Mapping PD-1 Immune Checkpoint Receptor Signalling Networks in T-Cells

Tumour cells can suppress immune cell activation by engaging inhibitory receptors, such as programmed cell death protein-1 (PD-1), on T-cells. Targeting these receptors with immune checkpoint inhibitors (ICIs) can stimulate the anti-tumour immune cell activity. Enlisting the immune system to fight cancer is an attractive concept, as it promises to be effective against many different types of cancer. Unfortunately, responses are highly variable and low in many tumour types (response rates < 12%). However, ICI responses tend to be durable asserting that the immune system can successfully eradicate or control cancer. Thus, ICIs are promising therapeutic agents, but can be improved by a deeper mechanistic understanding of their molecular mode of action. While T-cell receptor (TCR) signalling is well described, little is known about the signalling of immune checkpoints. This project aimed to dissect the signalling networks of PD-1 to better understand its role in controlling T-cell activation in order to improve ICI efficacy. To achieve this objective, a multi-omics approach was utilised. Interaction proteomic profiling, whole-cell proteomic profiling and phosphoproteomic profiling was performed in 1, T-cell lines activated in vitro by TCR ligation and 2, T-cell lines activated in vitro by combined TCR ligation and PD-1 ligation. To evaluate if signals from the receptor were transduced to the nucleus and activate transcriptional processes, the proteomics data were complemented by transcriptomics data generated by a collaborator. This analysis has yielded some novel aspects about PD-1 signalling. For instance, EZR and MSN were identified as novel binding partners of PD-1. A stable and dynamic interactome of PD-1 were defined in TCR and TCR + PD-1 ligated cells. The whole-cell proteome analysis revealed that changes in protein expression are rather small following TCR or PD-1 stimulation. However, PD-1 expressing cells showed increased abundance of RCSD1, F11R, CARMIL2, SPN and USF1, proteins which are important for various T-cell functions and represent possibly novel mediators of PD-1 inhibitory functions. Phosphoproteomic profiling captured a stable and dynamic phosphoproteome of PD-1. A statistically significant increase in the number of phosphorylation sites observed at 20 min of PD-1 stimulation suggests that the PD-1 pathway signals beyond its known interactor, the phosphatase SHP2. Integration of the phosphoproteomics data and transcriptomics data revealed that TCR and PD-1 have common targets which may be controlled in a reverse fashion by stimulatory TCR and inhibitory PD-1. Studying the binding partners of SHP2, confirmed the existence of a stable interactome and the enrichment of Notch signalling pathway in PD-1 expressing T-cells further represents a novel pathway possibly regulated by the immune checkpoint. Ingenuity Pathway Analysis was utilised to contextualise our findings. Enriched pathways are associated with recognised TCR and PD-1 functions including the organisation of the actin cytoskeleton and the regulation of metabolic pathways. The model system used in this study can recapitulate known signalling pathways (TCR, ERK-MAPK, mTOR, AMPK). Interestingly and importantly, an enrichment of the ATM DNA damage response pathway was captured in the phosphoproteomics data from PD-1 stimulated cells suggesting a previously undescribed involvement of PD-1 in DNA damage response pathways. In conclusion, multi-omics analysis provided insights into pathways that control the regulation of T-cell (de)activation. Comparison of data obtained from TCR and PD-1 ligated cells reveals that TCR signalling and PD-1 signalling impinge on similar targets. Moreover, PD-1 may be implicated in novel pathways, such as the ATM DNA damage response pathway. A deeper mechanistic understanding of immune checkpoints will help to improve ICIs application in patients in the clinics.