Die Rolle von Ceramiden und Sphingomyelinasen für Dynamische Membranveränderungen in T-Zellen

Previous work of our group has established a role of sphingomyelinases in the regulation of T cell responses to TCR or pathogen stimulation, and this became particularly evident at the level of actin cytoskeletal dynamics. The formation of lipid membrane microdomains is crucial for receptor clustering and signal induction, and therefore, ceramide accumulation by membrane sphingomyelin breakdown is needed for signalling- complex-assembly. Pathogen-induced overshooting of SMase activation substantially impacted the formation of membrane protrusions, with T cell spreading as well as a front/rear polarisation upon CD3/CD28 co-stimulation [103]. On the other hand, NSM activation is part of the physiological TCR signal [67], indicating that a spatiotemporally balanced NSM activation is crucial for its physiological function. It involves actin cytoskeletal reorganisation and T cell polarisation. These two functions are also of central importance in directional T cell migration and motility in tissues. This thesis aims on defining the role of NSM in compartmentalisation of the T cell membrane in polarisation and migration. Therefore, functional studies on the impact of NSM activity in these processes had to be complemented by the development of tools to study ceramide compartmentalisation in living T cells.Sphingolipide sind wichtige Komponenten der Plasmamembran, und besonders Ceramid stellt das Grundgerüst für komplexere Sphingolipide dar. Darüber hinaus bildet Ceramid Mikrodomänen aus, die für die Organisation von Rezeptoren, z. B. dem T-Zell-Rezeptorkomplex entscheidend sind und somit die Funktion von T-Zellen beeinflussen. In dieser Arbeit wurden neue azid-funktionalisierte Ceramide angewendet, die durch eine bio-orthogonale Click-Reaktion mit fluoreszenten Farbstoffmolekülen kovalent verbunden werden können. Dies ermöglicht die live-Verfolgung der Ceramide durch lebende und auch stimulierte Zellen, da die Aktivierbarkeit von T-Zellen durch die Zufütterung nicht beeinflusst wurde. Es konnte gezeigt werden, dass N$_3$-C$_6$-cer in die Plasmamembran interkaliert und sich in Mikrodomänen, die durch eine Aktivierung der ASM nach CD28 Bindung entstehen, bewegt. Darüber hinaus wurde N$_3$-C$_6$-cer aus dem Zentrum der immunologischen Synapse zwischen T-Zellen und dendritischen Zellen oder Mikrokügelchen ausgeschlossen, wie zuvor in fixierten und mit Antikörper gefärbten T-Zellen gezeigt wurde. In migrierenden T-Zellen sammelte sich das N$_3$-C$_6$-cer am hinteren Ende der Zelle und beeinflusste die Bewegung der Zellen nicht. Dies unterstreicht die Anwendbarkeit dieser neuen Methode, um die subzelluläre Verteilung von Ceramiden in Lebendzell-Experimenten zu untersuchen. Sphingomyelinasen beeinflussen durch ihre Funktion die Verhältnisse von Sphingolipiden in der Plasmamembran und haben so Einfluss auf die Zytoskelettdynamik, die Zellpolarisation und die Rezeptororganisation. Es wurde bereits zuvor gezeigt, dass die neutrale Sphingomyelinase wichtig ist für die T-Zellaktivierung. Nun wurde darüber hinaus ihre Rolle in der gerichteten Migration und Adhäsion dargestellt. In vivo Hemmung der NSM reduzierte die frühe Wanderung von T-Zellen in die Lymphknoten, und detaillierte in vitro Analysen zeigten, dass die basale Aktivität der neutralen Sphingomyelinase für die gerichtete Migration entlang eines SDF-1$\alpha$-Gradienten notwendig ist. Darüber hinaus ist ihre Funktion wichtig für die T-Zell Polarisierung und hier besonders die Organisation von CXCR4 und pERM. Außerdem spielt die neutralen Sphingomyelinase eine Rolle in der Polymerisierung von F-Aktin nach einer Stimulation der T-Zellen mit SDF-1$\alpha$. Auch die Adhäsion an das TNF$\alpha$/IFN$\gamma$-stimulierte Endothel sowie die Ausbildung und Organisation der offenen Form von LFA-1 hängen von der neutralen Sphingomyelinase ab. Für den Prozess der Transmigration war im Gegensatz hierzu nur die Funktion der sauren Sphingomyelinase von Bedeutung. Zusammenfassend konnte in dieser Arbeit die zentrale Rolle der Sphingomyelinasen für die T-Zell-Migration im ruhenden und stimulierten Zustand gezeigt werden. Die neutrale Sphingomyelinase reguliert die polarisierte Organisation von Rezeptoren und Zytoskelett-Komponenten, welche für eine gerichtete Migration und Adhäsion unabdingbar sind

