ABSTRACT
Chimeric antigen receptor (CAR) T cells have been used to successfully treat various blood cancers, but adverse effects have limited their potential. Here, we developed chimeric adaptor proteins (CAPs) and CAR tyrosine kinases (CAR-TKs) in which the intracellular ζ T cell receptor (TCRζ) chain was replaced with intracellular protein domains to stimulate signaling downstream of the TCRζ chain. CAPs contain adaptor domains and the kinase domain of ZAP70, whereas CAR-TKs contain only ZAP70 domains. We hypothesized that CAPs and CAR-TKs would be more potent than CARs because they would bypass both the steps that define the signaling threshold of TCRζ and the inhibitory regulation of upstream molecules. CAPs were too potent and exhibited high tonic signaling in vitro. In contrast, CAR-TKs exhibited high antitumor efficacy and significantly enhanced long-term tumor clearance in leukemia-bearing NSG mice as compared with the conventional CD19-28ζ-CAR-T cells. CAR-TKs were activated in a manner independent of the kinase Lck and displayed slower phosphorylation kinetics and prolonged signaling compared with the 28ζ-CAR. Lck inhibition attenuated CAR-TK cell exhaustion and improved long-term function. The distinct signaling properties of CAR-TKs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an antitumor chimeric receptor.
Subject(s)
Receptors, Antigen, T-Cell , Receptors, Chimeric Antigen , Signal Transduction , T-Lymphocytes , Animals , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/metabolism , Receptors, Chimeric Antigen/genetics , Humans , Signal Transduction/immunology , Mice , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , ZAP-70 Protein-Tyrosine Kinase/metabolism , ZAP-70 Protein-Tyrosine Kinase/genetics , ZAP-70 Protein-Tyrosine Kinase/immunology , Immunotherapy, Adoptive/methods , Mice, Inbred NOD , Cell Line, Tumor , PhosphorylationABSTRACT
Activation of the T cell antigen receptor (TCR) is a key step in initiating the adaptive immune response. Single-molecule localization techniques have been used to investigate the arrangement of proteins within the signaling complexes formed around activated TCRs, but a clear picture of nanoscale organization in stimulated T cells has not emerged. Here, we have improved the examination of T cell nanostructure by visualizing individual molecules of six different proteins in a single sample of activated Jurkat T cells using the multiplexed antibody-size limited direct stochastic optical reconstruction microscopy (madSTORM) technique. We formally define irregularly shaped regions of interest, compare areas where signaling complexes are concentrated with other areas, and improve the statistical analyses of the locations of molecules. We show that nanoscale organization of proteins is mainly confined to the areas with dense concentrations of TCR-based signaling complexes. However, randomly distributed molecules are also found in some areas containing concentrated signaling complexes. These results are consistent with the view that the proteins within signaling complexes are connected by numerous weak interactions, leading to flexible, dynamic, and mutable structures which produce large variations in the nanostructure found in activated T cells.
Subject(s)
Nanostructures , T-Lymphocytes , Receptors, Antigen, T-Cell , Cell Membrane , MicroscopyABSTRACT
We describe the first cases of germline biallelic null mutations in ARPC5, part of the Arp2/3 actin nucleator complex, in two unrelated patients presenting with recurrent and severe infections, early-onset autoimmunity, inflammation, and dysmorphisms. This defect compromises multiple cell lineages and functions, and when protein expression is reestablished in-vitro, the Arp2/3 complex conformation and functions are rescued. As part of the pathophysiological evaluation, we also show that interleukin (IL)-6 signaling is distinctively impacted in this syndrome. Disruption of IL-6 classical but not trans-signaling highlights their differential roles in the disease and offers perspectives for therapeutic molecular targets.
