CD28 shapes T cell receptor signaling by regulating Lck dynamics and ZAP70 activation.

Introduction: T cell activation requires T cell receptor (TCR) engagement by its specific ligand. This interaction initiates a series of proximal events including tyrosine phosphorylation of the CD3 and TCRζ chains, recruitment, and activation of the protein tyrosine kinases Lck and ZAP70, followed by recruitment of adapter and signaling proteins. CD28 co-stimulation is also required to generate a functional immune response.

CD28 Shapes T Cell Receptor Signaling by Regulating ZAP70 Activation and Lck Dynamics.

T cell activation requires T cell receptor (TCR) engagement, which initiates a series of proximal events including tyrosine phosphorylation of the CD3 and TCRζ chains, recruitment, and activation of the protein tyrosine kinases Lck and ZAP70, followed by recruitment of adapter and signaling proteins. CD28 co-stimulation is also required to generate a functional immune response. Currently we lack a full understanding of the molecular mechanism of CD28 activation.

Generation of antitumor chimeric antigen receptors incorporating T cell signaling motifs.

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.

Heterogeneity of Signaling Complex Nanostructure in T Cells Activated Via the T Cell Antigen Receptor.

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.

In vitro reconstitution reveals cooperative mechanisms of adapter protein-mediated activation of phospholipase C-γ1 in T cells.

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.

Microclusters as T Cell Signaling Hubs: Structure, Kinetics, and Regulation.

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.

Expression of a TMC6-TMC8-CIB1 heterotrimeric complex in lymphocytes is regulated by each of the components.

The genes encode a set of homologous transmembrane proteins whose functions are not well understood. Biallelic mutations in either or 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.

Bypassing ubiquitination enables LAT recycling to the cell surface and enhanced signaling in T cells.

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.

Pak1 Kinase Promotes Activated T Cell Trafficking by Regulating the Expression of L-Selectin and CCR7.

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.

TCR microclusters form spatially segregated domains and sequentially assemble in calcium-dependent kinetic steps.

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.

Cooperative assembly of a four-molecule signaling complex formed upon T cell antigen receptor activation.

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.

Timed Regulation of 3BP2 Induction Is Critical for Sustaining CD8 T Cell Expansion and Differentiation.

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.

Plasma membrane LAT activation precedes vesicular recruitment defining two phases of early T-cell activation.

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.

The Cish SH2 domain is essential for PLC-γ1 regulation in TCR stimulated CD8 T cells.

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.

Intensity and duration of TCR signaling is limited by p38 phosphorylation of ZAP-70 and destabilization of the signalosome.

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-70) had enhanced TCR proximal signaling and increased effector responses.

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM.

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.

Unexpected Cartilage Phenotype in CD4-Cre-Conditional SOS-Deficient Mice.

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 mice to CD4-Cre transgenic mice. We previously reported an effect of these mutations on T cell signaling and T cell migration.

Super-resolution Analysis of TCR-Dependent Signaling: Single-Molecule Localization Microscopy.

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.

Recruitment of calcineurin to the TCR positively regulates T cell activation.

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 (Lck). Loss of calcineurin activity impaired phosphorylation of Tyr493 of the tyrosine kinase ZAP-70 (ZAP-70), as well as some downstream pathways in a manner consistent with signaling in cells expressing Lck (Lck that cannot be phosphorylated) or Lck (a phosphomimetic mutant).

Development of nanoscale structure in LAT-based signaling complexes.

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.

madSTORM: a superresolution technique for large-scale multiplexing at single-molecule accuracy.

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.

Hierarchical nanostructure and synergy of multimolecular signalling complexes.

Signalling complexes are dynamic, multimolecular structures and sites for intracellular signal transduction. Although they play a crucial role in cellular activation, current research techniques fail to resolve their structure in intact cells. Here we present a multicolour, photoactivated localization microscopy approach for imaging multiple types of single molecules in fixed and live cells and statistical tools to determine the nanoscale organization, topology and synergy of molecular interactions in signalling complexes downstream of the T-cell antigen receptor.