Investigating ici-induced thyroid dysfunction: Initial immune dysregulation insights and 3D co-culture model with Nthy-Ori 3-1 and PBMC

Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, significantly improving survival rates across a range of malignancies by reinforcing T cells' ability to recognize and eradicate cancer cells. Despite their effectiveness, ICIs are frequently associated with immune-related adverse events (irAEs), the most prevalent of which is thyroid dysfunction (TD). Rarely, these toxicities may be severe enough to cause therapy to be postponed or terminated. Understanding the mechanisms underlying thyroid irAEs could provide valuable insights into both thyroid autoimmune diseases and the broader spectrum of irAEs linked to ICIs. This study addresses gaps in current research by immunophenotyping patients undergoing ICI therapy at two critical time points: before treatment and three weeks post-treatment. Comparative analyses were performed against control groups of healthy individuals and patients with Hashimoto's thyroiditis. Among the 9 patients studied, 2 developed TD within three weeks of PD-1 inhibitor treatment. Flow cytometry revealed significant increases in IFN-γ producing CTLs and NK cells, along with TNF-α producing B cells and its expression levels in NKT-like cells following the therapy (p<0.05). Notably, the increase in TNF-α producing B cells was more pronounced when compared to all study groups. These findings suggest that immune cell activation and subsequent cytokine production, aimed at combating tumor cells, may also serve as predictive biomarkers for irAEs, including ICI-induced TD. To improve the physiological relevance of in vitro studies, this research also introduces a novel 3D co-culture model using thyroid spheroids derived from the Nthy-Ori 3-1 cell line, co-cultured with patient-specific PBMCs. Initial results indicated no significant differences in thyroid cell viability between TD patients and controls in the 3D setting, contrasting with notable viability reductions in 2D co-cultures. Additionally, immunophenotyping performed on 3D co-cultures revealed a substantial increase in B cell populations of the patients on the third week of therapy compared to healthy controls (p=0.0500). The findings from both immunophenotyping and in vitro research deepen our understanding of conditions where the pathophysiology is poorly defined, as in the case of ICI-induced TD. Despite limitations related to the allogeneic nature of the approach, this study is pioneering in creating an in vitro model specifically designed to investigate ICI-related irAEs. It offers preliminary insights and establishes a methodological framework for future thyroid research.