Distinct Cytokine Signatures in Thyroiditis Induced by PD-1 or CTLA-4 Blockade: Insights from a New Mouse Model.

Background: The pathogenesis of thyroiditis caused by immune-checkpoint inhibitors (ICIs) such as antiprogrammed death receptor-1 (PD-1) and anticytotoxic T lymphocyte antigen-4 (CTLA-4) is incompletely understood. To gain mechanistic insights, we developed a mouse model of ICI-related thyroiditis and assessed the clinical, hormonal, and cytokine profiles. Methods: Forty NOD-H2 ^h4 mice, 112 days old at the start of the experiments, were divided into two sequential cohorts. In the first one (No. = 21), mice were injected with both anti-PD-1 and anti-CTLA-4 checkpoint inhibitors while drinking either regular water or iodine-supplemented water. In the second cohort (No. = 19), mice were injected with either anti-PD-1 or anti-CTLA-4 while drinking iodine-supplemented water. Mice were sacrificed two months after the initial injection to collect thyroid gland for histopathology (to assess thyroiditis severity) and flow cytometry (to identify immune cell subsets and tissue-resident memory T cell markers). Mice were also studied before sacrifice to determine thyroid area and structure (by ultrasound), thyroid function (serum total thyroxine, thyrotropin, thyroid antibodies), and cytokine profile (by bead-based Luminex technology). Results: Thyroiditis was more severe upon PD-1 than CTLA-4 blockade ( p  = 0.01) and significantly correlated with the number of CD45 ⁺ cells infiltrating the thyroid (cumulative odds ratio [OR] 1.2 [95% confidence interval, CI 1.1-1.3], p  < 0.001, that is 20% greater odds of a higher severity score for every 170-unit increase in CD45 infiltrating cells). Thyroiditis was instead more prevalent (100% vs. 63%, p  < 0.01) in the anti-CTLA-4 mice, which also showed a larger thyroid area (17 ± 8.2 mm) than those treated with anti-PD-1 (11 ± 4.2 mm) and controls ( p  < 0.01). Serum IL-6 was markedly increased upon PD-1 blockade (40 pg/mL at baseline, 198 pg/mL on day 172), an increase not seen in the anti-CTLA-4 group ( p  = 0.01). IL-6 mirrored thyroiditis severity, with highest serum values found in greatest histopathology scores (cumulative OR 1.1 [CI 1.02-1.15], p  = 0.009). GM-CSF and MIP1β increased more in the anti-CTLA-4 group ( p  < 0.001 for both), whereas the other cytokines did not differ among the treatment groups. Conclusions: The study reports a mouse model of thyroiditis induced by PD-1 blockade and, comparing it to the anti-CTLA-4 model, uncovers distinctive histopathological, sonographic, hormonal, and immunological features, offering biomarkers, such as serum IL-6, that could be used in the clinical setting.