Barbara Hausmann

Thymic selection threshold defined by compartmentalization of Ras/MAPK signalling

A healthy individual can mount an immune response to exogenous pathogens while avoiding an autoimmune attack on normal tissues. The ability to distinguish between self and non-self is called ‘immunological tolerance’ and, for T lymphocytes, involves the generation of a diverse pool of functional T cells through positive selection and the removal of overtly self-reactive thymocytes by negative selection during T-cell ontogeny. To elucidate how thymocytes arrive at these cell fate decisions, here we have identified ligands that define an extremely narrow gap spanning the threshold that distinguishes positive from negative selection. We show that, at the selection threshold, a small increase in ligand affinity for the T-cell antigen receptor leads to a marked change in the activation and subcellular localization of Ras and mitogen-activated protein kinase (MAPK) signalling intermediates and the induction of negative selection. The ability to compartmentalize signalling molecules differentially in the cell endows the thymocyte with the ability to convert a small change in analogue input (affinity) into a digital output (positive versus negative selection) and provides the basis for establishing central tolerance.

T Cell Affinity Regulates Asymmetric Division, Effector Cell Differentiation, and Tissue Pathology

The strength of interactions between T cell receptors and the peptide-major histocompatibility complex (pMHC) directly modulates T cell fitness, clonal expansion, and acquisition of effector properties. Here we show that asymmetric T cell division is an important mechanistic link between increased signal strength, effector differentiation, and the ability to induce tissue pathology. Recognition of pMHC above a threshold affinity drove responding T cells into asymmetric cell division. The ensuing proximal daughters underwent extensive division and differentiated into short-lived effector cells expressing the integrin VLA-4, allowing the activated T cell to infiltrate and mediate destruction of peripheral target tissues. In contrast, T cells activated by below-threshold antigens underwent symmetric division, leading to abortive clonal expansion and failure to fully differentiate into tissue-infiltrating effector cells. Antigen affinity and asymmetric division are important factors that regulate fate specification in CD8(+) T cells and predict the potential of a self-reactive T cell to mediate tissue pathology.

A constant affinity threshold for T cell tolerance

T cell tolerance depends on the T cell receptors affinity for peptide/major histocompatibility complex (MHC) ligand; this critical parameter determines whether a thymocyte will be included (positive selection) or excluded (negative selection) from the T cell repertoire. A quantitative analysis of ligand binding was performed using an experimental system permitting receptor–coreceptor interactions on live cells under physiological conditions. Using three transgenic mouse strains expressing distinct class I MHC–restricted T cell receptors, we determined the affinity that defines the threshold for negative selection. The affinity threshold for self-tolerance appears to be a constant for cytotoxic T lymphocytes.

The T Cell Receptor’s α-Chain Connecting Peptide Motif Promotes Close Approximation of the CD8 Coreceptor Allowing Efficient Signal Initiation

The CD8 coreceptor contributes to the recognition of peptide-MHC (pMHC) ligands by stabilizing the TCR-pMHC interaction and enabling efficient signaling initiation. It is unclear though, which structural elements of the TCR ensure a productive association of the coreceptor. The α-chain connecting peptide motif (α-CPM) is a highly conserved sequence of eight amino acids in the membrane proximal region of the TCR α-chain. TCRs lacking the α-CPM respond poorly to low-affinity pMHC ligands and are unable to induce positive thymic selection. In this study we show that CD8 participation in ligand binding is compromised in T lineage cells expressing mutant α-CPM TCRs, leading to a slight reduction in apparent affinity; however, this by itself does not explain the thymic selection defect. By fluorescence resonance energy transfer microscopy, we found that TCR-CD8 association was compromised for TCRs lacking the α-CPM. Although high-affinity (negative-selecting) pMHC ligands showed reduced TCR-CD8 interaction, low-affinity (positive-selecting) ligands completely failed to induce molecular approximation of the TCR and its coreceptor. Therefore, the α-CPM of a TCR is an important element in mediating CD8 approximation and signal initiation.