title: Recombinant Escherichia coli express a defined, cytoplasmic epitope that is efficiently processed in macrophage phagolysosomes for class II MHC presentation to T lymphocytes.
abstract: Although the processing of soluble Ag for presentation to T cells has been extensively studied in vitro, similar studies of phagocytic Ag processing have been limited. We have developed an in vitro model system to study the ability of macrophages to process recombinant Escherichia coli strain HB101 with cytoplasmic or surface expression of the well characterized T cell epitope of hen egg lysozyme (HEL) 52-61. This epitope was expressed within full length HEL or within a fusion protein containing the HEL epitope. Phagocytosis of E. coli with cytoplasmic expression of HEL or the HEL fusion protein resulted in strong presentation of HEL(52-61) to T cells. Surface-conjugated HEL was processed with even greater efficiency. Processing required viable macrophages, was inhibited by cytochalasin D, and was achieved within 20 min of bacterial contact with the macrophages. Within this time span, phagosomes containing bacteria fused with lysosomes, and the bacteria were extensively degraded. Uptake of as few as four bacteria per macrophage produced an Ag-specific T cell response. We conclude that bacterial compartmentalization of the antigenic epitope (cytoplasmic vs surface) had some effect on its processing, but that phagocytic Ag processing organelles contain extensive capacity to degrade internalized bacteria and liberate intracellular Ag epitopes for recycling and presentation, consistent with a central role for phagolysosomes. Thus, future recombinant bacterial vaccines may be effectively designed with T cell epitopes expressed either on the surface or within the bacterial cytoplasm.
authors.affiliation: Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110.
authors.forename: J D
authors.initials: JD
authors.lastname: Pfeifer
authors.forename: M J
authors.initials: MJ
authors.lastname: Wick
authors.forename: D G
authors.initials: DG
authors.lastname: Russell
authors.forename: S J
authors.initials: SJ
authors.lastname: Normark
authors.forename: C V
authors.initials: CV
authors.lastname: Harding
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology*; D001483:Base Sequence; D003593:Cytoplasm / Q000276:immunology; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000276:immunology*; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D008264:Macrophages / Q000276:immunology*; D051379:Mice; D008808:Mice, Inbred CBA; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics / Q000276:immunology; D010587:Phagocytosis; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000276:immunology
chemical_list: D000942:Antigens, Bacterial; D000939:Epitopes; D000949:Histocompatibility Antigens Class II; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D009113:Muramidase