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

Blood ◽  
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.

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

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1373-1373
Author(s):  
Zeping Zhou ◽  
Yunlong Wang ◽  
Renchi Yang ◽  
Huiyuan Li
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Dendritic Cells ◽  
Real Time ◽  
Cd4 T Cells ◽  
Immune Thrombocytopenia ◽  
Negative Control ◽  
Rt Pcr ◽  
Ifn Γ ◽  
Normal Controls

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.

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

Blood ◽  
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.

scholarly journals CD137 Agonist Induces Gastric Cancer Cell Apoptosis by Enhancing The Functions of CD8+ T Cells via NF-κB Signaling

2020 ◽  
Author(s):  
Ben-Shun Hu ◽  
Tian Tang ◽  
Tie-Long Wu ◽  
Ying-Yue Sheng ◽  
Yu-Zheng Xue
Keyword(s):  
Gastric Cancer ◽  
Flow Cytometry ◽  
T Cells ◽  
Real Time ◽  
Peripheral Blood ◽  
Cell Apoptosis ◽  
Nuclear Translocation ◽  
Granzyme B ◽  
Ifn Γ

Abstract Background: CD137 is identified as a target for tumor immunotherapy. However, the role of CD137 in gastric cancer (GC), especially in inducing GC cell apoptosis has not been studied yet. Methods: Foxp3+ and CD8+ T cells in GCs were investigated by immunohistochemistry (IHC). CD137 expression in GCs was detected by flow cytometry, IHC and immunofluorescence (IF). Peripheral blood mononuclear cells (PBMCs) and CD8+ T cells isolated from peripheral blood were stimulated with a CD137 agonist in vitro. CD8+ T cells proliferation and p65 expression were explored by flow cytometry. p65 nuclear translocation was analyzed by IF. IL-10, TGF-β, IFN-γ, Perforin and Granzyme B were detected by real-time quantitative PCR (real-time PCR). PBMCs and primary GC cells were cocultured and stimulated with the CD137 agonist in vitro. Apoptosis of the primary GC cells was detected by flow cytometry. Results: Our data demonstrated that GC tumors show characteristics of an immunosuppressive microenvironment. CD137 was predominantly expressed in CD8+ T cells in GCs and had a positive correlation with tumor cell differentiation. CD137 agonist promoted CD8+ T cells proliferation and increased the secretion of IFN-γ, Perforin and Granzyme B, which induced primary GC cell apoptosis. Mechanistically, this study found that CD137 agonist could induce NF-κB nuclear translocation in CD8+ T cells. Conclusion: Our results demonstrate that CD137 agonist can induce primary GC cell apoptosis by enhancing CD8+ T cells via activating NF-κB signaling.

LncRNA-ENST00000421645 promotes T cells to secrete IFN-γ by sponging PCM-1 in neurosyphilis

Epigenomics ◽  
2021 ◽  
Author(s):  
Wen-Na Liu ◽  
Kai-Xuan Wu ◽  
Xiao-Tong Wang ◽  
Li-Rong Lin ◽  
Man-Li Tong ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Peripheral Blood ◽  
Long Noncoding Rna ◽  
Noncoding Rna ◽  
Chip Assay ◽  
Rna Immunoprecipitation ◽  
Ifn Γ ◽  
Significant Expression

Aim: Neurosyphilis patients exhibited significant expression of long noncoding RNA (lncRNA) in peripheral blood T lymphocytes. In this study, we further clarified the role of lncRNA- ENST00000421645 in the pathogenic mechanism of neurosyphilis. Methods: lncRNA- ENST00000421645 was transfected into Jurkat-E6-1 cells, namely lentivirus (Lv)-1645 cells. RNA pull-down assay, flow cytometry, RT-qPCR, ELISA (Neobioscience Technology Co Ltd, Shenzhen, China) and RNA immunoprecipitation chip assay were used to analyze the function of lncRNA- ENST00000421645. Results: The expression of IFN-γ in Lv-1645 cells was significantly increased compared to that in Jurkat-E6-1 cells stimulated by phorbol-12-myristate-13-acetate (PMA). Then, it was suggested that lncRNA- ENST00000421645 interacts with PCM1 protein. Silencing PCM1 significantly increased the level of IFN-γ in Lv-1645 cells stimulated by PMA. Conclusion: This study revealed that lncRNA- ENST00000421645 mediates the production of IFN-γ by sponging PCM1 protein after PMA stimulation.

