The Role Of Voltage-gated Potassium Channel Kv1.3 Of T Cells In Aplastic Anemia

Aplastic anemia(AA) is a bone marrow failure disorder characterized by peripheral blood pancytopenia and bone marrow hypoplasia.Immune-mediated suppression of hematopoiesis is considered as the pivotal mechanism responsible for bone marrow failure in this disease.Based on the ability to express chemokine receptor CCR7 and the phosphatase CD45RA,T cells can be divided into na(l|¨)ve T cells,central memory T cells(T_CM cells),and effector memory T cells(T_EM cells).Oligoclonal T cells are described to expand in AA and detected to show mature memory/effector phenotypes, implicating memory T cells may participate in pathophysiologic process of AA.Potassium channels play an important role in T cell activation.Two lymphocyte potassium channels,the voltage-gated potassium channel Kv1.3 and the calcium-dependent potassium channel KCa3.1 could regulate the T cell activation by modulating membrane potential and Ca²⁺ signaling in T cells.In parallel with the differentiation from na(l|¨)ve into T_EM cells,the expression pattern of the lymphocyte potassium channels changes drastically.Kv1.3 channels are up-regulated in activated T_EM cells and become the dominant functional potassium channels.In contrast,in na(l|¨)ve T cells and T_CM cells,the number of Kv1.3 channels increases only modestly after activation,while KCa3.1 channels are up-regulated as the dominant functional potassium channels.Blocking Kv1.3 channels could inhibit the proliferation and function Of T_EM cells.In this study,we detected the frequency and function of na(l|¨)ve T cells,T_CM cells and T_EM cells in AA,investigated the expression of Kv1.3 channels in activated T cells from AA,evaluated the role of Kv1.3 channels in the activation of T cells and T-cell subsets from AA,and discussed the possibility of Kv1.3 channel as a therapeutic target for AA.Partâ… Frequency and function of na(l|¨)ve and memory T-cell subsets in aplastie anemiaObjective:To detect the frequency and function of na(l|¨)ve T cells,T_CM cells and T_EM cells in CD4⁺ and CD8⁺ T cells from bone marrow and peripheral blood of patients with AA.Methods:1.Peripheral blood mononuclear cells(PBMCs) and bone marrow mononuclear cells (BMMNCs) were separated by Ficoll-Hypaque density gradient centrifugation. The percentages of na(l|¨)ve T cells(CD45RA⁺CCR7⁺),T_CM cells(CD45RA^-CCR7⁺) and T_EM cells(CD45RA^-CCR7^-,CD8⁺ T cells also contain terminally differentiated CD45RA⁺CCR7^- T_EM cells) in CD4⁺ and CD8⁺ T cells in PBMCs and BMMNCs of patients with AA and controls were analyzed by flow cytometry.2.Isolated PBMCs and BMMNCs of patients with AA and controls were simulated with PMA and ionomycin in the presence of monesin for 5h.The expression of intracellular cytokine IFN-γin CD4⁺/CD8⁺ CCR7⁺ na(l|¨)ve/T_CM cells and CCR7^-T_EM cells were analyzed by flow cytometry.Results:1.Comparison of percentages of T-cell subsets in PBMCs and BMMNCs between patients with AA and controls:In the CD4⁺ T-cell population,significantly decreased percentages of na(l|¨)ve T cells were observed in PBMCs and BMMNCs of patients with AA compared with controls(p＜0.001).In contrast,the percentages of T_EM cells in PBMCs and BMMNCs were higher in patients with AA than in controls(p＜0.001).As far as CD8⁺ T-cell population was concerned, patients with AA still had decreased percentages of na(l|¨)ve T cells in PBMCs and BMMNCs compared with controls(p＜0.001).Meanwhile,both T_EM cells and terminal T_EM cells were increased in PBMCs and BMMNCs of patients with AA compared with controls(p＜0.001).There