Two Subsets of Naive T Helper Cells with Distinct T Cell Receptor Excision Circle Content in Human Adult Peripheral Blood

During ageing thymic function declines and is unable to meet the demand for peripheral T helper (Th) cell replenishment. Therefore, population maintenance of naive Th cells must be at least partly peripherally based. Such peripheral postthymic expansion of recent thymic emigrants (RTEs) during ageing consequently should lead to loss or dilution of T cell receptor excision circles (TRECs) from a subset of naive T cells. We have identified two subsets of naive Th cells in human adult peripheral blood characterized by a striking unequal content of TRECs, indicating different peripheral proliferative histories. TRECs are highly enriched in peripheral naive CD45RA+ Th cells coexpressing CD31 compared with peripheral naive CD45RA+ Th cells lacking CD31 expression, in which TRECs can hardly be detected. Furthermore we show that CD31−CD45RA+ Th cells account for increasing percentages of the naive peripheral Th cell pool during ageing but retain phenotypic and functional features of naive Th cells. As CD31 is lost upon T cell receptor (TCR) engagement in vitro, we hypothesize that TCR triggering is a prerequisite for homeostatically driven peripheral postthymic expansion of human naive RTEs. We describe here the identification of peripherally expanded naive Th cells in human adult blood characterized by the loss of CD31 expression and a highly reduced TREC content

Thymic -naive und central-naive human Th-lymphocytes

0. Titel, Inhaltsverzeichnis, Abkürzungsverzeichnis 1. Einleitung 1 2. Material und Methoden 15 3. Ergebnisse 36 4. Diskussion 77 5. Zusammenfassung 103 6\. Literaturverzeichnis 104 7. Danksagung 114 8. Publikationen 115Die große Vielfalt des TCR Repertoires der peripheren naiven T-Zellen gewährleistet eine angemessene Immunantwort gegenüber unbekannten, neuen Pathogenen. Die naive Th-Zell-Homöostase wird durch die postnatale Thymopoese und periphere Thymus-unabhängige Mechanismen reguliert. Das Zusammenspiel dieser Mechanismen verändert sich im Laufe des Alterns. Im ersten Teil dieser Arbeit wurden Expressionsanalysen des CD31-Moleküls auf peripheren T-Zellen durchgeführt. Hierbei konnte festgestellt werden, dass während des Alterns eine drastische Änderung hinsichtlich der CD31-Expression auf CD45RA+ Th- Zellen stattfindet. Der Prozentsatz von CD31+ CD45RA+ Th-Zellen nahm kontinuierlich von ca. 90% im Nabelschnurblut auf ca. 50% im peripheren Blut von über 60-Jährigen ab. Gleichzeitig nahm dementsprechend die Frequenz von CD31- CD45RA+ Th-Zellen zu. Untersuchungen bezüglich der Expression von Oberflächenmarkern, der Zytokinproduktion nach polyklonaler Kurzzeitstimulation und der Differenzierungs-fähigkeit zu proinflammatorischen Th-Zellen konnten zeigen, dass CD31+ und CD31- CD45RA+CD4+ Th-Zellen gleichermaßen die phänotypischen und funktionellen Eigenschaften von naiven Th-Zellen aufweisen. Weiterhin konnte festgestellt werden, dass innerhalb der CD31+ CD45RA+ Zellen TREC+ Zellen stark angereichert waren. Der TREC-Gehalt von CD31- CD45RA+ Th-Zellen war dagegen drastisch reduziert. Durch den Vergleich der Genexpressionsprofile von CD31+ und CD31-CD45RA+ Th-Zellen konnten mindestens 50 Gene unter 12.625 analysierten Genen identifiziert werden, die möglicherweise eine Rolle bei der Regulation der peripheren homöostatischen Proliferation von naiven Th-Zellen spielen. Die Ergebnisse dieser Arbeit implizieren, dass CD31- CD45RA+ Th-Zellen naive Th-Zellen repräsentieren, die in der Peripherie expandiert sind, um einer zu starken Abnahme des naiven Th-Zellpools nach Thymusinvolution entgegenzusteuern. Die hier erstmals beschriebene Unterscheidung der beiden peripheren naiven CD45RA+ Th-Zell-Subpopulationen anhand eines phänotypischen Markers ermöglicht erstmals weitere funktionelle und molekulare Analysen und gestattet die Identifizierung von Faktoren, die in den Homöostaseprozess involviert sind.The highly diverse TCR repertoire of peripheral naive Th-cells ensures an appropriate immune response against unknown, new pathogens. The homeostasis of naive Th-cells is regulated by postnatal thymopoiesis and peripheral thymic- independent mechanisms. The interplay between these mechanisms changes during ageing. At first expression studies of the CD31 molecule on peripheral T cells were performed. Thereby it was observed that drastic changes regarding the frequency of CD45RA+ Th cells coexpressing the CD31 molecule occur during ageing. CD31 coexpression was high (85?90%) in CD45RA+ Th-cells from cord blood and from children, but decreased constantly from 60-80% in 20 to 30 year-old adults down to approx. 50% in adults older than 60 years. In addition a concomitant age-dependant increase of peripheral CD45RA+ Th-cells lacking CD31 was detected. The analysis of surface and activation markers on CD31+ und CD31- CD45RA+ CD4+ Th-cells, the cytokine secretion profiles after polyclonal in vitro stimulation and the Th1-induction capacities of CD31+ und CD31- CD45RA+ CD4+ Th-cells revealed, that both subsets show equally the phenotypic and functional features of naive Th-cells. Furthermore quantitative TREC analysis displayed that CD31+ and CD31- CD45RA+ CD4+ Th-cells are characterized by a striking unequal content of TRECs. TRECs were highly enriched in CD31+ CD45RA+ cells, whereas CD45RA+ Th cells lacking CD31 expression were characterized by a drastically reduced content of TRECs. By comparing the gene expression profiles of CD31+ und CD31- CD45RA+ Th-cells, at least 50 out of 12.625 analysed genes were identified, which could play a role in regulating the peripheral homeostatic proliferation of naive Th-cells. The results of this study indicate, that CD31- CD45RA+ Th-cells represent naive Th-cells, which have undergone peripheral proliferation to compensate for the reduced thymic output after involution of the thymus. For the first time it is now possible to distinguish two subsets of naive Th-cells in human adult peripheral blood by means of a phenotypic marker. These results enable direct access and thereby further functional and molecular analysis of the two naive Th-cell subsets and permit the identification of factors involved in the process of homeostatic proliferation

