Project Summary One of the major challenges in vaccine development is access to adjuvants that promote long-lived pathogen specific CD4+ and CD8+ T cell responses and provide protective cell mediated immunity. Our previous studies identified a molecular adjuvant that uses CD40 and TLR agonists (CD40/TLR) that induces protective long- lived T cell populations in mice and primates but why this approach is so effective is unclear. We believe that if we can understand why this adjuvant is different from other adjuvants or infection induced responses then it will help in the rational design of vaccines to generate protective cell mediated immunity. We have documented that vaccine-elicited T cells (Tvax) are distinct from infection-elicited T cells (Tinf) and this includes a unique metabolic program and unanticipated requirement for the cytokine IL-27 for Tvax generation. In addition, the ability of a specialized subset of dendritic cells (cDC1), to produce IL-27 predicts the magnitude of vaccine- elicited CD8+ T cell expansion and memory formation. Our recent studies have identified an IL-27-dependent c-Myc transcriptional signature within Tvax that is associated with T cell proliferation and survival. We developed a computational model which mathematically recapitulates T cell expansion data derived from antigen challenge studies in vivo. This unsupervised analysis indicates that the ability of this adjuvant to rapidly promote T cell interactions with APC at the initiation of T cell priming is the major predictor of the magnitude and quality of the T cell response. Based on these data sets will use intravital imaging of T–DC interactions and the manipulation of IL-27, DC functions and c-Myc pathways to understand the mechanistic determinants of the combined CD40L/TLR adjuvant. These data sets will be integrated into a stochastic agent-based mathematical model to predict and validate the key events involved in Tvax formation. The proposed studies bring together the combined efforts of three productive laboratories and their respective expertise in adjuvant discovery, CD8+ T cell biology, cytokine and transcriptional networks, multi photon imaging, and computational modeling in order to understand the molecular basis for adjuvant-elicited cellular immunity.