The Role of Neutral Sphingomyelinase-2 (NSM2) in the Control of Neutral Lipid Storage in T Cells.

The accumulation of lipid droplets (LDs) and ceramides (Cer) is linked to non-alcoholic fatty liver disease (NAFLD), regularly co-existing with type 2 diabetes and decreased immune function. Chronic inflammation and increased disease severity in viral infections are the hallmarks of the obesity-related immunopathology. The upregulation of neutral sphingomyelinase-2 (NSM2) has shown to be associated with the pathology of obesity in tissues. Nevertheless, the role of sphingolipids and specifically of NSM2 in the regulation of immune cell response to a fatty acid (FA) rich environment is poorly studied. Here, we identified the presence of the LD marker protein perilipin 3 (PLIN3) in the intracellular nano-environment of NSM2 using the ascorbate peroxidase APEX2-catalyzed proximity-dependent biotin labeling method. In line with this, super-resolution structured illumination microscopy (SIM) shows NSM2 and PLIN3 co-localization in LD organelles in the presence of increased extracellular concentrations of oleic acid (OA). Furthermore, the association of enzymatically active NSM2 with isolated LDs correlates with increased Cer levels in these lipid storage organelles. NSM2 enzymatic activity is not required for NSM2 association with LDs, but negatively affects the LD numbers and cellular accumulation of long-chain unsaturated triacylglycerol (TAG) species. Concurrently, NSM2 expression promotes mitochondrial respiration and fatty acid oxidation (FAO) in response to increased OA levels, thereby shifting cells to a high energetic state. Importantly, endogenous NSM2 activity is crucial for primary human CD4+ T cell survival and proliferation in a FA rich environment. To conclude, our study shows a novel NSM2 intracellular localization to LDs and the role of enzymatically active NSM2 in metabolic response to enhanced FA concentrations in T cells.

Down-regulation of acid sphingomyelinase and neutral sphingomyelinase-2 inversely determines the cellular resistance to plasmalemmal injury by pore-forming toxins.

Bacterial pore-forming toxins compromise plasmalemmal integrity, leading to Ca2+ influx, leakage of the cytoplasm, and cell death. Such lesions can be repaired by microvesicular shedding or by the endocytic uptake of the injured membrane sites. Cells have at their disposal an entire toolbox of repair proteins for the identification and elimination of membrane lesions. Sphingomyelinases catalyze the breakdown of sphingomyelin into ceramide and phosphocholine. Sphingomyelin is predominantly localized in the outer leaflet, where it is hydrolyzed by acid sphingomyelinase (ASM) after lysosomal fusion with the plasma membrane. The magnesium-dependent neutral sphingomyelinase (NSM)-2 is found at the inner leaflet of the plasmalemma. Because either sphingomyelinase has been ascribed a role in the cellular stress response, we investigated their role in plasma membrane repair and cellular survival after treatment with the pore-forming toxins listeriolysin O (LLO) or pneumolysin (PLY). Jurkat T cells, in which ASM or NSM-2 was down-regulated [ASM knockdown (KD) or NSM-2 KD cells], showed inverse reactions to toxin-induced membrane damage: ASM KD cells displayed reduced toxin resistance, decreased viability, and defects in membrane repair. In contrast, the down-regulation of NSM-2 led to an increase in viability and enhanced plasmalemmal repair. Yet, in addition to the increased plasmalemmal repair, the enhanced toxin resistance of NSM-2 KD cells also appeared to be dependent on the activation of p38/MAPK, which was constitutively activated, whereas in ASM KD cells, the p38/MAPK activation was constitutively blunted.-Schoenauer, R., Larpin, Y., Babiychuk, E. B., Drücker, P., Babiychuk, V. S., Avota, E., Schneider-Schaulies, S., Schumacher, F., Kleuser, B., Köffel, R., Draeger, A. Down-regulation of acid sphingomyelinase and neutral sphingomyelinase-2 inversely determines the cellular resistance to plasmalemmal injury by pore-forming toxins.

