Integrating complementary approaches reveals antigen-reactive CD4⁺ T cell states after SARS-CoV-2 vaccination

Understanding antigen reactivity is crucial for characterizing CD4⁺ T helper (Th) cell fate, yet conventional peptide restimulation assays introduce phenotypic bias by activating cells ex vivo. By combining single-cell RNA and T cell receptor (TCR) sequencing on antigen-stimulated and unstimulated samples, clonotypes can be tracked across conditions to identify antigen-reactive CD4⁺ T cells while assessing their phenotypes in the unperturbed state. Using this "reverse phenotyping" strategy, complemented by DNA-barcoded peptide-HLA class II multimers and TCR similarity metrics, we tracked SARS-CoV-2 spike-reactive CD4⁺ T cells longitudinally after repeated mRNA vaccination. Without stimulation, reactive clones showed more Th-neutral features and less activated Th1-like states than after restimulation. Furthermore, transgenic TCR re-expression further separated antigen-specific from bystander-activated clones. These results demonstrate the complementary value of different methods to identify and characterize antigen-reactive CD4⁺ T cells, and highlight that cell state classification can differ when defined by phenotype versus function.