The Research On Rabs In Endogenous Antigen Presentation Of Dendritic Cell

As we known, dendritic cells(DC) are the most powerful professional antigen presenting cells in vivo. With the endogenous antigen presentation pathway, they can present virus- or self-derived antigens to initiate antigen-specific immune response, or maintain immunological tolerance. DCs are also play an important role in vaccine development as natural adjuvant. However, the research on endogenous presentation was mainly about somatic cell in the past, the endogenous presentation of DCs were rarely known.Endogenous antigen presentation is a cellular process involving protein synthesis, modification and transport, in which the vesicular transport is a central part. Membrane traffic generally includes four major steps: vesicle budding, vesicle delivery, vesicle tethering, and fusion of the vesicle membrane with that of the target compartment. Recently, advances in cell biology shown that Rabs use the guanine nucleotide- dependent switch mechanism to regulate intracelluar mambrane traffic. Furthermore, members of this large family of Ras like GTPases localized on distinct compartments, so they can be used to describe different membrane compartments transport. For example, Rab4 can be the marker of early endosomes; Rab5 modulates the transport between clathrin-coated pits and plasma membrane, while Rab7 is highly expressed in late endosomes. Therefore, Rabs can be used as a road sign to describe the complex intracellular transport pathways. Several researches recently shown that a few Rabs were related to antigen presentation, so indentifying Rabs relating to DCs'endogenous antigen presentation and finding out their functional relationship is important to understand the molecular mechanism of DCs'endogenous antigen presentation.Nowadays, the rapid development of siRNA interference technology makes genomewide screen of the Rabs possible. In our research, we used ovalbumin(OVA) as a model antigen and established an efficient endogenous antigen presentation assay system with a T cell hybridoma. Then, we constructed a siRNA lentivirus library against the 57 members of mouse Rab protein family. After screening endogenous antigen presentation of dendritic cell with the library, our data indicated that impairing Rab1B, Rab4A, Rab5B, Rab5C, Rab8A,Rab9,Rab11A,Rab19 or Rab24 decreased the level of endogenous antigen presentation of DCs.Concerning of intracellular locations of these Rab proteins from published data, we speculate: (1) Interference of transcription of Rab1B, Rab8A,Rab11A,Rab19 and Rab24, whose major function are regulating the transport between ER and Golgi, result in the inhibition of DCs'endogenous antigen presentation, and this result accords with the classic endogenous antigen presentation theory; (2) Rab4A,Rab5B and Rab5C control the transport from early endosomes to plasma membrance, while Rab9 control transport of late endosome. Their involvement indicate that DCs may have more than one pathway of endogenous antigen presentation besides the classic one; (3)Rab19 has an effect on endogenous antigen presentation of DC too, but its function is not known yet.Base on the results, we presume the existance of another endogenous antigen presentation pathway other than the classic one, and early endosome may be part of this pathway. Therefore, we chose Rab4 to testify the efficiency of siRNA library screen and to further investigate the possibility of a nontradational endogenous antigen presentation pathway.We transfected systhesis siRNA against Rab4 into DCs with liposome and found markly decrease of endogenous antigen presentation in the case of transient inhibition of Rab4. We also determined the role of Rab4 in endogenous antigen presenentation by transiently over expression several mutants in DCs. Our results indicate that over-expression dominnat-negative Rab4 mutant (Rab4S22N) inhibits the endogenous antigen presentaion in DC, whereas expression of the active Rab4 mutant (Rab4Q67L) does not. The intracellular distribution of Rab4 and mutants are different too. Active Rab4 mutant (Rab4Q67L) and wild type Rab4 protein are mainly aggregating under the plasma membrane, and the dominnat-negative Rab4 mutant are mainly aggregating around the nucelus. The results suggest that Rab4 participates in endogenous antigen presentation by changing its activity and location and partly testify the efficiency and accuracy of siRNA library screen. In all, we finded out some Rabs that related to the endogenous antigen presentation of DC using siRNA library. our results suggests the existance of two enougenous antigen presentation pathway in DCs. These data provide a founded research for therapies of virus infection and tumors.