Neisseria meningitidis Type IV Pili Trigger Ca2+-Dependent Lysosomal Trafficking of the Acid Sphingomyelinase To Enhance Surface Ceramide Levels.

Acid sphingomyelinase (ASM) is a lipid hydrolase that converts sphingomyelin to ceramide and that can be activated by various cellular stress mechanisms, including bacterial pathogens. Vesicle transportation or trafficking of ASM from the lysosomal compartment to the cell membrane is a prerequisite for its activation in response to bacterial infections; however, the effectors and mechanisms of ASM translocation and activation are poorly defined. Our recent work documented the key importance of ASM for Neisseria meningitidis uptake into human brain microvascular endothelial cells (HBMEC). We clearly identified OpcA to be one bacterial effector promoting ASM translocation and activity, though it became clear that additional bacterial components were involved, as up to 80% of ASM activity and ceramide generation was retained in cells infected with an opcA-deficient mutant. We hypothesized that N. meningitidis might use pilus components to promote the translocation of ASM into HBMEC. Indeed, we found that both live, piliated N. meningitidis and pilus-enriched fractions trigger transient ASM surface display, followed by the formation of ceramide-rich platforms (CRPs). By using indirect immunocytochemistry and direct stochastic optical reconstruction microscopy, we show that the overall number of CRPs with a size of ∼80 nm in the plasma membrane is significantly increased after exposure to pilus-enriched fractions. Infection with live bacteria as well as exposure to pilus-enriched fractions transiently increased cytosolic Ca2+ levels in HBMEC, and this was found to be important for ASM surface display mediated by lysosomal exocytosis, as depletion of cytosolic Ca2+ resulted in a significant decrease in ASM surface levels, ASM activity, and CRP formation.

The neutral sphingomyelinase 2 in T cell receptor signaling and polarity.

By hydrolyzing its substrate sphingomyelin at the cytosolic leaflet of cellular membranes, the neutral sphingomyelinase 2 (NSM2) generates microdomains which serve as docking sites for signaling proteins and thereby, functions to regulate signal relay. This has been particularly studied in cellular stress responses while the regulatory role of this enzyme in the immune cell compartment has only recently emerged. In T cells, phenotypic polarization by co-ordinated cytoskeletal remodeling is central to motility and interaction with endothelial or antigen-presenting cells during tissue recruitment or immune synapse formation, respectively. This review highlights studies adressing the role of NSM2 in T cell polarity in which the enzyme plays a major role in regulating cytoskeletal dynamics.

Inhibition of Acid Sphingomyelinase Allows for Selective Targeting of CD4+ Conventional versus Foxp3+ Regulatory T Cells.

CD4+ Foxp3+ regulatory T cells (Tregs) depend on CD28 signaling for their survival and function, a receptor that has been previously shown to activate the acid sphingomyelinase (Asm)/ceramide system. In this article, we show that the basal and CD28-induced Asm activity is higher in Tregs than in conventional CD4+ T cells (Tconvs) of wild-type (wt) mice. In Asm-deficient (Smpd1-/-; Asm-/-) mice, as compared with wt mice, the frequency of Tregs among CD4+ T cells, turnover of the effector molecule CTLA-4, and their suppressive activity in vitro were increased. The biological significance of these findings was confirmed in our Treg-sensitive mouse model of measles virus (MV) CNS infection, in which we observed more infected neurons and less MV-specific CD8+ T cells in brains of Asm-/- mice compared with wt mice. In addition to genetic deficiency, treatment of wt mice with the Asm inhibitor amitriptyline recapitulated the phenotype of Asm-deficient mice because it also increased the frequency of Tregs among CD4+ T cells. Reduced absolute cell numbers of Tconvs after inhibitor treatment in vivo and extensive in vitro experiments revealed that Tregs are more resistant toward Asm inhibitor-induced cell death than Tconvs. Mechanistically, IL-2 was capable of providing crucial survival signals to the Tregs upon inhibitor treatment in vitro, shifting the Treg/Tconv ratio to the Treg side. Thus, our data indicate that Asm-inhibiting drugs should be further evaluated for the therapy of inflammatory and autoimmune disorders.

