Antigen-reactive CD4 T cells after SARS-CoV-2 vaccination show divergent phenotypic states with or without restimulation [mixed samples]

Understanding T cell fate requires precise knowledge of antigen reactivity. For CD4+ T cells, conventional identification of antigen reactivity by peptide restimulation relies on cellular activation, but thereby also introduces a phenotypic bias. However, by performing single-cell RNA and TCR sequencing on both antigen-stimulated and unstimulated samples, clonotypes can be tracked across conditions to identify antigen-reactive CD4+ T cells and simultaneously assess their phenotypes in the unperturbed state. Using this “reverse phenotyping” strategy, complemented by DNA-barcoded peptide–HLA class II multimers, we here investigated the longitudinal phenotypic evolution of SARS-CoV-2 spike–reactive CD4+ T cells after repeated mRNA vaccination. Without stimulation, reactive clones showed more Th-neutral features and less of an activated Th1-like state than would be assessed after antigen restimulation, yet retained a distinct transcriptional state into the memory phase. These results uncover durable imprints in antigen-reactive CD4+ T cells and highlight that cell state classification can differ fundamentally when judged by phenotype versus function.