scholarly journals Single-Molecule, Super-Resolution, and Functional Analysis of G Protein-Coupled Receptor Behavior Within the T Cell Immunological Synapse

2021 ◽  
Vol 8 ◽  
Author(s):  
James H. Felce ◽  
Lucia Parolini ◽  
Erdinc Sezgin ◽  
Pablo F. Céspedes ◽  
Kseniya Korobchevskaya ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
G Protein ◽  
T Cell Activation ◽  
Chemokine Receptors ◽  
Cell Activation ◽  
Immunological Synapse ◽  
Super Resolution ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

A central process in immunity is the activation of T cells through interaction of T cell receptors (TCRs) with agonistic peptide-major histocompatibility complexes (pMHC) on the surface of antigen presenting cells (APCs). TCR-pMHC binding triggers the formation of an extensive contact between the two cells termed the immunological synapse, which acts as a platform for integration of multiple signals determining cellular outcomes, including those from multiple co-stimulatory/inhibitory receptors. Contributors to this include a number of chemokine receptors, notably CXC-chemokine receptor 4 (CXCR4), and other members of the G protein-coupled receptor (GPCR) family. Although best characterized as mediators of ligand-dependent chemotaxis, some chemokine receptors are also recruited to the synapse and contribute to signaling in the absence of ligation. How these and other GPCRs integrate within the dynamic structure of the synapse is unknown, as is how their normally migratory Gαi-coupled signaling is terminated upon recruitment. Here, we report the spatiotemporal organization of several GPCRs, focusing on CXCR4, and the G protein Gαi2 within the synapse of primary human CD4+ T cells on supported lipid bilayers, using standard- and super-resolution fluorescence microscopy. We find that CXCR4 undergoes orchestrated phases of reorganization, culminating in recruitment to the TCR-enriched center. This appears to be dependent on CXCR4 ubiquitination, and does not involve stable interactions with TCR microclusters, as viewed at the nanoscale. Disruption of this process by mutation impairs CXCR4 contributions to cellular activation. Gαi2 undergoes active exclusion from the synapse, partitioning from centrally-accumulated CXCR4. Using a CRISPR-Cas9 knockout screen, we identify several diverse GPCRs with contributions to T cell activation, most significantly the sphingosine-1-phosphate receptor S1PR1, and the oxysterol receptor GPR183. These, and other GPCRs, undergo organization similar to CXCR4; including initial exclusion, centripetal transport, and lack of receptor-TCR interactions. These constitute the first observations of GPCR dynamics within the synapse, and give insights into how these receptors may contribute to T cell activation. The observation of broad GPCR contributions to T cell activation also opens the possibility that modulating GPCR expression in response to cell status or environment may directly regulate responsiveness to pMHC.

scholarly journals A T-cell–redirecting bispecific G-protein–coupled receptor class 5 member D x CD3 antibody to treat multiple myeloma

Blood ◽  
2020 ◽  
Vol 135 (15) ◽  
pp. 1232-1243 ◽  
Author(s):  
Kodandaram Pillarisetti ◽  
Suzanne Edavettal ◽  
Mark Mendonça ◽  
Yingzhe Li ◽  
Mark Tornetta ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
G Protein ◽  
T Cell Activation ◽  
Cell Activation ◽  
Plasma Cells ◽  
Phase 1 ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

Abstract T-cell–mediated approaches have shown promise in myeloma treatment. However, there are currently a limited number of specific myeloma antigens that can be targeted, and multiple myeloma (MM) remains an incurable disease. G-protein–coupled receptor class 5 member D (GPRC5D) is expressed in MM and smoldering MM patient plasma cells. Here, we demonstrate that GPRC5D protein is present on the surface of MM cells and describe JNJ-64407564, a GPRC5DxCD3 bispecific antibody that recruits CD3+ T cells to GPRC5D+ MM cells and induces killing of GPRC5D+ cells. In vitro, JNJ-64407564 induced specific cytotoxicity of GPRC5D+ cells with concomitant T-cell activation and also killed plasma cells in MM patient samples ex vivo. JNJ-64407564 can recruit T cells and induce tumor regression in GPRC5D+ MM murine models, which coincide with T-cell infiltration at the tumor site. This antibody is also able to induce cytotoxicity of patient primary MM cells from bone marrow, which is the natural site of this disease. GPRC5D is a promising surface antigen for MM immunotherapy, and JNJ-64407564 is currently being evaluated in a phase 1 clinical trial in patients with relapsed or refractory MM (NCT03399799).

