Adequate immune response ensured by binary IL-2 and graded CD25 expression in a murine transfer model

The IL-2/IL-2Ralpha (CD25) axis is of central importance for the interplay of effector and regulatory T cells. Nevertheless, the question how different antigen loads are translated into appropriate IL-2 production to ensure adequate responses against pathogens remains largely unexplored. Here we find that at single cell level, IL-2 is binary (digital) and CD25 is graded expressed whereas at population level both parameters show graded expression correlating with the antigen amount. Combining in vivo data with a mathematical model we demonstrate that only this binary IL-2 expression ensures a wide linear antigen response range for Teff and Treg cells under real spatiotemporal conditions. Furthermore, at low antigen concentrations binary IL-2 expression safeguards by its spatial distribution selective STAT5 activation only of closely adjacent Treg cells regardless of their antigen specificity. These data show that the mode of IL-2 secretion is critical to tailor the adaptive immune response to the antigen amount

Digital NFATc2 Activation per Cell Transforms Graded T Cell Receptor Activation into an All-or-None IL-2 Expression

The expression of interleukin-2 (IL-2) is a key event in T helper (Th) lymphocyte activation, controlling both, the expansion and differentiation of effector Th cells as well as the activation of regulatory T cells. We demonstrate that the strength of TCR stimulation is translated into the frequency of memory Th cells expressing IL-2 but not into the amount of IL-2 per cell. This molecular switch decision for IL-2 expression per cell is located downstream of the cytosolic Ca2+ level. Here we show that in a single activated Th cell, NFATc2 activation is digital but NF-κB activation is graded after graded T cell receptor (TCR) signaling. Subsequently, NFATc2 translocates into the nucleus in an all-or-none fashion per cell, transforming the strength of TCR-stimulation into the number of nuclei positive for NFATc2 and IL-2 transcription. Thus, the described NFATc2 switch regulates the number of Th cells actively participating in an immune response

NFATc1 controls the cytotoxicity of CD8+ T cells

NFAT nuclear translocation has been shown to be required for CD8+ T cell cytokine production in response to viral infection. Here the authors show NFATc1 controls the cytotoxicity and metabolic switching of activated CD8+ T cells required for optimal response to bacteria and tumor cells

NFATc2 is a switch of t-cell-receptor dependent activation of human CD4+ Th cells

