@phdthesis{Boerner2020,
  author    = {B{\"o}rner, Kevin},
  title     = {How CLEC16A modifies the function of thymic epithelial cells},
  doi       = {10.25972/OPUS-20023},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-200230},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Genomweite Assoziationsstudien haben CLEC16A als ein Suszeptibilit{\"a}tsgen f{\"u}r Typ 1 Diabetes und weitere Autoimmunerkrankungen identifiziert. Die genaue Funktion von CLEC16A bleibt jedoch ungekl{\"a}rt. Studien zeigten, dass sowohl das Drosophila Ortholog ema als auch das murine Clec16a eine Rolle in Autophagie spielen. Autophagie tr{\"a}gt zur Beladung der MHC-Klasse-II Molek{\"u}le und somit der Antigenpr{\"a}sentation bei. Dar{\"u}ber hinaus konnten Studien belegen, dass Autophagie zur Antigenpr{\"a}sentation w{\"a}hrend der T-Zell Selektion in Thymus-Epithelzellen ben{\"o}tigt wird. Dies schl{\"a}gt eine m{\"o}gliche Funktion von CLEC16A in Thymus-Epithelzellen w{\"a}hrend der T-Zell Selektion vor. Außerdem berichteten Arbeiten, dass CLEC16A als quantitativer Trait Locus f{\"u}r seine Nachbargene fungiert und dass Clec16a KD in Langerhans Inseln im Pankreas die Insulinsekretion und den Glukosestoffwechsel beeintr{\"a}chtigt. Dieser Arbeit vorausgehend hatten Schuster et al. eine Clec16a KD NOD Maus generiert, welche vor spontanem autoimmunem Diabetes gesch{\"u}tzt war. F{\"u}r diese Arbeit wurde vermutet, dass CLEC16A als Suszeptibilit{\"a}tsgen f{\"u}r Typ 1 Diabetes den Prozess der Autophagie in Thymus-Epithelzellen beeintr{\"a}chtigt und somit Antigenpr{\"a}sentation und das T-Zell Repertoire beeinflusst. Um auf der Vorarbeit von Schuster et al. aufzubauen und diese zu erg{\"a}nzen, zielte diese Arbeit darauf ab, den Einfluss von CLEC16A auf Thymus-Epithelzellen zu untersuchen. Hierf{\"u}r wurde ein CLEC16A KD in menschlichen Zellen mittels RNA Interferenz erzeugt und Autophagie durch Immunoblotting untersucht. Zus{\"a}tzlich wurde die Entz{\"u}ndung im Pankreasgewebe von Clec16a KD NOD M{\"a}usen mittels H.E. F{\"a}rbung beurteilt und bewertet. Thymus-Transplanationen wurden durchgef{\"u}hrt, um zu sehen, ob der Einfluss von Clec16a KD T-Zell intrinsisch ist. Außerdem wurden intraperitoneale Glukosetoleranztests durchgef{\"u}hrt, um den Blutzuckerstoffwechsel in Clec16a KD M{\"a}usen zu beurteilen. Schließlich wurden mittels qPCR Expressionslevel der benachbarten Gene, wie zum Beispiel Dexi und Socs1, erhoben, um die Eigenschaften von CLEC16A als quantitativer Trait Locus einzuordnen. Gemeinsam mit den Ergebnissen von Schuster et al. kann diese Arbeit aufzeigen, dass Clec16a KD die Auspr{\"a}gung von Insulitis im Pankreas reduziert und Clec16a KD NOD M{\"a}use vor spontanem Autoimmundiabetes sch{\"u}tzt. Dieser Schutz vor Erkrankung wird durch beeintr{\"a}chtigte Autophagie in Thymus-Epithelzellen hervorgerufen, welche die T-Zell Selektion beeinflusst und die Reaktivit{\"a}t von T-Zellen reduziert. Der Einfluss des Clec16a KD ist innerhalb des Thymus wirksam. Der Blutzuckerstoffwechsel in Clec16a KD NOD M{\"a}usen bleibt unver{\"a}ndert und kann deshalb als Ursache f{\"u}r den Schutz vor Type 1 Diabetes ausgeschlossen werden. Clec16a und Dexi zeigen {\"a}hnliche Expressionslevel auf, dennoch ben{\"o}tigt es weitere detaillierte Studien, um eine Beziehung zwischen den beiden Genen etablieren zu k{\"o}nnen. Letztlich konnte die Beeintr{\"a}chtigung von Autophagie in menschlichen CLEC16A KD Zellen nachgewiesen werden, was bedeutet, dass die Funktion von CLEC16A evolution{\"a}r konserviert ist und ein m{\"o}glicher Zusammenhang zwischen CLEC16A Polymorphismen und einem erh{\"o}hten Risiko f{\"u}r Typ 1 Diabetes im Menschen besteht.},
  subject      = {Thymus},
  language  = {en}
}
@phdthesis{Riano2016,
  author    = {Ria{\~n}o, Rub{\´e}n Felipe},