Bak and Bax are non-redundant during infection- and DNA damage-induced apoptosis

Mitochondrial outer membrane permeabilization (MOMP) and release of mitochondrial intermembrane proteins like cytochrome c are critical steps in the control of apoptosis. Previous work has shown that MOMP depends on the functionally redundant multidomain proapoptotic proteins, Bak and Bax. Here we demonstrate that Bak and Bax are functionally non-redundant during Neisseria gonorrhoeae (Ngo)- and cisplatin-induced apoptosis. While the activation of Bak is caspase independent Bax activation needs Bak and active caspases. Silencing of either Bak or Bax resists both Ngo- and cisplatin- but not TNFα-induced apoptosis. Activation of Bak is required to release cytochrome c from the mitochondria; however, Bax is still required to activate effector caspases. Thus, both Bak and Bax are necessary to accomplish DNA damage and Ngo-induced apoptosis

Don't fear 'fear conditioning': Methodological considerations for the design and analysis of studies on human fear acquisition, extinction, and return of fear

The so-called 'replicability crisis' has sparked methodological discussions in many areas of science in general, and in psychology in particular. This has led to recent endeavours to promote the transparency, rigour, and ultimately, replicability of research. Originating from this zeitgeist, the challenge to discuss critical issues on terminology, design, methods, and analysis considerations in fear conditioning research is taken up by this work, which involved representatives from fourteen of the major human fear conditioning laboratories in Europe. This compendium is intended to provide a basis for the development of a common procedural and terminology framework for the field of human fear conditioning. Whenever possible, we give general recommendations. When this is not feasible, we provide evidence-based guidance for methodological decisions on study design, outcome measures, and analyses. Importantly, this work is also intended to raise awareness and initiate discussions on crucial questions with respect to data collection, processing, statistical analyses, the impact of subtle procedural changes, and data reporting specifically tailored to the research on fear conditioning