scholarly journals Temporal Analysis of T-Cell Receptor-Imposed Forces via Quantitative Single Molecule FRET Measurements

2020 ◽  
Author(s):  
Janett Göhring ◽  
Florian Kellner ◽  
Lukas Schrangl ◽  
René Platzer ◽  
Enrico Klotzsch ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lipid Bilayers ◽  
T Cell Activation ◽  
Single Molecule ◽  
Cell Activation ◽  
Antibody Fragment ◽  
Antigen Recognition ◽  
Single Chain ◽  
Single Chain Antibody

ABSTRACTMechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their magnitude, spread, and temporal behavior are still poorly defined. We here report a FRET-based sensor equipped with a TCR-reactive single chain antibody fragment, which was tethered to planar supported lipid bilayers (SLBs) and informs most directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level. When confronting T-cells with gel-phase SLBs we observed both prior and upon T-cell activation a single, well-resolvable force-peak of approximately 5 pN and force loading rates on the TCR of 1.5 pN per second. When facing fluid SLBs instead, T-cells still exerted tensile forces yet with threefold reduced magnitude and only prior to but not upon activation. Our findings do not only provide first truly molecular information on TCR-imposed forces within the immunological synapse, they also recalibrate their significance in antigen recognition.

scholarly journals Temporal analysis of T-cell receptor-imposed forces via quantitative single molecule FRET measurements

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Janett Göhring ◽  
Florian Kellner ◽  
Lukas Schrangl ◽  
René Platzer ◽  
Enrico Klotzsch ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lipid Bilayers ◽  
T Cell Activation ◽  
Single Molecule ◽  
Cell Activation ◽  
Antibody Fragment ◽  
Temporal Analysis ◽  
Single Chain ◽  
Single Chain Antibody

AbstractMechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their magnitude, spread, and temporal behavior are still poorly defined. We here report a FRET-based sensor equipped either with a TCR-reactive single chain antibody fragment or peptide-loaded MHC, the physiological TCR-ligand. The sensor was tethered to planar glass-supported lipid bilayers (SLBs) and informed most directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level. When confronting T-cells with gel-phase SLBs we observed both prior and upon T-cell activation a single, well-resolvable force-peak of approximately 5 pN and force loading rates on the TCR of 1.5 pN per second. When facing fluid-phase SLBs instead, T-cells still exerted tensile forces yet of threefold reduced magnitude and only prior to but not upon activation.

T-cell activation and cytokine production via a bispecific single-chain antibody fragment targeted to blood-stage malaria parasites

Blood ◽  
2003 ◽  
Vol 101 (6) ◽  
pp. 2300-2306 ◽  
Author(s):  
Shigeto Yoshida ◽  
Tominari Kobayashi ◽  
Hiroyuki Matsuoka ◽  
Chisato Seki ◽  
William L. Gosnell ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Recombinant Baculovirus ◽  
Antibody Fragment ◽  
Antibody Fragments ◽  
Therapeutic Drug ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Single Chain Antibody Fragment

A novel bispecific single-chain antibody fragment (biscFv) has been constructed to address the possibility of a new approach to malaria therapeutic drug development. The biscFv consists of 2 different single-chain antibody fragments linked by a flexible peptide linker (Gly4-Ser)3. Of the 2 scFv fragments, one is directed against a conserved epitope of the 19-kDa C-terminal fragment of the major surface protein of human malignant malaria parasite, Plasmodium falciparum, and the other is directed against the CD3 antigen of human T cells. The biscFv expressed by a recombinant baculovirus retained the antigen-binding properties of the corresponding univalent single-chain antibody fragments and formed a bridge between P falciparum and T cells. In cooperation with T cells, the biscFv specifically induced not only interferon γ and tumor necrosis factor α, but also a significant increase of merozoite phagocytosis and growth inhibition of P falciparum in vitro. Thus, the biscFv possesses highly selective malaria-targeting properties and stimulates T cells to induce cytokines, presumably resulting in activation of macrophages, neutrophils, and natural killer cells, and parasite killing in vivo.

