Kontrolle der Ausbildung der immunologischen Synapse von regulatorischen T Zellen

Regulatory T cells (Tregs) are a CD4+ T cell subset, which maintains tolerance towards self and harmless foreign antigens and is characterized by expression of the master transcription factor Foxp3. Besides this well studied Treg-specific feature, several effector molecules have been identified as preferentially but not exclusively expressed in Tregs. Activation via the T cell receptor (TCR) is crucial for T cell functionality, and there is accumulating evidence that TCR signaling is differentially organized in Tregs as compared to their counterparts, conventional T cells (Tconv). Accordingly, formation and composition of the immunological synapse (IS), built at the interface of interacting immune cells, differs between Tregs and Tconv. Quantitative phosphopeptide sequencing of Tregs and Tconv, which was recently performed in our laboratory, revealed a pre-activated phenotype of Tregs as compared to Tconv and identified differentially regulated phosphorylation sites between the two T cell subsets. Those might serve as promising candidates for targeted intervention of Treg activation. The present study (i) addressed the functional role of Themis, which is strongly under-represented in Tregs, for their suppressive capacity. Furthermore, in order to expand current knowledge about differential TCR signaling in Tregs and Tconv, (ii) phosphorylation kinetics of selected sites were performed. (iii) The functional impact of a novel phosphorylation site at Y523 of CalDAG GEFI (Calcium and DAG regulated guanine nucleotide exchange factor I), which was identified via the aforementioned phosphoproteomic approach, and (iv) the role of the protein itself for development and function of Tregs was investigated. In summary, selective targeting of Tregs by intervention at the signaling level might serve as promising future therapeutic strategy. Up to date, the major obstacle is the lack of specific tools, both to investigate intracellular signaling at the bench and to target selected signaling processes cell subset-specifically at the bedside.Regulatorische T-Zellen (Treg-Zellen) sind ein Teil der CD4+ T-Zellen und dienen der immunologischen Toleranz gegenüber “Selbst” und harmlosen Fremdantigenen. Das Hauptcharakteristikum von Treg-Zellen ist die Expression des bereits eingehend untersuchten Transkriptionsfaktors Foxp3. Neben Foxp3 wurden weitere Effektormoleküle identifiziert, die zwar vorwiegend, aber nicht ausschließlich von Treg-Zellen exprimiert werden, und daher nur bedingt der Unterscheidung von Treg-Zellen und konventionellen T-Zellen (Tconv-Zellen) dienen. Die Aktivierung über den T-Zellrezeptor (TCR) ist entscheidend für die Funktion aller T-Zellen, und es wird zunehmend deutlich, dass der intrazelluläre TCR Signalweg von Treg-Zellen und Tconv-Zellen divergent reguliert ist. Dementsprechend unterscheiden sich auch Formierung und Zusammensetzung der immunologischen Synapse, der Kontaktfläche zwischen interagierenden immunologischen Zellen, zwischen Treg- und Tconv-Zellen. Eine quantitative Bestimmung phosphorylierter Proteine beider T-Zellsorten, welche kürzlich in unserem Labor durchgeführt wurde, identifizierte diverse differentiell regulierte Phosphorylierungsstellen, welche vielversprechende Kandidaten zur spezifischen Intervention der Treg-Zell-Aktivierung darstellen. In der vorliegenden Arbeit wurde (i) die funktionelle Rolle von Themis, einem Protein mit geringerer Expression in Treg- als Tconv-Zellen, für den suppressiven Phänotyp von Treg-Zellen untersucht. Um die Identifizierung differentiell phosphorylierter Proteine voranzubringen, wurden (ii) Kinetiken ausgewählter Phosphorylierungsstellen generiert. Außerdem wurde (iii) die Funktion einer Phosphorylierung des Proteins CalDAG GEFI (Calcium and DAG regulated guanine nucleotide exchange factor I) untersucht und (iv) die generelle Rolle dieses Proteins in Treg-Zellen analysiert. Zusammenfassend stellt ein gezieltes Eingreifen in Treg-Zell-spezifische Signalwege eine vielversprechende Möglichkeit für zukünftige Therapiestrategien dar. Bisher ist hierbei das Fehlen spezifischer Hilfsmittel zur erfolgreichen Erforschung und zielgerichteten Adressierung der T-Zell-Signaltransduktion die größte Hürde

Effect of natural aging on the chemical composition of norway spruce, fir, and european oak wood.

