SummaryThis study was undertaken to investigate the role of CD4+FoxP3+ regulatory T cells (Tregs) in regulating neuroinflammation during viral Ag-challenge and re-challenge. CD8+lymphocytes persisting within tissues are designated tissue-resident memory T cells (TRM), within brain: bTRM. Reactivation of bTRMwith T cell epitope peptides generates rapid antiviral recall, but repeated stimulation leads to cumulative dysregulation of microglial activation, proliferation, and prolonged neurotoxic mediator production. Here, we show Tregs were recruited into murine brains following prime-CNS boost, but displayed altered phenotypes following repeated Ag-challenge. In response to repeated Ag, brain Tregs (bTregs) displayed inefficient immunosuppressive capacity, along with reduced expression of suppression of tumorigenicity 2 (ST2) and amphiregulin (Areg).Ex vivoAreg treatment revealed reduced production of neurotoxic mediators such as iNOS, IL-6, and IL-1β, and decreased microglial activation and proliferation. Taken together, these data indicate bTregs display an unstable phenotype and fail to control reactive gliosis in response to repeated Ag-challenge.