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 (ICI) such as anti--PD--1 and anti--CTLA--4 is incompletely understood. To gain mechanistic insights, we developed a mouse model of ICI--related thyroiditis and assessed clinical, hormonal, and cytokine profiles. METHODS Fourty NOD-H2h4 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-CLTA-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 2 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-cells markers). Mice were also studied before sacrifice to determine thyroid area and structure (ultrasound), thyroid function (serum total T4, TSH, thyroid antibodies), and cytokine profile (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 OR 1.2, 95% 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, 95% 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.