Effects of RNA Interference on CD70 in Dendritic Cells of Patients with Immune Thrombocytopenia

Abstract Objective: This study aims to assess the biological properties and functions of CD70 costimulatory molecule on dendritic cells (DCs) from patients with chronic immune thrombocytopenia (ITP). Methods: DCs were generated from monocytes isolated from peripheral blood, the immunophenotype markers were detected by flow cytometry. Chemically synthesized siRNA was then transferred into the cells by Lipofectamine 2000 to choose the most effective siRNA to block CD70 expression in three candidates. The mRNA expression and protein synthesis were analyzed by real-time RT-PCR and flow cytometry. The CD4+ T cells were co-cultured with DCs to assess the suppression capacity of DCs in the proliferation of CD4+ T cells and the production of IL-10 and IFN-γ by CD4+ T cells. The CD4+ T cells were co-cultured with DCs to assess the ability of DCs in the induction of Regulatory T cells (Tregs). Results: Dendritic cells were transfected with FITC-labelled scrambled siRNA for 4-6h. The transfection efficiency was average 60%. After transfection (48 h), the results of real-time RT-PCR analysis showed strong and specific downregulation of CD70 mRNA levels in transfected cells (p=0.046, siRNA1\2\3 Inhibition rates (74.5±6.1) %, (78.1±8.0) %, (90.1±2.5) % respectively, but not in negative control or in the absence of siRNA(p=0.157). The CD70-3 siRNA was extraordinarily effective in repressing the expression of CD70. The expression of CD70 on the DCs with siRNA3 was also significantly lower(p=0.043), but showed no difference in negative control (p=0.157). DCs from transfected group could inhibit the proliferation of PHA-activated CD4+ T cells than the negative control in ITP patients(p=0.008), but showed no difference in group of normal control (p=0.08). In chronic ITP patients and normal controls, the DCs from transfected group could inhibit the expression of IFN-γ compared with the group of negative control (p=0.018, p=0.043) and increase the expression of IL-10 (p=0.012, p=0.043). In chronic ITP patients and normal controls, the DCs from transfected group were defective in inducing the CD4+ T cell differentiating to CD4+CD25+CD127low Tregs compared to the negative control (p=0.012, p=0.043). Conclusions: In our study, siRNA is capable of inducing RNAi in DCs, it can knock down the CD70 gene expression specifically and effectively. The DCs from transfected group could inhibit the CD4+ T cells and induce the CD4+ T cells differentiating to Tregs. This approach is a useful tool by which costimulatory molecules of DC can be studied as well as a potential therapeutic option for autoimmune disease. Disclosures No relevant conflicts of interest to declare.

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Cytokine Production Profile of CD4+ T Cells From Patients with Immune Thrombocytopenia.

Blood ◽

10.1182/blood.v114.22.4460.4460 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4460-4460

Author(s):

Shanhua Zou ◽

Feng Li ◽

Weiguang Wang ◽

Yunfeng Cheng

Keyword(s):

T Cells ◽

Real Time ◽

Real Time Pcr ◽

Cd4 T Cells ◽

Healthy Subjects ◽

Immune Thrombocytopenia ◽

Plasma Concentrations ◽

Platelet Destruction ◽

Expression Levels ◽

Ifn Γ

Abstract Abstract 4460 Introduction To balance self-tolerance and immunity, the immune system depends on both activation mechanisms and down regulatory mechanisms. CD4+ T cells are important for the regulation of immune responses and they exert their effects through the secretion of cytokines. Immune thrombocytopenia(ITP) is a common hematologic disorder manifested by immune-mediated platelet destruction. ITP in adults often has a persistent course and frequently requires treatment to prevent bleeding. The mechanism of ITP has historically been attributed to platelet autoantibody production and the resultant platelet destruction. However, more recent evidence suggests that cell immunity pathogenesis plays an important role in ITP. Methods Real-time PCR assays was used for the quantification of mRNA of cytokines IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, IL-23, IL-17, IFN-γ, TNF-α and TGF-β in CD4+ T cells from adult ITP patients and healthy subjects. The mRNA of T-bet, GATA-3, RORγt, and FOXP3 were also analyzed using Real-time PCR. The plasma concentrations of IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, IL-23, IFN-γ, TNF and IL-17 were determined by enzyme-linked immunosorbent assay at the same time. Results The plasma concentrations of IL-2, IL-17 and IFN-γ in CD4+T cells were increased, while levels of IL-4, IL-6, IL-10 and TGF-β were decreased, comparing adult patients with ITP with healthy subjects. Patients with ITP presented significant increased mRNA expression levels on IL-2,IL-17,T-bet and RORγt. Conclusions In adult ITP patients, the expression levels of IL-2 and IL-17 were up-regulated, expression levels of TGF-β and IL-10 were down-regulated, suggesting that ITP is a Th1 and Th17 predominant disease although the precise mechanisms await further study. Disclosures: No relevant conflicts of interest to declare.