scholarly journals The Purinergic G Protein-Coupled Receptor 6 Inhibits Effector T Cell Activation in Allergic Pulmonary Inflammation

2011 ◽  
Vol 187 (3) ◽  
pp. 1486-1495 ◽  
Author(s):  
Giorgio Giannattasio ◽  
Shin Ohta ◽  
Joshua R. Boyce ◽  
Wei Xing ◽  
Barbara Balestrieri ◽  
...  
Keyword(s):  
T Cell ◽  
G Protein ◽  
T Cell Activation ◽  
Cell Activation ◽  
Pulmonary Inflammation ◽  
G Protein Coupled Receptor ◽  
Effector T Cell ◽  
G Protein Coupled

scholarly journals CD45 pre-exclusion from the tips of T cell microvilli prior to antigen recognition

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yunmin Jung ◽  
Lai Wen ◽  
Amnon Altman ◽  
Klaus Ley
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Immunological Synapse ◽  
Transmembrane Domain ◽  
Tyrosine Phosphatase ◽  
Adaptive Immune Response ◽  
Super Resolution ◽  
Cell Receptor

AbstractThe tyrosine phosphatase CD45 is a major gatekeeper for restraining T cell activation. Its exclusion from the immunological synapse (IS) is crucial for T cell receptor (TCR) signal transduction. Here, we use expansion super-resolution microscopy to reveal that CD45 is mostly pre-excluded from the tips of microvilli (MV) on primary T cells prior to antigen encounter. This pre-exclusion is diminished by depleting cholesterol or by engineering the transmembrane domain of CD45 to increase its membrane integration length, but is independent of the CD45 extracellular domain. We further show that brief MV-mediated contacts can induce Ca2+ influx in mouse antigen-specific T cells engaged by antigen-pulsed antigen presenting cells (APC). We propose that the scarcity of CD45 phosphatase activity at the tips of MV enables or facilitates TCR triggering from brief T cell-APC contacts before formation of a stable IS, and that these MV-mediated contacts represent the earliest step in the initiation of a T cell adaptive immune response.

scholarly journals CD45 pre-exclusion from the tips of microvilli establishes a phosphatase-free zone for early TCR triggering

2020 ◽  
Author(s):  
Yunmin Jung ◽  
Lai Wen ◽  
Amnon Altman ◽  
Klaus Ley
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Immunological Synapse ◽  
Transmembrane Domain ◽  
Tyrosine Phosphatase ◽  
Adaptive Immune Response ◽  
Super Resolution ◽  
Free Zone

SummaryThe tyrosine phosphatase CD45 is a major gatekeeper for restraining T cell activation. Its exclusion from the immunological synapse (IS) is crucial for TCR signal transduction. Here, we used expansion super-resolution microscopy to reveal that CD45 is pre-excluded from the tips of microvilli on primary T cells prior to antigen encounter. This pre-exclusion was diminished by depleting cholesterol or by engineering the transmembrane domain of CD45 to increase its membrane integration length, but was independent of the CD45 extracellular domain. We further show that brief microvilli-mediated contacts can induce Ca2+ influx in mouse antigen-specific T cells engaged by antigen-pulsed APCs. We propose that the absence of CD45 phosphatase activity at the tips of microvilli enables or facilitates TCR triggering from brief T cell-APC contacts before formation of a stable IS, and that these microvilli-mediated contacts represent the earliest step in the initiation of a T cell adaptive immune response.Graphical abstract

scholarly journals Periodic Membrane Potential and Ca2+ Oscillations in T Cells Forming an Immune Synapse

2020 ◽  
Vol 21 (5) ◽  
pp. 1568 ◽  
Author(s):  
Ferenc Papp ◽  
Peter Hajdu ◽  
Gabor Tajti ◽  
Agnes Toth ◽  
Eva Nagy ◽  
...  
Keyword(s):  
T Cells ◽  
Membrane Potential ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Calcium Release ◽  
Immunological Synapse ◽  
Level Control ◽  
Potential Oscillations ◽  
Immune Synapse

