Antigen-specific CD4⁺ T cells exhibit distinct transcriptional phenotypes in the lymph node and blood following vaccination in humans

SARS-CoV-2 infection and mRNA vaccination induce robust CD4⁺ T cell responses that are critical for the development of protective immunity. Here, we evaluated spike-specific CD4⁺ T cells in the blood and draining lymph node (dLN) of human subjects following BNT162b2 mRNA vaccination using single-cell transcriptomics. We analyze multiple spike-specific CD4⁺ T cell clonotypes, including novel clonotypes we define here using Trex, a new deep learning-based reverse epitope mapping method integrating single-cell T cell receptor (TCR) sequencing and transcriptomics to predict antigen-specificity. Human dLN spike-specific T follicular helper cells (T_FH) exhibited distinct phenotypes, including germinal center (GC)-T_FH and IL-10⁺ T_FH, that varied over time during the GC response. Paired TCR clonotype analysis revealed tissue-specific segregation of circulating and dLN clonotypes, despite numerous spike-specific clonotypes in each compartment. Analysis of a separate SARS-CoV-2 infection cohort revealed circulating spike-specific CD4⁺ T cell profiles distinct from those found following BNT162b2 vaccination. Our findings provide an atlas of human antigen-specific CD4⁺ T cell transcriptional phenotypes in the dLN and blood following vaccination or infection.