  title     = {BTN3A1 in the immune response of Vγ9Vδ2 T cells},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142084},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Human Vγ9Vδ2 T cells are the main γδ T cell subset in the circulation, accounting for up to 5\% of the total peripheral blood lymphocyte population. They have been suggested to be important in response to tumors and infections. Their immune mechanisms encompass cell killing via cytotoxicity and secretion of pro-inflammatory cytokines such as IFNγ and tumor necrosis factor (TNF). The main stimulators of Vγ9Vδ2 T cells are isopentenyl pyrophosphate (IPP) and (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), denominated phosphoantigens (PAg). A major advance in the understanding of PAg detection and Vγ9Vδ2 T cell activation has been the identification of the butyrophlin 3A (BTN3A) proteins as key mediators in these processes. In humans, three isoforms constitute the BTN3A family: BTN3A1, BTN3A2, and BTN3A3; and their genes are localized on the short arm of chromosome 6. The role of BTN3A1 has been highlighted by BTN3A-specific monoclonal antibody 20.1 (mAb 20.1), which has an agonist effect and causes proliferation, expansion, and activation of primary human Vγ9Vδ2 T cells. On the other hand, BTN3A-specific monoclonal antibody 103.2 (mAb 103.2) is antagonistic, inhibiting the Vγ9Vδ2 T cell response. The actual mechanism underlying both PAg- and mAb 20.1-mediated activation is not completely elucidated, but the importance of BTN3A1 is clear. The main objective of this dissertation was to characterize the role of BTN3A1 in the PAg-dependent and PAg-independent Vγ9Vδ2 T cell activation and to evaluate its contribution in the response to influeza A virus infected cells. This research work demonstrated, by using Vγ9Vδ2 TCR MOP-transduced murine cells (reporter cells), that human chromosome 6 (Chr6) is mandatory for PAg-induced stimulation, but not for stimulation with mAb 20.1. The reporter cells responded to mAb 20.1 in cultures with BTN3A1-transduced Chinese hamster ovary cells (CHO BTN3A1) as antigen presenting cells. Nevertheless, for PAg-dependent activation the presence of Chr6 in CHO BTN3A1 was mandatory. Although reporter cells expressing clonotypically different Vγ9Vδ2 TCRs showed similar PAg response, they clearly differed in the mAb 20.1 response. The reporter cell line transduced with Vγ9Vδ2 TCR D1C55 demonstrated essentially no response to mAb 20.1 compared to Vγ9Vδ2 TCR MOP cells. These findings were further supported by experiments performed with human PBMCs-derived Vγ9Vδ2 T cell clones. The results indicate heterogeneity in the PAg- and 20.1-dependent responses, in terms of CD25 and CD69 expression, among three different Vγ9Vδ2 T cells clones. Co-cultures of reporter cells with Raji RT1BI and PAg plus mAb 20.1 or single chain antibody 20.1 (sc 20.1) revealed no additive or synergistic activating effects. In contrast, mAb 20.1 or sc 20.1 inhibited the PAg-mediated activation of the reporter cells. The comparison of the relative contribution of the isoforms BTN3A2 and BTN3A3, in the activation of Vγ9Vδ2 T cells, was undertaken by overexpression of these isoforms in CHO cells. The results showed that BTN3A2 contributes to both PAg- and mAb-induced Vγ9Vδ2 T cell activation. On the contrary, BTN3A3 does not support PAg-mediated γδ T cell response. Additionally, mutations in the proposed PAg- and mAb 20.1-binding sites of the extracellular BTN3A1 domain were generated by means of site-directed mutagenesis. These mutations revoked the mAb 20.1-induced Vγ9Vδ2 T cell activation, but not that induced by PAg. Finally, co-cultures of Vγ9Vδ2 TCR MOP-transduced murine reporter cells with influenza A/PR/8/34-infected cells, or infection of PBMCs with this virus strain indicated that BTN3A1 might be dispensable for the Vγ9Vδ2 T cell response against influenza A. The data of this research work points out that: i) in addition to BTN3A1, other Chr6-encoded genes are necessary for Vγ9Vδ2 T cell activation with PAg; ii) clonotypical (CDR3) differences influence the PAg- and mAb 20.1-mediated