Distinct cytokine signatures in Anti-PD-1 or Anti-CTLA-4 induced thyroiditis: insights from a new mouse model

The pathogenesis of thyroiditis caused by immune checkpoint inhibitors (ICIs), 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. Forty‐three NOD‐H2h4 mice (24 M, 19 F), 112 days old at the time of the first i.p. injection, were divided into four experimental groups: 20 mice received combined anti‐PD‐1 and anti‐CLTA‐4; 8 only anti‐PD‐1; 8 only anti‐CTLA‐4; and 7 an isotype control. Mice were sacrificed on day 172 (2 months after the initial injection) to collect thyroid gland (for histopathology and flow cytometry) and spleen (for flow cytometry). Mice were also studied before sacrifice to determine thyroid area and structure (by ultrasound using a MS700 transducer), thyroid function (by serum total T4 and TSH using bead‐based Luminex technology), and the serum levels of numerous cytokines/chemokines (by Luminex).Thyroiditis was more severe in mice receiving anti‐PD‐1 than anti‐CTLA‐4 (p = 0.01), and associated significantly with the absolute number of CD45+ infiltrating cells (cumulative OR 1.25, 95% CI 1.1–1.4, p < 0.001). On the contrary, thyroiditis was more prevalent (100% vs 63%, p < 0.01) in the anti‐CTLA‐4 mice, which also showed a larger thyroid area (17 ± 8.2 vs 11 ± 4.2 mm2, p < 0.01) than those treated with anti‐PD‐1 and controls. Interestingly, mice treated with PD‐1 that developed thyroiditis showed a striking increase in systemic IL‐6 (40 pg/mL at baseline, 268 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 MIP1b increased more in the anti‐CTLA‐4 group (p < 0.001 for both), whereas the other cytokines/chemokines did not differ among the groups. The study reports the first 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. The study also offers biomarkers, such as serum IL‐6, that could be used in the clinical setting.