ABSTRACT Neutralizing antibodies are critical for protection from infectious diseases. The lymph node (LN) germinal center (GC) is the site where B cells undergo antibody affinity maturation and develop into long-lived plasma cells – key events that are required for the development of highly effective neutralizing antibodies following infection or vaccination. T follicular helper cells (TFH) are the CD4+ T cell subset responsible for providing B cell help during an ongoing GC. TFH are absolutely required for GC formation and maintenance. By extension, TFH are necessary to produce effective neutralizing antibodies following antigen exposure. While many aspects of the TFH response and TFH function have been examined in animal models, human TFH responses in the draining LN have only recently been explored with the novel application of an established technique: serial ultrasound-guided fine needle aspiration of draining LN following vaccination. With this technique, we recently demonstrated that the antigen-specific TFH response to SARS-CoV-2 spike (S) protein mRNA vaccination persists in the GC for more than 4 months following vaccination and correlates with the presence of S-specific GC B cells. Furthermore, we have noted substantial LN TFH transcriptional phenotypic changes suggestive of functional maturation over this prolonged GC time interval using single cell RNA-seq in a small preliminary cohort. In this proposal, we will expand upon these findings to address our primary hypothesis: human TFH phenotypic maturation occurs over time in the draining LN following vaccination and these phenotypic changes are associated with changes in TFH function. To explore this hypothesis, we propose three specific aims: 1) We will first establish that TFH phenotypic maturation occurs over time in multiple antigen-specific TFH populations that we will define and characterize from a cohort of fourteen COVID-19 mRNA vaccine recipients using single cell RNA-seq and ex vivo epitope identification methods. 2) We will determine how these antigen-specific TFH populations change in a tertiary immune response following COVID-19 mRNA vaccine “boost” and continued serial LN sampling of the same cohort participants. We will also ascertain if new antigen-specific TFH populations are recruited to the GC during a recall response. 3) Finally, we will verify that the profound phenotypic changes we observe lead to changes in the functional capacity of antigen-specific TFH to provide help to B cells using an ex vivo system of sorted human LN TFH and an in vivo murine adoptive transfer model. By addressing these aims, we will significantly enhance our understanding of the role that human TFH play in directing GC B cell responses to vaccination.

NARRATIVE Human T follicular helper cells function by providing essential support to B cells in draining lymph nodes allowing those B cells to produce highly effective neutralizing antibodies following vaccination or infection. The dynamics of human antigen-specific T follicular helper cell development, maturation and function are not well-understood. The work we propose will fill this knowledge gap and lead to a deeper understanding of the impact of human T follicular helper cell maturation on function in the setting of a very efficacious human lipid nanoparticle mRNA vaccine platform.