Heterogeneity of islet-infiltrating interleukin-21+ CD4 T cells in a mouse model of type 1 diabetes

Interleukin (IL)-21 is essential for type 1 diabetes (T1D) development in the NOD mouse model. IL-21-expressing CD4 T cells are present in pancreatic islets where they contribute to disease progression. However, little is known about their phenotype and differentiation states. To fill this gap, we generated a novel IL-21 reporter NOD strain to further characterize IL-21+ CD4 T cells in T1D. IL-21+ CD4 T cells accumulate in pancreatic islets and recognize ß-cell antigens. Single-cell RNA sequencing revealed that most CD4 T effector cells in islets actively express IL-21 and they are highly diabetogenic despite expressing multiple inhibitory molecules, including PD-1 and LAG3. Islet IL-21+ CD4 T cells segregate into four phenotypically and transcriptionally distinct differentiation states, less differentiated early effectors, Tfh-like cells, and two Th1 subsets. Trajectory analysis predicts that early effectors differentiate into both Tfh-like and terminal Th1 cells. We further demonstrated that intrinsic IL-27 signaling controls the differentiation of islet IL-21+ CD4 T cells, contributing to their helper function. Collectively, our study reveals the heterogeneity of islet-infiltrating IL-21+ CD4 T cells and indicates that both Tfh-like and Th1 subsets continuously produce IL-21 throughout their differentiation process, highlighting the important sources of IL-21 in T1D pathogenesis. Overall design: Islet-infiltrating CD4 T cells from NOD.Il21V/+ mice were isolated by fluorescence-activated cell sorting (FACS) according to the presence of absence of Venus signal and analyzed by scRNA-seq.