Vγ9Vδ2 T cell activation; iii) the PAg- and mAb 20.1-induced responses are not synergistic and interfere with each other; iv) BTN3A2 and BTN3A3 isoforms differ in the ability to support PAg- or mAb 20.1-dependent Vγ9Vδ2 T cell activation; v) the importance of the intracellular B30.2 domain of BTN3A1, in the Vγ9Vδ2 T cell activation, might be higher than that of the extracellular domain; and vi) in spite of the importance of BTN3A1 in the activation of Vγ9Vδ2 T cells, it is possible that many molecules with redundant functions are involved in the elimination of influenza virus infection by these cells. In summary, it is possible to hypothesize a model in which BTN3A1 detects prenyl pyrophosphates in the cytoplasm via its B30.2 domain and in association with another protein(s). The binding of PAg to this domain induces a multimerization of BTN3A1 or a conformational change of its extracellular domain (mimicked by mAb 20.1). These modifications might be recognized by the Vγ9Vδ2 TCR or by an associated T cell protein. In the case that the TCR directly recognizes BTN3A1, the intensity of the response will depend on the Vγ9Vδ2 TCR clonotype. Future research will allow to gain a better understanding of BTN3A1, its interaction with other proteins, its actual role in the activation of Vγ9Vδ2 T cells, and its importance in specific models of cancer or infection. This knowledge will be necessary to transform these cells into effective tools in the clinic.},
  subject      = {T-Lymphozyt},
  language  = {en}
}
@phdthesis{Dagvadorj2016,
  author    = {Dagvadorj, Nergui},
  title     = {Improvement of T-cell response against WT1-overexpressing leukemia by newly developed anti-hDEC205-WT1 antibody fusion proteins},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-149098},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Wilms tumor protein 1 (WT1) is a suitable target to develop an immunotherapeutic approach against high risk acute myeloid leukemia (AML), particularly their relapse after allogeneic hematopoietic stem cell transplantation (HSCT). As an intracellular protein traversing between nucleus and cytoplasm, recombinant expression of WT1 is difficult. Therefore, an induction of WT1-specific T-cell responses is mostly based on peptide vaccination as well as dendritic cell (DC) electroporation with mRNA encoding full-length protein to mount WT1-derived peptide variations presented to T cells. Alternatively, the WT1 peptide presentation could be broadened by forcing receptor-mediated endocytosis of DCs. In this study, antibody fusion proteins consisting of an antibody specific to the human DEC205 endocytic receptor and various fragments of WT1 (anti-hDEC205-WT1) were generated for a potential DC-targeted recombinant WT1 vaccine. Anti-hDEC205-WT1 antibody fusion proteins containing full-length or major parts of WT1 were not efficiently expressed and secreted due to their poor solubility and secretory capacity. However, small fragment-containing variants: anti-hDEC205-WT110-35, anti-hDEC205-WT191-138, anti-hDEC205-WT1223-273, and anti-hDEC205-WT1324-371 were obtained in good yields. Since three of these fusion proteins contain the most of the known immunogenic epitopes in their sequences, the anti-hDEC205-WT191-138, anti-hDEC205-WT1223-273, and anti-hDEC205-WT1324-371 were tested for their T-cell stimulatory capacities. Mature monocyte-derived DCs loaded with anti-hDEC205-WT191-138 could induce ex vivo T-cell responses in 12 of 16 blood samples collected from either healthy or HSC transplanted individuals compared to included controls (P < 0.01). Furthermore, these T cells could kill WT1-overexpressing THP-1 leukemia cells in vitro after expansion. In conclusion, alongside proving the difficulty in expression and purification of intracellular WT1 as a vaccine protein, our results from this work introduce an alternative therapeutic vaccine approach to improve an anti-leukemia immune response in the context of allogeneic HSCT and potentially beyond.},
