Preparing antigens suitable for cross-presentation assays in vitro and in vivo

Cross-presentation is defined as the ability of certain professional antigen-presenting cells to take up, process and present extracellular antigens on major histocompatibility class I (MHC-I) molecules to CD8+ T cells. The stimulation of naive cytotoxic CD8+ T cells by this process, termed cross-priming, is involved in many different responses, including those to tumors, pathogens, graft tissues, and self-antigens. Dendritic cells (DCs), a heterogeneous cell population, are endowed with the highest cross-priming capacity. Investigation of their cross-presentation capacities, important both for vaccination and for the induction of immune tolerance can be performed by in vivo and in vitro assays. In this chapter we describe the preparation of antigens that can be used to test cross-presentation via pinocytosis, receptor-mediated endocytosis, and phagocytosis.