Jérémie Rossy

Biotechnology Institute Thurgau

Tumor-specific T cells are frequently exhausted by chronic antigenic stimulation. We here report on a human antigen-specific ex vivo model to explore new therapeutic options for T cell immunotherapies. T cells generated with this model resemble tumor-infiltrating exhausted T cells on a phenotypic and transcriptional level. Using a targeted pooled C...

Background T cell activation leads to increased expression of the receptor for the iron transporter transferrin (TfR) to provide iron required for the cell differentiation and clonal expansion that takes place during the days after encounter with a cognate antigen. However, T cells mobilise TfR to their surface within minutes after activation, alth...

T cell activation requires engagement of a cognate antigen by the T cell receptor (TCR) and the co-stimulatory signal of CD28. Both TCR and CD28 aggregate into clusters at the plasma membrane of activated T cells. While the role of TCR clustering in T cell activation has been extensively investigated, little is known about how CD28 clustering contr...

Endocytic trafficking controls the density of molecules at the plasma membrane and by doing so, the cell surface profile, which in turn determines how cells interact with their environment. A full apprehension of any cellular process necessitates to understand how proteins associated with the plasma membrane are endocytosed, how they are sorted aft...

Endocytic trafficking relies on highly localized events in cell membranes. Endocytosis involves the gathering of protein (cargo/receptor) at distinct plasma membrane locations defined by specific lipid and protein compositions. Simultaneously, the molecular machinery that drives invagination and eventually scission of the endocytic vesicle assemble...

Dendritic cell (DC) aggresome-like induced structures (DALIS) are protein aggregates of polyubiquitylated proteins that form transiently during DC maturation. DALIS scatter randomly throughout the cytosol and serve as antigen storage sites synchronising DC maturation and antigen presentation. Maturation of DCs is accompanied by the induction of the...

T cell receptor phosphorylation by Lck is an essential step in T cell activation. It is known that the conformational states of Lck control enzymatic activity; however, the underlying principles of how Lck finds its substrate over the plasma membrane remain elusive. Here, single-particle tracking is paired with photoactivatable localization microsc...

: T cell activation is immediately followed by internalization of the T cell receptor (TCR). TCR endocytosis is required for T cell activation, but the mechanisms supporting removal of TCR from the cell surface remain incompletely understood. Here we report that TCR endocytosis is linked to the clathrin-independent carrier (CLIC) and GPI-enriched e...

The targeted endocytic recycling of the T cell receptor (TCR) to the immunological synapse is essential for T cell activation. Despite this, the mechanisms that underlie the sorting of internalised receptors into recycling endosomes remain poorly understood. To build a comprehensive picture of TCR recycling during T cell activation, we developed a...

T cell migration from blood to, and within lymphoid organs and tissue, as well as, T cell activation rely on complex biochemical signaling events. But T cell migration and activation also take place in distinct mechanical environments and lead to drastic morphological changes and reorganization of the acto-myosin cytoskeleton. In this review we dis...

T cell receptor (TCR) phosphorylation by Lck is an essential step in T cell activation. It is known the conformational states of Lck control enzymatic activity; however, the underlying principles of how Lck finds its substrate in the plasma membrane remain elusive. Here, single-particle tracking is paired with photoactivatable localization microsco...

Endocytosis of surface receptors and their polarized recycling back to the plasma membrane are central to many cellular processes, such as cell migration, cytokinesis, basolateral polarity of epithelial cells and T cell activation. Little is known about the mechanisms that control the organization of recycling endosomes and how they connect to rece...

Clustering of the T-cell receptor (TCR) is thought to initiate downstream signalling. However, the detection of protein clustering with high spatial and temporal resolution remains challenging. Here we establish a Förster resonance energy transfer (FRET) sensor, named CliF, which reports intermolecular associations of neighbouring proteins in live...

Membrane charge has a critical role in protein trafficking and signaling. However, quantification of the effective electrostatic potential of cellular membranes has remained challenging. We developed a fluorescence membrane charge sensor (MCS) that reports changes in the membrane charge of live cells via Förster resonance energy transfer (FRET). MC...