  subject      = {Akute myeloische Leuk{\"a}mie},
  language  = {en}
}
@phdthesis{Probst2015,
  author    = {Probst, Lilli Teresa},
  title     = {Immune cell function in the Clec16a Knock-down Mouse},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-122513},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Genome wide association studies (GWAS) have identified Clec16a as disease suscepti-bility gene for numerous auto-immune disorders in particular type 1 diabetes. In spite of this strong genetic link, the role of Clec16a for immune regulation continues to be largely unknown. To study the function of Clec16a in an environment susceptible to autoimmune diseases a Clec16a deficient non obese diabetic (NOD) mouse strain was generated by means of lentiviral RNA interference. Clec16a knock down (KD) mice prove to be strongly protected against developing type 1 diabetes, an effect that is mediated by hyporeactive T effector cells. T cell hyporeactivity seems to result from an impairment of proximal TCR signalling and its cause is likely to be external to T cells. Given evidence on the involvement of the Clec16a Drosophila ortholog ema in endo- and autophagosomal processes, alterations in peripheral and/or central antigen presenting cells appeared to be potential reasons for the observed T cell hyporeactivity. While we are not able to identify any changes in quantity and quality of peripheral antigen presenting cells due to Clec16a silencing activation status of thymic epithelial cells in Clec16a KD mice deviates from NOD WT. The findings presented here suggest that thymic T cell development is affected by Clec16a variation. Such a relationship could explain the genetic association between Clec16a variations in humans and susceptibility to immune-mediated diseases, yet further investigations are needed to confirm this notion.},
  language  = {en}
}
@phdthesis{Wegner2015,
  author    = {Wegner, Julia},
  title     = {Restoring tissue-like functionality in circulating CD8 T-cells: mechanistic studies and application in immunomonitoring of cancer patients},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-124177},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Peripheral blood mononuclear cells (PBMCs) are the only source of human lymphoid cells routinely available for immunologic research and for immunomonitoring of T-cell responses to microbial and tumor-associated antigens. However the large majority of human T-cells resides in tissues, especially in lymphatic organs, while only 1 \% of the body's T-cells circulate in the blood stream. Previous work in mice and humans had indicated that CD4 T-cells transiently lose antigen sensitivity when cellular contacts are lost, e.g. by leaving lymphoid organs such as lymph nodes (LNs) and entering the circulation. In this study, these findings were extended to CD8 T-cells. Thus, CD8 T-cell responses of the human tonsil show a significant drop in sensitivity to viral antigens if tissue-exit was simulated by keeping cells in dispersed culture at body temperature for two hours. Conversely, tissue-like functionality in blood-derived CD8 T-cells was restored by applying the simple and robust RESTORE protocol. Indeed, application of the RESTORE protocol, i.e. pre-culturing PBMCs for two days at a high cell density before initiation of antigenic stimulation, demonstrated that CD8 T-cell responses to a broad range of viral and to tumor-associated antigens are greatly underestimated, and sometimes even remain undetected if conventional, unprocessed PBMC cultures are used. The latter finding is particularly striking with regard to the appearance of Wilms tumor 1 (WT1)-specific CD8 T-cell responses in leukemia patients after allogeneic bone marrow transplantation. My studies on the mechanism of the RESTORE protocol show that HD preculture of PBMCs does not involve antigen-or cytokine-driven clonal expansion of