was no significant difference between patients with AA and controls in the percentages of CD4⁺ and CD8⁺ T_CM cells from PBMCs or BMMNCs.2.Comparison of percentages of T-cell subsets between PBMCs and BMMNCs: There were no significant differences of T-cell subsets in CD4⁺ T cells between PBMCs and BMMNCs in both patients with AA and controls.Concerning CD8⁺ T-cell population,significantly increased terminal T_EM cells and decreased na(l|)ve T cells were observed in BMMNCs compared with PBMCs in patients with AA (p=0.002,p＜0.001,respectively),but no difference was detected in controls.3.Comparison of percentages of T-cell subsets in BMMNCs and PBMCs between SAA and NSAA patients:In CD4⁺ and CD8⁺ T cell population of PBMCs and BMMNCs,no significant difference in percentages of na(l|¨)ve and memory T-cell subsets was detected between SAA and NSAA.4.The percentages of IFN-γ⁺ na(l|¨)ve and memory T-cell subsets in CD4⁺ and CD8⁺ T cells:In CD4⁺ and CD8⁺ T cells,the percentages of IFN-γ⁺CCR7^- T_EM cells from both PBMCs and BMMNCs were significantly higher than IFN-γ⁺CCR7⁺ na(l|¨)ve/T_CM cells in patients with AA as well as in controls(p＜0.001).5.Comparison of the expression of intracellular cytokine IFN-γin T-cell subsets between patients with AA and controls:Among total CD4⁺ or CD8⁺ T cells,the percentages of IFN-γ⁺CCR7^- T_EM cells were significantly higher from PBMCs and BMMNCs in patients with AA than that in controls(p＜0.001).The percentages of IFN-γproducers in CD4⁺CCR7^- T_EM cells from PBMCs or BMMNCs did not differ between patients with AA and controls;whereas the production of IFN-γin CD8⁺CCD7^- T_EM cells from both PBMCs and BMMNCs was significantly higher in patients with AA than controls(p＜0.001).Conclusions:CD4⁺ T_EM cells and CD8⁺ T_EM cells are increased in peripheral blood and bone marrow of AA,and CD8⁺ terminal T_EM cells are preferentially increased in bone marrow of AA.T_EM cells in AA have increased effector capacity compared with na(l|¨)ve/T_CM cells in AA and T_EM cells in controls.Increased T_EM cells,particularly in marrow of AA,may react as effector cells and participate in immune-mediated suppression of hematopoiesis in AA. Partâ…¡Expression of the voltage-gated potassium channel Kv1.3 in T cells of aplastic anemiaObjective:To investigate the expression of Kv1.3 channels in activated T cells from bone marrow of patients with AA.Methods:1.T cells were isolated from bone marrow of patients with AA and controls,and then stimulated for 72h with anti-CD3 antibody for T cell activation.2.Kv1.3 mRNA was detected in activated T cells of AA using real-time RT-PCR.3.Kv1.3 protein was detected in activated T cells of AA using western blot.4.The expression of Kv1.3 and CCR7 on activated T cells was detected by immunofluorescence microscopy.5.The percentages of na(l|¨)ve T cells,T_CM cells and T_EM cells in activated CD4⁺ and CD8⁺ T cells were analyzed by flow cytometry.Results:1.The expression of Kv1.3 mRNA in activated T cells was higher in patients with AA than in controls(p=0.001).2.The expresstion of Kv1.3 protein in activated T cells was higher in patients with AA than in controls(p=0.001).3.Immunofluorescence microscopy analysis revealed that activated T cells of patients with AA were mainly CCR7^- T_EM cells,having Kv1.3^high phenotype.In contrast,activated T cells of controls were mainly CCR7⁺ na(l|¨)ve/T_CM cells,and did not exhibit conspicuous membrane Kv1.3 staining.4.The percentages of T_EM cells in activated CD4⁺ T cells were higher in patients with AA than in controls(p＜0.001).The percentages of both T_EM cells and terminal T_EM cells in