Alternatively activated neutrophils limit T cell–driven neuroinflammation

Non-canonical myeloid cell populations are increasingly recognized as critical regulators of inflammation in neuroimmunological disease. Here, we investigate the role of alternatively activated neutrophils (aaN) in limiting encephalitogenic T cell responses during experimental autoimmune encephalomyelitis (EAE), a widely used model of multiple sclerosis. Arginase-1–expressing aaN were identified and characterized within central nervous system (CNS) infiltrates during EAE using flow cytometry, single-cell RNA sequencing, and fluorescent in situ hybridization (RNAscope) combined with immunohistochemistry. The immunomodulatory properties of aaN were evaluated in vitro using CD4⁺ T cell suppression assays and in vivo by adoptive transfer of ex vivo–generated aaN during the preclinical phase following encephalitogenic T-cell injection. aaN were consistently detected within the CNS throughout EAE and spatially co-localized with encephalitogenic T cells. Transcriptomic profiling of aaN revealed enrichment of pathways associated with regulation of T cell activation and immune suppression. CNS-derived aaN potently inhibited CD4⁺ T cell proliferation in vitro. Therapeutic augmentation of this population, via adoptive transfer of ex vivo–generated aaN into mice following the injection of encephalitogenic T cells, delayed clinical EAE onset, markedly reduced the accumulation of pathogenic T cells within CNS lesions, and significantly enhanced neuronal survival. Mechanistically, ex vivo–generated aaN suppressed T cell responses through a contact-dependent, PD-L1–independent pathway, indicating a previously unrecognized mode of neutrophil-mediated immunoregulation. These findings identify aaN as a previously underappreciated immunoregulatory population within the inflamed CNS that restrains pathogenic T cell responses and limits neuroinflammation during EAE. Collectively, our data support the therapeutic potential of strategies that augment aaN activity, including autologous aaN-based cell therapy or interventions that promote CNS homing, polarization, and persistence of endogenous aaN, as novel approaches for disease modification in multiple sclerosis.