Selective blockade of CD28 and not CTLA-4 with a single-chain Fv–α1-antitrypsin fusion antibody

Blood ◽  
2003 ◽  
Vol 102 (2) ◽  
pp. 564-570 ◽  
Author(s):  
Bernard Vanhove ◽  
Geneviève Laflamme ◽  
Flora Coulon ◽  
Marie Mougin ◽  
Patricia Vusio ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Antibody Fragment ◽  
Single Chain ◽  
Selective Blockade ◽  
Mhc Molecules ◽  
Single Chain Fv

Abstract B7-1 and B7-2 are costimulatory molecules expressed on antigen-presenting cells. The CD28/B7 costimulation pathway is critical for T-cell activation, proliferation, and Th polarization. Blocking both cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) and CD28 interactions with a CTLA-4/Ig fusion protein inhibits various immune-mediated processes in vivo, such as allograft rejection and autoimmunity. However, selective blockade of CD28 may represent a better strategy for immunosuppression than B7 blockade, because CTLA-4/B7 interactions have been shown to participate in the extinction of the T-cell receptor–mediated activation signal and to be required for the induction of immunologic tolerance. In addition, selective CD28 inhibition specifically decreases the activation of alloreactive and autoreactive T cells, but not the activation of T cells stimulated by exogenous antigens presented in the context of self major histocompatibility complex (MHC) molecules. CD28 blockade cannot be obtained with anti-CD28 dimeric antibodies, which cluster their target and promote T-cell costimulation, whereas monovalent Fab fragments can block CD28 and reduce alloreactivity. In this study, we report the construction of a monovalent single-chain Fv antibody fragment from a high-affinity antihuman CD28 antibody (CD28.3) that blocked adhesion of T cells to cells expressing the CD28 receptor CD80. Genetic fusion with the long-lived serum protein α1-antitrypsin led to an extended half-life without altering its binding characteristics. The anti-CD28 fusion molecule showed biologic activity as an immuno-suppressant by inhibiting T-cell activation and proliferation in a mixed lymphocyte reaction.

T-cell activation and B-cell depletion in chimpanzees treated with a bispecific anti-CD19/anti-CD3 single-chain antibody construct

2005 ◽  
Vol 55 (5) ◽  
pp. 503-514 ◽  
Author(s):  
Bernd Schlereth ◽  
Cornelia Quadt ◽  
Torsten Dreier ◽  
Peter Kufer ◽  
Grit Lorenczewski ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Depletion ◽  
B Cell Depletion ◽  
Single Chain ◽  
Single Chain Antibody

Preclinical evaluation of AMG 160, a next-generation bispecific T cell engager (BiTE) targeting the prostate-specific membrane antigen PSMA for metastatic castration-resistant prostate cancer (mCRPC).

2019 ◽  
Vol 37 (7_suppl) ◽  
pp. 301-301 ◽  
Author(s):  
Julie Bailis ◽  
Petra Deegen ◽  
Oliver Thomas ◽  
Pamela Bogner ◽  
Joachim Wahl ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Half Life ◽  
Cell Activation ◽  
Phase 1 ◽  
Xenograft Model ◽  
Cytokine Release ◽  
Single Chain

301 Background: mCRPC is a disease of high unmet medical need, especially for patients who fail novel hormonal therapies and chemotherapy. BiTE molecules provide an off the shelf therapy that activates a patient’s own immune system and redirects T cells to kill tumor cells. The BiTE mechanism of action is distinct from other immunotherapies and may unlock immune response in mCRPC. PSMA is a compelling BiTE target that is highly expressed on PCa compared to normal tissue and has increased expression in mCRPC. Methods: AMG 160 is a fully human, half-life extended (HLE) BiTE that targets PSMA on tumor cells and CD3 on T cells. AMG 160 comprises two tandem single chain variable fragments fused to an Fc domain. Results: AMG 160 binds human and non-human primate (NHP) PSMA and CD3, leading to T cell activation and proliferation and cytokine production. AMG 160 redirects T cells to kill PSMA-positive cancer cell lines in vitro, including those with low PSMA levels or androgen-independent signaling. Weekly dosing of AMG 160 induces significant antitumor activity in established PCa xenograft model. The pharmacokinetics (PK) and pharmacodynamics of AMG 160 were tested in NHP. AMG 160 treatment led to BiTE target engagement in vivo, including transient T cell activation and cytokine release in blood, and mixed cellular infiltrates in multiple organs known to express PSMA. AMG 160 treatment was well tolerated. Cytokine release associated with the first dose could be attenuated using a step dose regimen. The half-life of AMG 160 in NHP was about one week. Based on allometric scaling, the PK profile of AMG 160 may be projected to enable dosing every other week in humans. Conclusions: AMG 160 is a potent HLE BiTE with specificity for PSMA-positive tumor cells. A Phase 1 study is planned to evaluate the safety and efficacy of AMG 160 in patients with mCRPC.

