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Rac2 Negatively Regulates Cross Presentation of Dendritic Cells

Author: LiNa
Tutor: WuYuZhang
School: Third Military Medical University
Course: Immunology
Keywords: Dendritic cells Cross - submissions Rho
CLC: R392
Type: Master's thesis
Year: 2008
Downloads: 77
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Abstract

Dendritic cells (dendritic cells, DC) is the main antigen-presenting cells, now considered The DC cell antigen presentation in three ways: a DC phagocytosis of foreign antigens, and processed to form the epitope peptide, and the combined MHC II molecules, which activate CD4 T cells; another DC cells endogenously synthesized protein processing and MHC class I molecules combined with activation of CD8 T cells; Bevan proposed a new DC cell antigen presenting the way - - Cross submissions (cross presentation), it refers to the exogenous antigen processing, the load to the MHC class I molecules form complexes on the surface of antigen presenting cells, thereby activating the initial CD8 T cells caused by the process of the cellular immune response. Cross-presenting plays a very important role in monitoring the tumor tissue, as well as clear the virus can not infect antigen presenting cells. Learn more about the DC mechanism will help us find better anti-tumor and anti-viral pathway provides new clues for the immunotherapy of tumors and virus-infected cells cross presenting. But little adjustment mechanism of cross-presenting DC cell. Many previous studies showed that Rho GTPase involved in regulating many aspects of the DC cell physiology activities, including the remodeling of cell morphology, dendritic stretching phagocytosis of antigen and activation of T cells. Cdc42 and Rac1 affect the phagocytic capacity of DC cells to foreign antigens, wherein the DC cell maturation, activation of Cdc42 phagocytosis changes play an important role; RhoB affect DC cell surface MHC II molecule expression after LPS stimulation; Further, Rac1 inhibitory mutant transgenic mice DC phagocytosis of apoptotic cells decreased ability, thus affecting the cross-submissions. So we want to know the Rho family of proteins plays what role in the cross-presenting what mechanisms involved? Purpose: This study screened through a series of experiments and further cross-presenting role of Rho proteins explore the possible mechanism. RNAi interference screening six common Rho molecular methods: 1,: of RNAi screening method is conducive to different molecular level comparison, and provide clues for understanding the biological functions of the different cell. The efficiency of cross-presenting ability of DC cell; synthetic siRNAs this study were to interfere with the the DC cell in six common Rho molecule, then the use of the T cell hybridoma B3Z detection interference RNA interference and verified by real-time quantitative PCR method ; 2 expression Rac2 different nature mutant the DC cell cross presenting capability analysis: According to the molecular properties of Rho activation mutants and inhibitory mutant is often used as an effective means to study the molecular function of Rho. The method of the present study point mutation was constructed Rac2 two mutants - the activation mutants Rac2Q61L and the suppression type mutant Rac2D57N fusion expression, and the yellow fluorescent protein EYFP; efficient infection of dendritic cells by the lentiviral vector, DC2.4 to obtain a stable expression of EYFP-Rac2 mutants expression of different mutants DC cell cross presenting ability, and then detected by ELISA; 3, the molecular mechanism of: (1) phagocytic process Rac2 activation: fluorescence resonance energy transfer (F? rster Resonance Energy Transfer, FRET) is currently the most effective means to study the interaction between two protein molecules. Relative to the confocal microscope, it demonstrates not only the spatial distance between two molecules similar to better clarify the relationship between the two molecules from the function. Rac2 activation downstream of PAK molecular interactions with the use of lentiviral vectors, DC cells transfected with yellow fluorescent protein tagged Rac2 and red fluorescent protein-labeled PAK molecules using laser confocal microscope in phagocytic foreign activation of the molecules in the process of antigen Rac2 and positioned in the cell; (2) phagocytic experiment: the use of the DC cells express different Rac2 mutants observed transfection of different mutants DC cells foreign to the expression of the red fluorescent protein of bacteria Rac2 molecular phagocytosis antigen at different phases of the phagocytic capacity; presenting experimental endogenous (3): this the DC cell can endogenously expressed OVA protein, the epitope peptide with MHC class I molecules composite submissions to the DC cell surface, and then expressing EYFP-Rac2 mutant lentivirus infection DC-OVA cells, observed Rac2 DC-OVA cells endogenously presenting Rac2 molecular cross-presenting DC cells in affect aspects; Cd11c-EYFP-Rac2Q61L transgenic mice to establish: depth discussion and observation Rac2 molecular function of DC cells, we can in the DC cell-specific expression the EYFP-Rac2Q61L lentiviral vector to infect mouse embryo cells, obtained Cd11c-EYFP-Rac2Q61L transgenic mice, the platform for the establishment of the experiment provides an effective means for us to study the cross-presenting related molecules, thereby facilitating can be detected from the in vivo levels of the molecules on the cross-presenting ability. Conclusion: Through this study, we observed that: (1) the ability of the different Rho cross-presenting molecules on the DC cells differ, interference Rac2 expression of DC cell cross-presenting ability is significantly increased; (2) Construction of lentiviral vector Expression of activation and suppression type Rac2 mutant, and transfected DC cells, we found that overexpression activation mutant Rac2Q61L DC cells can effectively reduce the cross-presenting; (3) in the process of phagocytosis of foreign antigens, Rac2 activation occurs in the phagocytosis of the membrane portion of the foreign antigen, activated Preparation PAK molecules; (4) Rac2 DC phagocytosis does not affect the amount of the foreign antigen, and the endogenous presenting. Preliminary conclusions: Rac2 Unlike other Rho molecule, which negatively regulate dendritic cells in cross-presenting, and Rac2 activation mainly occurs in the membrane surrounding, through interaction with PAK molecules regulate the DC cells subsequent process the remaining stages may be provided with the acidification of the phagosome, the antigen degradation related rather than acting in cross-presenting. In this study, for the understanding of the molecular mechanisms laid the foundation for cross-presenting dendritic cells, and this negative regulator molecules the Rac2 of discovery for understanding the disease state, such as local tumor immune tolerance to provide new clues.

Rac2 Negatively Regulates Cross Presentation of Dendritic Cells

Abstract

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