The immunological synapse (IS) is a specialized contact area formed between a T cell and an antigen presenting cell (APC). Besides molecules directly involved in antigen recognition such as the TCR/CD3 complex, ion channels important in the membrane potential and intracellular free Ca2+ concentration control of T cells are also recruited into the IS. These are the voltage-gated Kv1.3 and Ca2+-activated KCa3.1 K+ channels and the calcium release-activated Ca2+ channel (CRAC). However, the consequence of this recruitment on membrane potential and Ca2+ level control is not known. Here we demonstrate that the membrane potential (MP) of murine T cells conjugated with APCs in an IS shows characteristic oscillations. We found that depolarization of the membrane by current injection or by increased extracellular K+ concentration produced membrane potential oscillations (MPO) significantly more frequently in conjugated T cells than in lone T cells. Furthermore, oscillation of the free intracellular Ca2+ concentration could also be observed more frequently in cells forming an IS than in lone cells. We suggest that in the IS the special arrangement of channels and the constrained space between the interacting cells creates a favorable environment for these oscillations, which may enhance the signaling process leading to T cell activation.

scholarly journals Casein kinase I delta controls centrosome positioning during T cell activation

2011 ◽  
Vol 195 (5) ◽  
pp. 781-797 ◽  
Author(s):  
Deborah Zyss ◽  
Hani Ebrahimi ◽  
Fanni Gergely
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Immunological Synapse ◽  
Casein Kinase ◽  
Cell Junction ◽  
Binding Motif ◽  
Casein Kinase I ◽  
Microtubule Growth

Although termed central body, the centrosome is located off-center in many polarized cells. T cell receptor (TCR) engagement by antigens induces a polarity switch in T cells. This leads to the recruitment of the centrosome to the immunological synapse (IS), a specialized cell–cell junction. Despite much recent progress, how TCR signaling triggers centrosome repositioning remains poorly understood. In this paper, we uncover a critical requirement for the centrosomal casein kinase I delta (CKIδ) in centrosome translocation to the IS. CKIδ binds and phosphorylates the microtubule plus-end–binding protein EB1. Moreover, a putative EB1-binding motif at the C terminus of CKIδ is required for centrosome translocation to the IS. We find that depletion of CKIδ in T lymphocytes and inhibition of CKI in epithelial cells reduce microtubule growth. Therefore, we propose that CKIδ–EB1 complexes contribute to the increase in microtubule growth speeds observed in polarized T cells, a mechanism that might serve to generate long-stable microtubules necessary for centrosome translocation.

scholarly journals Micro–adhesion rings surrounding TCR microclusters are essential for T cell activation

2016 ◽  
Vol 213 (8) ◽  
pp. 1609-1625 ◽  
Author(s):  
Akiko Hashimoto-Tane ◽  
Machie Sakuma ◽  
Hiroshi Ike ◽  
Tadashi Yokosuka ◽  
Yayoi Kimura ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adhesion Molecules ◽  
Focal Adhesion ◽  
T Cell Activation ◽  
Cell Activation ◽  
Immunological Synapse ◽  
Cell Receptor ◽  
Ring Structure ◽  
Cell Functions

The immunological synapse (IS) formed at the interface between T cells and antigen-presenting cells represents a hallmark of initiation of acquired immunity. T cell activation is initiated at T cell receptor (TCR) microclusters (MCs), in which TCRs and signaling molecules assemble at the interface before IS formation. We found that each TCR-MC was transiently bordered by a ring structure made of integrin and focal adhesion molecules in the early phase of activation, which is similar in structure to the IS in microscale. The micro–adhesion ring is composed of LFA-1, focal adhesion molecules paxillin and Pyk2, and myosin II (MyoII) and is supported by F-actin core and MyoII activity through LFA-1 outside-in signals. The formation of the micro–adhesion ring was transient but especially sustained upon weak TCR stimulation to recruit linker for activation of T cells (LAT) and SLP76. Perturbation of the micro–adhesion ring induced impairment of TCR-MC development and resulted in impaired cellular signaling and cell functions. Thus, the synapse-like structure composed of the core TCR-MC and surrounding micro–adhesion ring is a critical structure for initial T cell activation through integrin outside-in signals.

