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.