PBMC-06- Intracellular Cytokine Staining (ICS) of IFN-gamma, IL-4 and IL-17 on Human Peripheral Blood Mononuclear Cells (PBMC) v1

2021 ◽  
Author(s):  
Alessia Furgiuele ◽  
Emanuela Rasini ◽  
Maria Giulia Albizzati ◽  
Alessandra Luini ◽  
Marco Ferrari ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Brefeldin A ◽  
Intracellular Cytokine ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Ifn Γ

This present protocol is developed to analyze the frequency of IFN-γ-, IL-4- and IL-17-producing CD4+T cells, identified from ex vivo human peripheral blood mononuclear cells (PBMC). The frequencies of cytokine producing cells derived from activation of PBMC was induced trough the stimulus phorbol 12-myristate 13-acetate (PMA) and ionomycin. According onpreviously published protocols concentrations of stimulating substances were in the range from 10, to 50 ng/ml for PMA and 1 µg/ml for ionomycin (Gupta and Maecker, 2015; Foster et al., 2007; Freer and Rindi, 2013; https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.in.560751.pdf). The PMA concentrations of 10, 20 and 50 ng/ml were tested and finally the PMA concentration of 10 ng/ml was chosen since it was sufficient to obtain a frequency of cytokines comparable to that obtained with higher stimulus concentrations. PMA/ionomycin and brefeldin A are incubate together for a time of 5 h (Gupta and Maecker, 2015, Foster et al., 2007, Freer and Rindi, 2013, https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.in.560751.pdf). The protein secretion inhibitor brefeldin A, was used at the concentration of 10 µg/ml (Gupta and Maecker, 2015; Foster et al., 2007; Freer and Rindi, 2013). Cell concentrations may vary in a range from 2.5 x106 to 10 x106 cells/ml (Maecker, 2004; Freer and Rindi, 2013a; https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.in.560751.pdf). Concentration of 1x106 cells/ml, 4x106 cells/ml and 8x106cells/ml were tested. Cell tritation have shown a higher functional response proportional to the cell concentration when exposed to a fixed concentration of stimulants. Cell concentration of 8 milions/ml was selected in order to obtain the higher percentage of IFN-γ-, IL-4- and IL-17-producing CD4+T cells. In conclusion the present protocol provides that, for a optimal optimal percentage of IFN-γ-, IL-4- and IL-17-producing CD4+T cells as assessed by flow cytometry (Table 1), PBMC in a concentration 8 milions/ml were stimulated with PMA 10 ng/ml and ionomycin 1 µg/ml, and cultured for 5 h in presence of brefeldin A 10 µg/ml according to the procedure described in detail below. References Baran, J., Kowalczyk, D., Ozog, M., Zembala, M., 2001. Three-color flow cytometry detection of intracellular cytokines in peripheral blood mononuclear cells: Comparative analysis of phorbol myristate acetate-ionomycin and phytohemagglutinin stimulation. Clin. Diagn. Lab. Immunol. 8, 303–313. https://doi.org/10.1128/CDLI.8.2.303-313.2001 Foster, B., Prussin, C., Liu, F., Whitmire, J.K., Whitton, J.L., 2007. Detection of intracellular cytokines by flow cytometry. Curr. Protoc. Immunol. Chapter 6. https://doi.org/10.1002/0471142735.im0624s78 Freer, G., Rindi, L., 2013. Intracellular cytokine detection by fluorescence-activated flow cytometry: Basic principles and recent advances. Methods 61, 30–38. https://doi.org/10.1016/j.ymeth.2013.03.035 Gupta, S., Maecker, H., 2015. Intracellular Cytokine Staining (ICS) on Human Lymphocytes or Peripheral Blood Mononuclear Cells (PBMCs). BIO-PROTOCOL 5. https://doi.org/10.21769/bioprotoc.1442 Maecker, H.T., 2004. Cytokine flow cytometry. Methods Mol. Biol. 263, 95–108. https://doi.org/10.1385/1-59259-773-4:095 https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.us.560751.pdf BEFORE STARTING with this procedure Moreover, work under laminar flow hood when you are processing samples from the beginning to the end of the culture. Make sure you are using, sterile culture medium and sterile plastic disposable as well.

