Human axillary lymph node T follicular helper (Tfh) and Precursor‐Tfh cells exhibit functional flexibility following seasonal influenza vaccination

Objectives: T-cell subsets within human lymph nodes (LNs) post-vaccination to assess the potential for adaptability and flexibility in an immune response. Methods: Ultrasound-guided fine needle biopsies/aspirates were used to isolate T follicular helper (Tfh) and Precursor (Pre)-Tfh cells from the vaccine-draining and contralateral axillary LNs of five individuals at 5 days after influenza vaccination, followed by single-cell RNA sequencing, gene expression and T-cell receptor analysis. Results: Tfh and Pre-Tfh cells within five clusters with distinct gene expression profiles, designated: Resting, Activated migrating, B-cell-interacting Tfh, Proliferating and Cytotoxic, exhibited attributes of more than one T-cell lineage and expanded T-cell clones were present in more than one cluster, suggesting divergent differentiation into different fate lineages from a common precursor. Inferred pseudotime suggested a bifurcating trajectory rooted in the resting cluster and terminating at either the Proliferating or Cytotoxic clusters. This analysis predicted the transition of cells through activation states and the gain and loss of lineage attributes and effector functions. Enriched gene pathways along the pseudotime trajectories were consistent with these functional transitions and involvement in the immune response to vaccination. Conclusion: These results reveal the flexible potential within the Tfh lineage that could be leveraged to drive more efficient vaccine responses and inform rational vaccine design.