Human endogenous retrovirus envelope proteins target dendritic cells to suppress T-cell activation.

Though mostly defective, human endogenous retroviruses (HERV) can retain open reading frames, which are especially expressed in the placenta. There, the envelope (env) proteins of HERV-W (Syncytin-1), HERV-FRD (Syncytin-2), and HERV-K (HML-2) were implicated in tolerance against the semi-allogenic fetus. Here, we show that the known HERV env-binding receptors ASCT-1 and -2 and MFSD2 are expressed by DCs and T-cells. When used as effectors in coculture systems, CHO cells transfected to express Syncytin-1, -2, or HML-2 did not affect T-cell expansion or overall LPS-driven phenotypic DC maturation, however, promoted release of IL-12 and TNF-α rather than IL-10. In contrast, HERV env expressing choriocarcinoma cell lines suppressed T-cell proliferation and LPS-induced TNF-α and IL-12 release, however, promoted IL-10 accumulation, indicating that these effects might not rely on HERV env interactions. However, DCs conditioned by choriocarcinoma, but also transgenic CHO cells failed to promote allogenic T-cell expansion. This was associated with a loss of DC/T-cell conjugate frequencies, impaired Ca(2+) mobilization, and aberrant patterning of f-actin and tyrosine phosphorylated proteins in T-cells. Altogether, these findings suggest that HERV env proteins target T-cell activation indirectly by modulating the stimulatory activity of DCs.

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.

Neutral sphingomyelinase 2 is a key factor for PorB-dependent invasion of Neisseria gonorrhoeae.

Disseminated gonococcal infection (DGI) is a rare but serious complication caused by the spread of Neisseria gonorrhoeae in the human host. Gonococci associated with DGI mainly express the outer membrane protein PorBIA that binds to the scavenger receptor expressed on endothelial cells (SREC-I) and mediates bacterial uptake. We recently demonstrated that this interaction relies on intact membrane rafts that acquire SREC-I upon attachment of gonococci and initiates the signalling cascade that finally leads to the uptake of gonococci in epithelial cells. In this study, we analysed the role of sphingomyelinases and their breakdown product ceramide. Gonococcal infection induced increased levels of ceramide that was enriched at bacterial attachment sites. Interestingly, neutral but not acid sphingomyelinase was mandatory for PorBIA -mediated invasion into host cells. Neutral sphingomyelinase was required to recruit the PI3 kinase to caveolin and thereby activates the PI3 kinase-dependent downstream signalling leading to bacterial uptake. Thus, this study elucidates the initial signalling processes of bacterial invasion during DGI and demonstrates a novel role for neutral sphingomyelinase in the course of bacterial infections.

The PI3K pathway acting on alternative HIV-1 pre-mRNA splicing.

HIV-1 mediates pro-survival signals and prevents apoptosis via the phosphatidylinositol-3-kinase (PI3K) pathway. This pathway, however, also affects phosphorylation of serine-arginine (SR) proteins, a family of splicing regulatory factors balancing splice site selection. We now show that pharmacologic inhibition of PI3K signalling alters the HIV-1 splicing pattern of both minigene- and provirus-derived mRNAs. This indicates that HIV-1 might also promote PI3K signalling to balance processing of its transcripts by regulating phosphorylation of splicing regulatory proteins.

The role of sphingomyelin breakdown in measles virus immunmodulation.