CC-122 Repairs T Cell Activation in Chronic Lymphocytic Leukemia That Results in a Concomitant Increase in PD-1:PD-L1 and CTLA-4 Immune Checkpoint Expression at the Immunological Synapse

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1738-1738
Author(s):  
Benedetta Apollonio ◽  
Mariam Fanous ◽  
Mohamed-Reda Benmebarek ◽  
Stephen Devereux ◽  
Patrick Hagner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Cell Activation ◽  
Immunological Synapse ◽  
Lymphocytic Leukemia ◽  
Immune Synapse ◽  
Cell Synapse

Abstract Immunomodulatory drugs (IMiDs®) such as lenalidomide and immune checkpoint blockade (ICB) antibodies can enhance autologous anti-tumor T cell immunity and have the potential to elicit durable control of disease in B cell malignancies. These immunotherapies are likely to be most effective when employed in treatment combinations. Thus, the goal of pre-clinical research should be to reveal mechanisms of action (MOA) in the tumor microenvironment (TME) and identify biomarkers to guide development of combination therapy for patients. CC-122 is a novel first-in-class pleiotropic pathway modifier (PPM®) that has potent anti-proliferative, anti-angiogenic and immunomodulatory activities and is currently in Phase I clinical trials for lymphoma and chronic lymphocytic leukemia (CLL). Here, we have utilized the immunological synapse bioassay to examine T cell interactions with CLL tumor cells (modeling anti-tumor T cell responses in the TME) following CC-122 treatment and measure the expression of co-signaling complexes at the synapse. Conjugation assays and confocal imaging were used to visualize intercellular conjugate interactions and F-actin polymerization at the immune synapse between CD4+ and CD8+ T cells and autologous CLL tumor cells pulsed with superantigen (acting as antigen-presenting cells, APCs). Peripheral blood was obtained from treatment naive CLL patients (n=40) representative of disease heterogeneity. Treatment of both purified CLL cells and CD4+ or CD8+ T cells with CC-122 (0.01 - 1 μM for 24h) dramatically enhanced the number of T cells recognizing tumor cells (% conjugation) and increased the formation of F-actin immune synapses (area, μm2) compared to vehicle treated cells (P<.01). Notably, CC-122 treatment induced T cells to engage in multiple tumor cell synapse interactions that were more pronounced in restored CD8+ T cell lytic synapses. This immunomodulatory activity was detected across all CLL patient samples and drug concentrations tested. In addition, synapse strength as measured by total fluorescence intensity of F-actin per T cell:APC conjugate increased significantly with CC-122 (P<.01). A critical MOA of lenalidomide is activation of T cell immune synapse signaling. Here, our comparative studies revealed that CC-122 (0.1 - 1 μM) significantly enhanced autologous T cell synapse activity in CLL by 4 - 5 fold versus vehicle (P<.01), whereas lenalidomide (1 μM) enhanced activity by 3 fold vs vehicle. Moreover, CC-122 treatment resulted in increased expression and polarization of tyrosine-phosphorylated proteins at T cell synapses compared to lenalidomide and vehicle treatment (P<.01). This data provides evidence that CC-122 induces functional T cell synapses that control the assembly of signaling complexes between the T cell receptor (TCR) and the F-actin cytoskeletal layer. Following T cell recognition of APCs, co-signaling receptors co-localize at the immune synapse where they synergize with TCR signaling to promote (co-stimulatory receptors) or inhibit (co-inhibitory/'immune checkpoint' receptors) T cell activation. Quantitative image analysis studies revealed that restoration of T cell synapse activity with CC-122 was accompanied by an increased recruitment of inducible co-stimulator (ICOS) to the synapse that was dose-dependent (P<.01). CC-122 treatment also increased polarized expression of CTLA-4 and PD-1 immune checkpoint proteins at the synapse with PD-L1+ tumor cells. The observed up-regulation of co-inhibitory receptors led to combining CC-122 with anti-PD-L1, anti-PD-1 or anti-CTLA-4 blocking antibodies. Results show that these treatment combinations increased T cell synapse activity compared to using these immunotherapies alone (P<.01). In conclusion, our results demonstrate for the first time that CC-122 can activate T cell immune synapse signaling against autologous CLL tumor cells and this immunomodulatory capability is more potent than lenalidomide. We further show that CC-122 activation of T cells is associated with enhanced expression of the co-stimulatory receptor ICOS and co-inhibitory checkpoints CTLA-4 and PD-1 at the synapse site. Importantly, our pre-clinical data demonstrates that this regulatory feedback inhibition can be exploited by the addition of anti-PD-L1, anti-PD-1 or anti-CTLA-4 ICB to CC-122 to more optimally stimulate T cell activity against immunosuppressive tumor cells. Disclosures Hagner: Celgene: Employment, Equity Ownership. Pourdehnad:Celgene: Employment. Gandhi:Celgene: Employment, Equity Ownership. Ramsay:MedImmune: Research Funding; Celgene: Research Funding.

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