A generic cell surface ligand system for studying cell–cell recognition

Abstract

Dose-response experiments are a mainstay of receptor biology studies and can reveal valuable insights into receptor function. Such studies of receptors that bind cell surface ligands are currently limited by the difficulty in manipulating the surface density of ligands at a cell–cell interface. Here, we describe a generic cell surface ligand system that allows precise manipulation of cell surface ligand densities over several orders of magnitude. These densities are robustly quantifiable, a major advance over previous studies. We validate the system for a range of immunoreceptors, including the T-cell receptor (TCR), and show that this generic ligand stimulates via the TCR at a similar surface density as its native ligand. We also extend our work to the activation of chimeric antigen receptors. This novel system allows the effect of varying the surface density, valency, dimensions, and affinity of the ligand to be investigated. It can be readily broadened to other receptor–cell surface ligand interactions and will facilitate investigation into the activation of, and signal integration between, cell surface receptors.

Immunomodulatory Response of Toll-like Receptor Ligand–Peptide Conjugates in Food Allergy

“…We have analyzed the level of uptake of TLRn lig -Pp3 on monocyte-derived DCs (moDCs), using 10 nM of TLR4 lig -Pp3 ( 1 ) or TLR7 lig -Pp3 ( 2 ), both labeled with Alexa fluor 647, by flow cytometry and confocal microscopy (CM) . Flow cytometry measurements indicated that TLRn lig -Pp3 were uptaken by moDCs in a time-dependent manner, which is faster and higher for TLR4 lig -Pp3 compared to TLR7 lig -Pp3 (Figure A).…”

CD8 Co-Receptor Enhances T-Cell Activation without Any Effect on Initial Attachment

“…As previously, we used a human Jurkat T-cell line expressing the 1G4 TCR [ 27 ]. As this line does not express CD8 (CD8−), we used lentiviral transduction [ 28 ] to produce a version that expresses CD8αβ (CD8+). Cell receptor expression was checked with flow cytometry, using a LSRFortessa X20 flow cytometer (Beckton-Dickinson, Franklin Lakes, NJ, USA).…”

Power to the protein: enhancing and combining activities using the Spy toolbox

“…5C). 85 Target cells expressed a surface protein bearing a twin Strep-tag. Then, questions about cell-cell interactions in terms of ligand length, valency and affinity could be addressed in a modular fashion (without making a huge range of stably transfected cell clones), through titrating in a bridging molecule of SpyCatcher linked to Strep-Tactin (Fig.…”

“…5C). 85 Membrane decoration may also be applied for vaccine assembly: Outer Membrane Vesicles (OMVs) from attenuated Salmonella displaying a SpyTag-linked E. coli autotransporter facilitate display of SpyCatcher-linked immunogens (Fig. 5D).…”