Significance T-cell activation requires the translation of antigen binding to the T-cell receptor (TCR) into intracellular signaling. However, how antigen recognition and signal transduction are mechanistically linked is poorly understood. Here, we used single-molecule localization microscopy to link TCR clustering to signaling. We found that the l...

There is emerging evidence that exocytosis plays an important role in regulating T cell receptor (TCR) signaling. The trafficking molecules involved in lytic granule (LG) secretion in cytotoxic T lymphocytes (CTL) have been well-studied due to the immune disorder known as familial hemophagocytic lymphohistiocytosis (FHLH). However, the knowledge of...

Although it is recognized that lipids and membrane organization in T cells affect signaling and T cell activation, to what extent dietary lipids alter T cell responsiveness in the absence of obesity and inflammation is not known. In this study, we fed low-density lipoprotein receptor knockout mice a Western high-fat diet for 1 or 9 wk and examined...

Phosphorylation of the T cell receptor (TCR) by the kinase Lck is the first detectable signaling event upon antigen engagement. The distribution of Lck within the plasma membrane, its conformational state, kinase activity, and protein–protein interactions all contribute to determine how efficiently Lck phosphorylates the engaged TCR. Here, we used...

Annexin A6 (AnxA6) has been implicated in cell signalling by contributing to the organisation of the plasma membrane. Here, we examined whether AnxA6 regulates signalling and proliferation in T cells. We used a contact hypersensitivity model to immune challenge wild-type (WT) and AnxA6(-/-) mice and found that the in vivo proliferation of CD4+ T ce...

Viruses are often thought to have static structure, and they only remodel after the viruses have entered target cells. Here, we detected a size expansion of virus particles prior to viral entry using cryo-electron microscopy (cryo-EM) and single molecule fluorescence imaging. HIV expanded both under cell-free conditions with soluble receptor CD4 (s...

Measuring protein dynamics in the plasma membrane can provide insights into the mechanisms of receptor signaling and other cellular functions. To quantify protein dynamics on the single molecule level over the entire cell surface, sophisticated approaches such as single particle tracking (SPT), photo-activation localization microscopy (PALM) and fl...

Objective: Therapeutic interventions that increase plasma levels of high-density lipoproteins and apolipoprotein A-I (apoA-I) A-I, the major high-density lipoprotein apolipoprotein, improve glycemic control in people with type 2 diabetes mellitus. High-density lipoproteins and apoA-I also enhance insulin synthesis and secretion in isolated pancrea...

Cell membranes are a complex adaptive system: they are constantly re-organised in response to extra- and intracellular inputs and their local and global structure ultimately determines how, where and when these inputs are processed. This requires a tight coupling of signalling and membranes in localised and specialised compartments. While lipids ar...

We demonstrate a combined univariate and bivariate Getis and Franklin's local point pattern analysis method to investigate the co-clustering of membrane proteins in two-dimensional single-molecule localisation data. This method assesses the degree of clustering of each molecule relative to its own species and relative to a second species. Using sim...

Significance Cell-expressed integrins mediate adhesion with other cells and with extracellular matrix and are essential for embryonic development and for controlling leukocyte migration in later life. Integrin adhesion depends on conformational change leading to activation, although it remains unknown exactly how integrins alter their conformationa...

Identifying the three-dimensional molecular organization of subcellular organelles in intact cells has been challenging to date. Here we present an analysis approach for three-dimensional localization microscopy that can not only identify subcellular objects below the diffraction limit but also quantify their shape and volume. This approach is part...

Signalling endosomes represent a general mechanism to modulate and compartmentalise cell signalling, which is achieved by delineating specific spatial environments and connecting the plasma membrane with intracellular events. The molecular composition of vesicles, their targeting mechanisms and endocytic routes contribute to the outcome of signalli...

