Functional and structural correlates of T cell receptor specificity and cross-reactivity

An effective immune system depends upon a network of molecular interactions that detect and respond to pathogens and altered self. T cells are central to mammalian immunity, harnessing their T cell receptors (TCR) to discriminate between self and foreign or altered peptides presented on major histocompatibility complexes (pMHC). The TCR repertoire is dwarfed by a much greater landscape of foreign pMHC, requiring each TCR to recognise numerous peptides for sufficient immunological surveillance, a concept known as “cross-reactivity”. Important aspects of the molecular mechanisms underlying TCR cross-reactivity are unknown, prohibiting the accurate mapping of TCR to pMHC and vice versa. This imposes significant limitations on the applicability and safety of TCR-based therapies. The work described in this thesis seeks to characterise TCR cross-reactivity by probing TCR/pMHC interactions at scale using diverse pMHC yeast display libraries together with functional and biophysical assays. Leveraging the affinity-enhanced a3a TCR with its established cross-reactivity for self-antigens as a case study, the capacity to “de-engineer” cross-reactivity is explored by systematically reverting the engineered amino acid substitutions to the original TCR sequence. Characterisation of these novel TCR variants confirms that the cross-reactivity of the a3a TCR is driven by greater contributions of TCR:MHC relative to TCR:peptide contacts and further exacerbated by a peptide binding “hotspot”. Combining pMHC yeast display libraries with computational prediction pipelines characterises the agonist peptide landscapes of the TCR variants and identifies sequences displaying minimal cross-reactivity. Steps are also taken toward investigating the possibility of tuning T cell sensitivity without manipulating the extracellular TCR by altering the intracellular signalling domains. Lastly, in an attempt to establish an alternative to the classic X-ray crystallographic methods used to describe the structural components of the TCR/pMHC interaction, the structures of two physiological TCR/pMHC complexes are determined by cryo-EM.