Steady-state memory-phenotype conventional CD4⁺ T cells exacerbate autoimmune neuroinflammation in a bystander manner via the Bhlhe40/GM-CSF axis

Memory-phenotype (MP) CD4⁺ T cells are a substantial population of conventional T cells that exist in steady-state mice, yet their immunological roles in autoimmune disease remain unclear. In this work, we unveil a unique phenotype of MP CD4⁺ T cells determined by analyzing single-cell transcriptomic data and T cell receptor (TCR) repertoires. We found that steady-state MP CD4⁺ T cells in the spleen were composed of heterogeneous effector subpopulations and existed regardless of germ and food antigen exposure. Distinct subpopulations of MP CD4⁺ T cells were specifically activated by IL-1 family cytokines and STAT activators, revealing that the cells exerted TCR-independent bystander effector functions similar to innate lymphoid cells. In particular, CCR6^high subpopulation of MP CD4⁺ T cells were major responders to IL-23 and IL-1β without MOG_35-55 antigen reactivity, which gave them pathogenic Th17 characteristics and allowed them to contribute to autoimmune encephalomyelitis. We identified that Bhlhe40 in CCR6^high MP CD4⁺ T cells as a key regulator of GM-CSF expression through IL-23 and IL-1β signaling, contributing to central nervous system (CNS) pathology in experimental autoimmune encephalomyelitis. Collectively, our findings reveal the clearly distinct effector-like heterogeneity of MP CD4⁺ T cells in the steady state and indicate that CCR6^high MP CD4⁺ T cells exacerbate autoimmune neuroinflammation via the Bhlhe40/GM-CSF axis in a bystander manner.