Differentiation marker-negative CD4 ⁺ T cells persist after yellow fever virus vaccination and contribute to durable memory

Factors that contribute to durable immunological memory remain incompletely understood. In our longitudinal analyses of CD4 ⁺ T cell responses to the yellow fever virus (YFV) vaccine by peptide-MHC tetramers, we unexpectedly found naïve phenotype virus-specific CD4 ⁺ T cells that persisted months to years after immunization. These Marker negative T cells (T _MN ) lacked CD95, CXCR3, CD11a, and CD49d surface protein expression, distinguishing them from previously discovered stem-cell memory T cells. Functionally, they resembled genuine naïve T cells upon in vitro stimulation. Single-cell TCR sequencing detected expanded clonotypes within the T _MN subset and identified a shared repertoire with memory and effector T cells. T cells expressing T _MN -associated TCRs were rare before vaccination, suggesting their expansion following vaccination. Longitudinal tracking of YFV-specific responses over the subsequent years revealed superior stability of the T _MN subset and their association with the longevity of the overall population. The identification of these long-lived, antigen-experienced T cells may inform the design of durable T cell-based vaccines and engineered T cell therapies.