T follicular helper (TFH) cells are essential for establishing protective immunity in responses to infection and immunization, where they help high-affinity antibody production by germinal center (GC) B cells, generation of plasma cells and memory B cells. The TFH-B cell cooperativity thus constitutes the cornerstone of immunological memory formation induced by vaccines. Deciphering pathways and factors that promote TFH cell differentiation and function is a prerequisite for formulating more efficacious vaccines. In response to infection or immunization, activated CD4+ T cells undergo stage-wise differentiation to become functionally competent TFH cells. These stages include early stage TFH lineage specification to generate nascent TFH cells, GC response stage to mature to GC-TFH cells, and later stage formation of memory TFH cells. Vaccines are delivered as live-attenuated viruses, inactivated organisms, or protein subcomponents via different routes. It has not drawn enough attention whether all TFH cells are created equal by different vaccination approaches. We extended our in vivo studies to CD28 and ICOS costimulatory receptors, which are well-established TFH regulators. CD28-PYAP and ICOS-YMFM intracellular motifs are preferentially connected to PKCq/PDK1 and PI3K-Akt pathways, respectively. Current views hold that CD28 is critical for initiating and ICOS for sustaining TFH responses. Contrary to this belief, our preliminary data revealed that the ICOS-YMFM motif is essential for initiating Imm_TFH, while the CD28-PYAP motif was indispensable for sustaining Inf_TFH responses. These findings led to our central hypothesis that TFH cells have distinct molecular requirements depending on activating agents/approaches, where CD28 and ICOS motifs differentially regulate Inf_TFH and Imm_TFH cells in a stage-specific manner. To test this, we designed multi-layered functional and molecular studies in 3 specific aims: Specific Aim 1. To define differential requirements for CD28 and ICOS motifs in TFH fate specification. Specific Aim 2. To delineate CD28 and ICOS motif-controlled molecular circuits that promote GC-TFH cell maturation and B-cell help function. Specific Aim 3. To investigate the roles of CD28 and ICOS motifs in memory TFH persistence and recall response. Developing efficacious vaccines against infectious diseases is thus essential for protecting troops in service as well as veterans. Our proposed study directly addresses this need, by advancing the understanding of TFH cells at new frontiers. We expect to yield essential information on tailoring TFH activity to provide optimal B cell help, and hence provide paradigm-shifting rationales for enhancing vaccine design.