@phdthesis{Foeger2000, author = {F{\"o}ger, Niko}, title = {Costimulatory function of CD44}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-1186}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2000}, abstract = {T cell activation is supposed to require two signals via engagement of the TCR and a costimulatory molecule. However, the signaling cascade of costimulatory molecules has remained elusive. Here, I provide evidence that CD44 supports proliferation as well as apoptosis mainly, if not exclusively, by enhancing signal transduction via the TCR/CD3 complex. Blockade of CD44 interferes with mounting of an immune response. This has been demonstrated by the significantly decreased IL-2 production of a T helper line, when stimulated in the presence of a competing CD44 receptor globulin. To evaluate the underlying mechanism, CD44 was cross-linked by an immobilized antibody (IM7). Cross-linking of CD44 induces proliferation of peripheral T cells and apoptosis of thymocytes and a T helper line in the presence of subthreshold levels of anti-CD3. CD44-induced proliferation was accompanied by an upregulation of the activation markers CD25 and CD69 and an increased cytokine production. TCR-mediated apoptosis was accompanied by an upregulation of CD95 ligand and CD95 receptor, which could be greatly enhanced by costimulation via CD44. On the level of signal transduction, coligation of CD44 with CD3 resulted in a strong and sustained increase of early tyrosine phosphorylation events and upregulated downstream signal transduction pathways, such as the ras/ERK and the JNK signaling cascades. These pleiotropic effects of CD44 are due to its involvement in the most proximal events in TCR signaling, as demonstrated by a strong increase in the phosphorylation of the TCR z-chain and ZAP-70. Notably, cross-linking of CD44 was binding-site dependent and was only effective when supporting colocalization of the TCR/CD3 complex and CD44. Cross-linking of CD44 via immobilized IM7 also induced profound changes in cell morphology, characterized by strong adhesion, spreading and development of surface extensions, which were dependent on a functional tubulin and actin cytoskeleton. These cytoskeletal rearrangements were mediated by rac1, a small GTPase of the rho subfamily, and src-family kinases, two of which, fyn and lck, were found to be associated with CD44. By cross-linkage of CD44 these kinases were redistributed into so called lipid rafts. It is supposed that for T cell activation a relocation of the TCR/CD3 complex into the same membrane microdomains is required. The data are interpreted in the sense that the costimulatory function of CD44 relies on its cooperativity with the TCR. Most likely by recruitment of phosphokinases CD44 significantly lowers the threshold for the initiation of signaling via the TCR. The requirement for immobilized anti-CD44, the necessity for neighbouring anti-CD3 and the dependence on the binding site of CD44 strongly suggest that the costimulatory mechanism involves cytoskeletal rearrangements, which facilitate recruitment and redirection of src-family protein kinases in glycolipid enriched membrane microdomains.}, subject = {Antigen CD44}, language = {en} } @phdthesis{Seitz2007, author = {Seitz, Sabine}, title = {Identifikation und Charakterisierung von autoreaktiven humanen T-Zell-Rezeptor Molek{\"u}len}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-22511}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2007}, abstract = {Die Multiple Sklerose und die entz{\"u}ndlichen Muskelerkrankungen Polymyositis und Einschlussk{\"o}rperchenmyositis sind Autoimmunerkrankungen, in denen T-Lymphozyten in das Gehirn bzw. den Muskel eindringen und dort k{\"o}rpereigenes Gewebe zerst{\"o}ren. Die Pathogenese dieser Krankheiten ist bis jetzt noch nicht bekannt. Ziel dieser Arbeit war es, die ins Zielgewebe eingedrungenen autoaggressiven T-Lymphozyten aus gefrorenem Biopsiegewebe zu isolieren, ihren ab-T-Zell-Rezeptor zu