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Efficacy of Immunomodulatory Therapy with All-Trans Retinoid Acid for Adults with Chronic Immune Thrombocytopenia

Blood ◽

10.1182/blood.v120.21.3335.3335 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3335-3335

Author(s):

Lan Dai ◽

Zhaoyue Wang ◽

Ri Zhang ◽

Xia Bai ◽

Mingqing Zhu ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Peripheral Blood ◽

Immune Thrombocytopenia ◽

Pcr Analysis ◽

Therapeutic Effects ◽

Rt Pcr ◽

Platelet Destruction ◽

Prednisone Treatment ◽

Ifn Γ

Abstract Abstract 3335 Objective: Immune thrombocytopenia (ITP) is an immune-mediated disorder characterized by antibody- and cell-mediated platelet destruction and impaired platelet production. In addition of antibody-mediated platelet clearance, T-cell is also involved in platelet destruction or marrow suppression. During immune responses CD4+ T cells can differentiate into a range of cell types, including T helper type 1 (Th1), Th2, Th17 and regulatory T cells (Treg). The goal of the present study is to investigate the therapeutic effects of all-trans-retinoic acid (ATRA) plus prednisone treatment on adult refractory idiopathic thrombocytopenic purpura (RITP) and to further explore the underlying mechanisms. Methods: All 35 patients were treated with ATRA at fixed dose of 10 mg/tid plus prednisone and were started after discontinuation of other previous ITP treatment. Peripheral blood samples were collected from 35 RITP patients and 20 healthy subjects. The concentrations of the peripheral blood CD 4+ T cells (Th1, Th2, TH17, TREG) were analyzed by flow cytometry, the levels of cytokines were confirmed by enzyme-linked immunoassay (ELISA), and the expression of T-bet, GATA-3, RORγt, FOXP3 were detected by semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) in 35 RITP patients before and after treatments, with 20 normal individuals as respective controls. Results: The overall response rate of ATRA plus prednisone treatment in RITP patients was 54.3%, 28.6%(n=10) of patients with a complete response and 25.7%(n=9) of patients with a partial response. Among the 19 responders, the mean platelet count was 34+13×106/ml before ATRA therapy and 106+29×106/ml after treatment. No severe adverse effects happened. The levels of regulory T cells were significantly increased in patients after ATRA plus prednisone treatment (P < 0.05); the levels of Th1, Th2, Th17 were not significantly improved (P >0.05) in effective patients after treatments in flow cytometry analysis. The concentrations of IL-10, TGF-β was increased (P<0.05), whereas IFN-γ, IL-4, IL-17 factor showed no obvious change in the effective groups after treatment (P > 0.05). The expression levels of Foxp3 enhanced dramatically after treatment in real-time RT-PCR analysis (P < 0.05). However, the concentrations of T-bet, GATA-3 and ROR-γ showed no obvious change after the therapy in real-time RT-PCR analysis(P > 0.05). Conclusions: 54.3% of RITP patients recovered after ATRA plus prednisone treatments. The therapeutic effects of ATRA plus prednisone may be involved in the increased levels of regulory T cells in peripheral blood through increased expression of TGF-β, IL-10, and Foxp3. In comparison, the therapeutic effects may not be attributed to the expression of TH1, TH2, TH17, IFN-γ, IL-4, IL-17, T-bet, GATA-3, RORγt. Disclosures: No relevant conflicts of interest to declare.

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Splenic Stroma-Educated Regulatory Dendritic Cells Induce Apoptosis of Activated CD4 T Cells via Fas Ligand-Enhanced IFN-γ and Nitric Oxide

The Journal of Immunology ◽

10.4049/jimmunol.1101696 ◽

2011 ◽

Vol 188 (3) ◽

pp. 1168-1177 ◽

Cited By ~ 14

Author(s):

Xiongfei Xu ◽

Hai Yi ◽

Zhenhong Guo ◽

Cheng Qian ◽

Sheng Xia ◽

...

