Cd69 expression on regulatory t cells as potential predictor of cardiovascular events risk before immunotherapy

Abstract Background/Introduction Immunotherapies, specifically immune checkpoint inhibitors (ICIs), have revolutionised cancer treatment by enhancing antitumor immune responses through inhibition of regulatory T cells. However, these treatments can also induce immune-related adverse events (irAEs), including myocarditis, a rare but highly fatal complication. CD69 is a regulator of immune tolerance that promotes Treg function and inhibits Th17 differentiation. Reduced CD69 expression on Tregs has been associated with worse outcomes in several cardiovascular diseases. Purpose This sub-study of the immunotherapy registry of cardiovascular toxicity aims to characterize the immune profile of cancer patients (before and during treatment) to identify baseline biomarkers that allow stratification of patients according to their susceptibility to develop cardiotoxicities. Methods Patients were recruited from multiple centres, and peripheral blood samples were collected at baseline and at 1, 3, 6, and 12 months after initiation of immunotherapy. Flow cytometry was performed on PBMCs from 500 patients using an LSRFortessa (BD), with data analysed using FlowJo v10.1. Results Patients show no significant differences in total CD4 T-cell population after the immunotherapy treatment started. However, in the first weeks of treatment, a systemic lost of peripheral tolerance is observed due to a very significant drop in activated regulatory T cells (CD4+Foxp3+ CD69+). Moreover, there is a slow but steady increase in the cytokine-secreting T cells which becomes significant six months after the initiation of immunotherapy. Using bioinformatic analyses, patients were stratified into two groups based on baseline (before immunotherapy) CD69 expression on Tregs: low CD69 and high CD69. Further analyses show significant differences between these groups, with low CD69 at baseline patients exhibiting the utmost loss of CD69 expression 2–4 weeks after immunotherapy. This phenomenon of loss of peripheral tolerance usually overlaps temporally with the development of cardiovascular events. In fact, preliminary data suggest that patients with low CD69 expression at baseline present a twofold higher incidence of cardiovascular events compared with patients with high CD69 expression. Conclusion This study presents a new understanding of the dynamics in peripheral blood lymphocytes during cancer immunotherapy. Results suggest that the decrease in activated Tregs play a key role in maintaining immune homeostasis. Preliminary findings indicate that baseline CD69 expression on Tregs may serve as a biomarker to stratify patients according to their risk of developing cardiotoxicities. Data also show that immunotherapy treatment could lead to chronic inflammation in susceptible patients. Combining these findings with clinical data, we have the potential to identify biomarkers that could predict or prevent adverse events and ultimately improve patient outcomes. Figure 2