Subject(s)
Actin-Related Protein 2-3 Complex , Actins , Humans , Actin-Related Protein 2-3 Complex/genetics , Actin-Related Protein 2-3 Complex/metabolism , Actins/genetics , Actins/metabolism , Cell Movement , Germ-Line Mutation , Cytokines/geneticsABSTRACT
Activation of T cells upon engagement of the T cell antigen receptor rapidly leads to a number of phosphorylation and plasma membrane recruitment events. For example, translocation of phospholipase-Cγ1 (PLC-γ1) to the plasma membrane and its association with the transmembrane adapter protein LAT and two other adapter proteins, Gads and SLP-76, are critical events in the early T cell activation process. We have previously characterized the formation of a tetrameric LAT-Gads-SLP-76-PLC-γ1 complex by reconstitution in vitro and have also characterized the thermodynamics of tetramer formation. In the current study, we define how PLC-γ1 recruitment to liposomes, which serve as a plasma membrane surrogate, and PLC-γ1 activation are regulated both independently and additively by recruitment of PLC-γ1 to phosphorylated LAT, by formation of the LAT-Gads-SLP-76-PLC-γ1 tetramer, and by tyrosine phosphorylation of PLC-γ1. The recently solved structure of PLC-γ1 indicates that, in the resting state, several PLC-γ1 domains inhibit its enzymatic activity and contact with the plasma membrane. We propose the multiple cooperative steps that we observed likely lead to conformational alterations in the regulatory domains of PLC-γ1, enabling contact with its membrane substrate, disinhibition of PLC-γ1 enzymatic activity, and production of the phosphoinositide cleavage products necessary for T cell activation.
Subject(s)
Phospholipase C gamma , Signal Transduction , T-Lymphocytes , Enzyme Activation , Membrane Proteins/genetics , Membrane Proteins/metabolism , Phospholipase C gamma/genetics , Phospholipase C gamma/metabolism , Phosphorylation , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/enzymology , T-Lymphocytes/metabolismABSTRACT
The TMC genes encode a set of homologous transmembrane proteins whose functions are not well understood. Biallelic mutations in either TMC6 or TMC8 are detected in more than half of cases of the pre-malignant skin disease epidermodysplasia verruciformis (EV). It is controversial whether EV induced by mutations in TMC6 or TMC8 originates from keratinocyte or lymphocyte defects. Quantification of TMC6 and TMC8 RNA levels in various organs revealed that lymphoid tissues have the highest levels of expression of both genes, and custom antibodies confirmed protein expression in mouse lymphocytes. To study the function of these proteins we generated mice with targeted deletion mutant alleles of Tmc6 or Tmc8 Either TMC6 or TMC8 deficiency induced a reduction in apparent molecular weight and/or amount of the other TMC molecule. Co-immunoprecipitation experiments indicated that TMC6 and TMC8 formed a protein complex in mouse and human T cells. MS and biochemical analysis demonstrated that TMC6 and TMC8 additionally interacted with the CIB1 protein to form TMC6-TMC8-CIB1 trimers. We demonstrated that TMC6 and TMC8 regulated CIB1 levels by protecting CIB1 from ubiquitination and proteasomal degradation. Reciprocally, CIB1 was needed for stabilizing TMC6 and TMC8 levels. These results suggest why inactivating mutations in any of the three human genes leads to similar clinical presentations. We also demonstrated that TMC6 and TMC8 levels are drastically lower and the proteins are less active in regulating CIB1 in keratinocytes than in T cells. Our study suggests that defects in lymphocytes may contribute to the etiology and pathogenesis of EV.
Subject(s)
Calcium-Binding Proteins/metabolism , Membrane Proteins/metabolism , Multiprotein Complexes/metabolism , T-Lymphocytes/metabolism , Animals , Calcium-Binding Proteins/genetics , Humans , Jurkat Cells , Keratinocytes/cytology , Keratinocytes/metabolism , Membrane Proteins/genetics , Mice , Multiprotein Complexes/genetics , Proteolysis , T-Lymphocytes/cytology , UbiquitinationABSTRACT
LAT molecules defective in ubiquitination have an increased half-life and induce enhanced signaling when expressed in T cells. In this study, we have examined the role of ubiquitination in regulating LAT endocytosis, recycling, and degradation in resting and stimulated T cells. By tracking and comparing plasma membrane-labeled wild type and ubiquitination-resistant 2KR LAT, we find that ubiquitination promotes the degradation of surface LAT in T cells. Activation of T cells increases LAT ubiquitination and promotes trafficking of internalized LAT to lysosomes for degradation. Ubiquitination of LAT does not change internalization rates from the cell surface, but prevents efficient recycling of LAT to the surface of T cells. Our study demonstrates that surface LAT levels are tightly controlled by ubiquitination. LAT in unstimulated cells lacks ubiquitin allowing for increased LAT stability and efficient T cell activation upon TCR triggering; ubiquitination leads to efficient removal of LAT after activation.
