Conformational states of the kinase Lck regulate clustering in early T cell signaling.

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, active conformation inducing clustering and the closed, inactive conformation preventing clustering. In contrast, association with lipid domains and protein networks were not sufficient or necessary for Lck clustering. Conformation-driven Lck clustering was highly dynamic, so that TCR triggering resulted in Lck clusters that contained phosphorylated TCRs but excluded the phosphatase CD45. Our data suggest that Lck conformational states represent an intrinsic mechanism for the intermolecular organization of early T cell signaling.

Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events.

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 nor laterally transported to TCR activation sites, which suggested that these clusters do not participate in TCR signaling. Instead, TCR activation resulted in the recruitment and phosphorylation of Lat from subsynaptic vesicles. Studies of Lat mutants confirmed that recruitment preceded and was essential for phosphorylation and that both processes were independent of surface clustering of Lat. Our data suggest that TCR ligation preconditions the membrane for vesicle recruitment and bulk activation of the Lat signaling network.

Rapid increase in transferrin receptor recycling promotes adhesion during T cell activation.

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, although the reason and mechanism driving this process remain unclear. RESULTS: Here we show that T cells transiently increase endocytic uptake and recycling of TfR upon activation, thereby boosting their capacity to import iron. We demonstrate that increased TfR recycling is powered by a fast endocytic sorting pathway relying on the membrane proteins flotillins, Rab5- and Rab11a-positive endosomes. Our data further reveal that iron import is required for a non-canonical signalling pathway involving the kinases Zap70 and PAK, which controls adhesion of the integrin LFA-1 and eventually leads to conjugation with antigen-presenting cells. CONCLUSIONS: Altogether, our data suggest that T cells boost their iron importing capacity immediately upon activation to promote adhesion to antigen-presenting cells.

FAT10 localises in dendritic cell aggresome-like induced structures and contributes to their disassembly.

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 ubiquitin-like modifier FAT10 (also known as UBD), which localises to aggresomes, structures that are similar to DALIS. FAT10 is conjugated to substrate proteins and serves as a signal for their rapid and irreversible degradation by the 26S proteasome similar to, yet independently of ubiquitin, thereby contributing to antigen presentation. Here, we have investigated whether FAT10 is involved in the formation and turnover of DALIS, and whether proteins accumulating in DALIS can be modified through conjunction to FAT10 (FAT10ylated). We found that FAT10 localises to DALIS in maturing DCs and that this localisation occurs independently of its conjugation to substrates. Additionally, we investigated the DALIS turnover in FAT10-deficient and -proficient DCs, and observed FAT10-mediated disassembly of DALIS. Thus, we report further evidence that FAT10 is involved in antigen processing, which may provide a functional rationale as to why FAT10 is selectively induced upon DC maturation.

Conformational States Control Lck Switching between Free and Confined Diffusion Modes in T Cells.

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 microscopy to observe the diffusive modes of Lck in the plasma membrane. Individual Lck molecules switched between free and confined diffusion in both resting and stimulated T cells. Lck mutants locked in the open conformation were more confined than Lck mutants in the closed conformation. Further confinement of kinase-dead versions of Lck suggests that Lck confinement was not caused by phosphorylated substrates. Our data support a model in which confined diffusion of open Lck results in high local phosphorylation rates, and inactive, closed Lck diffuses freely to enable long-range distribution over the plasma membrane.

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination.

Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR-CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR-CD3 complexes. We found that only TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR-CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.

Prolonged Intake of Dietary Lipids Alters Membrane Structure and T Cell Responses in LDLr-/- Mice.

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 T cell responses in vivo along with T cell lipid composition, membrane order, and activation ex vivo. Our data showed that high levels of circulating lipids for a prolonged period elevated CD4(+) and CD8(+) T cell proliferation and resulted in an increased proportion of CD4(+) central-memory T cells within the draining lymph nodes following induction of contact hypersensitivity. In addition, the 9-wk Western high-fat diet elevated the total phospholipid content and monounsaturated fatty acid level, but decreased saturated phosphatidylcholine and sphingomyelin within the T cells. The altered lipid composition in the circulation, and of T cells, was also reflected by enhanced membrane order at the activation site of ex vivo activated T cells that corresponded to increased IL-2 mRNA levels. In conclusion, dietary lipids can modulate T cell lipid composition and responses in lipoprotein receptor knockout mice even in the absence of excess weight gain and a proinflammatory environment.