Keyword(s):

Nitric Oxide ◽

T Cells ◽

Dendritic Cells ◽

Cd4 T Cells ◽

Fas Ligand ◽

Ifn Γ ◽

Regulatory Dendritic Cells

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Human Dendritic Cells Stimulated via TLR7 and/or TLR8 Induce the Sequential Production of Il-10, IFN-γ, and IL-17A by Naive CD4+ T Cells

The Journal of Immunology ◽

10.4049/jimmunol.0801969 ◽

2009 ◽

Vol 182 (6) ◽

pp. 3372-3379 ◽

Cited By ~ 74

Author(s):

Vincent Lombardi ◽

Laurence Van Overtvelt ◽

Stéphane Horiot ◽

Philippe Moingeon

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Cd4 T Cells ◽

Ifn Γ ◽

Human Dendritic Cells

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Sunitinib Exerts In Vitro Immunomodulatory Activity on Sarcomas via Dendritic Cells and Synergizes With PD-1 Blockade

Frontiers in Immunology ◽

10.3389/fimmu.2021.577766 ◽

2021 ◽

Vol 12 ◽

Author(s):

Darina Ocadlikova ◽

Mariangela Lecciso ◽

Javier Martin Broto ◽

Katia Scotlandi ◽

Michele Cavo ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Cell Lines ◽

Effector T Cells ◽

Sarcoma Cell ◽

Ifn Γ ◽

Sarcoma Cells

BackgroundHigh-grade sarcomas are a heterogeneous group of aggressive tumors arising in bone and soft tissues. After relapse, treatment options are limited. The multi-targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and inhibitor of PD-1 (anti-PD-1) nivolumab have shown antitumor activity in selected subtypes. In this study, we examine the role of TKIs and PD-1 based therapy in in vitro cocultures of sarcoma.MethodsThe human osteosarcoma (SaOS-2) and synovial sarcoma (SYO-1) cell lines were treated with sunitinib. After cell death and proliferation assessment, expression of PD-L1 was analyzed by flow cytometry. Sunitinib-treated sarcoma cells were cocultured with dendritic cells (DCs), and the phenotype of mature DCs was determined by flow cytometry. Mature DCs were cultured with autologous T cells. PD-1 expression on T cells, their proliferation, T regulatory cell (Tregs) induction and IFN-γ production, before and after nivolumab exposure, were analyzed.ResultsAlong with its anti-proliferative and direct pro-apoptotic effect on sarcoma cell lines, sunitinib prompted PD-L1 upregulation on sarcoma cells. Interestingly, sunitinib-treated sarcoma cells drive DCs to full maturation and increase their capacity to induce sarcoma-reactive T cells to produce IFN-γ. Conversely, no effect on T cell proliferation and T cell subpopulation composition was observed. Moreover, both bone and synovial sarcoma cell lines induced Tregs through DCs but sunitinib treatment completely abrogated Treg induction. Finally, sarcoma cell lines induced PD-1 upregulation on both effector T cells and Tregs when loaded into DCs, providing a rationale for using PD-1 blockade. Indeed, PD-1 blockade by nivolumab synergized with sunitinib in inducing IFN-γ-producing effector T cells.ConclusionsTaken together, our in vitro data indicate that the treatment of sarcoma cells with sunitinib can exert significant changes on immune cell subsets toward immune activation, leading to DC-based cross-priming of IFN-γ-producing effector T cells and reduced Treg induction. PD-1 blockade with nivolumab has a synergistic effect with sunitinib, supporting the use of TKI and anti-PD-1 approach in sarcomas, and perhaps in other cancers. DC-targeted drugs, including toll-like receptor 3 inhibitors and CD47 inhibitors, are under development and our preclinical model might help to better design their clinical application.

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Abstract 001: Axl Expression by CD4+ T Lymphocytes Promotes Salt-Dependent Hypertension

Hypertension ◽

10.1161/hyp.64.suppl_1.001 ◽

2014 ◽

Vol 64 (suppl_1) ◽

Author(s):

Kristine M Wadosky ◽

Sri N Batchu ◽

Angie Hughson ◽

Kathy Donlon ◽

Craig N Morrell ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Cd4 T Cells ◽

