| Background:It has been reported that there are changes of dendritic cells(DC) and their subsets in some diseases including tuberculosis(TB) and changes of DC subsets in patients with pulmonary TB at different stages, but the relativity between the changes of DC subsets and Vδ2expressed low of γδ T cells is not clear. It has been demonstrated that Mycobacterium tuberculosis (Mtb) can suppress the function of DC. Mtb heat resistant antigen (Mtb-HAg), a kind of small peptides antigen, can preferentially stimulate human γδT cells. Mtb H37Ra ultrasound broken protein antigen is a kind of complicated ingredients raw protein which including both the structure of the bacteria proteins, such as the surface antigen of the bacteria and the cytoplasm antigen. Mtb secreted protein antigen is secreted by Mtb during the period of growth and gradually released into the bacteria cultures. Effects of these three kind of antigens on the DC maturation and on the DC regulation of γδ T cell immune function are also not clear. And the effects of DC induced by Mtb-HAg on γδ T cell secretion of IL-17and IFN-γ during the co-culture have not been reported.Objective:To investigate the changes of DC subsets in peripheral blood of TB patients with different diseases stages and the relativity between the changes of DC subsets and Vδ2expressed low of γδ T cells. To investigate the effects of mycobacterium tuberculosis heat resistant antigen (Mtb-HAg), ultrasound broken antigen and secreted protein antigen on the maturation and functions of DC and on the DC regulation of γδ T cell immune function. To investigate the effects of DC induced by MtbH-Ag on the self γδ T cells secreting IL-17and IFN-γ.Methods:1.Peripheral blood specimens of36healthy donors,139pulmonary TB patients,39pneumosilicosis patients and18vivax malaria patients were collected and stained with four colored fluorescent antibodies, followed by lysing red blood cells and washing cells, the percentages of mDC (Lin-HLA-DR+CDllc+) and pDC (Lin-HLA-DR+CD123+) were measured and analyzed by flow cytometry. According to the standard of diagnosis and therapy status, all the patients of TB were divied into initial treatment group and recurrent group.2. The three Mtb antigens were prepared in advance by our lab.3. ImDCs were derived from human monocytes of healthy donors in the presence of GM-CSF and IL-4. After DC had been incubated respectively with MtbH-Ag, ultrasound broken antigen and secreted protein antigen, molecules including CD83, CD86, HLA-DR, CD69and DC-specific ICAM-3grabbing non integrin (DC-SIGN) were detected by flow cytometry (FCM). The untreated DC and lipopolysaccharide (LPS) induced DC were set to be the controls.4. Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood by density gradient centrifugation, stimulated with MtbH-Ag, and cultured in IL-2containing media to generate effective γδ T cells enriched cell line that called as Mtb-AT. Meanwhile, another group was stimulated with Mtb-HAg, and cultured in IL-2, IL-23, IL-1β and TGF-β containing media to generate effective γδ T cells which secreting IL-17.5. The percentages of self γδ T cells secreting IL-17and IFN-γ were detected by FCM on the third days after self γδ T cells had been co-cultured with DC which had been treated with Mtb-HAg. On the sixth day of co-culture with DC induced by Mtb-HAg, ultrasound broken antigen and secreted protein antigen, FCM was used to measure the cells-proliferations of self γδ T cells which had been marked with CFSE(carboxy fluorescein diacetate succinimidyl ester).Results:1.The percentage of pDC(0.92±0.87)%of pulmonary TB patients(n=138) was significantly decreased compared to HD group(n=36)(1.94±0.36)%(p<0.05); there was no difference in proportion of mDC between two groups. The ratio of mDC/pDC in TB patients was significantly higher than that of HD group (p<0.05).2.The percentage of pDC(0.71±0.57)%of pneumosilicosis patients(n=39) was significantly decreased compared to HD group(n=36)(1.94±0.36)%(p<0.05); there was no difference in proportion of mDC between two groups. The ratio of mDC/pDC in pneumosilicosis patients was significantly higher than that of HD group (p<0.05).3.The percentage of pDC(0.34±0.43)%of vivax malaria patients(n=18) was significantly decreased compared to HD group(n=36)(1.94±0.36)%(p<0.05); there was no difference in proportion of mDC between two groups. The ratio of mDC/pDC in vivax malaria patients was significantly higher than that of HD group (p<0.05).4.The percentage of pDC(0.96±0.11)%of initial treatment group of PTB(n=111) was significantly decreased compared to HD group(n=36)(1.94±0.36)%(p<0.05); there was no difference in proportion