BNT162b2 mRNA Vaccination Activates Spike-Reactive Regulatory T Cells in Humans

Studies using mouse models of viral infection have shown that viral antigen-reactive regulatory T cells play multiple context-dependent roles, including attenuating the immune response against the pathogen and limiting inflammatory damage. By contrast, evidence for the presence and function of viral antigen-specific Tregs in humans remains very limited. Here, we investigated whether BNT162b2 mRNA COVID-19 vaccination activates spike (S)-reactive Tregs in healthy adults. We performed an integrated analysis combining scRNA-seq, TCR repertoire profiling, and flow cytometry of activation-induced marker (AIM) expressing S-reactive CD4+ T cells isolated from PBMCs cultured with trimeric S protein. We found that CD39+ cells co-expressing CTLA4 and TIGIT within the S-reactive AIM+ CD4+ T cell population were preferentially enriched for Tregs relative to their CD39- counterparts. In subjects vaccinated with BNT162b2 mRNA vaccine, the frequencies of CD39+CTLA4+ and CD39+TIGIT+ S-reactive Treg-like cells were significantly increased following vaccination. Using scRNA-seq, we identified two FoxP3+ Treg clusters among S-reactive CD4+ T cells: one characterized by IFN-activated gene signatures and another by high HLA class II, CD39 and CTLA4 expression. Both subsets were transcriptionally activated following vaccination, with upregulation of genes associated with T cell activation and inflammatory cytokines including TNF and IFNG. Consistent with these findings, flow cytometry detected increased proportions of CD39+ and FoxP3+ S-reactive Tregs expressing IFN-γ and/or TNF-α following vaccination. Collectively, these findings support a model in which BNT162b2 mRNA vaccination activates both an S-reactive effector T cell response and a Treg response, suggesting Treg co-induction may be critical for balancing antiviral immunity with immunoregulation.