| It is suggested that exogenous antigens are processed and presentated by antigen presentating cells(APCs) in MHC classs II restricted pathway, while endogenous antigens are processed in MHC class I restricted pathway. In certain conditions, APCs can internalize exogenous antigens and present them as MHC class I-bound peptides. This process has been termed"cross-presentation"and is essential for generating CD8+ T cell immunity to antigens expressed extensively in parenchymal cells (e.g. from mutations or tissue-tropic virues). Dendritic cells (DC) are unique among APCs in their efficient cross presentation capacity.To efficiently prime T cells, DC needs to undergo a process of maturation that implies a reduced potential for antigen uptake. This transition is accompanied by dramatic cell phenotype and cell morphology alteration. All of these changes are related to regulation of actin assembly, which in turn is mediated by the Rho GTPases family. The Rho GTPases family comprises at least 23 separate proteins, of which RhoA, Rac1 and Cdc42 are the best studied. Data from a number of laboratories over the past few years have revealed that members of the Rho family play a crucial role in cellular morphogenesis, endocytosis, migration, MHC class II restricted presentation and cross-presentation of DC. It is reported that the ability of DC to dramatically decrease their capacity for endocytosis upon maturation is relevant to downregulation levels of activated Cdc42. It is also suggested that activated form of Cdc42 and RhoA increase the ability of DC to present antigen within MHC class II restricted pathway. Consequently, inhibition of Rac1 results in a reduction in the capacity of DC for phagocytosis of apoptotic cells and cross-presentation.Rho GTPases share a high level of homology, despite the fact that their function can be very distinct. The most divergent region between these GTPases is the hypervariable C-terminal polybasic region. Emerging evidence highlights the fundamental role of C-terminal polybasic region in the physiological functions of Rho GTPases. Rac1, through its polybasic domain, was shown to bind and activate PAK, and also required for the activation of the neutrophil NADPH oxidase. The C-terminal polybasic region of Rac2 was shown to be required for correct intracellular localization of Rac2 protein and was essential for the biologic functions of Rac2. To study the role of these regions in cellular function mediated by Rho family proteins, researchers used Tat, a cell-penetrating peptide, to deliver the C-terminal polybasic region of Rho family into cells. It is demonstrated that the cell-permeable versions of the C-terminal polybasic region of Rho family acted rapidly and efficiently in transduced cells, and could directly interact with particular effectors of downstream signal pathway. Considering this, we hope to use the fusion peptides, comprised the C-terminal domain of different members of Rho family and Tat peptide, to test the role of C-terminal domain of different Rho family members on cross-presentation in DC.First, six fusion peptides that were composed of Tat and C-terminal domain of different members of Rho family and a Tat control peptide were synthetized, including Tat-Rac1, Tat-hRac2, Tat-mRac2, Tat-Rac3, Tat-RhoA and Tat-Cdc42 C-terminal peptides. Then, we tested the influences of different fusion peptides on cross-presentation by a characterized DC2.4 dendritic cell line. In our experiment, bacteria expressing dsRed-OVA144-386 fusion proteins (E.coli BL21/pYOVA) was used as a model antigen and an effective antigen cross-presentation assay system with DC2.4 cells was successfully established. We firstly found that Tat-Rac1 C-terminal peptide could significantly enhance the cross-presentation of DC2.4 cells.Next, the result of Tat-Rac1 C-terminal peptide obtained from DC2.4 cells was further confirmed. DCs were generated from mouse bone marrow and identified in several aspects, i.e. cell morphology, yield, purity, and molecules surface expression with stimulation of LPS. The results showed that most of the cells were immature DC, which possessed typical morphologic characteristics with about 80%~90% purity at day 7, then, after establishing an antigen cross-presentation assay system with BMDC, the influence of Tat-Rac1 C-terminal peptide on cross-presentation by BMDC was analyzed with E.coli BL21/pYOVA antigen. Similar results with DC2.4 cell were observed in this assay and cross-presentation by BMDC was also remarkably enhanced by the Tat-Rac1 C-terminal peptide.To further investigate the mechanism of promoted cross-presentation ability by DC caused by Tat-Rac1 C-terminal peptide, we analysed phagocytosis and maturation of DC by fluorescence microscope and fluorescence activated cell sorting. We observed the changes of phagocytosis with E.coli BL21/pYOVA antigen in DC after pulsing Tat-Rac1 C-terminal peptide. The results illustrated that phagocytosis of bacteria antigen was markedly facilitated by the Tat-Rac1 C-terminal peptide, which suggesting that enhanced antigen cross-presentation mediated by this fusion peptide maybe explained by the enhancement of capacity in antigen uptake. Then, we investigated the possible mechanism involved in the increased ability of antigen uptake by Tat-Rac1 C-terminal peptide. We used FITC labeled transferrin, OVA, E.coli opsonized by IgG antibodies, and dextran as model antigens and analyzed changes in uptake of these antigens by DC after pulsing Tat-Rac1 C-terminal peptide. Our results indicated that the peptide had no effect on the uptake of these four types of antigen. These results predicted that increased antigen uptake caused by this fusion peptide was not associated with some of the endocytic pathways, i.e. transferrin mediated endocytosis, mannose receptor mediated endocytosis, Fc receptor mediated phagocytosis, and macropinocytosis. To further analyze the influence of Tat-Rac1 C-terminal peptide on maturation of DC, we used a bacteria expressing OVA1-386 as an antigen and detected the surface molecules expression of peptide pulsed DC. Results showed that this peptide did not affect the DC maturation.In summary, the influences of different fusion peptides comprised the C-terminal domain of different members of Rho family and Tat peptide were assessed on cross-presentation by DC2.4 cells and the function of Tat-Rac1 C-terminal peptide was proved for the first time that it could significantly enhance the cross-presentation of DC, which was further confirmed within BMDC. Furthermore, the mechanism by which Tat-Rac1 C-terminal peptide affects cross-presentation of DC was analyzed. Our results demonstrated that phagocytosis of DC was remarkably increased by the Tat-Rac1 C-terminal peptide, which suggesting that enhanced cross-presentation by Tat-Rac1 C-terminal peptide in DC was associated with the enhancement of capacity in antigen uptake. And this effect was not due to transferrin mediated endocytosis, mannose receptor mediated endocytosis, Fc receptor mediated phagocytosis, and macropinocytosis, it is probably relevant to complement receptor mediated phagocytosis or clathrin-independ endocytic pathway. The present study provides a new theoretical foundation for further apprehension of mechanism in cross-presentation of DC. In addition, the Tat-Rac1 C-terminal peptide may have an important contribution to DC based immunotherapy. |
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