How T cells discriminate between antigens of different affinities
T lymphocytes are essential cells of the immune system, and have the ability to detect a wide range of antigenic peptides related to the major histocompatibility complex (MHC). However, they only initiate an appropriate response after recognizing high-affinity antigens from pathogens, whereas interaction with self peptides does not activate them. Through an extensive, time-resolved phosphoproteomic and interactomic analysis of the signaling events that occur in T cells upon stimulation with peptides of different affinity, scientists from the IPBS and the CIML have deciphered the mechanisms that govern this highly specific discrimination. These results are published in the journal Nature Immunology.
T cell activation, differentiation and phenotypic response depend on the specificity and strength of the signals delivered by the T cell receptor (TCR) upon recognition of an antigenic peptide. Although low-affinity self-peptides derived from host proteins can bind to the TCR, they do not elicit a response, even when present in high concentrations. Conversely, even small amounts of foreign antigenic peptides that bind to the TCR with high affinity are sufficient to fully activate T cells. To achieve such specificity, it has been postulated that the TCR uses a kinetic proofreading mechanism, based on the presence of multiple biochemical intermediate steps between antigen binding and a final irreversible step leading to full activation. This introduces a delay that allows only high-affinity antigens, which remain bound to the TCR long enough, to complete the series of signaling steps leading to T cell activation.
TCR stimulation promotes rapid and successive phosphorylation of many intracellular proteins, as well as protein-protein interactions and complex formation. To further characterize these mechanisms, and to identify key steps in antigenic discrimination, the scientists analyzed in a comprehensive manner all molecular events that occur in T cells when the TCR is engaged using peptide ligands of decreasing affinity. They applied mass spectrometry-based approaches to follow the phosphorylation kinetics of several thousand proteins, and the formation of complexes around different important molecules of the signaling pathway.
They were thus able to unbiasedly classify events independent of ligand affinity, those showing a response scaling with ligand affinity, and those elicited only by strong ligands. Some upstream molecular events appear to be completely unaffected by differences in ligand affinity, for example phosphorylation of TCR CD3 chains and recruitment of ZAP70 kinase to the TCR. Instead, differences between strong and weak ligands are reflected by changes in the final trans-autophosphorylation step of the ZAP70 catalytic domain, required for full kinase activation, and in LAT signalosome formation, which is severely impaired with weak ligands. The CD6 signalosome assembly is only partially affected, and it still contains inhibitory molecules that may play a role in attenuating TCR signals under conditions of stimulation with low-affinity ligands.
This study provides fundamental insights into how the series of signaling steps downstream of the TCR plays a role in antigenic discrimination, consistent with the kinetic replay model. This knowledge of TCR signaling mechanisms may be useful in the future for the rational design of chimeric T cell receptors used for cancer immunotherapy. It also provides a useful data resource for researchers, revealing new candidate molecules that may play a role in the TCR pathway.
T cell receptor signaling is triggered when the TCR binds to an MHC-related antigenic peptide, leading to phosphorylation of CD3 chains and recruitment of the ZAP70 kinase. (a) Stimulation with high-affinity ligands completes all steps of ZAP70 phosphorylation, leading to full activation of the kinase and formation of the LAT signalosome, orchestrating the T cell response. (b) In contrast, when the TCR binds to a lower affinity peptide presented by the MHC, the half-life of the interaction is not long enough to allow step 3 of ZAP70 activation to occur. Stimulation with weak peptides only allows the formation of the CD6 signalosome, which is capable of driving a negative feedback loop.
“Kinetic proofreading through the multi-step activation of the ZAP70 kinase underlies early T cell ligand discrimination” Voisinne, G., Locard-Paulet, M., Froment, C., Maturin, E., Menoita, M.G., Girard, L., Mellado, V., Burlet-Schiltz, O., Malissen, B.*, Gonzalez de Peredo, A.*, and Roncagalli, R.* Nature Immunology, August 13 2022