Generation And Analysis Of The Specific Cytotoxic T Lymphocytes Elicited By Dendritic Cells Loaded With The Purified EBV-LMP2A Protein In Vitro

Epstein-Barr virus (EBV), infecting over 95 percent of humans andpersisting for the lifetime of the person, belongs to the gama-herpesvirinaesubfamily and was known its relationship with human tumors early. SinceEBV DNA was detected in tissues from patients with nasopharyngealcarcinoma (NPC) in 1966, serological studies have confirmed EBVinfections are the crucial factors in the pathogenesis of NPC. NPC is amalignant tumor derived from the epithelial cells and endemic in theSoutheast Asia and South of China. NPC usually are not suitable forsurgery and tumor cells are not eliminated effectively and completely byradio therapeutic and chemotherapeutic methods. Relapse and metastasismay occur in a portion of NPC patients and side effect arising fromconventional therapeutic methods will affect self immune system greatly.Therefore, it is critically important to develop an immunotherapeuticmethod for NPC except for conventional therapy.Studies on immune system of NPC show that cytotoxicT lymphocytes(CTLs) specific for EBV antigens are crucial for eliminating tumor cells.CTL subtype can recognize and kill tumor cells directly and CD4+ andCDS+ T cells co-contribute it. However, suitable target antigen should beselect in immunotherapy based on CTL.Within NPC tumor cells, the viruses establishes a predominantly latentinfection, with laminated expression of three viral proteins, nuclearantigens 1 (EBNA 1) and three latent membrane proteins (LMPs 1, 2A and2B). EBNA1 can induce CTL specific for it and also produce CD4+regulatory Treg cells, which suppress immune response. CDS+ CTLresponses specific for EBNA1 epitopes are mainly restricted by HLA-B3501,B7. LMP1 has been confirmed to be consistence with itsability to transform rodent fibroblasts, and its target epitopes recognized byvirus-specific cytotoxic T cells (CTL) in NPC patients are easy to mutate.Responses detected in healthy virus carriers indicate that LMP1 is poorlyimmunogenic, thus, the most likely target antigen for a CD8 CTL-basetherapy is LMP2. LMP2 amino acid is more conserved and containsHLA-restrict CTL target epitopes which can elicit strong specific CTLresponse. Pre-CTL cells specific for EBV-LMP2 can be detected in the seraof NPC patients, but its number and response are obviously lower thanhealthy carders, which results in immune tolerance. Therefore, it's veryimportant to correct this low CTL-response status and elicit EBV-specificCTL in NPC patient is one of the key immunotherapeutic strategies.T cells can only recognize antigen peptide-MHC monocular complexpresented by antigen presenting cells (APCs). Dendritic cells (DC) areknown as the most powerful and the only APC which are capable ofactivating naive T cells and initiating primary immune response. Resultsfrom DC-based immunotherapy have revealed a promising approach forcancer treatment and become one of the hot points in immunotherapyresearch at present.In this study, DC was utilized as APC, and EBV-LMP2A was chosenas the target antigen of NPC immunotherapy. LMP2A stable expressioncell line was constructed by monocular and cellular biological methods,and was purified. We then loaded DC induced from monocytes of EBVhealthy carriers with the purified LMP2A protein. The loaded DC wereco-cultured with autologous CD4~+ and CD8~+ T cells separately to elicitEBV-specific CD4~+ and CD8~+ T cells which were further analyzed itsfunctions. The main methods and results are as following:PartⅠ. Establishment of EBV LMP2A gene recombinant retrovirusvector and L929 cell line stable expressing LMP2AThe full-length EBV LMP2A gene was generated by PCRamplification from pPICZ-α/LMP2A which contain complementnucleotide sequence of EBV LMP2A gene with a pair of primers. Afterdigestion with EcoRⅠ,BamHⅠ, PCR products were inserted downstream of LTR of retroviral vector pSIV-1 to obtain recombinant retroviralexpression vector pSIV-1/LMP2A. Recombinant retroviral expressionvector pSIV-1/LMP2A was detected positive by PCR, enzyme digestionwith EcoRⅠand BarnHⅠas well as sequence analysis. To produceretroviral virus, packing cells, 293T cells were co-transfected withrecombinant retroviral expression vector pSIV-1/LMP2A and two auxiliaryviral vectors pHIT456 and pHIT60 by lipofectAMINE2000. Colonies wereisolated by neomycin (G418) selection and obtained after 1 week toexpand. Supematants of cloned packing cells were harvested, whichcontain recombinant retrovirus. The PCR results showed that EBV LMP2Agene had been integrated into the genome of resistant 293T cells. Viraltitration was performed on NIH3T3 according to the instructions of themanufacturer yielding a titer of 5×10~5 infectious particles/ml. The PCRpositive results indicated that NIH3T3 genome had contained LMP2A gene.Indirect immunofluorescence showed that LMP2A gene had beenexpressed in the murine flbroblast, NIH3T3. Transduction rate was 91.3%determined by fluorescence activated cell sorter (FACS). Western blotresults showed a specific protein lane at 54KDa position. These resultssuggested that transfer of LMP2A could be efficiently mediated byrecombinant retrovirus. Western blot results also confirmed it. LMP2Aprotein was extracted and purified by His Bind Ni-NTA affinitychromatography column, the purity of over 90% LMP2A protein wasobtained.The purified recombinant protein LMP2A was used as an antigen todetect specific antibodies in the sera from NPC patients. 