Dynamics of Circulating Follicular Helper and Regulatory T-Cell Memory Induced by mRNA, Inactivated, and Live-Attenuated Vaccines.

OX40 signaling promotes the differentiation of memory CD4+ T cells into T follicular helper (Tfh) cells that support germinal center (GC) B-cell responses. Using an extended activation-induced marker (AIM) assay based on OX40 (CD134) and CD25 coexpression, we quantified antigen-specific circulating memory Tfh and T follicular regulatory (Tfr) cells following in vitro stimulation with influenza, SARS-CoV-2, or measles antigens. Responses were assessed longitudinally in recipients of inactivated influenza (n = 20) and mRNA COVID-19 (n = 13) vaccines and cross-sectionally in individuals vaccinated with the live-attenuated measles vaccine (n = 8). All three vaccines elicited humoral responses. Functional assays, performed when sufficient cell numbers were available, showed that sorted influenza-specific Tfh cells (OX40+CD25+CXCR5+), cocultured with autologous CD27+CD19+ memory B cells, promoted B-cell expansion and antibody secretion. This was accompanied by increased CXCL13, IL-21, soluble CD40L, and APRIL, consistent with effective T-cell help and B-cell activation. Comparative analyses revealed distinct patterns of follicular immune responses: influenza vaccination induced coordinated Tfh and Tfr responses, whereas mRNA COVID-19 vaccination generated increased Tfh but minimal Tfr cells. At peak response, measles and influenza vaccinees exhibited comparable Tfh and Tfr frequencies, while COVID-19 vaccinees showed similar Tfh but reduced Tfr proportions. Overall, the extended AIM assay represents a practical and minimally invasive approach for simultaneous monitoring of Tfh and Tfr populations in humans, providing a scalable framework to investigate follicular immune balance across vaccination settings. Beyond enabling sensitive detection of antigen-specific memory responses, this approach highlights the importance of assessing both helper and regulatory follicular compartments to better understand vaccine-induced immunity.