Rac2 Negatively Regulates Cross Presentation Of Dendritic Cells

Background: Dendritic cell (DC) is the main participator of antigen presentation. At present, it is supposed to be three presentation pathways: (1) Classical MHC I pathway which indicates that intracellularly synthesized antigens, for example of viral or tumor origin, are presented by MHC I molecules and activate CD8+ cytotoxic T cells; (2) Extracellular antigens are presented by professional antigen-presenting cells (APCs) with MHC II molecules to CD4+ T helper cells; (3) Cross presentation, which is raised by Bevan, is a process whereby professional APC, mainly dendritic cells (DC), prime CD8+ cytotoxic T cells by presenting antigens processed from proteins of other cells such as tumor cells or virus-infected cells. Cross presentation is proved to be an important route for the organism to effectively clear tumor and virus-infected cells. Therefore, the study of the mechanism under this phenomenon is meaningful and valuable. Until now, it is still not clear how DC effectively regulates cross presentation.Previous studies show that Rho GTPase regulates a lot of physiological processes in DC: dendrite extension, antigen uptake, antigen presentation and T cell priming. Rac1 and Cdc42 play an important role in phagocytosis during DC maturation. RhoB regulates MHC II expression at the surface of DC after LPS stimulation. The dominant-negative mutant of Rac1 diminishs the apoptotic cell uptake of DC in vivo and thus inhibits its cross presentation. What about other Rho GTPase in cross presentation?Objection: To screen the effective Rho in cross presentation of dendritic cells and to try to elucidate its possible role.Methods:1. RNAi screening the related Rho in cross presentation: High-throughput functional screening with a siRNA library is an efficient and useful approach to study the biological behavior of some cells. In our study, we used the synthesized siRNAs targeted six frequent Rho molecules in DC2.4 and IL-2 secretion by T cell hybridoma revealed the cross presentation ability of these interferenced cells. After that, the effective Rho in cross presentation of DC was screened. The interferenced efficiency was then analyzed by real-time PCR;2. Cross presentation assay of DCs over-expressing different mutants of Rac2: Based on the characteristics of Rho GTPase, constitutive active (ca) and negative dominant (dn) mutants are useful means to study the function of Rho in different cells. By site-directed mutagenesis, we constructed different mutants of Rac2 (the screened Rho)——the ca form Rac2Q61L and the dn form Rac2D57N which were then co-expressed with the enhanced yellow fluorescence protein (EYFP) in DC2.4 by lentivirus transfection. The transduction efficiency was investigated by FACS. Cross presentation assay of these DCs were analyzed by IL-2 ELISA;3. Molecular mechanism investigation:(1) The activation assay of Rac2 during phagocytosis: At present, F?rster Resonance Energy Transfer (FRET) is supposed to be the most effective method of studying the interaction between two proteins. We used this technology to observe the EYFP-Rac2wt activation during phagocytosis when the activated Rac2 would interact with the downstream PAK molecule;(2) The phagocytosis assay: By FACS, we measured the phagocytosis ability of DC2.4 transfected with different Rac2 mutants;(3) The endogenous presentation assay: We have built one kind of DC which expressed OVA peptides endogenously and assembled them to the MHC I molecules. By transfecting different mutants of Rac2 to this cell, we investigated the endogenous presentation of these cells;4. Generation and identification of Cd11c-EYFP-Rac2Q61L transgenic mice: In order to confirm the role of Rac2 ex vivo and in vivo, by the high-titer lentivirus transfecting the embryonic cells from C57BL/6 mice, we have generated the transgenic mice whose transfected gene Rac2Q61L expressed specifically in DC under the promoter Cd11c. It is a fundament for further analysis of Rac2's effect on cross presentation in vivo.Results & conclusions: (1) RNA interference experiment showed that Rac2-interferenced DC2.4 increased its ability of cross presentation other than some other Rho molecules; (2) When DC2.4 was transfected with constitutive active form of Rac2, cross presentation was down-regulated; (3) Moreover, Rac2, not like Rac1, had no influence on the phagocytosis and endogenous presentation; (4) By FRET, Rac2 activation could be observed around the membrane during the phagocytosis of exogenous antigens.We conclude that Rac2, not like some other Rho, has negative regulation in cross presentation and this regulation is not due to the changes of phagocytosis ability by DC, it is probably owing to the activation of PAK and then the downstream molecules would influence its function.