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.

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.

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.

A mobile endocytic network connects clathrin-independent receptor endocytosis to recycling and promotes T cell activation.

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 receptor endocytosis. Here, we follow the endocytic journey of the T cell receptor (TCR), from internalization at the plasma membrane to recycling back to the immunological synapse. We show that TCR triggering leads to its rapid uptake through a clathrin-independent pathway. Immediately after internalization, TCR is incorporated into a mobile and long-lived endocytic network demarked by the membrane-organizing proteins flotillins. Although flotillins are not required for TCR internalization, they are necessary for its recycling to the immunological synapse. We further show that flotillins are essential for T cell activation, supporting TCR nanoscale organization and signaling.

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.

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.

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.

PALM imaging and cluster analysis of protein heterogeneity at the cell surface.

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 receptor-pathway kinase Lck fused to the photo-convertible fluorescent protein tdEos (Lck(N10)-tdEos), clusters into sub-100 nm regions which cover approximately 7% of the cell surface. 2-channel PALM imaging of Lck(N10)-tdEos and the N-terminus of the kinase Src (Src(N15)-PS-CFP2) are demonstrated. Finally, T cell microclusters at the immune synapse are imaged at super-resolution using dSTORM, showing that conventional TIRF images contain unresolved, small clusters. These methods are generally applicable to other cell and fluorophore systems to quantify 2-D molecular clustering at nanometer scales.

Ezrin/radixin/moesin: versatile controllers of signaling molecules and of the cortical cytoskeleton.

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 interactions with the C-terminal region. The alpha-helical domain also contributes to masking of binding sites. A specific sequence of signaling events results in dissociation of these intramolecular interactions resulting in ERM activation. ERM molecules have been implicated in mediating actin-membrane linkage and in regulating signaling molecules. They are involved in cell membrane organization, cell migration, phagocytosis and apoptosis, and may also play cell-specific roles in tumor progression. Their precise involvement in these processes has yet to be elucidated.

Ezrin/moesin in motile Walker 256 carcinosarcoma cells: signal-dependent relocalization and role in migration.

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 migration. To test this hypothesis, we used specific siRNA probes that resulted in a downregulation of ezrin and moesin by about 70% and in a similar reduction in the fraction of migrating cells. Spontaneous polarization however was not affected, indicating a more subtle role of ezrin and moesin in migration. We provide furthermore evidence that endogenous ezrin and moesin colocalize with F-actin at the contracted tail of polarized cells, similar to ectopically expressed green fluorescent protein-tagged ezrin. Our results suggest that myosin light chain and ezrin are markers of front and tail, respectively, even in the absence of morphological polarization. We further show that endogenous ezrin and moesin are phosphorylated and that activities of PI-3 kinase, Rho and Rac, but not of Rho-kinase, are required for this C-terminal phosphorylation. Activation of protein kinase C in contrast suppressed phosphorylation of ezrin and moesin. Inhibition of ezrin phosphorylation prevented its membrane association.

Role of Rho, Rac, and Rho-kinase in phosphorylation of myosin light chain, development of polarity, and spontaneous migration of Walker 256 carcinosarcoma cells.

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 is significantly decreased, correlating with inhibition of cell polarization and migration. Transfection with a dominant-negative Rho-kinase mutant similarly inhibits cell polarization and MLC phosphorylation. Transfection with a dominant-active Rho-kinase mutant leads to significantly increased MLC phosphorylation, membrane blebbing, and inhibition of cell polarization. This Rho-kinase-induced membrane blebbing can be inhibited by Y-27632, ML-7, and blebbistatin. Unexpectedly, overactivation of RhoA has similar effects as its inhibition. Introduction of a bacterially expressed constitutively activated mutant protein (but not of wild-type RhoA) into the cells or transfection of cells with a constitutively active RhoA mutant both inhibit polarization and decrease MLC phosphorylation. Transfection of cells with constitutively active or dominant-negative Rac both abrogate polarity, and the latter inhibits MLC phosphorylation. Our findings suggest an important role of Rac, Rho/Rho-kinase, and MLCK in controlling myosin activity in Walker carcinosarcoma cells and show that an appropriate level of RhoA, Rac, and Rho-kinase activity is required to regulate cell polarity and migration.