Neutral Sphingomyelinase in Physiological and Measles Virus Induced T Cell Suppression

T cell paralysis is a main feature of measles virus (MV) induced immunosuppression. MV contact mediated activation of sphingomyelinases was found to contribute to MV interference with T cell actin reorganization. The role of these enzymes in MV-induced inhibition of T cell activation remained equally undefined as their general role in regulating immune synapse (IS) activity which relies on spatiotemporal membrane patterning. Our study for the first time reveals that transient activation of the neutral sphingomyelinase 2 (NSM2) occurs in physiological co-stimulation of primary T cells where ceramide accumulation is confined to the lamellum (where also NSM2 can be detected) and excluded from IS areas of high actin turnover. Genetic ablation of the enzyme is associated with T cell hyper-responsiveness as revealed by actin dynamics, tyrosine phosphorylation, Ca2+-mobilization and expansion indicating that NSM2 acts to suppress overshooting T cell responses. In line with its suppressive activity, exaggerated, prolonged NSM2 activation as occurring in co-stimulated T cells following MV exposure was associated with aberrant compartmentalization of ceramides, loss of spreading responses, interference with accumulation of tyrosine phosphorylated protein species and expansion. Altogether, this study for the first time reveals a role of NSM2 in physiological T cell stimulation which is dampening and can be abused by a virus, which promotes enhanced and prolonged NSM2 activation to cause pathological T cell suppression

Model of NSM-dependent T cell suppression.

<p>T cell co-stimulation induces transient activation of the NSM which, as ceramides, is confined at early stages to the lamellum (upper graph, black line, left sketch of the T cell IS). In the presence of MV, NSM is activated more rapidly and prolonged, and ceramides are not excluded from the IS center (upper graph, red line, right sketch of the T cell IS). Based on genetic ablation studies, under physiologic conditions, the NSM appears to dampen overshooting T cell activation by regulating the speed of early T cell activation as defined by the typical parameters listed. When inappropriately (time, magnitude) activated as occurring by MV T cell contact, the dampening role of NSM is exaggerated and associated with actin cytoskeletal paralysis, but also contributes to MV-induced loss of certain p-tyr protein species and expansion. Therefore, the ability of MV to cause NSM induction in a contact dependent manner accounts for major features of MV-mediated T cell suppression.</p

NSM acts to dampen initiation of T cell activation.

<p>A–C. T cells transfected with NSM2 (NSMKD) or control siRNA (CTRL) were seeded onto co-stimulatory slides and analyzed for f-actin distribution (A, B), adherence (A), cell area (A, B) and formation of extended lamellar protrusions (C) after 5 min (A) or the time intervals indicated (B,C) by confocal (A, B) or scanning electron microscopy (C; veils marked by arrowheads; at least 50 cells were recruited per culture into the analysis). Representative examples of the quantitative analyses are depicted in the left panels. Size bars: A, B: 10 µm, C, 1 µm. D. Left panels: pERK or pAkt induction following αãCD3/CD28 activation (min) in CRTL and NSMKD T cells (signals for pAkt and pERK and the respective protein loading controls were quantified by AIDA software and are expressed as ‚fold induction', middle panels) Right panel: Ca²⁺ mobilization was determined over time in NSMKD (open histogram) versus CTRL T cells (filled histogram) following α-CD3/CD28 activation by flow cytometry. Representative examples out of at least each 3 are shown. E. Proliferative responses of human primary NSMKD (white bars) or CTRL T cells (black bars) 48 h and 72 h following αãCD3/CD28 activation (left panel) or <i>Smpd3</i>-deficient or –sufficient mouse splenocytes 72 h following co-culture with syngenic, superantigen-loaded bone marrow derived DCs (right panel).</p

Ceramide and NSM2 accumulate within the lamellum in co-stimulated T cells.

