Coexistence of multivalent and monovalent TCRs explains high sensitivity and wide range of response

Abstract

A long-standing paradox in the study of T cell antigen recognition is that of the high specificity–low affinity T cell receptor (TCR)–major histocompatibility complex peptide (MHCp) interaction. The existence of multivalent TCRs could resolve this paradox because they can simultaneously improve the avidity observed for monovalent interactions and allow for cooperative effects. We have studied the stoichiometry of the TCR by Blue Native–polyacrylamide gel electrophoresis and found that the TCR exists as a mixture of monovalent (αβγɛδɛζζ) and multivalent complexes with two or more ligand-binding TCRα/β subunits. The coexistence of monovalent and multivalent complexes was confirmed by electron microscopy after label fracture of intact T cells, thus ruling out any possible artifact caused by detergent solubilization. We found that although only the multivalent complexes become phosphorylated at low antigen doses, both multivalent and monovalent TCRs are phosphorylated at higher doses. Thus, the multivalent TCRs could be responsible for sensing low concentrations of antigen, whereas the monovalent TCRs could be responsible for dose-response effects at high concentrations, conditions in which the multivalent TCRs are saturated. Thus, besides resolving TCR stoichiometry, these data can explain how T cells respond to a wide range of MHCp concentrations while maintaining high sensitivity.

Synthetic Multivalent Ligands as Probes of Signal Transduction

“…Also unknown is the role of simple monovalent ligation of individual TCRs. [212] If individual or small groups of TCRs can activate signaling, the purpose of the synapse is unclear. Multivalent ligands will undoubtedly play prominent roles in addressing these fundamental issues.…”

Cognate Peptide–MHC Complexes Are Expressed as Tightly Apposed Nanoclusters in Virus-Infected Cells To Allow TCR Crosslinking

“…However, the pMHC-TCR interaction is of low affinity (4-6), making it difficult to explain how T cells achieve high sensitivity. We previously showed that the TCR was organized in nanoclusters prior to Ag engagement and that this clustering endowed T cells with higher sensitivity (7,8). Furthermore, it has long been recognized that experimental TCR ligands, such as TCR-specific Abs and soluble pMHC complexes, need to be at least bivalent to be capable of stimulating T cells (9,10).…”

“…We used our previously established approach of cell surface replica generation of fixed cells labeled with primary Abs and colloidal gold-conjugated secondary reagents in combination with transmission electron microscopy (7,8). This approach permits molecular resolution of the cell surface localization of endogenous proteins and, in contrast to most high-resolution light microscopy techniques, visualizes the cell surface membrane opposite to the one in contact with the experimental support.…”

“…In the case of labeling with the 25D1.16 mAb (mouse IgG1), a bridging hamster anti-mouse IgG was used to overcome the low affinity of protein A for murine IgG1 Abs. Replica generation was done as previously described (7,8). Replicas were analyzed on a JEOL 1010 transmission electron microscope operating at 80 kV, and images were acquired with a TemCAM F416 camera operated by EM-MENU software (Tietz Video and Image Processing Systems).…”

Kidins220/ARMS Associates with B-Raf and the TCR, Promoting Sustained Erk Signaling in T Cells

“…Stimulation was done with 5 mM pervanadate; a mixture of the anti-CD3ε Ab 145-2C11 and the anti-TCRb Ab 597-H57, both at 5 mg/ml (referred to as anti-TCR in the text); 25 nM H2K d -pepABA tetramers; or 20 nM pigeon cytochrome C peptide-loaded DCEK cells (APCs) for different time points at 37˚C. Where indicated, cells were lysed in 1 ml lysis buffer containing 0.3% Brij96, as described (30). Immunoprecipitation (IP), SDS-PAGE, and Western blotting (WB) were done using standard procedures, as described (31).…”