Measles virus (MV) efficiently causes generalized immunosuppression which accounts to a major extent for cases of measles-asscociated severe morbidity and mortality. MV infections alter many functions of antigen presenting cells (APC) (dendritic cells (DCs)) and lymphocytes, yet many molecular targets of the virus remain poorly defined. Cellular interactions and effector functions of DCs and lymphocytes are regulated by surface receptors. Associating with other proteins involved in cell signaling, receptors form part of receptosomes that respond to and transmit external signals through dynamic interctions with the cytoskeleton. Alterations in the composition and metabolism of membrane sphingolipids have a substantial impact on both processes. In this review we focus on the regulation of sphingomyelinase activity and ceramide release in cells exposed to MV and discuss the immunosuppressive role of sphingomyelin breakdown induced by MV.

Membrane dynamics and interactions in measles virus dendritic cell infections.

Viral entry, compartmentalization and transmission depend on the formation of membrane lipid/protein microdomains concentrating receptors and signalosomes. Dendritic cells (DCs) are prime targets for measles virus (MV) infection, and this interaction promotes immune activation and generalized immunosuppression, yet also MV transport to secondary lymphatics where transmission to T cells occurs. In addition to MV trapping, DC-SIGN interaction can enhance MV uptake by activating cellular sphingomyelinases and, thereby, vertical surface transport of its entry receptor CD150. To exploit DCs as Trojan horses for transport, MV promotes DC maturation accompanied by mobilization, and restrictions of viral replication in these cells may support this process. MV-infected DCs are unable to support formation of functional immune synapses with conjugating T cells and signalling via viral glycoproteins or repulsive ligands (such as semaphorins) plays a key role in the induction of T-cell paralysis. In the absence of antigen recognition, MV transmission from infected DCs to T cells most likely involves formation of polyconjugates which concentrate viral structural proteins, viral receptors and with components enhancing either viral uptake or conjugate stability. Because DCs barely support production of infectious MV particles, these organized interfaces are likely to represent virological synapses essential for MV transmission.

Measles virus transmission from dendritic cells to T cells: formation of synapse-like interfaces concentrating viral and cellular components.

Transmission of measles virus (MV) to T cells by its early CD150(+) target cells is considered to be crucial for viral dissemination within the hematopoietic compartment. Using cocultures involving monocyte-derived dendritic cells (DCs) and T cells, we now show that T cells acquire MV most efficiently from cis-infected DCs rather than DCs having trapped MV (trans-infection). Transmission involves interactions of the viral glycoprotein H with its receptor CD150 and is therefore more efficient to preactivated T cells. In addition to rare association with actin-rich filopodial structures, the formation of contact interfaces consistent with that of virological synapses (VS) was observed where viral proteins accumulated and CD150 was redistributed in an actin-dependent manner. In addition to these molecules, activated LFA-1, DC-SIGN, CD81, and phosphorylated ezrin-radixin-moesin proteins, which also mark the HIV VS, redistributed toward the MV VS. Most interestingly, moesin and substance P receptor, both implicated earlier in assisting MV entry or cell-to-cell transmission, also partitioned to the transmission structure. Altogether, the MV VS shares important similarities to the HIV VS in concentrating cellular components potentially regulating actin dynamics, conjugate stability, and membrane fusion as required for efficient entry of MV into target T cells.

DC-SIGN mediated sphingomyelinase-activation and ceramide generation is essential for enhancement of viral uptake in dendritic cells.