White Blood Cells ◽

Interleukin 4 ◽

Th2 Cells ◽

Salt Hypertension ◽

Ifn Γ

Introduction: Our laboratory has shown that Axl, a receptor tyrosine kinase, is important in both vascular and immune functions during deoxycorticosterone acetate (DOCA)-salt hypertension. We hypothesized that Axl activity specifically in T lymphocytes could explain the dependence of hypertension on Axl. Methods and Results: We did adoptive transfers of either Axl+/+ or Axl-/- CD4+ T cells to RAG1-/- mice that lack mature T cells. Once CD4+ T cell repopulations were confirmed, we induced DOCA-salt hypertension for 6 weeks. Systolic blood pressure (BP, mmHg) increased by 20±5 in Axl+/+RAG-/- mice after DOCA-salt, but Axl-/- RAG-/- mice had increases in BP by only 6+3 after 6 weeks of DOCA-salt. We isolated naïve CD4+ T cells from both Axl+/+ and Axl-/- littermates and primed them under either Th1 or Th2 polarizing conditions in culture. Production of interferon gamma (IFN-γ ng/mL) was significantly decreased (-23%, p<0.05) in Axl-/- (396±23) compared to Axl+/+ (512±42) under Th1-priming. However, Axl had no effect on interleukin 4 (IL-4, ng/mL) production under Th2 polarizing conditions. Intracellular staining of the Th1/Th2 cells with IFN-γ and IL-4 antibodies by flow cytometry confirmed expression of cytokines in culture media. Complete blood counts showed that Axl-/- mice had significantly lower white blood cells due to decreased numbers of lymphocytes (4.5±0.7x10 9 ) compared to Axl+/+ mice (7.8±0.7x10 9 ). We found a higher population of AnnexinV (marker of early apoptosis)-positive peripheral leukocytes in Axl-/- mice (10±1%) compared to Axl+/+ (4±1%) by flow cytometry; while the percentages of dead cells (~10%) were similar between Axl+/+ and Axl-/- mice. Conclusions: Altogether we show that expression of Axl by T cells drives salt-induced hypertension. The mechanism of Axl-dependent effects on T cells occurs via T-cell-dependent expression of the pro-inflammatory cytokine IFN-γ. In addition, Axl plays a role in inhibiting lymphocyte apoptosis in the circulation. Future work will focus on how Axl expression in T cells affects T cell-dependent vascular remodeling during hypertension.

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Transcription Factor BACH2 Inhibits AP1 Proteins JunB and FosL1 in Umbilical Cord Blood (UCB) CD4+ T-Cells.

Blood ◽

10.1182/blood.v108.11.1743.1743 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1743-1743

Author(s):

Mathew L. Lesniewski ◽

Laura R. Fanning ◽

Margeret Kozik ◽

Richard P. Weitzel ◽

Yeal Hegerfeldt ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Transcription Factor ◽

T Cell ◽

Cd4 T Cells ◽

Mrna Levels ◽

Rt Pcr ◽

Tnf Α ◽

Ifn Γ ◽

Sirna Knockdown

Abstract Introduction: Umbilical cord blood (UCB) CD4+ T-cells have been shown to express significant levels of BACH2 transcription factor protein compared to adult blood (AB) CD4+ T-cells. Previously, NFAT1 siRNA knockdown of UCB T-cells exhibited a significantly higher BACH2 mRNA expression, and IFN-γ, TNF-α. and CTLA-4 mRNA levels were significantly suppressed. BACH2, a member of the b-Zip family, has been shown to act as a heterodimer with the bZip protein MafK, as a transcriptional inhibitor via recruitment of a histone deacetylase class II complex (HDAC II) in differentiating B-cells, and neurons. Due to observed inverse expression of BACH2 and NFAT1 in UCB CD4+ T-cells, we hypothesized that BACH2 may regulate transcription factors known to bind with NFAT1 including AP-1 proteins JunB and FosL1. We tested this by siRNA knockdown of BACH2 in primary UCB-derived CD4+ T-cells. Key developmental transcription factors JUNB, FosL1, NFAT1 and downstream IFN-γ, and TNF-α were mRNA analyzed. Methods: UCB T-cells were purified using autoMACs system (Miltenyi). After overnight culture, T-cells were transfected with BACH2 siRNA (Dharmacon) using Amaxa Nucleofector system (Amaxa Inc). Both siRNA treated and control cells were incubated in media for 18 hours, and then stimulated using anti-CD3/anti-CD28 antibodies (BD BioScience). Aliquots of cells were collected at specified time points post-stimulation for protein and total RNA isolation. The relative change in mRNA levels for BACH2, JUNB, FosL1, IFN-γ, NFAT1, and TNF-α were determined by Lightcycler SybrGreen real time RT-PCR system (Roche). siRNA knockdown of BACH2 protein in transfected UCB T-cells was confirmed by western blot. Results: Real-time RT-PCR of BACH2 siRNA treated UCB CD4+ T-cells stimulated with anti-CD3/CD28 antibodies and analyzed after 6 hrs of stimulation showed a 4 log increase in FosL1 and NFAT1 mRNA, a 3 log increase in JunB mRNA, a 5 log increase in IFN-γ as compared to stimulated control UCB T-cells. TNF-α mRNA was decreased by 5 logs in BACH2 siRNA treated UCB T-cells as compared to control. CD3/CD28 stimulated untransfected UCB T-cells were previously shown to have decrease expression of NFAT1, JunB, FosL1, IFN-γ, and TNF-α, and in UCB T-cells compared to stimulated AB T-cells. Conclusions: BACH2 expression correlates with an inhibition of expression of AP1 transcription regulatory proteins in UCB T-cells during primary CD3/CD28 stimulation. The complete activation of the T-cell requires the activation of AP1 by CD28 pathway otherwise the antigen presenting cell signals the T-cell to enter anergy. In UCB CD4+ T-cells express BACH2, which acts as a transcriptional inhibitor of two critical AP1 genes, JUNB and FosL1, which mediate the CD28 co-stimulatory pathway. These results further suggests that expression of BACH2 in UCB T-cells may contribute to lower incidence of alloreactivity observed in leukemia patients receiving UCB stem cells compared to AB bone marrow stem cells and thus leads to low GVHD, and contribute to the weak Th1 response seen in stimulated UCB T-cells by reduced amounts of AP1 protein available for activating the T-cell.