<p>A. Ceramide and f-actin were co-detected in T cells 15 min after seeding onto co-stimulatory slides (left panels, with intensities indicated by false colour representation, bottom panels). Size bar: 5 µm. Subcellular distribution of actin (in red) and ceramide (in green) within the lamellum (LM) or the lamellipodium (LP) are representatively shown as profiles (middle panel, intensity profile plane indicated by the arrow) or blow ups imaged at lower or higher z planes (distance approximately 600 nm)(right panels, enlargements of boxed area middle panel). B. NSM2 and f-actin were co-detected 24 h following nucleofection of p-NSM2-GFP (NSM2: green, f-actin: red). Subcellular distribution of NSM2-GFP is shown after deconvolution and 3D reconstruction (right panel). Size bar: 5 µm.</p

MV causes early superactivation of NSM and ceramide accumulation within stimulatory interfaces.

<p>A. NSM activity was determined in membrane extracts isolated from T cells exposed to MV or MOCK prior to αãCD3/CD28 activation for the time intervals indicated. Time point ‚0 and 15 min' (right) measured NSM activity in T cells exposed to MV but not stimulated by αãCD3/CD28 ligation. B. Ceramide (green) and f-actin (red) were detected in T cells pre-exposed to bSMase, MOCK or MV and seeded onto co-stimulatory slides for 15 min. Representative examples of 50 cells analyzed per culture are shown after 3D deconvolution. C. The percentage of conjugates excluding ceramide from the interface between αãCD3/CD28 coated beads (marked by asterisks) and CTRL or NSMKD T cells pre-exposed to MV or MOCK was determined 15 min following conjugate formation, fixation and staining by α-ceramide antibody. D. 24 h following p-NSM2-GFP nucleofection, T cells were exposed to MOCK or MV, conjugated to αãCD3/CD28 coated beads and the frequency of conjugates excluding NSM2-GFP from the interface in both cultures was determined. C, D: Left panels show examples for interface exclusion/inclusion (boxed areas), at least 100 conjugates/per culture were recruited for quantitation in three independent experiments. DIC: differential interference contrast. Size bars: A, B: 5 µm, C: 10 µm.</p

ASM activation is ablated, while NSM activity is induced early in co-stimulated primary human T cells.

<p>A. ASM activity was determined in membrane extracts of T cells activated by α-CD28, α-CD3 or α-CD3/CD28 over time. B. Surface ceramide was detected on T cells activated by ligation of CD3, CD28 or CD3/CD28 by flow cytometry C. NSM activity in membrane extracts of T cells 96 h following transfection of control (CTRL) or NSM2 siRNA (NSMKD). D. ASM activity in membrane extracts of αãCD3/CD28 co-stimulated T cells (left) and extrafacial ceramide display (FACS staining) (right, upper graphs: unstimulated, CTRL or NSMKD T cells, upper right corner values represent mean fluorescence intensities; bottom graph: kinetics of ceramide surface display on CTRL versus NSMKD co-stimulated T cells. E. NSM activity was determined in membrane extracts of T cells activated by α-CD28, α-CD3 or α-CD3/CD28. F. ASM and NSM activity were determined over time in α-CD3/CD28 co-stimulated T cells.</p

NSM activation contributes to MV interference with T cell early activation and expansion.

<p>A. Overall tyrosine phosphorylation was determined at the time intervals indicated following αãCD3/CD28 activation in CTRL and NSMKD cells pre-exposed to MOCK or MV (MOI 1,2) by Western Blotting (where GAPDH detection served as loading control). p-tyr protein species detectably affected by MV interference are starred (*). Right: densitometric analysis of p-tyr profiles of all p-tyr species was performed for each individual lane, quantified using AIDA software and standardized to the signal intensities in unstimulated MOCK treated cells. B. Proliferation efficiencies of CTRL and NSMKD cells pre-exposed to MOCK (each: white symbols) or MV (each: black symbols) at the MOIs indicated (or corresponding protein amounts of MOCK) were determined after 48 h by incorporation of [³H]-thymidine.</p

MV-mediated NSM activation accounts for the loss of spreading responses.

<p>Cell areas were determined in T cells where NSM activity was genetically (NSMKD, A) or pharmacologically (pre-exposure to GW4869, B) ablated prior to MOCK or MV exposure and subsequent seeding onto co-stimulatory slides for 15 min. Spreading responses were detected by staining for f-actin alone (A) or co-detection with LAT used to detect microclusters (B). Frequencies of CTRL or NSMKD cells adhering to the co-stimulatory slides after exposure to MV or MOCK were determined (A). Size bars: A, B: 5 µm.</p