As pattern recognition receptor on dendritic cells (DCs), DC-SIGN binds carbohydrate structures on its pathogen ligands and essentially determines host pathogen interactions because it both skews T cell responses and enhances pathogen uptake for cis infection and/or T cell trans-infection. How these processes are initiated at the plasma membrane level is poorly understood. We now show that DC-SIGN ligation on DCs by antibodies, mannan or measles virus (MV) causes rapid activation of neutral and acid sphingomyelinases followed by accumulation of ceramides in the outer membrane leaflet. SMase activation is important in promoting DC-SIGN signaling, but also for enhancement of MV uptake into DCs. DC-SIGN-dependent SMase activation induces efficient, transient recruitment of CD150, which functions both as MV uptake receptor and microbial sensor, from an intracellular Lamp-1+ storage compartment shared with acid sphingomyelinase (ASM) within a few minutes. Subsequently, CD150 is displayed at the cell surface and co-clusters with DC-SIGN. Thus, DC-SIGN ligation initiates SMase-dependent formation of ceramide-enriched membrane microdomains which promote vertical segregation of CD150 from intracellular storage compartments along with ASM. Given the ability to promote receptor and signalosome co-segration into (or exclusion from) ceramide enriched microdomains which provide a favorable environment for membrane fusion, DC-SIGN-dependent SMase activation may be of general importance for modes and efficiency of pathogen uptake into DCs, and their routing to specific compartments, but also for modulating T cell responses.

Preculture of PBMCs at high cell density increases sensitivity of T-cell responses, revealing cytokine release by CD28 superagonist TGN1412.

Human volunteers receiving TGN1412, a humanized CD28-specific monoclonal antibody, experienced a life-threatening cytokine release syndrome during a recent trial. Preclinical tests using human PBMCs had failed to announce the rapid release of TNF, IFN-γ, and other toxic cytokines in response to this CD28 "superagonist" (CD28SA). CD28SA activate T-lymphocytes by ligating CD28 without overt engagement of the TCR. They do, however, depend on "tonic" TCR signals, which they amplify. Here we show that short-term preculture of PBMCs at high, but not at low, cell density results in massive cytokine release during subsequent stimulation with soluble TGN1412. Restoration of reactivity was cell-contact dependent, involved functional maturation of both monocytes and T cells, was sensitive to blockade by HLA-specific mAb, and was associated with TCR polarization and tyrosine phosphorylation. CD4 effector memory T cells were identified as the main source of proinflammatory cytokines. Importantly, responses to other T-cell activating agents, including microbial antigens, were also enhanced if PBMCs were first allowed to interact under tissue-like conditions. We provide a protocol, which strongly improves reactivity of circulating T cells to soluble stimulants, thereby allowing for more reliable preclinical testing of both activating and inhibitory immunomodulatory drugs.

Measles virus modulates dendritic cell/T-cell communication at the level of plexinA1/neuropilin-1 recruitment and activity.

Measles virus (MV)-infected DC fail to promote T-cell expansion, and this could explain important aspects of measles immunosuppression. The efficiency of the immune synapse (IS) is determined by the formation of stable, stimulatory conjugates involving a spatially and timely controlled architecture. PlexinA1 (plexA1) and its co-receptor neuropilin (NP-1) have been implicated in IS efficiency, while their repulsive ligand, SEMA3A, likely acts in terminating T-cell activation. Conjugates involving MV-infected DC and T cells are unstable and not stimulatory, and thus we addressed the potential role of plexA1/NP-1 and semaphorins (SEMAs) in this system. MV does not grossly affect expression levels of plexA1/NP-1 on T cells or DC, yet prevents their recruitment towards stimulatory interfaces. Moreover, MV infection promoted early release of SEMA3A from DC, which caused loss of actin based protrusions on T cells as did the plexA4 ligand SEMA6A. SEMA3A/6A differentially modulated chemokinetic migration of T cells and conjugation with allogeneic DC. Thus, MV targets SEMA receptor function both at the level of IS recruitment, and by promoting a timely inappropriate release of their repulsive ligand, SEMA3A. To the best of our knowledge, this is the first example of viral targeting of SEMA receptor function in the IS.

Induction of membrane ceramides: a novel strategy to interfere with T lymphocyte cytoskeletal reorganisation in viral immunosuppression.