Dynamic control of ß1 integrin adhesion by the plexinD1-sema3E axis.

Plexins and semaphorins comprise a large family of receptor-ligand pairs controlling cell guidance in nervous, immune, and vascular systems. How plexin regulation of neurite outgrowth, lymphoid trafficking, and vascular endothelial cell branching is linked to integrin function, central to most directed movement, remains unclear. Here we show that on developing thymocytes, plexinD1 controls surface topology of nanometer-scaled ß1 integrin adhesion domains in cis, whereas its ligation by sema3E in trans regulates individual ß1 integrin catch bonds. Loss of plexinD1 expression reduces ß1 integrin clustering, thereby diminishing avidity, whereas sema3E ligation shortens individual integrin bond lifetimes under force to reduce stability. Consequently, both decreased expression of plexinD1 during developmental progression and a thymic medulla-emanating sema3E gradient enhance thymocyte movement toward the medulla, thus enforcing the orchestrated lymphoid trafficking required for effective immune repertoire selection. Our results demonstrate plexin-tunable molecular features of integrin adhesion with broad implications for many cellular processes.

Annexin A6 regulates interleukin-2-mediated T-cell proliferation.

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 cells, but not CD8(+) T cells, was impaired in AnxA6(-/-) relative to WT mice. However, T-cell migration and signalling through the T-cell receptor ex vivo was similar between T cells isolated from AnxA6(-/-) and WT mice. In contrast, interleukin-2 (IL-2) signalling was reduced in AnxA6(-/-) compared with WT T cells. Further, AnxA6-deficient T cells had reduced membrane order and cholesterol levels. Taken together, our data suggest that AnxA6 regulates IL-2 homeostasis and sensitivity in T cells by sustaining a lipid raft-like membrane environment.

Apolipoprotein A-I increases insulin secretion and production from pancreatic ß-cells via a G-protein-cAMP-PKA-FoxO1-dependent mechanism.

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 pancreatic islets and clonal ß-cell lines. This study identifies the signaling pathways that mediate these effects. APPROACH AND RESULTS: Incubation with apoA-I increased cAMP accumulation in Ins-1E cells in a concentration-dependent manner. The increase in cAMP levels was inhibited by preincubating the cells with the cell-permeable, transmembrane adenylate cyclase inhibitor, 2'5' dideoxyadenosine, but not with KH7, which inhibits soluble adenylyl cyclases. Incubation of Ins-1E cells with apoA-I resulted in colocalization of ATP-binding cassette transporter A1 with the Gαs subunit of a heterotrimeric G-protein and a Gαs subunit-dependent increase in insulin secretion. Incubation of Ins-1E cells with apoA-I also increased protein kinase A phosphorylation and reduced the nuclear localization of forkhead box protein O1 (FoxO1). Preincubation of Ins-1E cells with the protein kinase A-specific inhibitors, H89 and PKI amide, prevented apoA-I from increasing insulin secretion and mediating the nuclear exclusion of FoxO1. Transfection of Ins-1E cells with a mutated FoxO1 that is restricted to the nucleus confirmed the requirement for FoxO1 nuclear exclusion by blocking insulin secretion in apoA-I-treated Ins-1E cells. ApoA-I also increased Irs1, Irs2, Ins1, Ins2, and Pdx1 mRNA levels. CONCLUSIONS: ApoA-I increases insulin synthesis and secretion via a heterotrimeric G-protein-cAMP-protein kinase A-FoxO1-dependent mechanism that involves transmembrane adenylyl cyclases and increased transcription of key insulin response and ß-cell survival genes.

