Antigens targeted to the Leishmania phagolysosome are processed for CD4+ T cell recognition

Processing of antigen for recognition by class II-restricted CD4+ T cells occurs within acidic compartments of the antigen-presenting cell. The exact nature of this compartment has yet to be precisely defined, however, but may vary depending upon the cell type studied and the antigen used. The acidic compartments of macrophages are also responsible for the degradation of ingested micro-organisms and play host to others which are adapted to an intracellular existence. To determine whether the phagolysosome (PL) formed in activated macrophages after ingestion of Leishmania parasites is also a site for entry of antigen into the class II presentation pathway, we have used the approach of genetic transformation. Hence, Leishmania were transfected with the genes for the protein antigens ovalbumin (OVA) and beta-galactosidase (beta-gal) and after infection were able to deliver these antigens specifically into the PL. Delivery of antigen to this site resulted in the ability of infected macrophages to present these antigens to antigen-specific CD4+ T cells. After taking into account the absolute levels of antigen uptake by macrophages, a 4-h processing period for OVA delivered by this or a soluble route led to equivalent levels of T cell activation. Unlike macrophages pulsed with soluble OVA, those with PL-targeted OVA still retained the ability to stimulate T cells after a 24-h processing period. This enhanced lifespan of antigen in macrophages corresponded to the kinetics of degradation of the parasite, suggesting slow release of antigen into the processing pathway. beta-gal presentation from the PL was tenfold less efficient under the same conditions. In addition to providing the first information on antigen processing in a protozoan PL, these studies highlight the usefulness of genetically transformed parasites for these types of studies.