Silencing of T cell activation and function is a highly efficient strategy of immunosuppression induced by pathogens. By promoting formation of membrane microdomains essential for clustering of receptors and signalling platforms in the plasma membrane, ceramides accumulating as a result of membrane sphingomyelin breakdown are not only essential for assembly of signalling complexes and pathogen entry, but also act as signalling modulators, e. g. by regulating relay of phosphatidyl-inositol-3-kinase (PI3K) signalling. Their role in T lymphocyte functions has not been addressed as yet. We now show that measles virus (MV), which interacts with the surface of T cells and thereby efficiently interferes with stimulated dynamic reorganisation of their actin cytoskeleton, causes ceramide accumulation in human T cells in a neutral (NSM) and acid (ASM) sphingomyelinase-dependent manner. Ceramides induced by MV, but also bacterial sphingomyelinase, efficiently interfered with formation of membrane protrusions and T cell spreading and front/rear polarisation in response to beta1 integrin ligation or alphaCD3/CD28 activation, and this was rescued upon pharmacological or genetic ablation of ASM/NSM activity. Moreover, membrane ceramide accumulation downmodulated chemokine-induced T cell motility on fibronectin. Altogether, these findings highlight an as yet unrecognised concept of pathogens able to cause membrane ceramide accumulation to target essential processes in T cell activation and function by preventing stimulated actin cytoskeletal dynamics.

Detection of Functionalized Sphingolipid Analogs in Detergent-Resistant Membranes of Immune Cells.

The analysis of protein enrichment in the detergent-resistant membranes (DRMs) isolated from immune cells enables us to analyze a link between the membrane lipid dynamics and cell activation. Here, we describe the fractionation of detergent-resistant membranes and the correlative analysis of the enrichment of T cell receptor (TCR) and ω-azido-modified synthetic ceramide in those fractions upon TCR stimulation.

The Manifold Roles of Sphingolipids in Viral Infections.

Sphingolipids are essential components of eukaryotic cells. In this review, we want to exemplarily illustrate what is known about the interactions of sphingolipids with various viruses at different steps of their replication cycles. This includes structural interactions during entry at the plasma membrane or endosomal membranes, early interactions leading to sphingolipid-mediated signal transduction, interactions with internal membranes and lipids during replication, and interactions during virus assembly and budding. Targeted interventions in sphingolipid metabolism - as far as they can be tolerated by cells and organisms - may open novel possibilities to support antiviral therapies. Human immunodeficiency virus type 1 (HIV-1) infections have intensively been studied, but for other viral infections, such as influenza A virus (IAV), measles virus (MV), hepatitis C virus (HCV), dengue virus, Ebola virus, and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), investigations are still in their beginnings. As many inhibitors of sphingolipid metabolism are already in clinical use against other diseases, repurposing studies for applications in some viral infections appear to be a promising approach.

Measles virus-induced immunosuppression: from effectors to mechanisms.

Immunosuppression is the major cause of infant death associated with acute measles. Hallmarks of this generalized modulation of immune functions include: (1) lymphopenia, (2) a prolonged cytokine imbalance consistent with suppression of cellular immunity to secondary infections and (3) silencing of peripheral blood lymphocytes that fail to expand in response to ex vivo stimulation. Lymphopenia results from depletion of T cells by mechanisms also involving MV infection, and expression of the major MV receptor CD150 plays an important role for targeting these cells. Virus transfer to T cells is thought to be mediated by dendritic cells (DCs), which are considered as central to the induction of T-cell silencing and functional skewing. MV interaction modulates functional differentiation of DCs, and thereby expression pattern of costimulatory molecules and soluble mediators. Moreover, MV proteins expressed by these cells actively silence T cells by interfering with signaling pathways essential for T-cell activation. As an essential component of this interference, the MV glycoprotein complex activates cellular sphingomyelinases in a contact-dependent manner, and these are effective at preventing stimulated rearrangements of the actin cytoskeleton and thereby morphological and functional polarization and motility of T cells.

Neutral Sphingomyelinase-2 (NSM 2) Controls T Cell Metabolic Homeostasis and Reprogramming During Activation.

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.