| Objective:T-SPOT.TB(T-SPOT)assay is widely used for detection of Mycobacterium tuberculosis infection that based on the detection of Mycobacterium tuberculosisspecific IFN-γ-secreting T cells(ISCs)in PBMCs.Recently,high frequencies of lowdensity granulocytes(LDGs)were found in the PBMCs of tuberculosis patients.Whether these LDGs will affect the detection of T-SPOT has not been investigated.Therefore,we investigated the impact of LDGs on T-SPOT assay and related mechanism.Methods:1.Peripheral blood mononuclear cells(PBMCs)from tuberculosis(TB)patients were isolated,the frequencies of LDGs in PBMCs were detected by Flow Cytometry(FCM).PBMCs were divided into two groups,high LDGs frequency group(LDGs >3%)and low LDGs frequency group(LDGs ≤ 3%)and T-SPOT assay was performed.To analysis the correlations between the frequencies of LDGs and the results of T-SPOT,the positive rates of T-SPOT and the number of ISCs in these two groups were compared.2.To investigate the effect of removing LDGs on T-SPOT assay,PBMCs were isolated from TB patients,and the frequencies of LDGs were detected by FCM.Samples with high LDGs frequency(LDGs > 3%)were selected and divided into two parts,one of which were directly detected by T-SPOT assay.In another part,LDGs were removed by immune-magnetic positive isolation and T-SPOT assay was then performed with the purified mononuclear cells.3.To investigate the effect of exogenous addition LDGs on T-SPOT assay,LDGs were purified by negative magnetic sorting.Purified LDGs were added into autologous PBMCs to a final percentage of 11–30%.Then,these PBMCs with exogenous LDGs and another untreated autologous PBMCs were detected by T-SPOT simultaneously.4.To investigate the effect of LDGs on interferon-γ(IFN-γ)production by T lymphocytes,PBMCs from TB patients were isolated and stimulated with TB antigen,and the frequencies of LDGs in PBMCs and the production of IFN-γ in T cells were detected by FCM.5.PBMCs from TB patients and normal density neutrophils(NDGs)from healthy individuals were isolated,and the expression of negative costimulatory molecules on LDGs from TB patients and NDGs from healthy individuals were detected by FCM and compared.6.To investigate the effect of blocking programmed death ligand 1(PD-L1)on PBMCs on T-SPOT assay,PBMCs from TB patients with high LDGs frequency(LDGs > %)were selected and divided into three parts.One part was treated with monoclonal antibody against PD-L1,and LDGs were removed by immune-magnetic positive isolation in another part,and the last part was left untreated.The three parts were simultaneously detected by T-SPOT.7.To investigate the effect of blocking PD-L1 on LDGs alone on T-SPOT.LDGs were purified and divided into two parts,one of which was treated with monoclonal antibody against PD-L1 and the other was left untreated.The two parts of LDGs were added to autologous PBMCs separately and T-SPOT assay was performed with those mixture.Results:1.The number of ISCs in response to both ESAT-6(25.41±3.987 vs 55.73±7.078,P=0.0002)and CFP-10(39.87±4.893 vs 64.82±7.941,P=0.0064)stimulation and the total ISCs in response to ESAT-6 and CFP-10(65.28±7.985 vs 120.5±13.84,P=0.0004)were significantly lower in high LDGs frequency group than those of low LDGs frequency group.The positive rate of T-SPOT assay in high LDGs frequency group was significantly lower than that of low LDGs frequency group(P=0.015).2.LDGs do not secrete IFN-γ under the stimulation of Mtb antigens and PHA.3.After the removal of LDGs,the numbers of the ISCs significantly increased in response to both ESAT-6(25.72 ±5.137 vs 39.94±6.520,P = 0.0049)and CFP-10(18.39±4.281 vs 40.83±8.988,P = 0.0055)stimulation.The total ISCs in response to ESAT-6 and CFP-10 were also significantly increased in the condition of LDGs removal(44.11±8.407 vs 80.78±14.69,P = 0.0041).In addition,the positive rate of TSPOT assay increased from 77.78 to 94.44% after the removal of LDGs,but the difference was not significant(P = 0.1482)because the number of negative specimens was relatively small.4.When compared to the untreated group,exogenous addition of LDGs significantly decrease the numbers of ISCs both in response to ESAT-6(25.32±5.456 vs 9.455±2.397,P = 0.0008)and CFP-10(35.95±8.188 vs 17.09±6.922,P = 0.0027).The total ISCs in response to ESAT-6 and CFP-10 were also significantly decreased in the condition of LDGs addition(61.27±11.89 vs 26.55±7.699,P = 0.0008).The positive rate of T-SPOT assay decreased from 100.00 to 59.09% after the addition of LDGs(P= 0.0008).5.The frequencies of IFN-γ-producing T cells were significantly lower in patients with high LDGs frequency than those with low LDGs frequency,both in response to ESAT-6(0.4743±0.09035 vs 1.058±0.2680,P = 0.0088)and CFP-10 stimulation(0.5479±0.1017 vs 0.9953±0.1260,P = 0.0082).Correlation analysis showed that,in response to ESAT-6 stimulation,the frequencies of IFN-γ-producing T cells were negatively correlated with the frequencies of LDGs(rho=-0.43,P=0.0207).6.There was no significant difference in the levels of IL-10 in the supernatant of PBMCs with different LDG ratios in response to both ESAT-6 and CFP-10 stimulation.The frequency of PD-L1-positive cells in LDGs was significantly increased than that in NDGs(28.26±4.521 vs 5.721±1.227,P = 0.0072).No significant difference was observed in the frequency of PD-1-expressing cells,TIM3-expressing cells,and TIGIT-expressing cells between LDGs and NDGs.7.When compared to the untreated group,blockade of the PD-L1 by monoclonal antibody significantly increased the number of ISCs,both in response to ESAT-6(17.09±6.388 vs 31.91±8.065,P=0.0042)and CFP-10(45.55±16.52 vs 57.00±20.31,P=0.0176).The number of ISCs in α-PD-L1-treated group had no significant difference compared to LDG-removed group.However,we found an increase in the total number of ISCs in LDG-removed group compared to the α-PD-L1-treated group(98.91±27.36 vs 88.91±27.36,P=0.0165).8.When compared to the addition of untreated LDGs,the addition of PD-L1antibody-treated LDGs significantly increased the number of ISCs in PBMCs,both in response to ESAT-6(11.85±2.655 vs 18.15±4.223,P=0.0073)and CFP-10(39.69±13.69 vs 46.92±14.66,P=0.0038).Conclusion:LDGs can inhibit the production of interferon-γ in T cells and decrease the positive rated of T-SPOT assay via highly expressed PD-L1.Pretreatment with anti-PD-L1 monoclonal antibody can effectively improve the positive rate of T-SPOT. |
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