The Effect Of Mycobacterium Tuberculosis Specific Antigen On Cytokine Production Of Human Peripheral γδT Cells

Background: TB is a serious global public health problem. Our country is one of22high TB burden countries around the world, and one of27multi-drug resistant TB(MDR-TB) and extensively drug-resistant TB (XDR-TB) popular seriously countriesin the world. Therefore, the situation of TB prevention and control is still quite serious.Studies have confirmed that gamma delta T cells have a strong response activity toMycobacterium tuberculosis antigens stimulate, suggested that γδT cells may beassociated with the protective immune function of host resistance to TB. But themechanism of γδ T cells in nonspecific and/or specific anti-tuberculosis immuneresponse is not fully clear. The γδ T cells which produce IL-17may also be involvedin the onset of TB. Under what terms and regulation γδ T cells produce theinflammatory cytokine IL-17, there has a certain academic significance of ourunderstanding of the pathogenesis of TB. We usually use nonspecific stimulusPMA+Ionomycin to determinate the IFN-γ produced cells by detecting theintracellular cytokines by flow cytometry, the reports of antigen specificity Mtb-HAgstimulate γδ T cells to produce the determination of INF-γ are rarely. Enzyme-linkedimmune spot test(T.SPOT-TB) reflect the number of T lymphocytes which releaseIFN-γ by detecting IFN-γ density, accurate judgment weather there is tuberculosisbacterium infection. It has high sensitivity and specificity, but the reports of cytokinesproduced by γδ T cells are rarely. TNF-α is an important factor involved in theimmune response and immune pathogenesis process of tuberculosis, less researchabout it’s content in the peripheral blood of TB infection condition is known.Objective: To detect the change of IFN-γ、IL-17and TNF-α produced by T cell subset which separated from peripheral blood mononuclear cell of healthy people andTB patients and stimulated by nonspecific stimuli(PMA+Iono、 PHA)and specificantigen Mtb-HAg, describe the difference of innate immunity and adaptive immunityof γδ T cells between healthy people and TB patients, provide new indicators toevaluate different specific immune state of TB patients.Methods:1To detect IFN-γ and IL-17producing γδ T cells which stimulated with differentstimulants by Flow cytometryHealthy human peripheral blood were stimulated and cultured with PMA+Iono for6hours, or Mtb-HAg, Mtb-HAg+IL-23, PHA, PHA+IL-23for16hours, Monensinwere added in all groups for last4hours. The cultured blood samples were labeledwith fluorescent monoclonal antibodies to surface molecules and intracellularcytokine, percentages of IFN-γ or IL-17producing γδ T cells were detected by flowcytometry. The percentages of IFN-γ or IL-17producing αβ T cells were detected as acomparison at the same time.2To detect production of IFN-γ by T cell subgroups stimulated by different stimulantsby ELISPOT2.1PBMC were isolated from peripheral blood of healthy people, put100000cells/well into the well that has already packaged by IFN-γ antibody, control groupsjoin PHA, mycobacterium tuberculosis antigen A+mycobacterium tuberculosisantigen B, take out sort of the remaining cells which without αβ T cells byimmunomagnetic beads, accurate the number of γδ T cells by fluorescence staining,put this kind of cells15000cells/well,30000cells/well,60000cells/well into theplate at37℃, after20hours incubation, washed, incubated color, finally computedIFN-γ spots number.2.2PBMC were isolated from peripheral blood of healthy people, put100000cells/well into the well that has already packaged by IFN-γ antibody, control groupsjoin PHA, mycobacterium tuberculosis antigen A+mycobacterium tuberculosisantigen B, take out sort of the remaining cells which without αβ T cells byimmunomagnetic beads, accurate the number of γδ T cells by fluorescence staining,put this kind of cells30000-50000cells/well into the plate at37℃, after20hoursincubation, washed, incubated color, finally computed IFN-γ spots number. 3To detect IFN-γ and TNF-α producing γδ T cells which stimulated with differentstimuli by Flow cytometryPeripheral blood of healthy human and TB patients were stimulated and cultured withPMA+Iono for6hours, or Mtb-HAg, PHA for16hours, Monensin were added in allgroups for last4hours. The cultured blood sample were labeled with fluorescentmonoclonal antibodies to surface molecules and intracellular cytokine, the percentageof IFN-γ or TNF-α producing T cells were detected by flow cytometry. Thepercentages of IFN-γ or IL-17producing αβ T cells were detected as a comparison atthe same time.Results:1.1After stimulated by different stimulants, both αβ T cells and γδ T cells producedIFN-γ, while the γδ T cells which produced IFN-γ was higher than αβ T cells whichproduced IFN-γ. In peripheral blood of healthy people, the Mtb-HAg stimulated group,the γδ T cells which produced IFN-γ was higher than αβ T cells which producedIFN-γ significantly, the ratios were significant difference (p <0.01).1.2After stimulated by different stimulants, both αβ T cells and γδ T cells producedIL-17. In peripheral blood of healthy people, the PMA stimulated group, Mtb-HAgstimulated group, Mtb-HAg+IL-23stimulated group and PHA stimulated group,peripheral blood of healthy people, the γδ T cells which produced IL-17was higherthan αβ T cells which produced IL-17significantly, the ratios were significantdifference (p <0.05).2When Mtb-HAg was used to stimulate the γδ T cells of peripheral blood to generateIFN-γ spots by in ELISPOT, it’s better to put30000-50000γδ+T cells/well.3.1After stimulated by different stimulants, both αβ T cells and γδ T cells producedIFN-γ, while the γδ T cells which produced IFN-γ was higher than αβ T cells whichproduced IFN-γ. In PMA group, INF-γ produced by αβ T cells of healthy people arehigher than INF-γ produced by αβ T cells of TB patients, the difference wasstatistically significant (p<0.05). In PMA group, Mtb-HAg group and PHA group,INF-γ produced by γδ T cells of healthy people are higher than INF-γ produced by γδT cells of TB patients, the difference was statistically significant (p<0.05, p<0.01andp<0.05).3.2After stimulated by different stimulants, both αβ T cells and γδ T cells producedTNF-α. In PMA group and PHA group, TNF-α produced by αβ T cells of healthy people was higher than TNF-α produced by αβ T cells of TB patients, the differencewas statistically significant (p<0.05). In PMA group and PHA group, TNF-α producedby γδ T cells of healthy people are higher than TNF-αproduced byγδ T cells of TBpatients, the difference was statistically significant (p<0.05and p<0.05).Conclusion：1Stimulated with Mtb-HAg, IFN-γ producing cells of γδ T cells was higher than thatof αβ T cells in healthy people.2IFN-γ producing cells of γδ T cells of healthy people were higher than that of TBpatients.3IL-17producing cells of γδ T cells of healthy people were higher than that of αβ Tcells of healthy people.4TNF-α producing cells of γδ T cells were higher than that of αβ T cells in bothhealthy people and TB patients.5TNF-α producing cells of γδ T cells of healthy people stimulated nonspecific stimuliwere higher than that of TB patients.