Antigen regulation of t cell receptor signalling and immune checkpoint expression in health and disease

Binding of cognate antigen to the T cell receptor (TCR) results in downstream signalling, which controls T cell activation and effector function. It is still however controversial how antigen dose and affinity, as well as over-expression of immune checkpoints in the tumour microenvironment (TME) affect such processes. The novel Nr4a3-Timer of cell kinetics and activity (Tocky) system was used for the temporal analysis of CD8+ T cell activation through distal TCR signalling analyses. It was first found that expression of two downstream TCR genes respond differently to signalling; Nr4a1 requires shorter, weaker signals than Nr4a3. Using the OTI TCR transgenic system and modified peptides, it was established that TCR affinity affects the antigen dose required to trigger expression of IFNγ and PD-1 as well as affecting maximum response, by altering TCR signalling dynamics. Finally, the in vivo effects of the TME on T cell activation, immune checkpoints and IFNγ expression was investigated, resulting in a set of findings that provide an overview of T cell activation and checkpoint expression in response to tumour antigens. This project therefore broadens our understanding of how antigen regulates CD8+ T cell biology and proposes future lines of investigation to understand anti-tumour T cell responses

Exploring BenzylethoxyAryl Urea Scaffolds for Multitarget Immunomodulation Therapies

Thirteen benzylethoxyaryl ureas have been synthesized and biologically evaluated as multitarget inhibitors of VEGFR-2 and PD-L1 proteins to overcome resistance phenomena offered by cancer. The antiproliferative activity of these molecules on several tumor cell lines (HT-29 and A549), on the endothelial cell line HMEC-1, on immune cells (Jurkat T) and on the non-tumor cell line HEK-293 has been determined. Selective indexes (SI) have been also determined and compounds bearing p-substituted phenyl urea unit together with a diaryl carbamate exhibited high SI values. Further studies on these selected compounds to determine their potential as small molecule immune potentiators (SMIPs) and as antitumor agents have been performed. From these studies, we have concluded that the designed ureas have good tumor antiangiogenic properties, exhibit good inhibition of CD11b expression, and regulate pathways involved in CD8 T-cell activity. These properties suggest that these compounds could be potentially useful in the development of new cancer immune treatments

Antigen and checkpoint receptor engagement recalibrates T cell receptor signal strength

How T cell receptor (TCR) signal strength modulates T cell function and to what extent this is modified by immune checkpoint blockade (ICB) are key questions in immunology. Using Nr4a3-Tocky mice, we characterized early quantitative and qualitative changes that occur in CD4(+) T cells in relation to TCR signaling strength. We captured how dose- and time-dependent programming of distinct co-inhibitory receptors rapidly recalibrates T cell activation thresholds and visualized the immediate effects of ICB on T cell re-activation. Our findings reveal that anti-PD1 immunotherapy leads to an increased TCR signal strength. We defined a strong TCR signal metric of five genes upregulated by anti-PD1 in T cells (TCR.strong), which was superior to a canonical T cell activation gene signature in stratifying melanoma patient outcomes to anti-PD1 therapy. Our study therefore reveals how analysis of TCR signal strength—and its manipulation—can provide powerful metrics for monitoring outcomes to immunotherapy