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

Blood ◽  
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.

scholarly journals Insufficient Phosphorylation of STAT5 in Tregs Inhibits the Expression of BLIMP-1, Leading to a Lack of Tregs in Pediatric Aplastic Anemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2183-2183
Author(s):  
Lifen Huang ◽  
Junbin Huang ◽  
Chengming Zhu ◽  
Hongman Xue ◽  
Chi Kong Li ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Aplastic Anemia ◽  
Inflammatory Cytokines ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Healthy Controls ◽  
Healthy Donors ◽  
Ifn Γ ◽  
Low Expression

Abstract Aplastic anemia (AA) is a group of bone marrow failure diseases characterized by three-line blood cell reduction and decreased myeloproliferation. It is believed that T cell immune disorder is the leading cause of the disease, especially the number and functional damage of regulatory T cells (Tregs). BLIMP-1 is a transcription factor encoded by PRDM1 gene, which is indispensable for Tregs. The expression of BLIMP-1 is mainly induced by the IL-2/STAT5 signaling pathway. However, the level of phosphorylation of STAT5 and the expression of BLIMP-1 in Tregs from patients with AA has not been revealed, and the mechanism of Tregs damage in AA has not yet been clarified. In the present study, we collected peripheral blood from 10 newly diagnosed AA children and 10 age-matched healthy donors. We observed that the ratio of Tregs/lymphocytes and Tregs/CD4 + T cells decreased significantly in AA patients, compared with healthy controls by flow cytometry. In addition, we found significantly elevated levels of inflammatory cytokines IL-2, IL-6, and IFN-γ, but decreased levels of anti-inflammatory cytokines IL-10 and TGF-β in plasma of children with AA, compared with healthy controls. Quantitative real-time PCR showed decreased transcriptional level of BLIMP-1 in peripheral blood mononuclear cells (PBMC) from children with AA, compared with healthy donors. We used flow cytometry to detect the protein level of BLIMP-1 in Tregs and found that the level of BLIMP-1 in Tregs in the peripheral blood of children with AA was significantly lower than that of healthy donors. The correlation analysis showed that the percentage of BLIMP-1 + Tregs was positively correlated with the ratio of Tregs/CD4 + T cells (r=0.829, p＜0.001), the plasma level of IL-10 (r=0.492, p=0.027), and TGF-β (r=0.482, p=0.030), suggesting that low expression level of BLIMP-1 in Tregs may lead to decreased number of Tregs in peripheral blood and declined levels of IL-10 and TGF-β in children with AA. When stimulated IL-2, the level of pSTAT5 in CD4 + T cells of children with AA was significantly reduced compared with that of healthy donors. The level of pSTAT5 in CD4 + T cells was also positively correlated with the ratio of Tregs/CD4 + T cells (r= 0.575, p= 0.008) and the expression of BLIMP-1 in Tregs (r=0.693, p＜0.0001)，suggesting that STAT5 signal is poorly activated in pediatric AA, and it may be an important cause for the low expression of BLIMP-1 in Tregs and the decrease in the number of Tregs in children with AA. Furthermore, we constructed an AA mouse model by co-administering IFN-γ and busulfan for 10 consecutive days. These mice exhibited decreased ratio of Tregs/CD4 +T cells in the spleen and lower BLIMP-1 in Tregs, compared with controls. Also, we isolated Tregs with immunomagnetic beads from spleens of mice and observed that the level of IL-2-stimulated pSTAT5 was lower in isolated Tregs from AA mice than controls. These phenotypes were partially rescued by intervention of IL-2-JES6-1, an antibody complex tends to promote the proliferation of Tregs in mice, while inhibiting the proliferation of effector T cells. IL-2-JES6-1 increased the level of pSTAT5 and the expression of BLIMP-1 in Tregs from spleen of the AA mice, and elevated the ratio of Tregs/CD4 + T cells in the spleen. In conclusion, we found that Tregs from AA patients have impaired phosphorylation of STAT5 and insufficient expression of BLIMP-1, which may contribute to the pathogenesis of AA. Disclosures No relevant conflicts of interest to declare.