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Study on Mechanism of Human Marrow Mesenchymal Stem Cell in Treating Patients with Aplastic Anemia.

Blood ◽

10.1182/blood.v110.11.4099.4099 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4099-4099

Author(s):

Zhenhua Qiao ◽

Xiujuan Zhao

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Aplastic Anemia ◽

Hematopoietic Cell ◽

Control Group ◽

Rt Pcr ◽

Tnf Α ◽

Ifn Γ ◽

Tgf Β1

Abstract Objective: To explore mechanism of human marrow mesenchymal stem cells (MSCs) in treating patients with aplastic anemia(AA). Methods: MSCs in patients with aplastic anemia(AA) and the control group were separated with Percoll(1.073g/m L) and cultured in low glucose DMEM. Then, observed their morphologies,checked their molecule surface antigen by flow cytometry and examined the process of adipogenic differention. The mononuclear cells (MNC)of marrow in patients with AA were enriched based 1.077g/L density centrifuge and cultured in the 1640 medium. (1)MSC in control group and MNC in AA group were co-cultured with or without cytokines. The function of supporting hematopoiesis for MSC was to be observed in single confluence layer after plating by counting the total cells and the clones in every well every week. Then analyzed the dynamics of proliferation. T cells were harvested by using nylon column. MSC in control group and T cells in AA group were co-cultured. The proliferation of T cell was measured by MTT method. The CD25,CD69,CD4,CD8,Annexin-V expression rates of CD3+T cells were analyzed by flow cytometry .The gene and protein of IL-2, IL-4,IL-10,TNF-α,IFN-γ,TGF-β1 were examined by RT-PCR and ELISA respectively. MSC treated to the model of AA, by the examination of peripheral hemogram, bone marrow biopsy, pathological section of spleen. Results: There was no significant difference between control group MSC and AA-MSC in morphologies but adipogenic differentiation in AA patients is earlier than controls. The clones of CFU-GM in group(MSC)(78.46±3.58)/2×105 cells, after 14 days cultured was significantly higher than(9.21±4.32)/2×105 cells in group(CK + DMEM medium), while lower than (99.32±4.34)/2×105 cells in group(MSC+CK). (1)the Treg cells (TCD4+CD25+) in AA group (2.01±1.21)/ 2×105 was significantly lower than (4.43±1.67)/2×105 cells in control group, while(5.43±2.31) / 2×105 in group (MSC+AAT) was no more than (4.43±1.67)/2×105 cells in control group. (2) MSCs significantly inhibited T cell proliferation (P< 0. O5)by MTT. (3) RT-PCR and ELISA analysis showed that MSCs induced the expression of IL-4, IL-10, TGF-β1 and decreased significantly the expression of IL-2, TNF-α, IFN -γ in T cells of AA. the model of AA treated by MSCs showed improvements in 3 blood components greatly(p<0.05), Bone marrow proliferated and restored to the normal level, hematopoietic cell increased obviously (hematopoietic cell capacity was more than 40%), and atrophied spleen restore to normality. Conclusions: morphologies of AA’MSC had no evident different with the control but was more easy adipogenic differention. aplastic anemia belongs to autoimmune diseases in which T cells effect organ-specific destruction. The fundamental mechanism of MSC in treating AA should be potential to promote hematopoietic cell proliferation by adjusting immunity.

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