| It is supposed that exogenous and endogenous antigens are presented in two different pathways classified as MHC class I pathway and MHC class II pathway. Classical MHC class I pathway indicates that intracellular synthesized antigens, for example of viral or tumor origin, are presented by MHC I molecules and activate CD8+ cytotoxic T cells. Classical MHC class II pathway indicates extracellular antigens are presented by professional antigen-presenting cells (APCs) with MHC II molecules to CD4+ T helper cells. However, two pathways are not completely separated. In fact, exogenous antigens can also be presented in MHC class I restricted pathway. This process now called cross presentation, is first discovered by Bevan`s research in CD8+ T cell response to an exogenous cellular antigen (minor histocompatibility antigens from transplanted cells) in 1976. In 1992, Sigal group reported that this antigen presentation pathway play an important role in antiviral immune response at physiological situation. More and More evidences demonstrate that cross presentation is an important route for priming CD8+ T cells response and effectively clearing tumor, virus-infected cells and intracellular microorganism. On the other hand, this antigen presentation pathway is involved in maintaining immune tolerance in vivo and participate the process of autoimmune diseases such as type I diabetes and asthma. Further investigations on the mechanisms of cross presentation may provide opportunities to develop new immunotherapy strategies for autoimmune disease and vaccine.Dendritic cells are major antigen cross presentation cells in vivo as dendritic cell deleting transgenic mouse lack the ability to response to virus and cellular antigens. Cross presentation of dendritic cells is a complex process which includes antigens uptake, phagosome formation and finally MHC I-peptide complexes translocation onto plasma membrane. The mechanisms of this intracellular transport process were explored by many researches. Rodriguez A reported that soluble antigens are transport to cytosol and enter the classic MHC I restricted pathway, while the mechanisms of exogenous antigen transport from phagosome to cytosol remains uncovered. Houde M. and Guermonprez P. proposed that ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells. However this model recently has been refuted by the Tourset`s data. It is a reasonable consideration that detailed understandings of antigen cross presentation would benefit from a comprehensive analysis of membrane transport related proteins in participating in the process.In eukaryotes, membrane trafficking and fusion is under control of Rab GTPase and SNAREs. Rabs are small G proteins that are characterized by their specific localization on intracellular organelles. In the extensive studies on Rab functions, it have been found that specific Rab GTPases transposition between membrane and cytosol through its GDP/GTP binding switch and regulate the vesicles transport in time and spatial fashion. On the other hand, Rab protein anchor at membrane and recruit different effectors and mediate intracellular membrane trafficking and organelle-targeted membrane fusion. Recently, phagosome proteome revealed that some Rabs are recruiting to phagosome and participating phagosome formation.In this study, using ovalbumin expressing bacteria as a particle antigen, we screened an RNAi library against 57 mouse Rab GTPases, and identified twelve Rab proteins that associated with cross-presentation in dendritic cells. Further studies with fluorescent protein tagged Rabs and Rab27a found Rab3b, 3c, 32 positive compartments extend from the juxtaposition of phagosome to the plasma membrane, suggesting that that these vesicles participating the transport from phagosome to plasma. Analysis the characteristics of Rab3b, 3c, 32 compartments revealed that these vesicles are LAMP+, Tfn+. These data suggest that a previously unrecognized lysosome related organelles (LRO) participate in exogenous antigen cross-presentation. To explore the function of these LROs, the co-localization of fluorescent protein tagged MHC class I molecules with Rab3b, 3c was analyzed by confocal microscope and found that a fraction of MHC class I molecules were stored at the Rabs (3b, 3c) positive compartment, which was resistant to BFA treatment and labeled as recycled MHC class I molecules byβ2 microglobulin. However, the concentration of MHC class I in Rab3b, 3c LROs was inhibited by silencing Rab32. The results suggested that silencing Rab32 block the transport of recycled MHC class I to Rab3b,3c LROs, which in return, affect antigen cross presentation. To investigate the function of Rab32 in vivo, dendritic cell specific Rab32 silencing transgenic mouse was established by using lentiviral vector expressing shRNA under CD11c promoter. The 50% decreased expression of Rab32 in dendritc cells derived from Rab32 silencing transgenic mouse was analyzed by realtime PCR. The efficency of bacteria antigen cross presentation by dendritic cells derived from Rab32 silencing transgenic mouse was also analyzed.A threefold decrease in the effiency of bacteria antigen cross presentation by Rab32 silencing dendritic cells was obtained, whereas the MHC class II restricted antigen presentation was unaffected. These data suggested that Rab32 expression in dendritic cells is critical for the efficient cross presentation of at least bacteria antigens, but not required for MHC class II restricted antigen presentation. Some reported that impaired function of dendritic cells may induce the abnormal phenotype of T cells. Then the phenotype of T cells was analyzed in Rab32 silencing transgenic mouse. Both T cell subsets and function was normal. But whether the function of T cells remains unaffected after antigen challenged need further investigation. |
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