukemia zinc finger (PLZF) is expressed in CD44hi, but not in CD44lo, CD4+ T cells. Transgenic expression of PLZF during T cell development and in CD4+ and CD8+ T cells induced a T cell intrinsic program leading to an increase in peripheral CD44hi MP CD4+ and CD8+ T cells and a corresponding decrease of naïve CD44lo T cells. The MP CD4+ and CD8+ T cells produced IFNγ upon PMA/ionomycin stimulation, thus showing innate-like function. Changes in the naïve versus memory-like subset distribution were already evident in single-positive thymocytes, indicating PLZF-induced T cell developmental alterations. In addition, CD1d-restricted natural killer T cells in PLZF transgenic mice showed impaired development and were severely reduced in the periphery. Finally, after anti-CD3/CD28 stimulation, CD4+ transgenic T cells showed reduced IL-2 and IFNγ production but increased IL-4 secretion as a result of enhanced IL-4 production of the CD44hiCD62L+ subset. Our data indicate that PLZF is a novel regulator of the development of CD44hi MP T cells with a characteristic partial innate-like phenotype