Mycobacterium leprae 65hsp antigen expressed from a retroviral vector in a macrophage cell line is presented to T cells in association with MHC class II in addition to MHC class I.
Mycobacterium leprae lives free in the cytoplasm in infected macrophages. To test if an M. leprae antigen released into the cytoplasm would associate with major histocompatibility complex (MHC) class II we introduced the gene encoding the 65 kDa heat-shock protein (ML65hsp) into a retroviral shuttle vector (pZIPNeoSV(X)) and transfected the murine macrophage cell line J774G8. S1 nuclease mapping and Western blot analysis of the transfected cell line (CJ11) showed that specific messenger RNA and ML65hsp antigen were stably expressed. Presence of antigen at the cell surface was demonstrated by flow cytometric analysis with specific monoclonal antibodies (mAb). Antigen-specific T lymphocytes were stimulated by CJ11 cells to proliferate and release interleukins (IL-2 and IL-3). These responses were blocked by mAbs specific for either MHC class II or for the mycobacterial antigen. The endogenous antigen was also recognised by MHC class I-dependent cytotoxic T cells; cytotoxicity was inhibited by mAbs against either MHC class I molecules or ML65hsp. Thus, production of ML65hsp within the host cytoplasm resulted in association of the antigen with both MHC class I and MHC class II antigen-presenting structures and evoked both lymphocyte proliferation and cytotoxicity towards the antigen-presenting cell. These findings may be relevant to the development of recombinant subunit vaccines against intracellular pathogens.
Silva
C L
CL
Laboratory for Leprosy and Mycobacteria Research, National Institute for Medical Research, Mill Hill, London, U.K.
Palacios
A
A
Colston
M J
MJ
Lowrie
D B
DB
D016475:3T3 Cells; D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D002460:Cell Line; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000276:immunology*; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D000949:Histocompatibility Antigens Class II / Q000276:immunology; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D009166:Mycobacterium leprae / Q000235:genetics / Q000276:immunology*; D012190:Retroviridae / Q000235:genetics; D013601:T-Lymphocytes / Q000276:immunology*; D014162:Transfection
D000942:Antigens, Bacterial; D006360:Heat-Shock Proteins; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II