Cells continuously communicate with the surrounding environment employing variety of signaling molecules and pathways to integrate and transport the information in the cell. An example of signaling initiation is binding of extracellular ligand to its receptor at the plasma membrane. This initializes enzymatic reactions leading to the formation of b...

It is now recognized that the plasma membrane is not homogeneous but instead contains a variety of membrane microdomains. These include lipid microdomains (lipid rafts) and protein clusters which exist on a range of size and time scales but are often small and short-lived. The small size and dynamic nature of membrane domains has made them difficul...

Phosphorylation of the T cell antigen receptor (TCR) by the tyrosine kinase Lck is an essential step in the activation of T cells. Because Lck is constitutively active, spatial organization may regulate TCR signaling. Here we found that Lck distributions on the molecular level were controlled by the conformational states of Lck, with the open, acti...

Engagement of the T cell antigen receptor (TCR) triggers signaling pathways that lead to T cell selection, differentiation and clonal expansion. Superimposed onto the biochemical network is a spatial organization that describes individual receptor molecules, dimers, oligomers and higher order structures. Here we discuss recent findings and new conc...

T cell signaling begins with the ligation of the T cell antigen receptor (TCR) by a cognate peptide and the phosphorylation of the receptor’s immunoreceptor tyrosine-based activation motif domains by the kinase Lck. However, the canonical receptor model is insufficient to explain how the constitutively active kinase Lck can discriminate between non...

The visualization of viral proteins has been hindered by the resolution limit of conventional fluorescent microscopes, as the dimension of any single fluorescent signal is often greater than most virion particles. Super-resolution microscopy has the potential to unveil the distribution of proteins at the resolution approaching electron microscopy w...

T cell activation begins with the formation of signaling complexes at the cell surface involving among others, the T cell antigen receptor (TCR), the Src family kinase Lck and the adaptor protein, linker for activation of T cells (LAT). We have established photo-activatable localization microscopy (PALM), stochastic optical reconstruction microscop...

Different types of membrane microdomains (rafts) have been postulated to be present in the rear and front of polarized migrating T-lymphocytes. Disruption of rafts by cholesterol sequestration prevents T-cell polarization and migration. Reggie/flotillin-1 and -2 are two highly homologous proteins that are thought to shape membrane microdomains. We...

Engaged T cell antigen receptors (TCRs) initiate signaling through the adaptor protein Lat. In quiescent T cells, Lat is segregated into clusters on the cell surface, which raises the question of how TCR triggering initiates signaling. Using super-resolution fluorescence microscopy, we found that pre-existing Lat domains were neither phosphorylated...

The authors employed photoactivatable localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM) imaging and image analysis based on Ripley's K -function to quantify the distribution and heterogeneity of proteins at the cell plasma membrane. The membrane targeting sequence of the N-terminal region of the T cell...

Background: Neutrophils polarize and migrate in response to chemokines. Different types of membrane microdomains (rafts) have been postulated to be present in rear and front of polarized leukocytes and disruption of rafts by cholesterol sequestration prevents leukocyte polarization. Reggie/flotillin-1 and -2 are two highly homologous proteins that...

Ezrin, radixin and moesin (ERM) proteins are widely distributed proteins located in the cellular cortex, in microvilli and adherens junctions. They feature an N-terminal membrane binding domain linked by an alpha-helical domain to the C-terminal actin-binding domain. In the dormant state, binding sites in the N-terminal domain are masked by interac...

Rat Walker 256 carcinosarcoma cells spontaneously develop front-tail polarity and migrate in the absence of added stimuli. Constitutive activation of phosphatidylinositol-3 kinase (PI 3-kinase), Rac, Rho and Rho kinase are essential for these processes. Ezrin and moesin are putative targets of these signaling pathways leading to spontaneous migrati...

As previously shown, constitutive activation of the small GTPase Rho and its downstream target Rho-kinase is crucial for spontaneous migration of Walker carcinosarcoma cells. We now show that after treatment of cells with either the Rho inhibitor C3 exoenzyme or the Rho-kinase inhibitor Y-27632, constitutive myosin light chain (MLC) phosphorylation...