Subject(s)
Lymphocyte Activation/physiology , Ubiquitination/physiology , Adaptor Proteins, Signal Transducing/metabolism , Cell Line , Endocytosis/physiology , Humans , Immunoblotting , Lysosomes/metabolism , Microscopy, Confocal , Phosphorylation/physiology , Signal Transduction/physiologyABSTRACT
When T cell receptors (TCRs) engage with stimulatory ligands, one of the first microscopically visible events is the formation of microclusters at the site of T cell activation. Since the discovery of these structures almost 20 years ago, they have been studied extensively in live cells using confocal and total internal reflection fluorescence (TIRF) microscopy. However, due to limits in image resolution and acquisition speed, the spatial relationships of signaling components within microclusters, the kinetics of their assembly and disassembly, and the role of vesicular trafficking in microcluster formation and maintenance were not finely characterized. In this review, we will summarize how new microscopy techniques have revealed novel insights into the assembly of these structures. The sub-diffraction organization of microclusters as well as the finely dissected kinetics of recruitment and disassociation of molecules from microclusters will be discussed. The role of cell surface molecules in microcluster formation and the kinetics of molecular recruitment via intracellular vesicular trafficking to microclusters is described. Finally, the role of post-translational modifications such as ubiquitination in the downregulation of cell surface signaling molecules is also discussed. These results will be related to the role of these structures and processes in T cell activation.
ABSTRACT
Normal function of the adaptive immune system requires trafficking of T cells between the blood and lymphoid organs. Lymphocyte homing to lymph nodes requires that they cross endothelial barriers present in blood vessels and lymphatics. This multi-step process requires a remodeling of the lymphocyte plasma membrane, which is mediated by the dynamic re-arrangement of the actin cytoskeleton. Pak1 plays a central role in cell morphology, adhesion and migration in various cell types. Here we demonstrate that Pak1 is required for activated CD4+ T cell trafficking to lymph nodes. Pak1 deficiency in T cells causes a defect in the transcription of CCR7 and L-selectin, thereby altering lymphocyte trafficking. Additionally, we report an increase in L-selectin shedding in Pak1-deficient T cells, which correlates with a decrease in the recruitment of calmodulin to the cytoplasmic tail of L-selectin during T cell activation. Overall, our findings demonstrate that by regulating the expression of two major lymph node homing molecules, L-selectin and CCR7, Pak1 mediates activated CD4+ T cell trafficking.
Subject(s)
Gene Expression Regulation , L-Selectin/genetics , Lymphocyte Activation/immunology , Receptors, CCR7/genetics , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , p21-Activated Kinases/metabolism , Animals , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cell Movement , Forkhead Box Protein O1/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , L-Selectin/metabolism , Lymph Nodes/immunology , Lymph Nodes/metabolism , Lymphocyte Activation/genetics , Mice , Mice, Knockout , Receptors, CCR7/metabolism , Signal Transduction , Transcription, GeneticABSTRACT
Engagement of the T cell receptor (TCR) by stimulatory ligand results in the rapid formation of microclusters at sites of T cell activation. Whereas microclusters have been studied extensively using confocal microscopy, the spatial and kinetic relationships of their signaling components have not been well characterized due to limits in image resolution and acquisition speed. Here we show, using TIRF-SIM to examine the organization of microclusters at sub-diffraction resolution, the presence of two spatially distinct domains composed of ZAP70-bound TCR and LAT-associated signaling complex. Kinetic analysis of microcluster assembly reveal surprising delays between the stepwise recruitment of ZAP70 and signaling proteins to the TCR, as well as distinct patterns in their disassociation. These delays are regulated by intracellular calcium flux downstream of T cell activation. Our results reveal novel insights into the spatial and kinetic regulation of TCR microcluster formation and T cell activation.
Subject(s)
Calcium/metabolism , Lymphocyte Activation/physiology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/immunology , ZAP-70 Protein-Tyrosine Kinase/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Calcium/immunology , Feedback, Physiological , Gene Knockout Techniques , Humans , Image Processing, Computer-Assisted , Intravital Microscopy/methods , Jurkat Cells , Kinetics , Leukocytes, Mononuclear , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microscopy, Fluorescence , Primary Cell Culture , Protein Domains/physiology , Receptors, Antigen, T-Cell/immunology , Signal Transduction/immunology , T-Lymphocytes/metabolism , ZAP-70 Protein-Tyrosine Kinase/immunologyABSTRACT
The T cell antigen receptor encounters foreign antigen during the immune response. Receptor engagement leads to activation of specific protein tyrosine kinases, which then phosphorylate multiple enzymes and adapter proteins. One such enzyme, phospholipase-Cγ1, is responsible for cleavage of a plasma membrane lipid substrate, a phosphoinositide, into two second messengers, diacylglycerol, which activates several enzymes including protein kinase C, and an inositol phosphate, which induces intracellular calcium elevation. In T cells, phospholipase-Cγ1 is recruited to the plasma membrane as part of a four-protein complex containing three adapter molecules. We have used recombinant proteins and synthetic phosphopeptides to reconstitute this quaternary complex in vitro. Extending biophysical tools to study concurrent interactions of the four protein components, we demonstrated the formation and determined the composition of the quaternary complex using multisignal analytical ultracentrifugation, and we characterized the thermodynamic driving forces of assembly by isothermal calorimetry. We demonstrate that the four proteins reversibly associate in a circular arrangement of binding interfaces, each protein interacting with two others. Three interactions are of high affinity, and the fourth is of low affinity, with the assembly of the quaternary complex exhibiting significant enthalpy-entropy compensation as in an entropic switch. Formation of this protein complex enables subsequent recruitment of additional molecules needed to activate phospholipase-Cγ1. Understanding the formation of this complex is fundamental to full characterization of a central pathway in T cell activation. Such knowledge is critical to developing ways in which this pathway can be selectively inhibited.
