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Objective To explore the impact of rituximab on Th17 cells and related cytokines in patients with diffuse large B-cell lymphoma (DLBCL) in vitro and its significance.Methods 20 cases of DLBCL untreated patients and 20 healthy subjects were enrolled in the name of DLBCL group and health control group, respectively.4 peripheral blood samples were collected from every case to separate peripheral blood mononuclear cells (PBMCs), which were assigned to 4 subgroups according to different culture conditions: blank subgroup(subgroup A), rituximab subgroup (subgroup B), rituximab and serum subgroup (subgroup C) and polarization subgroup (subgroup D) (added IL-6 and TGF-β).After cultured in vitro, the percentage of Th17 cells in each subgroup was tested by flow cytometry, and the cytokine IL-17 in the abovementioned culture fluid was measured by enzyme-linked immunosorbent assay (ELISA).Results In health control group, the percentage of Th17 cells and the level of IL-17 in subgroup D [(17.12 ± 4.90) ％ and (45.735±10.012) pg/ml] were significantly higher than those in subgroup A, B, C (P ＜ 0.05), and there was no difference in each other subgroup A, B, C (P ＞ 0.05).The percentage of Th17 cells and the level of IL-17 in the DLBCL subgroup A were significantly lower than those in health control subgroup A [(0.69±0.24) ％ and (6.012±1.312) pg/ml vs (2.43±0.61) ％ and (8.217±1.681) pg/ml (P ＜ 0.05)].In DLBCL group, after cultured with rituximab, the percentages of Th17 cells in subgroup B, C, D were (2.34±0.48) ％, (2.31±0.53) ％ and (16.92±4.81) ％, and the levels of IL-17 were (7.944±1.538) pg/ml, (7.957±1.533) pg/ml and (44.417±9.881) pg/ml, respectively, which were all significantly higher than those in subgroup A.Besides, the percentage of Th17 cells and the level of IL-17 in DLBCL subgroup D were significantly higher than those in subgroup B, C (P ＜ 0.05), while there was no difference between subgroup B and subgroup C.Conclusion Experiments in vitro confirmed that the percentage of Th17 cells in PBMCs of DLBCL patients was lower than that in healthy persons, and rituximab could elevate the percentage of Th17 cells in PBMCs of DLBCL patients.
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Objective To explore the effect of recombinant human granulocyte colony stimulating factor (rhG-CSF) mobilization on TH17/Treg cells and its impact on suppressor of cytokine signaling-3 (SOCS3) gene expression in CD4+ T cells in donors' peripheral blood.Method Sixteen donors were injected subcutaneously with rhG-CSF 5 μg/kg every day for 5 consecutive days for peripheral blood stem cells mobilization.At the first 0,3,5 day,the mononuclear cclls (MNCs) in peripheral blood or graft and serum specimens were taken.The CD4 + T cells in MNCs were sorted using immuno-magnetic beads.The ratio of TH 17 and Treg cells in MNCs,cytokines concentrations of IL-17A,IL-21,ID23 and TGFβ1 in serum,and SODC3 gene expression in CD4+ T cells were detected by using flow cytometry,ELISA,and reverse transcription real-time quantitative PCR (RT-qPCR),respectively.Results (1)The ratio of Th17 cells (CD3+ CD8 CD17+) and Treg cells (CD4+ CD25+ Foxp3+) in MNCs in peripheral blood and graft at the first 0,3 and 5 days after mobilization was (2.69 ± 0.81) ％,(0.91 ± 0.33) ％,(0.35 ± 0.12) ％,(0.21 ± 0.05) ％,and (0.56 ± 0.24) ％,(0.72 ± 0.22％),(1.59 ± 0.54) ％,(3.38 ± 0.52) ％,respectively,showing a significant declining and increasing trend respectively (P＜0.05); (2)The cytokine concentrations in serum at the first 0,3 and 5 days after mobilization were 7.33 ± 0.89,5.78 ± 1.03 and 3.32 ± 0.84 μg/L for IL-17A; 124.56 ± 15.18,117.12 ± 14.45 and 64.94 ± 11.25 μg/L for IL-21 ; 183.52 ± 59.35,280.49 ± 69.75 and 393.62 ± 57.25μg/L for TGF-β1 (P＜0.01) ; and 45.89 ± 6.95,46.25 ± 7.44 and 47.45 ± 10.75 μg/L for IL-23,respectively.The IL-17A and IL-21 concentrations showed significant declining trend,contrarily TGF-β1 with an increasing trend,while IL-23 concentration had no change.After rhG-CSF mobilization,the SOCS3 gene expression in CD4 + T cells of peripheral blood and graft at the first 0,3,5 days was gradually increased.Conclusion rhG-CSF suppresses Th17 cells and promotes regulatory T cells generation,meanwhile decreases IL-17A and IL-21 and elevates serum TGF-β1 concentrations,and contributes to CD4 + T cells differentiation to Tregs,probably by elevating SOSC3 gene expression in CD4+ T cells.
