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bioRxiv · 10.1101/2025.11.19.688845

Antigen-reactive CD4+ T cells after SARS-CoV-2 vaccination show divergent phenotypic states with or without restimulation bias

Kocher, K.·Drost, F.·Schuelein, C.·Spriewald, B.·Schubert, B.·Schober, K.
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Abstract

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. However, by performing single-cell RNA and T cell receptor (TCR) sequencing on both antigen-stimulated and unstimulated samples, clonotypes can be tracked across conditions to identify antigen-reactive CD4+ T cells and simultaneously be assessed for their phenotypes in the unperturbed state. Using this reverse phenotyping strategy, complemented by DNA-barcoded peptide-HLA (pHLA) class II multimers, we here 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 of an activated Th1-like state than would be assessed after antigen restimulation. Furthermore, transgenic TCR re-expression guided separation of antigen-specific from bystander-activated clones. These results uncover unbiased phenotypes of antigen-reactive CD4+ T cells and highlight that cell state classification can differ fundamentally when judged by phenotype versus function.

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Kocher, K., Drost, F., Schuelein, C., Spriewald, B., Schubert, B., Schober, K.. 2025-11-21. Antigen-reactive CD4+ T cells after SARS-CoV-2 vaccination show divergent phenotypic states with or without restimulation bias. https://doi.org/10.1101/2025.11.19.688845

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