Maturation Of Dendritic Cells And Enhanced CTL Reaction Induced By Mannosylated RLD2 Efficiently

The nature of antigens and functional state of dendritic cells (DCs) are important in antigen presentation. The ability of DCs for the induction of T cell responses is promoted by maturation. It has been confirmed that mannose receptors mediate highly efficient endocytosis and presentation of mannosylated proteins. In the present study, L2 domain of ErbB2 ectodomain was expressed in E. coli, purified and mannosylated. The maturation and functional capacity of DCs induced by mannosylated L2 (mL2) protein were investigated. The results indicated that L2 protein could induce DC maturation, which was accompanied by elevated expression of MHC and costimulatory molecules. The effect of mL2 protein on DC maturation was more remarkable than that of non-mannosylated L2 proteins. Uptake of mL2 antigens by DCs was more efficient. Furthermore, the T cells can be stimulated to proliferate in vitro and secrete Th1 and Th2 cytokines. Higher levels of both IFN-γ and IL-10 were detected from the T cells stimulated by mL2-pulsed DCs. suggesting a concurrent activation of CD4~+ and CD8~+ T cells. The results demonstrated that L2 domain of ErbB2 receptor is an immunodominant molecule. The mannosylated L2 domain of ErbB2 can induce enhanced maturation and functional capacity of DCs. It may become an effective strategy to induce anti-ErbB2 response.In this study, four subjects were investigated:1. Soluble Expression and purification of HER-2/neu RLD2The co-expression of TrxA and soluble RLD2 (L2 ) was realized by transforming recombinated vecter pTIG-Trx A/RLD2 into BL21 (DE3) . Western-blotting analysis showed that the expressed protein could react with the specific antibody against proto-oncoprotein HER-2/neu. With the application of DEAE-Sepharose FF column and cobalt-based affinity resins, the purity of RLD2 obtained was over 85%. The purifiedprotein was further subject to mass spectrography molecular weight monitoring, and the molecular weight of RLD2 protein was identical to the expectation. By using TrxA expression system, the soluble expression of RLD2 protein was obtained with high efficiency, which will make it possible to further study of protein mannosylation.2. Mannosylation of HER-2/neu RLD2The purified HER-2/neu RLD2 protein was mannosylated by chemical method in vitro. RLD2 neoglycoprotein was identified by MALDI-TOF-MS. With the application of resorcinol-sulfuric acid method, the chemical compounds attached to RLD2 protein were vertified to be the sugar molecule. Futhermore, L2 or mL2 were conjugated with FITC.3. Isolation of DC from human PBMCMonocytes were isolated from the peripheral blood of healthy donors. Adherent cells were induced by GM-CSF and IL-4. After LPS induction following GM-CSF and IL-4 stimulation, DCs expressed high level of the costimulatory molecules (CD86 and CD40), CD83 and MHC class 11 molecules.4. The effect of mannosylation on uptake of antigen and maturation of DC Immuatured DCs (iDCs) incubated with FITC-conjugated L2 or mL2 proteins werestudied by flow cytometry and confocal microscopy. The uptake of proteins by iDCs was assessed by measuring mean fluorescence intensity. For evaluating DC maturation induced by L2 or mL2, immature DCs were pulsed with L2 or mL2 for 48 hours, stained for CD83, CD86 and MHC class II and analyzed by FACS. The result show that mL2 protein can induce the maturation of DC and enhance the uptake of antigens of DC.5. T cell anti-tumor immunity stimulated by DCDC activated by mL2 or L2 were employed to stimulate T lymphocytes, and MTT assay was performed to assess T lymphocyte proliferating activity. Specific cytotoxicity against SKBR-3 tumor cells was measured using cytotoxicity Detection Kit (lactate dehydrogenase, LDH). Secretion of INF-γ and 1L-10 levels was detected by double sandwich-ELIS A Kits. The results demonstrated that mL2 priming DC can stimulate high tumor cell lysis by T cells. High level of INF-γ and IL-10 was secreted by T cells stimulated by mL2 protein pulsed DC.Our results demonstrated that L2 domain of ErbB2 is an immunodominant molecule.