scholarly journals Early T cell receptor signals globally modulate ligand:receptor affinities during antigen discrimination

2017 ◽  
Vol 114 (46) ◽  
pp. 12190-12195 ◽  
Author(s):  
Rafal M. Pielak ◽  
Geoff P. O’Donoghue ◽  
Jenny J. Lin ◽  
Katherine N. Alfieri ◽  
Nicole C. Fay ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Single Molecule ◽  
Cell Activation ◽  
Cell Receptor ◽  
Physical Constraints ◽  
Molecular Binding ◽  
Antigen Presenting

Antigen discrimination by T cells occurs at the junction between a T cell and an antigen-presenting cell. Juxtacrine binding between numerous adhesion, signaling, and costimulatory molecules defines both the topographical and lateral geometry of this cell–cell interface, within which T cell receptor (TCR) and peptide major histocompatibility complex (pMHC) interact. These physical constraints on receptor and ligand movement have significant potential to modulate their molecular binding properties. Here, we monitor individual ligand:receptor binding and unbinding events in space and time by single-molecule imaging in live primary T cells for a range of different pMHC ligands and surface densities. Direct observations of pMHC:TCR and CD80:CD28 binding events reveal that the in situ affinity of both pMHC and CD80 ligands for their respective receptors is modulated by the steady-state number of agonist pMHC:TCR interactions experienced by the cell. By resolving every single pMHC:TCR interaction it is evident that this cooperativity is accomplished by increasing the kinetic on-rate without altering the off-rate and has a component that is not spatially localized. Furthermore, positive cooperativity is observed under conditions where the T cell activation probability is low. This TCR-mediated feedback is a global effect on the intercellular junction. It is triggered by the first few individual pMHC:TCR binding events and effectively increases the efficiency of TCR scanning for antigen before the T cell is committed to activation.

scholarly journals Trapping or slowing the diffusion of T cell receptors at close contacts initiates T cell signaling

2021 ◽  
Vol 118 (39) ◽  
pp. e2024250118 ◽  
Author(s):  
Kevin Y. Chen ◽  
Edward Jenkins ◽  
Markus Körbel ◽  
Aleks Ponjavic ◽  
Anna H. Lippert ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lipid Bilayers ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Receptor ◽  
Stochastic Simulations ◽  
T Cell Signaling ◽  
Extracellular Region ◽  
Close Contacts

T cell activation is initiated by T cell receptor (TCR) phosphorylation. This requires the local depletion of large receptor-type phosphatases from “close contacts” formed when T cells interact with surfaces presenting agonistic TCR ligands, but exactly how the ligands potentiate signaling is unclear. It has been proposed that TCR ligands could enhance receptor phosphorylation and signaling just by holding TCRs in phosphatase-depleted close contacts, but this has not been directly tested. We devised simple methods to move the TCR in and out of close contacts formed by T cells interacting with supported lipid bilayers (SLBs) and to slow the receptor’s diffusion in the contacts, using a series of anti-CD3ε Fab- and ligand-based adducts of the receptor. TCRs engaging a Fab extended with the large extracellular region of CD45 were excluded from contacts and produced no signaling. Conversely, allowing the extended Fab to become tethered to the SLB trapped the TCR in the close contacts, leading to very strong signaling. Importantly, attaching untethered anti-CD3ε Fab or peptide/MHC ligands, each of which were largely inactive in solution but both of which reduced TCR diffusion in close contacts approximately fivefold, also initiated signaling during cell/SLB contact. Our findings indicate that holding TCRs in close contacts or simply slowing their diffusion in phosphatase-depleted regions of the cell surface suffices to initiate signaling, effects we could reproduce in single-particle stochastic simulations. Our study shows that the TCR is preconfigured for signaling in a way that allows it to be triggered by ligands acting simply as receptor “traps.”