Method for co-cluster analysis in multichannel single-molecule localisation data.

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 simulated data, we show that this approach can quantify the degree of cluster overlap in multichannel point patterns. The method is validated using photo-activated localisation microscopy and direct stochastic optical reconstruction microscopy data of the proteins Lck and CD45 at the T cell immunological synapse. Analysing co-clustering in this manner is generalizable to higher numbers of fluorescent species and to three-dimensional or live cell data sets.

Quantitative analysis of three-dimensional fluorescence localization microscopy data.

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 particularly useful to map the topography of the plasma membrane and measure protein distribution within an undulating membrane.

Deletion of SNX9 alleviates CD8 T cell exhaustion for effective cellular cancer immunotherapy.

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 CRISPR-Cas9 screen and individual gene knockout validation experiments, we uncover sorting nexin-9 (SNX9) as a mediator of T cell exhaustion. Upon TCR/CD28 stimulation, deletion of SNX9 in CD8 T cells decreases PLCγ1, Ca2+, and NFATc2-mediated T cell signaling and reduces expression of NR4A1/3 and TOX. SNX9 knockout enhances memory differentiation and IFNγ secretion of adoptively transferred T cells and results in improved anti-tumor efficacy of human chimeric antigen receptor T cells in vivo. Our findings highlight that targeting SNX9 is a strategy to prevent T cell exhaustion and enhance anti-tumor immunity.

HIV taken by STORM: super-resolution fluorescence microscopy of a viral infection.

BACKGROUND: 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 without relying on morphological features of existing characteristics of the biological specimen that are needed in EM. RESULTS: Using direct stochastic optical reconstruction microscopy (dSTORM) to achieve a lateral resolution of 15-20 nm, we quantified the 2-D molecular distribution of the major structural proteins of the infectious human immunodeficiency virus type 1 (HIV-1) before and after infection of lymphoid cells. We determined that the HIV-1 matrix and capsid proteins undergo restructuring soon after HIV-1 infection. CONCLUSIONS: This study provides the proof-of-concept for the use of dSTORM to visualize the changes in the molecular distribution of viral proteins during an infection.

SNX9-induced membrane tubulation regulates CD28 cluster stability and signalling.

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 contributes to CD28 signalling. Here, we report that upon CD28 triggering, the BAR-domain protein sorting nexin 9 (SNX9) is recruited to CD28 clusters at the immunological synapse. Using three-dimensional correlative light and electron microscopy, we show that SNX9 generates membrane tubulation out of CD28 clusters. Our data further reveal that CD28 clusters are in fact dynamic structures and that SNX9 regulates their stability as well as CD28 phosphorylation and the resulting production of the cytokine IL-2. In summary, our work suggests a model in which SNX9-mediated tubulation generates a membrane environment that promotes CD28 triggering and downstream signalling events.

Dynamic reorganization of flotillins in chemokine-stimulated human T-lymphocytes.

BACKGROUND: 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 have previously demonstrated the enrichment of flotillins in the uropod of human neutrophils. We have now investigated mechanisms involved in chemokine-induced flotillin reorganization in human T-lymphocytes, and possible roles of flotillins in lymphocyte polarization. RESULTS: We studied flotillin reorganization and lateral mobility at the plasma membrane using immunofluorescence staining and FRAP (fluorescence recovery after photobleaching). We show that flotillins redistribute early upon chemokine stimulation, and form very stable caps in the uropods of human peripheral blood T-lymphocytes, colocalizing with the adhesion molecule PSGL-1 and activated ezrin/radixin/moesin (ERM) proteins. Chemokine-induced formation of stable flotillin caps requires integrity and dynamics of the actin cytoskeleton, but is not abolished by inhibitors suppressing Rho-kinase or myosin II activity. Tagged flotillin-2 and flotillin-1 coexpressed in T-lymphocytes, but not singly expressed proteins, colocalize in stable caps at the tips of uropods. Lateral mobility of coexpressed flotillins at the plasma membrane is already partially restricted in the absence of chemokine. Incubation with chemokine results in almost complete immobilization of flotillins. Capping is abolished when wild-type flotillin-1 is coexpressed with a mutant of flotillin-2 (G2A) that is unable to interact with the plasma membrane, or with a deletion mutant of flotillin-2 that lacks a putative actin-binding domain. Wild-type flotillin-2 in contrast forms caps when coexpressed with a mutant of flotillin-1 unable to interact with membranes. Transfection of T-lymphocytes with flotillin-2-G2A reduces cell polarization and uropod recruitment of endogenous flotillin-1 and PSGL-1. CONCLUSIONS: Our data suggest that stable flotillin cap formation in the rear of polarized T-lymphocytes requires flotillin heterooligomer formation, as well as direct F-actin interactions of flotillin-2 and raft/membrane association of flotillin-2, but not -1. Our data also implicate flotillin-rich actin-dependent membrane microdomains in T-lymphocyte uropod formation.

