Measuring antigen presentation in mouse brain endothelial cells ex vivo and in vitro

Having demonstrated that brain endothelial cells cross-present parasite antigen in mouse experimental cerebral malaria, the authors present ex vivo and in vitro protocols to measure this antigen presentation, generalizable to other disease contexts. We have recently demonstrated that brain endothelial cells cross-present parasite antigen during mouse experimental cerebral malaria (ECM). Here we describe a 2-d protocol to detect cross-presentation by isolating the brain microvessels and incubating them with a reporter cell line that expresses lacZ upon detection of the relevant peptide–major histocompatibility complex. After X-gal staining, a typical positive result consists of hundreds of blue spots, compared with fewer than 20 spots from a naive brain. The assay is generalizable to other disease contexts by using reporter cells that express appropriate specific T cell receptors. Also described is the protocol for culturing endothelial cells from brain microvessels isolated from naive mice. After 7–10 d, an in vitro cross-presentation assay can be performed by adding interferon-γ, antigen (e.g., Plasmodium berghei –infected red blood cells) and reporter cells in sequence over 3 d. This is useful for comparing different antigen forms or for probing the effects of various interventions.