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Objective To understand the changes of Th17 cells and related cytokines in diffuse large B-cell lymphoma (DLBCL) patients treated with rituximab and its significances.Methods Patients were assigned to 4 groups,there were 20 cases in the control group,31 cases in the initial treatment group,31 cases in CHOP group and 25 cases in RCHOP group.The percentage of Th17 cells in the peripheral blood of each group was tested by flow cytometry,the related cytokines IL-17,IL-21,IL-23,TGF-β in the peripheral blood were measured by enzyme-linked immunosorbent assay.Results The percentage of Th17 cells and the levels of IL-17,IL-21,IL-23 in the initial treatment group [(0.67±0.21) ％,(5.929±1.342) pg/ml,(130.632±17.945)pg/ml,(51.681±9.808) pg/ml] and the CHOP-CR group [(1.07±0.37) ％,(6.526±0.538) pg/ml,(132.119±7.700)pg/ml,(50.245±7.668) pg/ml] were both significantly lower than those in the control group[(2.53±0.63) ％,(8.435±2.031) pg/ml,(149.265±12.316) pg/ml,(55.303±7.778) pg/ml] (P ＜ 0.05).The level of TGF-β in the initial treatment group [(370.615±98.444) pg/ml] was significantly higher than that in the control group [(311.895±73.365) pg/ml] (P ＜ 0.05).The percentage of Th17 cells and the levels of IL-17,IL-21,IL-23 in the RCHOP-CR group [(2.38±0.59) ％,(7.724±0.780) pg/ml,(148.412±7.355) pg/ml,(55.668±7.532) pg/ml] were significantly higher than those in the initial treatment group [(0.67±0.21) ％,(5.929±1.342) pg/ml,(130.632±17.945) pg/ml,(51.681±9.808) pg/ml] and the CHOP-CR group [(1.07±0.37) ％,(6.526±0.538) pg/ml,(132.119±7.700) pg/ml,(50.245±7.668) pg/ml] (P ＜ 0.05).The level of TGF-β in the RCHOP-CR group[(283.904±59.223) pg/ml] was significantly lower than that in the CHOP-CR group [(341.481±95.597) pg/ml] (P ＜ 0.05).Conclusion Th17 cells might be negatively correlated with the DLBCL development,the reduced IL-23 and elevated TGF-β might suppress the differentiation of Th17 cells.Rituximab could elevate the percentage of Th17 cells in DLBCL patients,and it is related with the effect of chemotherapy.
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Objective To explore clinical features of chemotherapy-induced graft-versus-host disease and to observe the dynamical changes of Th17/Treg ratio and related cytokines in one relapsed acute T-lymphoblastic leukemia after allo HSCT.Methods Twenty health volunteers were healthy controls.One patient achieved complete haematologic remission after two courses of chemotherapy and underwent matched HLA identical sibling allogenic peripheral blood stem cell transplantation,donor cell implanted state,disease recurrence and graft-versus-host disease were observed and Th17/Treg cells and its related factors in the peripheral blood were detected in different periods dynamically changes by methods of flow cytometry and ELISA.Results The patient achieved complete donor chimerism (FDC) at day +27 after transplantation.Hematopoietic function was fully recovered at day +34.Chronic GVHD (cGVHD) occurred at day +110.Thereafter,extramedullary relapse occurred in cGVHD state at day +264.After one course of Hyper-CVAD chemotherapy,patient complicated overlap syndrome with cGVHD of oral cavity ulcer and degree Ⅳ intestinal aGVHD at day +294 and day +347,respectively.Th17 cell ratio in CD+4 T cells (1.7 ％) in the overlap syndrome slightly increased,compared with control group [(0.56±0.35) ％].The ratio of Treg cells in CD+4 T cells (4.66 ％) with cGVHD increased compared with normal control group [(0.59±0.33) ％],recurrence (0.39 ％),and hematopoietic stem cell implantation (1.15 ％),but the ratio of Treg cells increased significantly when patient complicated overlap syndrome (7.83 ％).The ratios of Th17/Treg were more than 1 at relapse stage and less than 1 at GVHD stage.The IL-17A level in serum significantly increased in cGVHD (6.114 pg/ml)and overlap syndrome (6.805 pg/ml) stage compared with normal control group [(5.19±0.77) pg/ml].TGF-β1 levels were significantly higher at different periods after transplantation compared with control group [(48.81±4.9) ng/ml].Conclusion Chemotherapy can induce GVHD in relapsed acute leukemia patients after hematopoietic stem cell transplantation,and the imbalance of the Th17/Treg cells and its cytokines maybe related with disease relapse and GVHD.