Quantitative visualization of endocytic trafficking through photoactivation of fluorescent proteins.

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 understanding how proteins associated with the plasma membrane are endocytosed, how they are sorted after internalization, and if and how they are recycled to the plasma membrane. To date, it is still difficult to experimentally gain access to this information, even more to do it in a quantitative way. Here we present a toolset based on photoactivation of fluorescent proteins that enabled us to generate quantitative information on endocytosis, incorporation into sorting and recycling endosomes, delivery from endosomes to the plasma membrane, and on the type of vesicles performing intracellular transport. We illustrate these approaches by revealing striking differences in the endocytic trafficking of T-cell receptor and CD4, which bind to the same molecule at the surface of antigen-presenting cells during T-cell activation.

Membrane Heterogeneity Controls Cellular Endocytic Trafficking.

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 assembles at the very same place on the inner leaflet of the membrane. It is membrane heterogeneity - the existence of specific lipid and protein domains in localized regions of membranes - that creates the distinct molecular identity required for an endocytic event to occur precisely when and where it is required rather than at some random location within the plasma membrane. Accumulating evidence leads us to believe that the trafficking fate of internalized proteins is sealed following endocytosis, as this distinct membrane identity is preserved through the endocytic pathway, upon fusion of endocytic vesicles with early and sorting endosomes. In fact, just like at the plasma membrane, multiple domains coexist at the surface of these endosomes, regulating local membrane tubulation, fission and sorting to recycling pathways or to the trans-Golgi network via late endosomes. From here, membrane heterogeneity ensures that fusion events between intracellular vesicles and larger compartments are spatially regulated to promote the transport of cargoes to their intracellular destination.

Cdc42 Couples T Cell Receptor Endocytosis to GRAF1-Mediated Tubular Invaginations of the Plasma Membrane.

: 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 endocytic compartments (GEEC) endocytic pathway. We show that unlike the canonical clathrin cargo transferrin or the adaptor protein Lat, internalized TCR accumulates in tubules shaped by the small GTPase Cdc42 and the Bin/amphiphysin/Rvs (BAR) domain containing protein GRAF1 in T cells. Preventing GRAF1-positive tubules to mature into endocytic vesicles by expressing a constitutively active Cdc42 impairs the endocytosis of TCR, while having no consequence on the uptake of transferrin. Together, our data reveal a link between TCR internalization and the CLIC/GEEC endocytic route supported by Cdc42 and GRAF1.

Flotillins promote T cell receptor sorting through a fast Rab5-Rab11 endocytic recycling axis.

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 suite of new imaging and quantification tools centred on photoactivation of fluorescent proteins. We show that the membrane-organising proteins, flotillin-1 and -2, are required for TCR to reach Rab5-positive endosomes immediately after endocytosis and for transfer from Rab5- to Rab11a-positive compartments. We further observe that after sorting into in Rab11a-positive vesicles, TCR recycles to the plasma membrane independent of flotillin expression. Our data suggest a mechanism whereby flotillins delineate a fast Rab5-Rab11a endocytic recycling axis and functionally contribute to regulate the spatial organisation of these endosomes.