Membrane Association Transforms an Inert Anti-TCRβ Fab’ Ligand into a Potent T Cell Receptor Agonist

Abstract

The natural peptide-major histocompatibility complex (pMHC) ligand for T cell receptors (TCRs) is inactive from solution yet capable of activating T cells at single-molecule levels when membrane-associated. This distinctive feature stems from the mechanism of TCR activation, which is thought to involve steric phosphatase exclusion as well as direct mechanical forces. It is possible to defeat this mechanism and activate T cells with solution ligands by cross-linking pMHC or using multivalent antibodies to TCR. However, these widely used strategies activate TCRs through a nonphysiological mechanism and can produce different activation profiles than natural, monovalent, membrane-associated pMHC. Here, we introduce a strictly monovalent anti-TCRb H57 Fab' ligand that, when coupled to a supported lipid bilayer via DNA complementation, triggers TCRs and activates nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT) with a similar potency to pMHC in primary murine T cells. Importantly, like monovalent pMHC and unlike bivalent antibodies, monovalent Fab'-DNA triggers TCRs only when physically coupled to the membrane, and only around 100 individual Fab':TCR interactions are necessary to stimulate early T cell activation.

Height, but not binding epitope, affects the potency of synthetic TCR agonists

“…with molecular displacement, !, corresponding delay time, !, and diffusion coefficient, D. D values for 2C11 Fab'-DNA and 17A2 Fab'-DNA were measured to be 2.49 ± 0.02 µm 2 /s and 2.83 ± 0.02 µm 2 /s, respectively, in close agreement with the previously reported value of D = 2.43 ± 0.02 µm 2 /s for H57 Fab'-DNA on comparable supported bilayers (25).…”

“…Fab'-DNA constructs were synthesized by digesting three commercially available anti-murine TCR antibodies: H57-597; 145-2C11; and 17A2, purifying the monovalent Fab' fragments, and conjugating those fragments to DNA oligonucleotides labeled with a single Atto647N dye, as described previously (see Materials and Methods for details) (Fig. S1 and S2 in the Supporting Material) (25).…”

“…We find that the intermembrane space established by DNA tether length dramatically affects Fab’-DNA potency, with larger spacing leading to less potent cellular activation, but that varying the Fab’ binding epitope has essentially no effect. T cell activation was monitored by imaging translocation of a fluorescent reporter for the nuclear factor for the activation of T cells (NFAT) from the cytosol to the nucleus (19, 20, 25). Localized formation of protein condensates of linker for the activation of T cells (LAT) also showed the same trend.…”

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

“…1A-D), as we anticipated this to give rise to stable TCR-bonds, which unlike pMHC-TCR interactions sustain the tensile forces applied in the synapse without rupture. It is known that monomeric membrane-bound H57 activates T-cells with similar activation thresholds as agonistic pMHC (32,33). Second, we also functionalized the force sensor with MHC class II molecule IE k loaded with a moth cytochrome c (MCC, IE k /MCC) peptide (MFS-IE k /MCC; for construction see Supplementary Fig.…”

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

“…1a–d ), as we anticipated this to give rise to stable TCR-bonds, which unlike pMHC–TCR interactions sustain the tensile forces applied in the synapse without rupture. It is known that monomeric membrane-bound H57 activates T-cells with similar activation thresholds as agonistic pMHC 35 , 36 . Second, we also functionalized the force sensor with MHC class II molecule IE k loaded with a moth cytochrome c (MCC, IE k /MCC) peptide (MFS-IE k /MCC; for construction see Supplementary Fig.…”