The Immune Regulatory Functions Of Circulating CD5+ B Cells Are Impaired In Adult Patients With Primary Immune Thrombocytopenia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3526-3526
Author(s):  
Fanli Hua ◽  
Feng Li ◽  
Shanhua Zou ◽  
Weiguang Wang ◽  
Yunfeng Cheng
Keyword(s):  
Cell Culture ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Healthy Subjects ◽  
Immune Thrombocytopenia ◽  
Ifn Γ ◽  
Regulatory Functions

Abstract Background and Objective Immune thrombocytopenia (ITP) is a typical autoimmune-mediated bleeding disorder in which isolated thrombocytopenia is resulted from increased platelet clearance and/or impaired thrombocytogenesis. In addition to producing antibodies and acting as antigen presenting cells, B cells have been proven to play an important role in regulating immune response mainly via production of interleukin 10 (IL-10). CD5+ B cells are able to produce IL-10, and CD5 is deemed as a hallmark of regulatory B cells. We have previously demonstrated insufficient IL-10 secretion by CD5+ B cells in patients with ITP. The objective of the current study was to investigate the alteration in regulatory functions of peripheral blood CD5+B cells in ITP patients. Methods Peripheral blood CD5+ B cells, CD5- B cells and CD4+ T cells were magnetically isolated from 7 adult patients with newly diagnosed ITP and 10 healthy controls. CD4+ T cells were labeled with CFSE (5(6)-Carboxyfluorescein N-hydroxysuccinimidyl ester) before cultured in RPMI1640 in the presence of CD3mAb/CD3mAb/IL-2 for 72 hours with or without CD5+/CD5- B cells at gradient B/T ratios. The proliferation of CD4+ T cells was assessed by the dilution of CFSE expression on a flow cytometry. After 72-hour culture, cells were stained with APC-CD4/FITC-Annexin-V/PI to evaluate the apoptosis of CD4+T cells. The concentrations of IL-4 and IFN-γ in cell culture supernatants were determined by ELISA. Results Purified CD5+ B cells from healthy subjects were capable of suppressing the proliferation of CD4+ T cells in a dose-dependent manner when co-cultured for 72 hours. The percentage of CD4+ T cells that underwent proliferation showed a statistically significant reduction (72.8 ± 9.4% vs 61.6 ± 12.8%; p = 0.039) in the presence of CD5+ B cells at 1:3 B/T ratio. With the B/T ratio increased to 1:1, the proportion of proliferating CD4+ T cells decreased to 43.7 ± 16.4% (p < 0.001). The apoptosis of CD4+ T cells was promoted by CD5+ B cells as the percentage of apoptotic CD4+ T cells was increased when co-cultured with CD5+ B cells at 1:1 B/T ratio (3.75 ± 0.82% vs 6.03 ± 2.51%; p = 0.014). In contrast, CD5- B cells did not suppress the proliferation of CD4+ T cells, quite contrary, the proportion of proliferating CD4+ T cells was slightly increased, though not statistically significant, when co-cultured at 1:1 with CD5- B cells for 72 hours as compared with CD4+ T cell cultured alone (72.8 ± 9.4% vs 78.9 ± 8.5%; p = 0.145). No effect of CD5- B cells on the apoptosis of CD4+T cells was observed. In ITP patients, CD5+ B cells showed compromised regulatory functions as the proliferation of CD4+ T cells was not altered by CD5+ B cells until the B/T ratio increased to 3:1 (1:1 B/T: 74.6 ± 12.5% vs 72.1 ± 16.2%, p = 0.752; 3:1 B/T: 74.6 ± 12.5% vs 60.7 ± 10.3%, p = 0.042, respectively). The ability of CD5+ B cells to promote the apoptosis of CD4+ T cells was also impaired in ITP patients: no difference in the percentage of apoptotic CD4+ T cells was found when CD5+ B cells (1:1 ratio of B to T) were added to the T cell culture system (2.97 ± 1.07% vs 3.24 ± 1.45%; p = 0.699). We also evaluated the ability of CD5+ B cells to suppress the secretion of IFN-γ, one of the Th1-type cytokines. In healthy subjects, the IFN-γ secretion by CD4+ T cells was decreased by CD5+ B cells at a minimal B/T ratio of 1:5 (2427.99 ± 632.62 pg/mL vs 1734.41 ± 507.16 pg/mL; p = 0.014) and dramatically dropped to 395.22 ± 136.54 pg/mL when the ratio of B/T increased to 1:1 (p < 0.001). The secretion of IL-4, a representative cytokine of Th2 subset, was not influenced by CD5+ B cells. CD5+ B cells from ITP patients showed no capacity for suppressing the IFN-γ secretion until the ratio of B/T increased as high as to 1:1 (3060.02 ± 493.98 pg/mL vs 2394.57 ± 417.42 pg/mL; p = 0.019) and, even under the condition of 5:1 B/T ratio, CD4+ T cells from ITP patients retained about 30% of the ability of IFN-γ secretion (1067.37 ± 499.70 pg/mL; p < 0.001 ). Conclusions CD5+ B cells from normal controls are competent for inhibiting the proliferation of CD4+ T cells and suppressing the secretion of IFN-γ by CD4+ T cells, suggesting potent immune regulatory functions of CD5+ B cells. While in patients with ITP, CD5+ B cells are severely functionally impaired, indicating that the immune regulatory dysfunctions in CD5+ B cells may participate in the pathogenesis of ITP. Disclosures: No relevant conflicts of interest to declare.

