Clonally-Expanded, Thyrotoxic Autoimmune Mediator CD8+ T cells Driven by IL21 Contribute to Checkpoint Inhibitor Thyroiditis

Autoimmune toxicity occurs in up to 60% of patients treated with immune checkpoint inhibitor (ICI) cancer therapy and is an increasing clinical challenge with the expanding use of these treatments. To date, human immunopathogenic studies of immune related adverse events (IRAEs) have relied upon sampling of circulating peripheral blood cells rather than affected tissues. Here, we directly obtained thyroid specimens from subjects with ICI-thyroiditis, one of the most common IRAEs, and compared immune infiltrates to those from subjects with spontaneous autoimmune Hashimoto's thyroiditis (HT) or no thyroid disease. Single cell RNA sequencing revealed a dominant, clonally expanded population of thyroid-infiltrating cytotoxic CXCR6+ CD8+ T cells ("CD8+ autoimmune mediators") present in ICI-thyroiditis, but not HT or healthy controls. Furthermore, we identified a crucial role for interleukin 21, a cytokine secreted by intrathyroidal T follicular (Tfh) and T peripheral helper (Tph) cells, as a driver of these thyrotoxic CD8+ autoimmune mediators. In the presence of IL21, human CD8+ T cells acquired the autoimmune mediator phenotype with upregulation of cytotoxic molecules (IFNγ, granzyme); the chemokine receptor CXCR6; and thyrotoxic capacity. We validated these findings in vivo using a novel mouse model of IRAEs, and further demonstrated that genetic blockade of IL21 signaling protected ICI-treated mice from thyroid immune infiltration. Taken together these studies reveal novel mechanisms and therapeutic targets by which IL21+ Tfh/Tph cells drive thyrotoxic CD8+ autoimmune mediators for the development of IRAEs in humans. ### Competing Interest Statement A.R. has received honoraria from consulting with Amgen, Bristol-Myers Squibb, Chugai, Genentech, Merck, Novartis, Roche, Sanofi and Vedanta, is or has been a member of the scientific advisory board and holds stock in Advaxis, Appia, Apricity, Arcus, Compugen, CytomX, Highlight, ImaginAb, Isoplexis, Kalthera, Kite-Gilead, Merus, PACT Pharma, Pluto, RAPT, Rgenix, Synthekine and Tango, has received research funding from Agilent and from Bristol-Myers Squibb through Stand Up to Cancer (SU2C), and patent royalties from Arsenal Bio. A.D. has received consulted for Bristol-Myers Squibb, AstraZeneca, Radmetrix, Seattle Genetics, Janssen, PACT Pharma, Merck, Roche/Genetech, Exelixis, Dyania Health, and has received research funding Kite/Gilead, AstraZeneca, Roche/Genetech, BMS, Merck, Jounce Therapeutics, Infinity Pharmaceuticals, Seattle Genetics. All other authors declare that they have no competing interests.