Strong influenza-induced T(FH) generation requires CD4 effectors to recognize antigen locally and receive signals from continuing infection

While influenza infection induces robust, long-lasting, antibody responses and protection, including the T follicular helper cells (T(FH)) required to drive B cell germinal center (GC) responses, most influenza vaccines do not. We investigated the mechanisms that drive strong T(FH) responses during infection. Infection induces viral replication and antigen (Ag) presentation lasting through the CD4 effector phase, but Ag and pathogen recognition receptor signals are short-lived after vaccination. We analyzed the need for both infection and Ag presentation at the effector phase, using an in vivo sequential transfer model to time their availability. Differentiation of CD4 effectors into T(FH) and GC-T(FH) required that they recognize Ag locally in the site of T(FH) development, at the effector phase, but did not depend on specific Ag-presenting cells (APCs). In addition, concurrent signals from infection were necessary even when sufficient Ag was presented. Providing these signals with a second dose of live attenuated influenza vaccine at the effector phase drove T(FH) and GC-T(FH) development equivalent to live infection. The results suggest that vaccine approaches can induce strong T(FH) development that supports GC responses akin to infection, if they supply these effector phase signals at the right time and site. We suggest that these requirements create a checkpoint that ensures T(FH) only develop fully when infection is still ongoing, thereby avoiding unnecessary, potentially autoimmune, responses.