Evaluating the repertoire of immune checkpoint markers expressed on peripheral and ascites CD8+ T cells in ovarian cancer.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 5571-5571 ◽  
Author(s):  
Stephanie Gaillard ◽  
Chelsae Dumbauld ◽  
Alyssa Bilewski ◽  
Jessie A Ehrisman ◽  
Angeles Alvarez Secord ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Flow Cytometry ◽  
T Cells ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Peripheral Blood ◽  
Ascites Fluid ◽  
High Dimensional ◽  
Inhibitory Receptors ◽  
Ifn Γ

5571 Background: Understanding the immune checkpoint marker repertoire can facilitate development of therapeutic strategies to improve efficacy of immune-based therapies. We used a novel high-dimensional flow cytometry panel to determine co-expression patterns of immune checkpoint markers and effector function of CD8+ T cells from peripheral blood and ascites of patients newly diagnosed with ovarian cancer. Methods: Peripheral blood and ascites samples were collected from patients with epithelial ovarian cancer (n=8). Cells isolated from peripheral blood and ascites were used for immune profiling by multiparameter flow cytometry of 5 inhibitory receptors (PD-1, LAG-3, TIM-3, TIGIT, and BTLA) on CD8+ T cells, along with 4 functional parameters (production of each of the following: TNF-α, IFN-γ, IL-2, and upregulation of CD107a). A complementary multiplex analysis on plasma and ascites fluid was performed to quantify 14 soluble checkpoint markers. Results: The concentrations of soluble PD-1, TIM-3, LAG-3, CTLA-4, BTLA, IDO, and CD137 were increased in ascites fluid compared to plasma from patients with ovarian cancer. Ascites CD8+ T cells co-express higher levels of inhibitory receptors than peripheral CD8+ T cells. In total, CD8+ T cells in ascites retained the ability to produce effector functions at levels similar to peripheral blood. However, IFN-γ production was retained in PD-1 only expressing CD8+ T cells and decreased in CD8+ T cells co-expressing multiple receptors. Conclusions: High-dimensional flow cytometry allowed for the phenotypic and functional characterization of CD8+ T cells from ovarian cancer patients. The profile of receptor co-expression was distinct in peripheral blood compared to ascites. Collectively, our study suggests that co-expression of factors beyond PD-1 influences CD8+ T cell activity. Thus blocking PD-1 and PD-L1 alone may not be sufficient for CD8+ T cells expressing multiple inhibitory receptors.

scholarly journals Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

2021 ◽  
Vol 11 (13) ◽  
pp. 5776
Author(s):  
Varvara G. Blinova ◽  
Natalia S. Novachly ◽  
Sofya N. Gippius ◽  
Abdullah Hilal ◽  
Yulia A. Gladilina ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Inflammatory Reactions ◽  
Effector Cells ◽  
Cycle Regulation ◽  
Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

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