of mDC between two groups. The ratio of mDC/pDC in initial treatment group of PTB was significantly higher than that of HD group (p<0.05).5.The percentage of pDC(0.77±0.13)%of recurrent PTB patients(n=27) was significantly decreased compared to HD group(n=36)(1.94±0.36)%(p<0.05); there was no difference in proportion of mDC between two groups. The ratio of mDC/pDC in recurrent TB patients was significantly higher than that of HD group (p<0.05).6. There were no differences in the percentages of pDC, mDC and the ratio of mDC to pDC between the initial treatment group of PTB and the recurrent PTB patients.7. There were no relativities between the changes of pDC, mDC, mDC/pDC and the vδ2expressed low of γδ T cells among the PTB patients.8. ImDCs could be derived from peripheral monocytes in the presence of GM-CSF and IL-4, which expressed CD11c high and CD14low.9.The proportion of CD83、CD86、HLA-DR、CD69and DC-SIGN on DC induced by LPS were significantly higher than the untreated DC.10.(1)The proportion of CD83、CD86、HLA-DR、CD69on DC induced by Mtb-HAg were were significantly higher than the untreated DC (p<0.05,p<0.01,p<0.01, p<0.05),The proportion of DC-SGN had no significant changes (p>0.05).(2)Only the proportion of HLA-DR、CD69on DC induced by ultrasound broken antigen were significantly higher than the untreated DC (p<0.01,p<0.01), the proportion of CD83、CD86and DC-SIGN had no significant changes (p>0.05). (3)The proportion of CD86、HLA-DR、CD69on DC induced by secreted protein antigen were significantly higher than the untreated DC (p<0.01,p<0.01,p<0.01), the proportion of CD83and DC-SIGN had no significant changes (p>0.05).11.(1)Compared with DC induced by LPS, the proportion of CD83, CD86, HLA-DR, CD69and DC-SIGN on DC induced by MtbH-Ag had no significant changes (p>0.05).(2)The proportion of CD86and CD69on DC induced by ultrasound broken antigen were increased significantly (p<0.05,p<0.05). The rest three molecules had no significant changes (p>0.05).(3)The proportion of DC-SIGN on DC induced by secreted protein antigen was increased significantly (p<0.01). The rest four molecules had no significant changes (p>0.05).12.PI (proliferative index) assaies of all the groups of self γδ T cells co-cultured with three Mtb antigens treated DCs were analyzed:the PIs of the self γδ T cells co-cultured with Mtb-HAg/ultrasound broken antigen/secreted protein antigen induced DC were higher than that of negative controls (p<0.01). Compared with the PI of the self γδ T cells co-cultured with LPS induced DC, there were no significant changes among the groups.13. The proportion of IFN-y secreting cells of self γδ T cells which co-cultured with the DC induced by Mtb-HAg were analyzed. Compared with the proportion of IFN-y secreting cells of self γδ T cells induced only by IL-2(negative controls), the proportions of IFN-y secreting cells of self γδ T cells induced by Mtb-HAg (positive controls)/Mtb-HAg+CK (test groups) were significantly increased (p<0.01, p<0.01). The proportion of IFN-y secreting cells of self γδ T cells had no significant changes among the groups (p>0.05).Conclusions:1. Compared to HD group,the proportions of pDC in PTB patients, pneumosilicosis patients, vivax malaria patients are significantly lower, there are no difference in the proportions of mDC between the each two groups, but the ratio of mDC to pDC in the groups mentioned above are higher than that in HD group.2. Compared to HD group, the proportions of pDC in initial treatment group of PTB and recurrent PTB patients are significantly lower, there are no differences in the proportions of mDC between the each two groups, but the ratio of mDC to pDC in the groups mentioned above are higher than that in HD group.3. There are no relativities between the changes of DC subsets and Vδ2expressed low of γδ T cells among the PTB patients.4. The maturative phenotypes of DC can be specifically up regulated in the presence of Mtb-HAg, and Mtb-HAg treating can promote the mature of DC. The maturative phenotypes of DC could be partially nonspecifically up regulated in the presence of Mtb ultrasound broken antigen/secreted protein antigen.5. Mtb-HAg, ltrasound broken antigen and secreted protein antigen treating can enhance the capacity of DC to advance proliferation of self γδ T cells.6. Compared with DC induced by LPS, the elevation of the proportion of DC-SIGN on DC induced by Mtb secreted protein antigen may be concerned with the half mature state of DC in the presence of secreted protein antigen.7. DC induced by Mtb-HAg have no effects in promoting self γδ T cells secreting IFN-γ and IL-17. |
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