Subject(s)
Multiprotein Complexes/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Receptors, Antigen, T-Cell/metabolism , Signal Transduction/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 7/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Cell Membrane/metabolism , Humans , Membrane Lipids/metabolism , Membrane Proteins/metabolism , Phospholipase C gamma/metabolism , Phosphoproteins/metabolism , Phosphorylation , Recombinant Proteins , Thermodynamics , Tissue Plasminogen Activator/metabolismABSTRACT
Successful anti-viral response requires the sustained activation and expansion of CD8+ T cells for periods that far exceed the time limit of physical T cell interaction with antigen-presenting cells (APCs). The expanding CD8+ T cell pool generates the effector and memory cell populations that provide viral clearance and long-term immunity, respectively. Here, we demonstrate that 3BP2 is recruited in cytoplasmic microclusters and nucleates a signaling complex that facilitates MHC:peptide-independent activation of signaling pathways downstream of the TCR. We show that induction of the adaptor molecule 3BP2 is a sensor of TCR signal strength and is critical for sustaining CD8+ T cell proliferation and regulating effector and memory differentiation.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , CD8-Positive T-Lymphocytes/immunology , Cell Proliferation , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Differentiation , Cells, Cultured , Humans , Jurkat Cells , Lymphocyte Activation , Mice , Receptors, Antigen, T-Cell/immunology , Signal TransductionABSTRACT
The relative importance of plasma membrane-localized LAT versus vesicular LAT for microcluster formation and T-cell receptor (TCR) activation is unclear. Here, we show the sequence of events in LAT microcluster formation and vesicle delivery, using lattice light sheet microscopy to image a T cell from the earliest point of activation. A kinetic lag occurs between LAT microcluster formation and vesicular pool recruitment to the synapse. Correlative 3D light and electron microscopy show an absence of vesicles at microclusters at early times, but an abundance of vesicles as activation proceeds. Using TIRF-SIM to look at the activated T-cell surface with high resolution, we capture directed vesicle movement between microclusters on microtubules. We propose a model in which cell surface LAT is recruited rapidly and phosphorylated at sites of T-cell activation, while the vesicular pool is subsequently recruited and dynamically interacts with microclusters.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Cell Membrane/immunology , Cytoplasmic Vesicles/immunology , Lymphocyte Activation/genetics , Membrane Proteins/genetics , Microtubules/immunology , Receptors, Antigen, T-Cell/genetics , Adaptor Proteins, Signal Transducing/immunology , Cell Membrane/ultrastructure , Cytoplasmic Vesicles/ultrastructure , GRB2 Adaptor Protein/genetics , GRB2 Adaptor Protein/immunology , Gene Expression Regulation , Genes, Reporter , Humans , Immunological Synapses/metabolism , Immunological Synapses/ultrastructure , Jurkat Cells , Membrane Proteins/immunology , Microscopy, Fluorescence , Microtubules/ultrastructure , Phosphorylation , R-SNARE Proteins/genetics , R-SNARE Proteins/immunology , Receptors, Antigen, T-Cell/immunology , Signal Transduction , Time-Lapse Imaging , ZAP-70 Protein-Tyrosine Kinase/genetics , ZAP-70 Protein-Tyrosine Kinase/immunologyABSTRACT
Cish, participates within a multi-molecular E3 ubiquitin ligase complex, which ubiquitinates target proteins. It has an inhibitory effect on T cell activation mediated by PLC-γ1 regulation, and it functions as a potent checkpoint in CD8+ T cell tumor immunotherapy. To study the structural and functional relationships between Cish and PLC-γ1 during CD8+ T cell activation, we tested mutants of the Cish-SH2 (R107K) and D/BC (L222Q, C226Q) domains. We confirmed that Cish-SH2-specific binding was essential for PLC-γ1 ubiquitination and degradation. This domain was essential for the Cish-mediated inhibition of Ca2+ release upon TCR stimulation. No effect on inhibition of cytokine release was observed with SH2 or D/BC mutants, although the absence of Cish led to an increased release of IFN-γ and TNF-α. Using imaging we showed that Cish was expressed mostly in the cytoplasm and we did not see any Cish clustering at the plasma membrane upon stimulation. We conclude that the Cish-SH2 domain is essential for PLC-γ1 regulation in TCR-stimulated CD8+ T cells.