T-cells. Moreover, the gain in antigen sensitivity cannot be explained by a decreased activity of regulatory T-cells during the preculture step. The increased antigen sensitivity of CD8 T-cells from HD precultures of PBMCs is associated with tonic T-cell receptor signaling as indicated by enhanced tyrosine phosphorylation of the CD3 ζ chains and the tyrosine kinase Lck, thereby preparing T-cells for full responses. The upregulation of genes involved in aerobic glycolysis in "restored" CD8 memory T-cells relative to fresh cells might be an essential requirement for increased T-cell functionality including the regulation of IFN-γ production. Taken together, the RESTORE protocol, which was initially described for the CD4 T-cell response to the antibody TGN1412 permits a more meaningful monitoring of CD8 T-cell responses to viral infections and tumors. Furthermore, when generating T-cell lines for adoptive T-cell therapy, the RESTORE protocol allows the generation of CD8 T-cell lines with an improved representation of clones responding to low antigen concentrations.},
  subject      = {Antigen CD8},
  language  = {en}
}
@phdthesis{Mahmood2015,
  author    = {Mahmood, Zafar},
  title     = {Effect of cytokine inhibition on peripheral memory B cells in patients with Rheumatoid arthtritis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-117334},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Objective: Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory autoimmune disease. Enhanced B cell activity has been proposed in the pathogenesis of RA along with different pro-inflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α), critically involved in chronic inflammation. Biological agents targeting these cytokines IL-6 and TNF-α have considerably advanced treatment of autoimmunity. Enhanced B cell activity, particularly memory B cells gained particularly interest in evaluating response during therapies from biologics. Human peripheral memory B cells can be distinguished by the phenotypic expression of CD27 and IgD defining three major B cell subpopulations: CD27+IgD+ pre-switch, CD27+IgD- post-switch and CD27-IgD- double negative (DN) memory B cells. Therefore, we analyzed different memory populations during cytokine inhibition by using tocilizumab (anti-IL-6R, TCZ) and adalimumab (anti-TNF-α, ADA), with focus on DN B cells Suspended. DN B cells lacking the conventional memory marker CD27, but due to their mutational Ig repertoire (IgR) considered in the memory compartment. However, only scare data are available for this DN subpopulation in RA. Methods: Phenotype analysis of activation markers (CD95 and ki-67) of B cell and their subsets were compared in RA patients (median age ~56 years) and in HD. DN memory B cells were phenotypically analyzed from RA patients during IL-6R or TNF-α inhibition at baseline week 12, week 24 and 1 year. Single B cell PCR approach was used to study Ig- receptors VH genes and isotype specific genes. Nonparametric Wilcoxon matched pair test and Mann-Whitney U test was used for statistical analysis by using GraphPadPrism 5. Univariate logistic regression was used to calculate odd ratios and correlation using Pearson r using SPSS statistics 22. Results: Surface and intracellular staining of B cells showed a significantly higher percentage of CD95 and ki-67 expressions in RA, which was highest in post-switch memory B cells followed by pre-switch and DN memory B cells. During cytokines (IL-6R \& TNF-α) inhibition, both CD95 and ki-67 expression were significantly reduced at week 12 and 24 along with reduction in their clinical parameters like DAS28, CRP, ESR. Furthermore, the phenotypic analysis in 107 RA patients and 49 healthy donors (HD) showed a significantly expanded population of DN B cells in RA which contain a heterogeneous mixture of IgA, IgG and IgM expressing cells with a clear dominance of IgG+ cells. Pre-therapy analysis of rearranged IgR sequences from patients (n=9) revealed that DN B cells carry rearranged heavy chain gene sequences with a diversified mutational pattern consistent with memory B cells. In contrast to tumor necrosis factor alpha (TNF-alpha) inhibition, a significant reduction in mutational frequency of BCR gene rearrangements at week 12, 24 and 1 year (p < 0.0001) was observed by in vivo IL-6R inhibition. These changes were observed for all BCR isotypes IgG, IgA and IgM at week 12, 24 and 1 year (p < 0.0001). IgA-RF, IgA serum level and IgA+ DN B cells decreased significantly (p < 0.05) at week 12 and week 24 during TCZ. Patients with a good European league against rheumatism (EULAR) response to TCZ had less DN B cells at baseline as compared to moderate responders (p = 0.006). Univariate logistic regression analysis revealed that the frequency of DN B cells at baseline is inversely correlated to a subsequent good EULAR response (p = 0.024) with an odds ratio of 1.48 (95\% confidence interval as 1.05-2.06). Conclusion: Both anti-TNF-α and anti-IL-6R could reduce higher B cell activity and improve disease activity tremendously in RA patients. The heterogeneous DN B cell compartment is expanded in RA and dominated by IgG isotype. TCZ can modulate the mutational status of DN Ig isotype receptors over 1 year. Interestingly, the frequency of DN B cells in RA may serve as a baseline predictor of subsequent EULAR response to TCZ.},
  subject      = {Arthrosis deformans},
  language  = {en}
}
@phdthesis{Ehmann2015,
  author    = {Ehmann, Nadine},
  title     = {Linking the active zone ultrastructure to function in Drosophila},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-118186},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Accurate information transfer between neurons governs proper brain function. At chemical synapses, communication is mediated via neurotransmitter release from specialized presynaptic intercellular contact sites, so called active zones. Their molecular composition constitutes a precisely arranged framework that sets the stage for synaptic communication. Active zones contain a variety of proteins that deliver the speed, accuracy and plasticity inherent to neurotransmission. Though, how the molecular arrangement of these proteins influences active zone output is still ambiguous. Elucidating the nanoscopic organization of AZs has been hindered by the diffraction-limited resolution of conventional light microscopy, which is insufficient to resolve the active zone architecture on the nanometer scale. Recently, super-resolution techniques entered the field of neuroscience, which yield the capacity to bridge the gap in resolution between light and electron microscopy without losing molecular specificity. Here, localization microscopy methods are of special interest, as they can potentially deliver quantitative information about molecular distributions, even giving absolute numbers of proteins present within cellular nanodomains. This thesis puts forward an approach based on conventional immunohistochemistry to quantify endogenous protein organizations in situ by employing direct stochastic optical reconstruction microscopy (dSTORM). Focussing on Bruchpilot (Brp) as a major component of Drosophila active zones, the results show that the cytomatrix at the active zone is composed of units, which comprise on average ~137 Brp molecules, most of which are arranged in approximately 15 heptameric clusters. To test for a quantitative relationship between active zone ultrastructure and synaptic output, Drosophila mutants and electrophysiology were employed. The findings indicate that the precise spatial arrangement of Brp reflects properties of short-term plasticity and distinguishes distinct mechanistic causes of synaptic depression. Moreover, functional diversification could be connected to a heretofore unrecognized ultrastructural gradient along a Drosophila motor neuron.},
  subject      = {Taufliege},
  language  = {en}
}