Analyzing trogocytosis of T lymphocytes by flow cytometry and confocal microscopy.

Here, we present a protocol to examine the mechanisms underlying the intercellular transfer of transmembrane molecules, termed trogocytosis, and the fate of transferred molecules. We describe the steps needed from T lymphocyte isolation, via co-culture with cells expressing the ligand of interest, to cell harvest and subsequent staining for flow cytometry and confocal microscopy. Furthermore, we showcase critical parameters and pitfalls, which allow easy adaptation of the protocol to investigate trogocytosis of various cell surface receptors in different cell types. For complete details on the use and execution of this protocol, please refer to Zink and Rohr.1.

Inhibition of acid sphingomyelinase increases regulatory T cells in humans.

Genetic deficiency for acid sphingomyelinase or its pharmacological inhibition has been shown to increase Foxp3+ regulatory T-cell frequencies among CD4+ T cells in mice. We now investigated whether pharmacological targeting of the acid sphingomyelinase, which catalyzes the cleavage of sphingomyelin to ceramide and phosphorylcholine, also allows to manipulate relative CD4+ Foxp3+ regulatory T-cell frequencies in humans. Pharmacological acid sphingomyelinase inhibition with antidepressants like sertraline, but not those without an inhibitory effect on acid sphingomyelinase activity like citalopram, increased the frequency of Foxp3+ regulatory T cell among human CD4+ T cells in vitro. In an observational prospective clinical study with patients suffering from major depression, we observed that acid sphingomyelinase-inhibiting antidepressants induced a stronger relative increase in the frequency of CD4+ Foxp3+ regulatory T cells in peripheral blood than acid sphingomyelinase-non- or weakly inhibiting antidepressants. This was particularly true for CD45RA- CD25high effector CD4+ Foxp3+ regulatory T cells. Mechanistically, our data indicate that the positive effect of acid sphingomyelinase inhibition on CD4+ Foxp3+ regulatory T cells required CD28 co-stimulation, suggesting that enhanced CD28 co-stimulation was the driver of the observed increase in the frequency of Foxp3+ regulatory T cells among human CD4+ T cells. In summary, the widely induced pharmacological inhibition of acid sphingomyelinase activity in patients leads to an increase in Foxp3+ regulatory T-cell frequencies among CD4+ T cells in humans both in vivo and in vitro.

Translational Approaches Targeting Ceramide Generation From Sphingomyelin in T Cells to Modulate Immunity in Humans.

In T cells, as in all other cells of the body, sphingolipids form important structural components of membranes. Due to metabolic modifications, sphingolipids additionally play an active part in the signaling of cell surface receptors of T cells like the T cell receptor or the co-stimulatory molecule CD28. Moreover, the sphingolipid composition of their membranes crucially affects the integrity and function of subcellular compartments such as the lysosome. Previously, studying sphingolipid metabolism has been severely hampered by the limited number of analytical methods/model systems available. Besides well-established high resolution mass spectrometry new tools are now available like novel minimally modified sphingolipid subspecies for click chemistry as well as recently generated mouse mutants with deficiencies/overexpression of sphingolipid-modifying enzymes. Making use of these tools we and others discovered that the sphingolipid sphingomyelin is metabolized to ceramide to different degrees in distinct T cell subpopulations of mice and humans. This knowledge has already been translated into novel immunomodulatory approaches in mice and will in the future hopefully also be applicable to humans. In this paper we are, thus, summarizing the most recent findings on the impact of sphingolipid metabolism on T cell activation, differentiation, and effector functions. Moreover, we are discussing the therapeutic concepts arising from these insights and drugs or drug candidates which are already in clinical use or could be developed for clinical use in patients with diseases as distant as major depression and chronic viral infection.

The Activity of the Neutral Sphingomyelinase Is Important in T Cell Recruitment and Directional Migration.

Breakdown of sphingomyelin as catalyzed by the activity of sphingomyelinases profoundly affects biophysical properties of cellular membranes which is particularly important with regard to compartmentalization of surface receptors and their signaling relay. As it is activated both upon TCR ligation and co-stimulation in a spatiotemporally controlled manner, the neutral sphingomyelinase (NSM) has proven to be important in T cell activation, where it appears to play a particularly important role in cytoskeletal reorganization and cell polarization. Because these are important parameters in directional T cell migration and motility in tissues, we analyzed the role of the NSM in these processes. Pharmacological inhibition of NSM interfered with early lymph node homing of T cells in vivo indicating that the enzyme impacts on endothelial adhesion, transendothelial migration, sensing of chemokine gradients or, at a cellular level, acquisition of a polarized phenotype. NSM inhibition reduced adhesion of T cells to TNF-α/IFN-γ activated, but not resting endothelial cells, most likely via inhibiting high-affinity LFA-1 clustering. NSM activity proved to be highly important in directional T cell motility in response to SDF1-α, indicating that their ability to sense and translate chemokine gradients might be NSM dependent. In fact, pharmacological or genetic NSM ablation interfered with T cell polarization both at an overall morphological level and redistribution of CXCR4 and pERM proteins on endothelial cells or fibronectin, as well as with F-actin polymerization in response to SDF1-α stimulation, indicating that efficient directional perception and signaling relay depend on NSM activity. Altogether, these data support a central role of the NSM in T cell recruitment and migration both under homeostatic and inflamed conditions by regulating polarized redistribution of receptors and their coupling to the cytoskeleton.