Activation of T Cells by Bispecific Single-Chain Construct MT103 (MEDI-538) Is Strictly Target Cell-Dependent.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3889-3889
Author(s):  
Klaus Brischwein ◽  
Scott A. Hammond ◽  
Larissa Parr ◽  
Schlereth Bernd ◽  
Mathias Locher ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Target Cell ◽  
Cell Activation ◽  
Cell Lysis ◽  
Target Cells ◽  
Single Chain ◽  
Activation Markers ◽  
Human T Cells

Abstract Background: Bispecific antibodies have been extensively studied in vitro and in vivo for their use in redirected tumor cell lysis. A particular challenge of bispecific antibody constructs recognizing the CD3 signaling complex is to achieve a controlled polyclonal activation of T-cells that, ideally, is entirely dependent on the presence of target cells. If this is not the case, systemic production of inflammatory cytokines and secondary endothelial reactions may occur as side effects, as are observed with the murine anti-human CD3e antibody OKT-3 (muromab, Orthoclone®). Here we present evidence that MT103 (or MEDI-538), a bispecific single chain antibody of the BiTE class that targets CD19 and CD3, induces T-cell activation exclusively in the presence of target cells. Material and methods: Peripheral blood mononuclear cells from healthy donors were prepared by Ficoll density centrifugation. PBMC were incubated for 24 hours with MT103 in presence or absence of specific target cells. Target cell lysis was determined by measurement of adenylate kinase activity released from lysed cells. De novo expression of activation markers CD69 and CD25 on T-cells was assessed by flow cytometry using directly conjugated monoclonal antibodies, and the concentration of cytokines in the supernatant was determined by a commercial FACS-based bead array. Results: MT103 was analyzed for conditional T-cell activation. In the presence of target-expressing cell lines, low picomolar concentrations of MT103 were sufficient to stimulate a high percentage of peripheral human T-cells to express cytokines and surface activation markers, to enter into the cell cycle and to induce redirected lysis of target cells. However, in the absence of target cells, the BiTE molecules no longer detectably activated human T-cells even at concentrations exceeding the ED50 for redirected lysis and conditional T-cell activation by more than five orders of magnitude. Conclusion: Our data show that T-cell activation by MT103 is highly conditional in that it is strictly dependent on the presence.

The effect of dexamethasone on polyclonal T cell activation and redirected target cell lysis as induced by a CD19/CD3-bispecific single-chain antibody construct

2007 ◽  
Vol 56 (10) ◽  
pp. 1551-1563 ◽  
Author(s):  
Christian Brandl ◽  
Cornelia Haas ◽  
Sandrine d’Argouges ◽  
Tanja Fisch ◽  
Peter Kufer ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Target Cell ◽  
Cell Activation ◽  
Cell Lysis ◽  
Single Chain ◽  
Single Chain Antibody

scholarly journals Interaction of CD31 with a heterophilic counterreceptor involved in downregulation of human T cell responses.

1996 ◽  
Vol 184 (1) ◽  
pp. 41-50 ◽  
Author(s):  
E Prager ◽  
R Sunder-Plassmann ◽  
C Hansmann ◽  
C Koch ◽  
W Holter ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Types ◽  
Interleukin 4 ◽  
Single Chain ◽  
Necrosis Factor Alpha ◽  
Human T Cell

CD31 is a 130-kD glycoprotein of the immunoglobulin (Ig) superfamily expressed on the surface of endothelial cells, platelets, and several leukocyte subsets. Previous reports indicated that CD31 can mediate intercellular adhesion via both homophilic and heterophilic interaction mechanisms. Using a soluble recombinant CD31-Ig fusion protein (CD31 receptor globulin [Rg]), we demonstrate here that human CD31- T lymphocytes and CD4+CD31- T cell clones express a heterophilic CD31 ligand that is upregulated 18 h after activation. Interaction of CD31Rg with CD31- T helper cell (Th) clones was divalent cation independent but could be blocked by heparin, thus indicating that the CD31 counterreceptor on T cells can be distinguished from the ligands identified on other cell types. Moreover, a single chain protein of 120 kD was precipitated by CD31Rg from the lysates of CD31- Th clones. CD31Rg completely downregulated the proliferative response and cytokine production (interleukin-4, interferon-gamma, and tumor necrosis factor-alpha) of CD31- Th clones when the cells were maximally stimulated via immobilized CD3 monoclonal antibody. These results suggest that interaction of CD31 with a heterophilic counterreceptor on T lymphocytes can interfere with a positive regulatory pathway of T cell activation, or directly signal T cells to downregulate immune function.

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