Understanding immune-related toxicities through multifacet profiling

Immune checkpoint inhibitor therapy is a new type of cancer treatment that has significantly improved the outcomes for many patients with widespread or locally advanced cancer. The immune system normally works to prevent cancer by recognising and destroying cancerous cells. However, many types of cancer use mechanisms to escape this immune surveillance. One way they do so is by reducing the activity of anti-cancer immune cells using cell-surface signalling molecules called immune checkpoints. When these immune checkpoints are engaged by cancer cells they render the immune cells inactive. This then allows tumours to grow unchecked. Immune checkpoint inhibitors block these immune checkpoints to reactivate anti-cancer immune cells. Given their success, these drugs have been licensed to treat an increasing range of cancer types. Their use is also expanding in the adjuvant setting, where patients receive immunotherapy after surgery to reduce the risk of their cancer returning. Thus, an ever-increasing number of patients receive these medications.
Unfortunately, immune checkpoint inhibition is associated with potentially serious side effects. These are called immune-related adverse events (irAEs). They result from harmful overactivation of immune cells, which attack healthy tissues rather than cancer cells. irAEs affect up to 95% of cancer patients treated with the strongest form of immunotherapy, which combines two immune checkpoint inhibitors. Up to 50% of patients will experience irAEs that require either treatment with powerful immune-suppressing steroids or discontinuation of their immunotherapy. Both carry a risk of worse prognosis of their cancer, whilst steroids can also cause significant other side effects.
We therefore require a better understanding of the mechanisms behind irAEs to develop improved treatments that target the irAE more specifically without compromising anti-cancer effects. Currently, how and why irAEs arise is very poorly understood. We do not know why some patients develop severe or even deadly irAEs whilst others develop no irAEs at all. We have also not identified the key types of immune cells and signalling molecules involved in irAEs. This is urgently required to identify potential new therapeutic targets.
Our research project is designed to address these key issues. We will be working with blood and tissue samples from patients receiving immune checkpoint inhibitors. We are focussing on skin, as it is very commonly affected by irAEs. The first blood sample is taken before patients start treatment. We then take repeat samples during treatment and when irAEs develop. We will identify what types of immune cells and messaging molecules arise in the blood of irAE patients compared to non-irAE patients. One key hypothesis is that irAEs patients have a defect in specific immune cells, which control the activity of other "effector" immune cells. The controller immune cells we will assess are T-regulatory cells, which control cell-mediated immune responses, and follicular T-cells, which control antibody-mediated immune responses. We will measure their numbers and test whether cells from irAE patients are impaired in their ability to control immune activation.
Skin biopsies from patients with and without skin irAEs will be analysed with a powerful technique called single cell RNA sequencing. This allows us to identify all the immune cells within the skin sample and what inflammatory messenger pathways are active in them. This can lead to the identification of novel therapeutic targets to switch the skin inflammation back off.
Finally, we will explore whether it is possible to predict if an individual patient is likely to develop irAEs before they start immunotherapy. This has the potential to help personalise treatment. Patients at high risk of irAEs could be monitored more closely or potentially receive less intense treatment to avoid serious side effects.

Aim:
Elucidate the pathogenesis and risk factors of immune related adverse events (irAEs) due to immune checkpoint inhibitor (ICI) therapy.
Materials:
Tissue, blood, and clinical data from patients undergoing standard of care ICI therapy at The Royal Marsden Hospital. Material and clinical data are derived from the EXACT (understanding immunE related toXicities through multifACeT profiling) trial (REC 21/WM/0251).
Objectives and assays:
1) Define the inflammatory infiltrate and molecular pathways of ICI-induced skin inflammation (single cell RNA sequencing)

2a) Elucidate whether irAEs are associated with derangements in circulating activated T-regulatory cells (Tregs) (mass cytometry)
2b) Test if Tregs derived from irAE patients using fluorescence activated cell sorting are functionally impaired in co-culture suppression assays compared to Tregs isolated from ICI-treated patients without irAEs

3a) Elucidate whether irAEs are associated with derangements in circulating T-follicular regulatory (cTfr) and helper (cTfh) cell populations (mass cytometry)
3b) Test whether cTfr and cTfh imbalances in irAE patients correlate with enhanced humoral autoimmunity assayed by multiplex auto-antibody quantification

4) Screen for biomarkers that might predict the development of irAEs and generate further hypotheses of irAE pathogenesis. The exploratory analysis will encompass:
- Mass cytometric assessment of peripheral blood mononuclear cell populations (data from objective 2 and 3)
- Quantification of circulating auto-antibodies (multiplex microarray from objective 3)
- Quantification of circulating cytokines (multiplex electrochemiluminescence immunoassay)

This work has the potential to identify novel therapeutic targets to treat irAEs and generate fundamental insights into mechanisms of loss of immunological self-tolerance, which result in autoimmunity.