| Objective:To investigate the effect of low density granulocytes(LDGs)in patients with rheumatoid arthritis(RA)on interferon-γ-secreting T lymphocytes(ISCs)detected by T-SPOT.TB assay and its related mechanisms.Methods:1.The peripheral blood of RA patients and healthy people was collected,and the mononuclear cells(PBMCs)layer cells were isolated by density gradient centrifugation.The proportion of LDGs in PBMCs of the two groups was analyzed by flow cytometry.2.All subjects who had their T-SPOT.TB tested at the First Affiliated Hospital of Nanchang University from May 2020 to July 2021 were retrospectively analyzed,and the difference between the positive rate of T-SPOT.TB test in RA patients and the overall positive rate was compared.3.PBMCs were isolated from peripheral blood of RA patients who had recently received T-SPOT.TB assay with at least 1 spot in any of the test wells.The proportion of LDGs was detected by flow cytometry,and PBMCs were divided into a low proportion of LDGs group(LDG ≤ 5%)and a high proportion of LDGs group(LDG>5%).T-SPOT.TB detection was performed respectively to compare the difference of T-SPOT.TB detection results between the two groups,and explore the correlation between the proportion of LDGs and the results of T-SPOT.TB detection.4.Peripheral blood was collected from RA patients who had recently received T-SPOT.TB assay with at least 1 spot in any of the test wells.According to the proportion of LDGs,the following treatment was performed:(1)PBMCs with a high proportion of LDGs(LDG>5%)were selected and divided into two groups:(1)LDGs removal group,LDGs were removed by immunomagnetic beads;(2)Control group,without treatment.(2)PBMCs with a low proportion of LDGs(LDG ≤ 5%)were selected and divided into two groups:(1)adding group,adding purified LDGs to PBMCs;(2)Control group,without treatment.On the one hand,the cells of each group were taken for T-SPOT.TB detection at the same time to analyze the effect of removal or exogenous addition of LDGs on T-SPOT.TB.On the other hand,phytohemagglutinin(PHA)was added to PBMCs before and after LDGs removal,and then flow cytometry was used to detect the level of IFN-γ in T lymphocytes in each group to explore the effect of LDGs on the secretion of IFN-γ by T lymphocytes.5.PBMCs of RA patients,normal density neutrophils(NDGs)of RA patients,and NDGs of healthy people were obtained by density gradient centrifugation.The expression of lectin-type oxidized low-density lipoprotein receptor-1(LOX-1)on RA-LDG and RA-NDG was analyzed by flow cytometry,and the proportion of polymorphonuclear myeloid-derived inhibitory cells(PMN-MDSCs)was explored.Meanwhile,the expression of negative costimulatory molecules CD155,T cell activation V domain Ig inhibitor(VISTA),and programmed death ligand 1(PD-L1)on the cell surface of RA-LDG,RA-NDG,and HC-NDG were analyzed by flow cytometry.6.Peripheral blood was collected from RA patients and PBMCs were isolated.PBMCs with a high proportion of LDGs(LDGs>5%)were selected and divided into three groups:(1)Control group,without treatment;(2)Removal group,LDGs in PBMCs were removed by immunomagnetic beads;(3)PD-L1 blocking group,PBMCs were blocked by anti-PD-L1 monoclonal antibody.T-SPOT.TB assay was performed to explore the effect of PD-L1 blockade on the results of T-SPOT.TB assay.7.PBMCs were isolated from the peripheral blood of RA patients.PBMCs with a low proportion of LDGs(LDG≤5%)were selected and divided into three groups:(1)control group,no other treatment;(2)In addition group,PBMCs were added with untreated purified LDGs;(3)LDG-PD-L1 blocking group,purified LDGs were treated with anti-PD-L1 monoclonal antibody for 1h and then added to PBMCs.T-SPOT.TB was performed to explore the effect of PD-L1 expression on the results of T-SPOT.TB.8.Peripheral blood of RA patients was collected and PBMCs were isolated.PBMCs with a high proportion of LDGs(LDGs>5%)were selected and divided into two groups:(1)Removal group,LDGs were removed by immunomagnetic bead positive selection;(2)Control group,no other treatment.Cells in the two groups were stimulated by adding specific antigens of Mycobacterium tuberculosis into the culture hole according to T-SPOT.TB detection method and cultured for 18-20 h.The culture supernatant was collected and the levels of arginase 1(Arg-1)and transforming growth factor β(TGF-β)were detected by enzyme-linked immunosorbent assay(ELISA).On this basis,the correlation between the proportion of LDGs and the levels of Arg-1 and TGF-β in the culture supernatant was analyzed.Results:1.The proportion of LDGs in PBMCs of RA patients was significantly higher than that of healthy controls(9.795±4.893 vs 0.760±0.617).2.The positive rate of T-SPOT.TB in RA patients was significantly lower than that of T-SPOT.TB in general(29.79% vs 43.28%,P<0.0001).3.The T-SPOT.TB positive rate in the group with a high percentage of LDGs was lower than that in the group with a low percentage of LDGs(33.33% vs 