72.29% patientswere positive for anti-LMP2A IgG. These studies demonstrate the potentialsignificance of LMP2A-specific Abs for the diagnosis and prognosis ofEBV-associated malignancies, especially of NPC.PartⅡ. Induction and identification of dendritic cells from humanperipheral blood monoeytes and load of DC with EBV-LMP2Apurified proteinThe monocytes were isolated from PBMC of a healthy EBV carrier and cultured with the cytokines (rhGM-CSF, rhIL-4), followed by additional 2days with TNF-α, yielding mature DCs. The morphological changes wereobserved by confocal analysis. The surface molecule of DCsIncluding CD80 and MHC classⅡwere measured by FACS. The resultsshowed that immature could be obtained from PBMC cultured in themedium containing GM-CSF and IL-4. DCs relative specific markerCDla, CD83 and co-stimulational monocular CDS0, HLA-DR wereexpressed low, while monocyte specific marker CD14 was relative high.When the immature DCs were cultured for additional two days in thepresence of TNF-α, mature DCs were harvested and express high level ofCD1a,CD83 and CD80,HLA-DR, while CD14 express low. DCs typicalcharacteristics were observed by morphological microscope. In addition,mature DC had potent ablility to stimulate the proliferation of allogeneic Tcells.Immature DCs were loaded with EBV-LMP2A purified protein atdifferent concentrations. The ability of DCs capture antigen was observedby confocal analysis and a suitable protein concentration was selected byFACS. The alteration of surface markers on mature DC including CDla,CD83, CD80 and HLA-DR was detected by means of FACS before andafter loaded with suitable LMP2A protein. The stimulatory effect of DCson proliferation of T lymphocytes was examined by ~3H-TdR incorporation.The results showed that above 80% of mature DC were expressed LMP2Aprotein after loaded with LMP2A protein at the concentration of 40μg/ml.No significant changes in the surface markers and the cytomorpholosis ofmature DC were examined during loading protein. It showed that DCsloaded antigen had no influence to the differentiation and maturation. DCsstill have strong potential to stimulate the proliferation of allogeneic T cells.The study could be the foundation of the immunotherapy with DC.PartⅢ. Generation and cytotoxieity assay of EBV LMP2A-specificCTL elicited by DC loaded with LMP2A protein in vitroImmuno-magnetic beads were used to prepare CD4~+ and CD8~+T cells.Median purity of the cell preparations was determined by flow cytometryanalysis and was routinely more than 95%. The proliferation of autologous CD4~+ cells, CD8~+T cells stimulated with Unloaded DCs (UL-DC) andloaded DCs (L-DC) was measured by methods of ~3H-TdR incorporation.The proliferative responses of CD8~+T and CD4~+ T cells to L-DC weresignificantly increased compared to those from UL-DC (P＜0.001,P＜0.001). The results demonstrate that DCs capture LMP2A protein andpresent their Ags to autologous T cells and LMP2A protein containsMHC-ⅠandⅡclass restricted epitopes. We tested the IFN-γand IL-10secretion from UL-DC and L-DC stimulated CD4~+T cells by ELISA andfound that L-DC stimulated CD4~+T secreted higher levels of IFN-γthanthose from UL-DC(P=0.034), but the level of IL-10 was rarely detected ineither group.EBV LMP2A gene recombinant retrovirus vector pGEZ-Term/LMP2A was established which contains GFP report gene. 293T cells wereco-transfected with recombinant retroviral expression vectorpGEZ-Term/LMP2A and two auxiliary viral vectors pHIT456 and pHIT60by lipofectAMINE2000. Colonies were isolated by zeocine. Supernatantsof cloned packing cells were harvested, which contain recombinantretrovirus. The monocytes were isolated from PBMC and incubated withGM-CSF and IL-4 medium. After a 24-h incubation, the cells weretransduced with retrovirus supernatants and polybrene at a finalconcentration of 8μg/ml. The supematant were replaced with GM-CSFand IL-4 medium 2-3h later. The transduction procedure was repeated threetimes. After 6 days later, TNF-αwas added and mature DCs wereharvested after additional 2 days culture. The surface molecule of DCs andthe efficiency of LMP2A expression were measured by FACS. The resultsshowed that 56.4% of DCs can express LMP2A determined by FACS.Immuno-magnetic beads were used to prepare EBV-LMP2A specificCD8~+ T cells, which were then restimulated with autologous DC loadedwith LMP2A protein. Cytotoxicity of LMP2A specific CTL wasdetermined with LDH release assay, the target cells are autologous DCtransduced with recombinant retrovirus pGEZ-Term/LMP2A,autologousLCL and K562 cells separately. We evaluated IFN-γ-producing cells bothin LMP2A-specific CD4~+ T cells and CD8~+ T cells induced by L-DCstimulated with recombinant retrovirus pGEZ-Term/LMP2A by FACS. CD4~+ T cells and CD8~+ T cells induced by L-DC had significantly higherfrequency of circulating LMP2A-specific IFN-γ-producing CD4~+ T cellsand CD8~+ T cells, compared to controls (P＜0.01).In summary, the results above suggested that LMP2A can beexpressed stably in L929 cell line, LMP2A may be of prognostic valuein NPC patients; LMP2A-specific CTL could be induced by DC loadedwith LMP2A protein in healthy EBV carriers in vitro and elicitcytotoxicity to autologous DC tranceduced with recombinantretrovirus pGEZ-Term/LMP2A. Such results could be the foundationfor the further immunotherapy of NPC in clinical trails; moreover,this research provides experimental and theoretical basis for othervirus related tumor immunotherapy.