Titelblatt und Inhaltsverzeichnis Abkürzungsverzeichnis Einleitung Material und Methoden Ergebnisse Diskussion Zusammenfassung Summary LiteraturverzeichnisDie IL-2 Expression von CD4+ Th-Zellen nach spezifischer TCR Stimulation ist ein Schlüsselereignis der adaptiven Immunantwort. Eine Fehlregulation des Zytokins IL-2 hat fundamentale Auswirkungen auf die Homöostase des adaptiven Immunsystems in vivo. Während die transkriptionelle Regulation des IL-2 Promotors hinreichend erforscht wurde, sind die zellulären Entscheidungsprozesse der Signaltransduktion, die zur Bildung von IL-2 führen, kaum bekannt. In dieser Arbeit wurde gezeigt, dass die transkriptionelle Induktion von IL-2 innerhalb einer peripheren humanen CD4+ Th-Zellpopulation während der initialen mitogenen Th-Zellaktivierung modulierbar war, und einem binären und nicht graduellen Entscheidungsprozess unterlag. Die graduelle Stimulation oder graduelle Inhibierung der TCR-abhängigen Aktivierung von Th- Zellen resultierte in einer Veränderung der Frequenz der IL-2 produzierenden Zellen und nicht in einer Veränderung der Intensität der IL-2 Produktion pro Zelle. Der Entscheidungsprozess dieser binären IL-2 Expression wurde in dieser Arbeit erstmalig und auf Einzelzellebene untersucht. Hierzu wurden die IL-2 Produzenten und die IL-2 Nichtproduzenten einer humanen CD4+ Th-Zellpopulation anhand des IL-2 Sekretionsassays separiert. Darüber hinaus wurde eine Methode zur durchflusszytometrischen Detektion der aktivierten Transkriptionsfaktoren NFATc2 und NF-kB (p65) in isolierten intakten Zellkernen aus humanen Th-Zellen etabliert. Mit dieser Arbeit konnte belegt werden, dass NFATc2 als ein zellulärer Schalter der Ca2+/Calcineurin-abhängigen T-Zellaktivierung funktioniert. Die IL-2 Produktion zeigte eine große Abhängigkeit von der intrazellulären exprimierten NFATc2 Menge und resultierte infolge eines binären Aktivierungs- und nukleären Translokationsprofils von NFATc2 in einem binären IL-2 Expressionsprofil in individuellen Zellen. Im Gegensatz dazu wurde für die Aktivierung von NF-kB (p65) eine graduelles nukleäres Translokationsprofil detektiert. Durch mathematische Modellierung der vorliegenden Ergebnisse konnte zudem eine binäre Geninduktion als Folge einer Schalteraktivierung von NFATc2 in Reaktion auf eine graduelle Stimulation mit Ionomycin simuliert werden, für die bereits die kooperative Dephosphorylierung von 7 der 13 Serin-Phosphorylierungsstellen am NFATc2 Molekül als ausreichend für die Induktion einer binären IL-2 Expression ermittelt werden konnte. Damit kann der hier beschriebene NFATc2 Schalter als ein Übersetzer der Signalstärke bei der T-Zellaktivierung in die effektive Anzahl an aktivierten, zytokinproduzierenden Th-Zellen angesehen werden, der als ein allgemeiner Kontrollpunkt in Th-Zellen die Qualität der adaptiven Immunantwort mitbestimmt.Expression of IL-2 by activated T helper cells is a key event of the adaptive immune response after antigenic stimulation. The dysregulation of IL-2 is of fundamental consequence for the homeostasis of the immune system in vivo. While the molecular analysis of IL-2 trans-criptional regulation has been extensively studied the cellular decision processes leading to IL-2 expression are not known. In this study is shown that in human periphal CD4+ Th cells the transcriptional response of IL-2 can be modulated after initial mitogenic stimulation and is followed by a binary and not a graded decision process. The titration of the Ca2+ ionophore ionomycin or inhibition of mitogenic stimulation by the classical calcineurin inhibitor cyclosporine A only changed the frequency of IL-2 producing cells but not the intensity of IL-2 production per cell. The molecular decision process leading to the binary IL-2 expression pattern shown here was examined for the first time and on individual cells. On this account, the IL-2 capture assay was used to separate IL-2 producing and non-producing cells after stimulation. For single cell analysis, a method for cytometrical detection of the activated transcription factors NFATc2 and NF-kB (p65) in isolated nuclei was developed. The results proved NFATc2 as a cellular switch of Ca2+/calcineurin dependent T cell activa-tion. The IL-2 production showed great dependancy on the intracellular level of expressed NFATc2 and results in binary IL-2 expression pattern after all-or-non activation of NFATc2 in individual cells. Graded stimulations by titration with Ionomycin or CsA lead to an all-or-non NFATc2 but graded NF-kB (p65) nuclear translocation per cell. Mathematical modeling predicts that co- operativity of at least 7 of the 13 NFATc2 dephosphorylation sites after graded stimulation by ionomycin would be sufficient to induce a binary IL-2 expression. By translating the strength of antigenic T cell stimulation into the frequency of cytokine producing Th cells, the described NFATc2 switch is a general hub for productive adaptive immune response

Berechnung von kompressiblen und inkompressiblen Stroemungsgrenzschichten unter Beruecksichtigung von Laengs- und Querkruemmung der Wand

SIGLEDEGerman

Impact of the Covid‑19 pandemic on the performance of machine learning algorithms for predicting perioperative mortality

Background Machine-learning models are susceptible to external influences which can result in performance deterioration. The aim of our study was to elucidate the impact of a sudden shift in covariates, like the one caused by the Covid-19 pandemic, on model performance. Methods After ethical approval and registration in Clinical Trials (NCT04092933, initial release 17/09/2019), we developed different models for the prediction of perioperative mortality based on preoperative data: one for the pre-pandemic data period until March 2020, one including data before the pandemic and from the first wave until May 2020, and one that covers the complete period before and during the pandemic until October 2021. We applied XGBoost as well as a Deep Learning neural network (DL). Performance metrics of each model during the different pandemic phases were determined, and XGBoost models were analysed for changes in feature importance. Results XGBoost and DL provided similar performance on the pre-pandemic data with respect to area under receiver operating characteristic (AUROC, 0.951 vs. 0.942) and area under precision-recall curve (AUPR, 0.144 vs. 0.187). Validation in patient cohorts of the different pandemic waves showed high fluctuations in performance from both AUROC and AUPR for DL, whereas the XGBoost models seemed more stable. Change in variable frequencies with onset of the pandemic were visible in age, ASA score, and the higher proportion of emergency operations, among others. Age consistently showed the highest information gain. Models based on pre-pandemic data performed worse during the first pandemic wave (AUROC 0.914 for XGBoost and DL) whereas models augmented with data from the first wave lacked performance after the first wave (AUROC 0.907 for XGBoost and 0.747 for DL). The deterioration was also visible in AUPR, which worsened by over 50% in both XGBoost and DL in the first phase after re-training. Conclusions A sudden shift in data impacts model performance. Re-training the model with updated data may cause degradation in predictive accuracy if the changes are only transient. Too early re-training should therefore be avoided, and close model surveillance is necessary