48.61%,P=0.0842).4.After LDGs removal,the number of ISCs was significantly increased(0.951±0.476 vs 2.113±1.040,P=0.0017).The Mean fluorescence intensity(MFI)of intracellular IFN-γ showed that the level of IFN-γ in T lymphocytes was also significantly increased(597.2±280.4 vs 670.0±323.8,P=0.0040).As compared with the control group,the number of ISCs was significantly increased in response to both ESAT-6(9.190±21.87 vs 21.43±31.70,P=0.0005)and CFP-10(2.643±4.951 vs10.52±12.61,P<0.0001)stimulation after LDG removal,as was also the total ISCs in the sum of the two specific antigen stimulation(11.83±23.01 vs 31.95±40.61,P<0.0001).Furthermore,as a result of LDG removal,the positive rates of T-SPOT increased from 38.10% to 85.71%(38.10% vs 85.71%,P < 0.0001).Exogenous LDGs addition reduced the number of ISCs in response to both ESAT-6(19.40 ±28.88 vs 10.10 ± 20.09,P=0.0017)and CFP-10(14.10 ± 43.84 vs 10.25 ± 37.86,P=0.0409)stimulation,and the total number of ISCs in the sum of the ESTA-6 and CFP-10(33.50 ± 59.24 vs 20.35 ± 49.71,P=0.0004)stimulation was also considerably decreased when compared to the control group.As a result of the addition of LDGs to the T-SPOT test,the positive rate decreased from 65.00% to40.00%,although this difference did not reach statistical significance(P=0.2049).Results that did not show spots before and after LDGs removal were excluded from the analysis.5.The LDGs of RA patients contained a high proportion of polymorphonuclear myeloid inhibitory cells(PMN-MDSCs),significantly higher than NDGs(63.38 ±10.95 vs 27.00±11.51,P < 0.0001).6.PD-L1 expression was significantly increased on LDGs when compared with autologous NDGs from patients with RA(30.52±19.39 vs 6.450±5.504,P<0.0001).One-way ANOVA showed that the expression of PD-L1 in LDGs of RA patients was significantly higher than that in autologous NDGs(32.72±20.53 vs 5.757±5.085,P<0.0001)and HC NDGs(32.72±20.53 vs 11.70±4.658,P<0.0001).No significant difference can be seen in the frequency of CD155 and VISTA expression between LDGs and NDGs.7.As compared with the control group,blocking PD-L1 in PBMCs significantly restored the ISC numbers response to both ESAT-6(3.118±4.337 vs 8.647±8.526,P=0.0017)and CFP-10(2.000±3.325 vs 4.529±5.304,P=0.0087)stimulation after LDG removal,as was also the total ISCs in the sum of the two specific antigen stimulation(5.118±6.397 vs 13.18±10.92,P=0.0002).However,the number of ISCs in the PD-L1 blockade group remained significantly lower than the LDGs removal group,in response to both ESAT-6(15.29±17.75 vs 8.647±8.526,P=0.0527)and CFP-10(9.706±8.770 vs 4.529±5.304,P=0.0050)stimulation after LDG removal,as was also the total ISCs in the sum of the two specific antigen stimulation(25.00±23.63 vs 13.18±10.92,P=0.0155).8.As compared with the addition of untreated LDGs,the addition of PD-L1-blocked LDGs significantly restored the ISC numbers response to both ESAT-6(5.643±12.06 vs 8.500±15.70,P=0.2262)and CFP-10(13.93±44.58 vs15.00±45.58,P=0.1358)stimulation after LDG removal,as was also the total ISCs in the sum of the two specific antigen stimulation(19.57±55.79 vs 23.50±57.97,P=0.1094).However,after the addition of PD-L1-blocked LDGs,the number of ISCs remained significantly lower than the control group,in response to both ESAT-6(10.71±17.06 vs 8.500±15.70,P=0.0396)and CFP-10(19.07±51.58 vs 15.00±45.58,P=0.1821)stimulation after LDG removal,as was also the total ISCs in the sum of the two specific antigen stimulation(29.79±65.29 vs 23.50±57.97,P=0.0694).9.Upon removal of LDGs from PBMCs,the level of Arg-1 in the supernatant was significantly reduced in response to both ESAT-6(452.4±442.8 vs 273.6±261.6,P=0.0315)and CFP-10(599.1±443.5 vs 495.0±490.2,P=0.0151)stimulation,while the number of ISCs was increased.A higher concentration of Arg-1 was also present in the culture supernatant of LDGs alone(735.3±562.7 ng/ml).In addition,the level of TGF-β in the supernatant was significantly reduced in response to both ESAT-6(66.77±49.53 vs 16.35±6.726,P=0.0042)and CFP-10(61.79±33.98 vs 16.02±6.273,P=0.0005)stimulation,while the number of ISCs was increased.Further studies showed that the proportion of LDGs in PBMCs positively correlated with the concentration of both Arg-1 and TGF-β in the culture supernatant.Conclusion:1.The level of LDGs in the peripheral blood of patients with rheumatoid arthritis is significantly increased,and it can inhibit the secretion of IFN-γ by T lymphocytes and reduce the positive rate of T-SPOT.TB test.2.LDGs in the peripheral blood of patients with rheumatoid arthritis have the phenotype of PMN-MDSCs and highly express PD-L1.The inhibitory effect of LDGS on the secretion of IFN-γ by T cells is partially mediated by the high expression of PD-L1 on the surface.3.LDGs highly expressed Arg-1 and TGF-β in the peripheral blood of RA patients,and these two immunosuppressive molecules may be related to the function of LDGs in inhibiting T cell activation. |
PDF Full Text Request