analysieren und diesen rekombinant zu exprimieren. Folgeversuche mit den TZR-Transfektanten sollten erste Hinweise auf ein m{\"o}gliches Antigen liefern. Um autoaggressive T-Zellen von irrelevanten zu unterscheiden, konzentrierten wir uns auf Zellen, die zum einen im Zielgewebe klonal expandiert vorlagen, zum anderen einen direkten morphologischen Kontakt mit der Zielzelle aufwiesen. Wir untersuchten das T-Zell-Repertoire verschiedener Patienten auf klonal expandierte Populationen durch CDR3-Spektratyping und isolierten positiv gef{\"a}rbte Kandidatenzellen durch Lasermikrodissektion aus dem Gewebe. Anschließend wurden die T-Zell-Rezeptoren mit einer speziellen, im Rahmen der Arbeit entwickelten Einzelzell-Muliplex-RT-PCR analysiert. Bisher war es nicht m{\"o}glich, die a- und b-Kette desselben T-Zell-Rezeptors aus Biopsiematerial zu identifizieren. Dies lag an der geringen Anzahl verf{\"u}gbarer anti-a-Ketten-Antik{\"o}rper sowie an der wesentlich h{\"o}heren Variabilit{\"a}t der a-Ketten-Gene. Aus dem Biopsiegewebe eines Patienten isolierten wir 23 ab-TZR-P{\"a}rchen, welche alle die identische expandierte TZR-b-Kette zeigten. 20 der 23 Zellen wiesen eine identische a-Kette auf. Die Dominanz des TZR ist ein Hinweis auf die pathogene Relevanz dieses T-Zell-Klons. Die anderen drei Zellen zeigten drei unterschiedliche funktionelle a-Ketten. Aus der Biopsie eines zweiten Patienten isolierten wir zwei weitere ab-TZR-P{\"a}rchen. Die vier Rezeptoren des ersten Patienten wurden in einer T-Zell Hybridom Zellinie exprimiert. Vorl{\"a}ufige Versuche, ein m{\"o}gliches Antigen zu detektieren, zeigten, dass es sich wahrscheinlich weder um ein muskelspezifisches Antigen noch um ein ubiquit{\"a}r exprimiertes Selbst-Antigen handelt. Die hier beschriebene Methode der Isolierung und Analyse von autoaggressiven T-Lymphozyten kann man nicht nur zur Untersuchung des TZR-ab-Repertoires von Myositis- oder MS-Patienten einsetzen. Sie ist ebenfalls geeignet, andere Autoimmunerkrankungen sowie Tumor- oder Infektionserkrankungen zu untersuchen.}, subject = {Autoaggressionskrankheit}, language = {de} } @article{DeLiraRamanSchulzeetal.2020, author = {De Lira, Maria Nathalia and Raman, Sudha Janaki and Schulze, Almut and Schneider-Schaulies, Sibylle and Avota, Elita}, title = {Neutral Sphingomyelinase-2 (NSM 2) Controls T Cell Metabolic Homeostasis and Reprogramming During Activation}, series = {Frontiers in Molecular Biosciences}, volume = {7}, journal = {Frontiers in Molecular Biosciences}, issn = {2296-889X}, doi = {10.3389/fmolb.2020.00217}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-211311}, year = {2020}, abstract = {Neutral sphingomyelinase-2 (NSM2) is a member of a superfamily of enzymes responsible for conversion of sphingomyelin into phosphocholine and ceramide at the cytosolic leaflet of the plasma membrane. Upon specific ablation of NSM2, T cells proved to be hyper-responsive to CD3/CD28 co-stimulation, indicating that the enzyme acts to dampen early overshooting activation of these cells. It remained unclear whether hyper-reactivity of NSM2-deficient T cells is supported by a deregulated metabolic activity in these cells. Here, we demonstrate that ablation of NSM2 activity affects metabolism of the quiescent CD4\(^+\) T cells which accumulate ATP in mitochondria and increase basal glycolytic activity. This supports enhanced production of total ATP and metabolic switch early after TCR/CD28 stimulation. Most interestingly, increased metabolic activity in resting NSM2-deficient T cells does not support sustained response upon stimulation. While elevated under steady-state conditions in NSM2-deficient CD4\(^+\) T cells, the mTORC1 pathway regulating mitochondria size, oxidative phosphorylation, and ATP production is impaired after 24 h of stimulation. Taken together, the absence of NSM2 promotes a hyperactive metabolic state in unstimulated CD4\(^+\) T cells yet fails to support sustained T cell responses upon antigenic stimulation.}, language = {en} }