<p>The innate immune system is increasingly recognized as a contributor to the development of type 1 diabetes (T1D), but the role of natural killer (NK) cells remains largely unclear. Here, we identify an expanded subset of transcriptionally active CD226⁺CD56^dimCD16⁺ NK cells at the onset of T1D that contracts in remission. Using single-cell RNA sequencing integrated with cross-sectional and longitudinal analyses in patients with T1D, we show that CD226⁺ NK cell frequency correlates with disease progression. CD226⁺ NK cells exhibit enhanced cytotoxicity, inflammation and glucose metabolism. Mechanistically, CD161⁺CD4⁺ T cells promote pathogenic NK cell generation through interleukin-21 (IL-21) and mTOR signalling. Inhibition of this pathway by CD226 blockade, IL-21 receptor fusion protein, IL-21 knockout or mTOR inhibition attenuates NK cell activation, reduces pancreatic infiltration and delays diabetes onset in female mice. Our data reveal a mechanistic link, bridging adaptive and innate immunity, in the progression and remission of T1D that could potentially be exploited in T1D immunotherapy.</p>