Immunization route dictates cross-priming efficiency and impacts the optimal timing of adjuvant delivery

Delivery of cell-associated antigen represents an important strategy for vaccination. While many experimental models have been developed in order to define the critical parameters for efficient cross-priming, few have utilized quantitative methods that permit the study of the endogenous repertoire. Comparing different strategies of immunization, we report that local delivery of cell-associated antigen results in delayed T cell cross-priming due to the increased time required for antigen capture and presentation. In comparison, deliv-ery of disseminated antigen resulted in rapid T cell priming. Surprisingly, local injection of cell-associated antigen, while slower, resulted in the differentiation of a more robust, polyfunctional, effector response. We also evaluated the combination of cell-associated antigen with poly I:C delivery and observed an immunization route-specific effect regard-ing the optimal timing of innate immune stimulation.These studies highlight the importance of considering the timing and persistence of antigen presentation, and suggest that intra-dermal injection with delayed adjuvant delivery is the optimal strategy for achieving CD8+T cell cross-priming.