Mode of T cell priming durably shapes the TCR repertoire, effector function and α4β1 integrin expression of human virus-specific CD4 ⁺ T cells

The generation of an effective T cell response against an antigen depends on the recognition of the antigen by the T cell receptor (TCR), followed by T cell priming, initiating coordinated biophysical, biochemical and proliferative changes that drive differentiation into effector and memory clones. The immunological environments in which priming occurs, such as natural infection or vaccination, influences the quality and persistence of memory T cells, but the long-term impacts remain incompletely understood. Here, we investigate how the mode of priming shapes durable antigen-specific CD4 ⁺ T cell memory, utilising two cohorts 3-4 years after initial antigen encounter: individuals recovered from SARS-CoV-2 infection and infection-naive individuals who received a SARS-CoV-2 vaccination. Using ex vivo single-cell RNA sequencing, paired TCR sequencing and in vitro functional analyses, we characterise the transcriptional, clonal and functional profile of Spike-specific CD4 ⁺ T cells. Across both cohorts, CD4 ⁺ T cell responses against spike epitopes S _166-180 , S _751-765 and S _866-880, were immunodominant, with shared public TCR clonotypes indicating conserved antigen-recognition regardless of mode of priming. Despite this shared specificity, infection-primed individuals exhibited greater TCR repertoire diversity and lower CDR3αβ sequence convergence. Transcriptionally, infection-primed cells exhibited a more cytotoxic and effector phenotype, while vaccine-primed cells preferentially adopted T follicular helper (Tfh)- and Th1-associated phenotypes. Infection-primed individuals also displayed enrichment of cell adhesion and integrin signalling pathways, with a greater proportion of spike-specific CD4 ⁺ T cells expressing α4β1 integrin subunits, consistent with enhanced migratory and effector potential. Collectively, our findings demonstrate that the mode of antigen priming may influence the long-term CD4 ⁺ T cell memory states, influencing TCR repertoire diversity, functional differentiation and tissue-homing potential, years after the initial immune response.