Subject(s)
CD8-Positive T-Lymphocytes/metabolism , Phospholipase C gamma/metabolism , Receptors, Antigen, T-Cell/metabolism , Suppressor of Cytokine Signaling Proteins/metabolism , src Homology Domains , Animals , CD8-Positive T-Lymphocytes/immunology , Calcium/metabolism , Cell Line , Cytokines/metabolism , Gene Expression , Humans , Lymphocyte Activation , Mice , Mice, Knockout , Phospholipase C gamma/chemistry , Phospholipase C gamma/genetics , Protein Binding , Protein Interaction Domains and Motifs , Signal Transduction , Suppressor of Cytokine Signaling Proteins/chemistry , Suppressor of Cytokine Signaling Proteins/geneticsABSTRACT
ZAP-70 is a tyrosine kinase that is essential for initiation of T cell antigen receptor (TCR) signaling. We have found that T cell p38 MAP kinase (MAPK), which is directly phosphorylated and activated by ZAP-70 downstream of the TCR, in turn phosphorylates Thr-293 in the interdomain B region of ZAP-70. Mutant T cells expressing ZAP-70 with an alanine substitution at this residue (ZAP-70T293A) had enhanced TCR proximal signaling and increased effector responses. Lack of ZAP-70T293 phosphorylation increased association of ZAP-70 with the TCR and prolonged the existence of TCR signaling microclusters. These results identify a tight negative feedback loop in which ZAP-70-activated p38 reciprocally phosphorylates ZAP-70 and destabilizes the signaling complex.
Subject(s)
Genes, T-Cell Receptor/physiology , ZAP-70 Protein-Tyrosine Kinase/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Amino Acid Sequence , Gene Expression Regulation , Humans , Jurkat Cells , Phosphorylation , Signal Transduction , ZAP-70 Protein-Tyrosine Kinase/genetics , p38 Mitogen-Activated Protein Kinases/geneticsABSTRACT
Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.
Subject(s)
Fluorescent Antibody Technique/methods , Microscopy, Fluorescence/methods , Single Molecule Imaging/methods , T-Lymphocytes/cytology , Fluorescent Dyes/chemistry , Humans , Jurkat Cells , Lymphocyte Activation , Receptors, Antigen, T-Cell/metabolism , Synaptosomal-Associated Protein 25/metabolism , T-Lymphocytes/metabolismABSTRACT
RAS signaling is central to many cellular processes and SOS proteins promote RAS activation. To investigate the role of SOS proteins in T cell biology, we crossed Sos1f/fSos2-/- mice to CD4-Cre transgenic mice. We previously reported an effect of these mutations on T cell signaling and T cell migration. Unexpectedly, we observed nodules on the joints of greater than 90% of these mutant mice at 5 months of age, especially on the carpal joints. As the mice aged further, some also displayed joint stiffness, hind limb paralysis, and lameness. Histological analysis indicated that the abnormal growth in joints originated from dysplastic chondrocytes. Second harmonic generation imaging of the carpal nodules revealed that nodules were encased by rich collagen fibrous networks. Nodules formed in mice also deficient in RAG2, indicating that conventional T cells, which undergo rearrangement of the T cell antigen receptor, are not required for this phenotype. CD4-Cre expression in a subset of cells, either immune lineage cells (e.g., non-conventional T cells) or non-immune lineage cells (e.g., chondrocytes) likely mediates the dramatic phenotype observed in this study. Disruptions of genes in the RAS signaling pathway are especially likely to cause this phenotype. These results also serve as a cautionary tale to those intending to use CD4-Cre transgenic mice to specifically delete genes in conventional T cells.
