Project summary: We and others have demonstrated that antigens derived from infectious viral infections persist in the host for extended periods of time, well beyond the time in which the infection is cleared from the host. Our lab has specifically identified that antigens derived from both vaccination and viral infections persist or are archived by the host lymphatic endothelial cells (LEC)s, identifying the source of archived antigens. We have published that this archived antigen maintains a more effector like pool of antigen specific memory cells which enhances the clearance of a secondary infectious challenge. Identification of key mechanisms involved in antigen archiving during vaccination is critical for our understanding of enhanced protective immunity to vaccination. While we have established many important criteria for antigen archiving and protective immunity, in this renewal application we aim to dive deeper into the cell types involved and the processes required. We aim to better appreciate how the expression of subset specific genes, now discovered in both lymphatic endothelial cells and dendritic cells, may be required for antigen handling, the implications of which could affect antigen archiving and protective immunity. We have established a novel methodology leveraging the 10x genomics platform to identify DNA-antigen conjugates for the study of antigen dispersal over long periods of time. With this methodology and technology in hand we now have the capability to accurately and faithfully measure cell types that acquire antigens as well as the exact number of antigens within each cell over time. Using these studies we have identified several novel findings based on antigen amount and transcriptional signature to lead us to the hypothesis that specific LECs and DCs, based on their transcriptional program, contribute to the acquisition, retention and exchange of antigens. Furthermore, this handling of antigens by LECs and DCs can be manipulated by other inflammatory events that cause antigen release and presentation, and as a result, improve immune responses to secondary and heterologous infections.