Identification of RORg(+)T Cells as Key Players in Thyroid Autoimmunity From Checkpoint Immunotherapy

Purpose: Immune checkpoint inhibitors (ICI) are powerful new cancer therapies that leverage the body’s own immune system to attack cancer cells. Unfortunately, their use may be limited by the development of immune-related adverse events (IrAE) in up to 60% of patients. Thyroiditis is a common IrAE, with shared and distinct features from spontaneous thyroid autoimmunity, i.e. Hashimoto thyroiditis (HT). The cause of IrAE remains unknown, however, recent data suggest that toxicity can be uncoupled from anti-tumor effects. Methods: We developed a novel mouse model to study mechanisms of IrAE, in which ICI (anti-PD-1 and/or anti-CTLA-4) treatment leads to multi-organ immune infiltrates, including thyroiditis. To understand immune changes occurring with ICI-autoimmunity, we first evaluated changes in the frequency and activation status of different immune cells in our mice using immunohistochemistry (IHC) and flow cytometry. Then we confirmed these findings in peripheral blood and thyroid fine needle aspiration (FNA) specimens from patients with ICI-thyroiditis, HT, or no IrAE, using flow cytometry and single cell RNA sequencing (scRNAseq) techniques. Results: In our mouse model, ICI treatment of autoimmune-prone non-obese diabetic mice induces multi-organ autoimmunity. Modeling ICI-IrAE observed in humans, our mice developed increased immune infiltrates in multiple tissues (e.g. thyroid, colon, liver, lung), autoantibodies, and acceleration of underlying autoimmune risk (i.e. diabetes). Increased frequency of autoimmune disease was seen with combination (anti-PD-1 + anti-CTLA-4) vs. single agent ICI. We found increased IL-17A(+) T cells in secondary lymphoid tissues of ICI-treated mice, a cytokine produced by RORγ (+) Th17 and Tc17 cells and associated with autoimmunity. IHC studies on thyroid infiltrates showed accumulation of CD4(+) and CD8(+) T cells and macrophages in ICI-treated vs. isotype control mice. This finding was confirmed by flow cytometry analyses of thyroid-infiltrating leukocytes in ICI-thyroiditis mice, which showed significantly increased T cells, specifically RORγ (+) T cells, and rare B220(+) B, CD11b(+) myeloid, or NKp46(+) NK cells. In patients with ICI-thyroiditis, thyroid FNA showed that thyroid immune infiltrates were predominately T cells. scRNAseq studies in patients with ICI-thyroiditis showed enrichment of Th17 and Tc17 (RORγ (+) IL23R(+) CD161(+)) T cells, compared to ICI-treated patients without IrAE. Conclusion: We have identified a role for RORγ (+) Th17 and Tc17 cells in thyroid autoimmunity from ICI using a newly developed mouse model of ICI-associated IrAE and translational studies in patients with ICI-thyroiditis. Th17 and Tc17 cells have previously been associated with spontaneous autoimmune disease, including HT, but have not yet been characterized in IrAE. These cells provide a potential therapeutic target for prevention of endocrine IrAE from ICI.