Direct Visualization of Peptide/MHC Complexes at the Surface and in the Intracellular Compartments of Cells Infected In Vivo by Leishmania major
Creators: name: Muraille, Eric
name: Gounon, Pierre
name: Cazareth, Julie
name: Hoebeke, Johan
name: Lippuner, Christoph
name: Davalos-Misslitz, Ana
name: Aebischer, Toni
name: Muller, Sylviane
name: Glaichenhaus, Nicolas
name: Mougneau, Evelyne
Publisher: name: Robert Koch-Institut, Infektionskrankheiten / Erreger
Year: 2010
Subjects: subject: Amino Acid Sequence
subject: Animals
subject: Mice
subject: Peptide Fragments/chemistry
subject: Mice Inbred BALB C
subject: Cell Compartmentation/immunology
subject: Cell Membrane/immunology
subject: Cell Membrane/metabolism
subject: Cells Cultured
subject: Fluorescent Antibody Technique/methods
subject: Histocompatibility Antigens Class II/immunology
subject: Histocompatibility Antigens Class II/metabolism
subject: Intracellular Membranes/metabolism
subject: Leishmania major/immunology
subject: Leishmaniasis Cutaneous/immunology
subject: Leishmaniasis Cutaneous/metabolism
subject: Mice Inbred C3H
subject: Mice Inbred C57BL
subject: Mice Inbred NOD
subject: Peptide Fragments/immunology
subject: Peptide Fragments/metabolism
subject: Phagosomes/immunology
subject: Phagosomes/metabolism
subject: 610 Medizin

description: Protozoa and bacteria infect various types of phagocytic cells including macrophages, monocytes, dendritic cells and eosinophils. However, it is not clear which of these cells process and present microbial antigens in vivo and in which cellular compartments parasite peptides are loaded onto Major Histocompatibility Complex molecules. To address these issues, we have infected susceptible BALB/c (H-2d) mice with a recombinant Leishmania major parasite expressing a fluorescent tracer. To directly visualize the antigen presenting cells that present parasite-derived peptides to CD4+ T cells, we have generated a monoclonal antibody that reacts to an antigenic peptide derived from the parasite LACK antigen bound to I-Ad Major Histocompatibility Complex class II molecule. Immunogold electron microscopic analysis of in vivo infected cells showed that intracellular I-Ad/LACK complexes were present in the membrane of amastigote-containing phagosomes in dendritic cells, eosinophils and macrophages/monocytes. In both dendritic cells and macrophages, these complexes were also present in smaller vesicles that did not contain amastigote. The presence of I-Ad/LACK complexes at the surface of dendritic cells, but neither on the plasma membrane of macrophages nor eosinophils was independently confirmed by flow cytometry and by incubating sorted phagocytes with highly sensitive LACK-specific hybridomas. Altogether, our results suggest that peptides derived from Leishmania proteins are loaded onto Major Histocompatibility Complex class II molecules in the phagosomes of infected phagocytes. Although these complexes are transported to the cell surface in dendritic cells, therefore allowing the stimulation of parasite-specific CD4+ T cells, this does not occur in other phagocytic cells. To our knowledge, this is the first study in which Major Histocompatibility Complex class II molecules bound to peptides derived from a parasite protein have been visualized within and at the surface of cells that were infected in vivo.