activated CD8⁺ T cells were higher in patients with AA than in controls(p＜0.001,p=0.001,respectively).Decreased percentages of na(l|¨)ve T cells were observed in activated CD4⁺ and CD8⁺ T cells of patients with AA compared with controls(p＜0.001).There was no significant difference between patients with AA and controls in the percentages of CD4⁺ and CD8⁺ T_CM cells. Conclusions:Kv1.3 expression is elevated at mRNA and protein levels in activated T cells from bone marrow of patients with AA.Activated T_EM cells in AA have Kv1.3^high phenotype.Higher Kv1.3 expression in the increased T_EM cells of AA causes the elevated Kv1.3 expression in activated T cells.Partâ…¢Effects of blocking Kv1.3 channels on proliferation and function of T cells and T-cell subsets in aplastic anemiaObjective:To evaluate the effects of blocking Kv1.3 channels on proliferation and function of T cells and T-cell subsets from bone marrow of patients with AA.Methods:1.T cells were isolated from bone marrow of patients with AA,and then stimulated by anti-CD3 antibody with ShK or without ShK for 72h.2.The effects of blocking Kv1.3 channels with ShK on T cell proliferation were measured by CCK-8 assay.Concentration of IFN-γand IL-4 in supernatant was measured by ELISA.3.CFSE staining was used to investigate the effects of blocking Kv1.3 channels with ShK on proliferation of different T-cell subsets.T cells were labelled with fluorochrome CFSE.After stimulated for 72h,the proliferation of CD4⁺/CD8⁺ CCR7⁺ na(l|¨)ve/T_CM cells and CCR7^- T_EM cells was analyzed by analyzing the dilution of CFSE signal in the FL1 channel by flow cytometry.4.Intracellular cytokine production staining was used to detect the effects of blocking Kv1.3 channels with ShK on function of different T-cell subsets.The expression of intracellular cytokine IFN-γ,and IL-4 in CD4⁺/CD8⁺ CCR7⁺ na(l|¨)ve/T_CM cells and CCR7^- T_EM cells was analyzed by flow cytometry.Results:1.As shown by CCK-8 analysis,the proliferation of T cells treated with ShK was significantly decreased in patients with AA compared with T cells stimulated by anti-CD3 without ShK(p＜0.001).2.The IFN-γproduction of T cells from patients with AA reduced significantly in the presence of ShK(p＜0.001).The suppression of IL-4 production induced by ShK was unremarkable in AA.3.CFSE^low cells represented the proliferating cells.In the presence of ShK,the percentages of CFSE^low cells reduced significantly in CD4⁺CCR7^- T_EM cells and CD8⁺CCR7^- T_EM cells of AA(p＜0.0001).But ShK exerted unremarkable inhibitory effects on the percentages of CFSE^low cells in CD4⁺CCR7⁺ na(l|¨)ve/T_CM cells and CD8⁺CCR7⁺ na(l|¨)ve/T_CM cells.4.IFN-γand IL-4-producing CD4⁺/CD8⁺ T cells were mainly CCR7^- T_EM cells. When T cells were stimulated by anti-CD3 in the presence of ShK,the percentages of IFN-γ⁺ cells in CD4⁺CCR7^- T_EM cells and CD8⁺CCR7^- T_EM was decreased significantly(p=0.002,p＜0.001,respectively).But ShK exhibited unremarkable inhibitory effects upon intercellular IFN-γexpression in CD4⁺CCR7⁺ na(l|¨)ve/T_CM cells and CD8⁺CCR7⁺ na(l|¨)ve/T_CM cells.Little intracellular IL-4 expressed in T cells from AA and the inhibition of ShK was unremarkable.Conclusions:Blocking Kv1.3 channels could inhibit the proliferation and function of T cells in bone marrow of AA.And inhibitory effects are mainly on the proliferation and type-1 response of T_EM cells.Kv1.3 channels are the dominant functional potassium channels of the activated T_EM cells in AA.Therapy targeting to Kv1.3 channels in T_EM cells may improve current immunosuppressive therapies for AA without influencing the whole immune system.