The chemical composition of a number of naturally aged construction wood samples of spruce, fir and oak was investigated. The content of lignin, polysaccharide, and extractives was determined using wet chemical and chromatographic methods. Differences to non-aged wood samples were found particularly found on oak wood samples with regard to lignin and polysaccharide content as well as to cellulose crystallinity. The composition of wood extractives of aged and non-aged wood samples revealed degradation processes due to oxidation and slight hydrolysis. The investigation showed that the relative content of the structural wood constituents depends on the sample age as well as the conditions they were aged under. The different degradation rates of those structural components may lead to contradictory results regarding their contents at certain degrees of aging. Cellulose crystallinity values are affected by extractives content

The guanine-nucleotide exchange factor CalDAG GEFI fine-tunes functional properties of regulatory T cells

Using quantitative phosphopeptide sequencing of unstimulated versus stimulated primary murine Foxp3(+) regulatory and Foxp3(-) conventional T cells (Tregs and Tconv, respectively), we detected a novel and differentially regulated tyrosine phosphorylation site within the C1 domain of the guanine-nucleotide exchange factor CalDAG GEFI. We hypothesized that the Treg-specific and activation-dependent reduced phosphorylation at Y523 allows binding of CalDAG GEFI to diacylglycerol, thereby impacting the formation of a Treg-specific immunological synapse. However, diacylglycerol binding assays of phosphomutant C1 domains of CalDAG GEFI could not confirm this hypothesis. Moreover, CalDAG GEFI(-/-) mice displayed normal Treg numbers in thymus and secondary lymphoid organs, and CalDAG GEFI(-/-) Tregs showed unaltered in vitro suppressive capacity when compared to CalDAG GEFI(+/+) Tregs. Interestingly, when tested in vivo, CalDAG GEFI(-/-) Tregs displayed a slightly reduced suppressive ability in the transfer colitis model when compared to CalDAG GEFI(+/+) Tregs. Additionally, CRISPR-Cas9-generated CalDAG GEFI(-/-) Jurkat T cell clones showed reduced adhesion to ICAM-1 and fibronectin when compared to CalDAG GEFI-competent Jurkat T cells. Therefore, we speculate that deficiency in CalDAG GEFI impairs adherence of Tregs to antigen-presenting cells, thereby impeding formation of a fully functional immunological synapse, which finally results in a reduced suppressive potential

Yersinia pseudotuberculosis supports Th17 differentiation and limits de novo regulatory T cell induction by directly interfering with T cell receptor signaling

Adaptive immunity critically contributes to control acute infection with enteropathogenic Yersinia pseudotuberculosis; however, the role of CD4(+) T cell subsets in establishing infection and allowing pathogen persistence remains elusive. Here, we assessed the modulatory capacity of Y. pseudotuberculosis on CD4(+) T cell differentiation. Using in vivo assays, we report that infection with Y. pseudotuberculosis resulted in enhanced priming of IL-17-producing T cells (Th17 cells), whereas induction of Foxp3(+) regulatory T cells (Tregs) was severely disrupted in gut-draining mesenteric lymph nodes (mLNs), in line with altered frequencies of tolerogenic and proinflammatory dendritic cell (DC) subsets within mLNs. Additionally, by using a DC-free in vitro system, we could demonstrate that Y. pseudotuberculosis can directly modulate T cell receptor (TCR) downstream signaling within naïve CD4(+) T cells and Tregs via injection of effector molecules through the type III secretion system, thereby affecting their functional properties. Importantly, modulation of naïve CD4(+) T cells by Y. pseudotuberculosis resulted in an enhanced Th17 differentiation and decreased induction of Foxp3(+) Tregs in vitro. These findings shed light to the adjustment of the Th17-Treg axis in response to acute Y. pseudotuberculosis infection and highlight the direct modulation of CD4(+) T cell subsets by altering their TCR downstream signaling

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers.DFG, 389687267, Kompartimentalisierung, Aufrechterhaltung und Reaktivierung humaner Gedächtnis-T-Lymphozyten aus Knochenmark und peripherem BlutDFG, 80750187, SFB 841: Leberentzündungen: Infektion, Immunregulation und KonsequenzenEC/H2020/800924/EU/International Cancer Research Fellowships - 2/iCARE-2DFG, 252623821, Die Rolle von follikulären T-Helferzellen in T-Helferzell-Differenzierung, Funktion und PlastizitätDFG, 390873048, EXC 2151: ImmunoSensation2 - the immune sensory syste

Activated protein C protects from GvHD via PAR2/PAR3 signalling in regulatory T-cells

Graft-vs.-host disease (GvHD) is a major complication of allogenic hematopoietic stem-cell(HSC) transplantation. GvHD is associated with loss of endothelial thrombomodulin, but the relevance of this for the adaptive immune response to transplanted HSCs remains unknown. Here we show that the protease-activated protein C (aPC), which is generated by thrombomodulin, ameliorates GvHD aPC restricts allogenic T-cell activation via the protease activated receptor (PAR)2/PAR3 heterodimer on regulatory T-cells (Tregs, CD4+FOXP3+). Preincubation of pan T-cells with aPC prior to transplantation increases the frequency of Tregs and protects from GvHD. Preincubation of human T-cells (HLA-DR4−CD4+) with aPC prior to transplantation into humanized (NSG-AB°DR4) mice ameliorates graft-vs.-host disease. The protective effect of aPC on GvHD does not compromise the graft vs. leukaemia effect in two independent tumor cell models. Ex vivo preincubation of T-cells with aPC, aPC-based therapies, or targeting PAR2/PAR3 on T-cells may provide a safe and effective approach to mitigate GvHD

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.ISSN:0014-2980ISSN:1521-414