| Background: The tuberculosis(TB) is a complex process that the dynamic interaction between Mycobacterium tuberculosis(Mtb) with immune cells of host. The mechanism of human’s immunity against Mtb infection and the pathogenesis of TB has not been fully elucidated. In recent years, it has been confirmed that, in numerous experimental study and clinical observations, the γδ T cells play the innate immunity like role in the human immunity to Mtb infection,. Researchers have reported that γδ T cells recognize a synthetic peptide sequence. Our recently work of this laboratory has obtained some peptide sequences that can be recognized by γδ T cells, by the phage display peptide library. γδ T cells can be activated by the synthetic peptide fragments. It confirmed that γδ T cells can directly bind and teconize the epitope peptides like B cells.But whether γδ T cells can bind and recognize the B cell epitope peptides has not been reported. In this study, we cultured human peripheral blood mononuclear cells(PBMC)with the synthesized peptide fragments derived from B cell and T cell epitopic sequences, and to investigate the specific stimulation of the B cell epitope peptides of Mycobacterium tuberculosis antigen(Mtb-Ag) on human peripheral γδ T cell activation and proliferation that were detected by flow cytometry. We intend to search for more epitope peptides from M.tb that are specific to γδ T cells.Objective: To investigate the specific stimulatory effect of the B cell and T cell epitope peptides of Mtb antigen on human peripheral γδ T cell proliferation.Method: 1. We selected the sequences of B cell epitope peptides from Mtb-Ag that were reported in literatures and γδ T cell epitope peptides that recently identified in this laboratory to synthesize six peptides of B cell epitopes(BP1-BP6) and seven peptides ofγδ T cell epitopes(TP13, TP14, TP15, TP16, TP18, TP19, TP23). The synthetic peptide fragments were dissolved in PBS to 1 mg / ml, and sterile filtered for ready use.2. The ELISA microplates were coated with these B cell epitopes. The serum fromhealthy individuals and TB patients were diluted by 1:3 and 1:9, and then put in the plates. After 2 hours, the Goat anti Human Ig G-HRP were added, the plates were washed, and then the tetramethylbenzidine(TMB) were added to color development.The microplate reader was used to detect the absorbance(OD Value) of each well. The OD Values were calculated and compared with negative control group.3. The 24-well culture plates were coated with these peptides. The PBMCs were isolated from peripheral blood of healthy individuals and stained with CFSE, followed cultured for 12 days in the IL-2 containing medium. Mtb heat resisted antigen(Mtb-HAg) group as positive control and IL-2 only group as negative control.4. The cultured cells in each group were collected, and the cells’ surface antigen ware stained with fluorescent conjugated amtobodies, including anti-CD3, anti-TCRγδ,anit-Vδ1, anti-Vδ2, etc.. The percentages and proliferation index of γδ T cells and the percentages of γδ T cells, and subsets of Vδ1 and Vδ2 were determined by flow cytometry. The data were analyzed by Flowjo software.5. We collected the γδ T cells that were stimulated with the epitope peptide and expanded in culture. The expanded cells were divided into three groups, and stimulated with same peptide, unrelated peptide, and without any peptide in equal amounts,respectively, for 18 hours. The percentages of CD69 express of γδ T cells were detected by staining with fluorescent antibodies and flow cytometry. Another set of expanded cells in three groups were stimulated with same peptides for 18 hours and with monesin(1 μg/ml) for more 4 hours. The percentage of γδ T cells that secreting IFN–γ were detected with flow cytometry.6. We collected the γδ T cells stimulated with the epitope peptides. Using the RT-PCR and scanning technique, we analyzed the distribution characteristics of TCR-Vδ-CDR3 gene fragment..Results: 1. The ELISA microplates were coated with these B cell epitopes. The serum from healthy individuals(16 samples) and TB(4 samples) were diluted by 1:3 and 1:9.The OD Value(OD450) of total samples was 2 times to the cut off value(COV). So the antibody from serum can be combined with the six B-cell epitope peptides.2. The B cell and T cell epitope peptides of Mtb-Ag added to the healthy adult PBMC,and then we observed proliferation of γδ T cells. We detected the percentage of PBMC(CD3+, TCRγδ+). By using Wilcoxon signed rank test for paired comparison of negative control group(4.4), the percentages of γδ T cells in cultured PBMCs with BP2(5.8),BP3(6.2), BP4(6.3), BP5(6.3), TP13(5.4), TP14(5.8), TP15(5.6), TP16(5.8) and Mtb-HAg(38.3) increased significantly in 22 samples(P value are 0.008, 0.044, 0.007,0.049, 0.044, 0.003, 0.022, 0.005 and 0.000).3. The proliferation index of γδ T cell in cultured PBMCs(CD3+, TCRγδ+, CFSE-Low)by the above method with BP2(3.9), BP4(3.7), BP5(4.2), BP6(3.7), TP14(3.7) and TP15(3.8) increased significantly in 7 samples compared with negative control group(2.0)(P value are 0.018, 0.018, 0.028, 0.028, 0.018 and 0.018),. Meanwhile, the proliferation index of αβ T cells in cultured PBMCs(CD3+, TCRγδ-, CFSE-Low) by the above method of none of groups in 7 samples increased significantly compared with negative control group(P>0.05).4. The percentage of CD3+TCRγδ+Vδ1+ cells and CD3+TCRγδVδ2+ cells in the expanded cells from PBMC that cultured with peptides in 12 day were detected with flow cytometry. The result showed that percentage of γδ+Vδ1+ cells in BP1(5.8),BP2(5.6), BP3(5.4), TP13(4.8), TP15(5.4), TP18(4.8), and TP23(7.1) group increased significantly in 8 samples compared with negative control group(3.4)(P value are 0.05,0.05, 0.05, 0.036, 0.036, 0.036 and 0.036). And the result of Vδ2 subset show that BP6(45.0)group compare with negative control(50.1) group reduced significantly in 27samples(P value is 0.039). It descript the BP6 nondominant stimulate Vδ2 subgroups.But the Mtb-HAg group compare with negative control(50.1) group increased significantly in 27 samples(P value is 0.000).5. The epitopes expanded γδ T cells were re-stimulated with same epitope peptides, the percentage of γδ T cells that expressed CD69 and secreting IFN-γ increased compared with the control group.6. The epitopes expanded γδ T cells were collected, and the TCR Vd-CDR3 gene fragment was analyzed by RT-PCR assay and gene spectratyping. We found that the TCR Vδ-CDR3 gene fragment districution of the γδ T cells that stimulated with epitope peptides was in oligoclonal pattern, compared to the control group in which that of Mtb-HAg stimulated γδ T cells showed in polyclonal distribution pattern.Conclusion: 1. The B cell and T cell epitope peptides from Mtb Antigens are capable of stimulating the γδ T cell proliferation specifically in vitro.2. Restimulation of same epitope peptides on the epitope peptides stimulated andexpanded γδ T cells leads to upregulation of expression of activation molecule CD69,and induction of production of effective cytokine IFN-γ.3. CDR3 gene fragment distribution of TCRVd of Mtb Ag epitope peptides stimulated and expanded γδ T cells showes oligoclonal pattern.4. It might suggest that the Mtb-Ag epitope petides stimulated and expanded γδ T cells are clonal specificity. |
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