ABSTRACT
Single-molecule localization microscopy (SMLM) comprises methods that produce super-resolution images from molecular locations of single molecules. These techniques mathematically determine the center of a diffraction-limited spot produced by a fluorescent molecule, which represents the most likely location of the molecule. Only a small cohort of well-separated molecules is visualized in a single image, and then many images are obtained from a single sample. The localizations from all the images are combined to produce a super-resolution picture of the sample. Here we describe the application of two methods, photoactivation localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM), to the study of signaling microclusters in T cells.
Subject(s)
Molecular Imaging/methods , Receptors, Antigen, T-Cell/immunology , Signal Transduction/immunology , T-Lymphocytes/immunology , Humans , Jurkat CellsABSTRACT
Calcineurin is a phosphatase whose primary targets in T cells are NFAT transcription factors, and inhibition of calcineurin activity by treatment with cyclosporin A (CsA) or FK506 is a cornerstone of immunosuppressive therapies. Here we found that calcineurin was recruited to the T cell antigen receptor (TCR) signaling complex, where it reversed inhibitory phosphorylation of the tyrosine kinase Lck on Ser59 (LckS59). Loss of calcineurin activity impaired phosphorylation of Tyr493 of the tyrosine kinase ZAP-70 (ZAP-70Y493), as well as some downstream pathways in a manner consistent with signaling in cells expressing LckS59A (Lck that cannot be phosphorylated) or LckS59E (a phosphomimetic mutant). Notably, CsA inhibited integrin-LFA-1-dependent and NFAT-independent adhesion of T cells to the intercellular adhesion molecule ICAM-1, with little effect on cells expressing mutant Lck. These results provide new understanding of how widely used immunosuppressive drugs interfere with essential processes in the immune response.
Subject(s)
Calcineurin/metabolism , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/immunology , ZAP-70 Protein-Tyrosine Kinase/metabolism , Animals , Cell Adhesion/drug effects , Cyclosporine/pharmacology , Humans , Immunosuppressive Agents/pharmacology , Intercellular Adhesion Molecule-1/metabolism , Jurkat Cells , Lymphocyte Activation/drug effects , Lymphocyte Function-Associated Antigen-1/metabolism , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics , Mice , Mice, Transgenic , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Phosphorylation/drug effects , Phosphorylation/genetics , Protein Binding , Signal Transduction , T-Lymphocytes/drug effects , Tacrolimus/pharmacologyABSTRACT
The adapter molecule linker for activation of T cells (LAT) plays a crucial role in forming signaling complexes induced by stimulation of the T cell receptor (TCR). These multi-molecular complexes are dynamic structures that activate highly regulated signaling pathways. Previously, we have demonstrated nanoscale structure in LAT-based complexes where the adapter SLP-76 (also known as LCP2) localizes to the periphery of LAT clusters. In this study, we show that initially LAT and SLP-76 are randomly dispersed throughout the clusters that form upon TCR engagement. The segregation of LAT and SLP-76 develops near the end of the spreading process. The local concentration of LAT also increases at the same time. Both changes require TCR activation and an intact actin cytoskeleton. These results demonstrate that the nanoscale organization of LAT-based signaling complexes is dynamic and indicates that different kinds of LAT-based complexes appear at different times during T cell activation.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Membrane Proteins/metabolism , Nanostructures/chemistry , Phosphoproteins/metabolism , Signal Transduction , Actin Cytoskeleton/metabolism , Green Fluorescent Proteins/metabolism , Humans , Jurkat Cells , Lymphocyte Activation , Membrane Fluidity , Microscopy , Receptors, Antigen, T-Cell/metabolismABSTRACT
Investigation of heterogeneous cellular structures using single-molecule localization microscopy has been limited by poorly defined localization accuracy and inadequate multiplexing capacity. Using fluorescent nanodiamonds as fiducial markers, we define and achieve localization precision required for single-molecule accuracy in dSTORM images. Coupled with this advance, our new multiplexing strategy, madSTORM, allows accurate targeting of multiple molecules using sequential binding and elution of fluorescent antibodies. madSTORM is used on an activated T-cell to localize 25 epitopes, 14 of which are on components of the same multimolecular T-cell receptor complex. We obtain an average localization precision of 2.6 nm, alignment error of 2.0 nm, and <0.01% cross-talk. Combining these technical advances affords the ability to move beyond obtaining superresolved structures to defining spatial relationships among constituent molecules within structures. Probing the molecular topology of complex signaling cascades and other heterogeneous networks is feasible with madSTORM.