scholarly journals Induction of CD4+CD25+ Regulatory T Cells from In Vitro Grown Human Mononuclear Cells by Sparteine Sulfate and Harpagoside

Biology ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 211 ◽  
Author(s):  
Nour Z. Atwany ◽  
Seyedeh-Khadijeh Hashemi ◽  
Manju Nidagodu Jayakumar ◽  
Mitzi Nagarkatti ◽  
Prakash Nagarkatti ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Mononuclear Cells ◽  
Cultured Cells ◽  
Inflammatory Responses ◽  
Human Peripheral Blood ◽  
Stimulation Index ◽  
Tgf Beta ◽  
Peripheral Blood Mononuclear

Regulatory T cells (Tregs) are key players in the regulation of inflammatory responses. In this study, two natural molecules, namely, sparteine sulfate (SS) and harpagoside (Harp), were investigated for their ability to induce Tregs in human peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from healthy volunteers and grown in the presence or absence of ConA, with TGF-beta, SS or Harp. Expression of the mRNA of FoxP3, TGF-beta, IL-10 and GAPDH was assessed via q-PCR. The expression of Treg markers including CD4, CD25, CD127 and FoxP3 was measured via flow cytometry. The secretion of IL-10 and TGF-beta by cultured cells was assessed by ELISA. Furthermore, the suppressive role of SS and Harp on PBMCs in vitro was tested via allogeneic mixed lymphocyte reaction (MLR). Data obtained show that both compounds increased the expression of FoxP3, TGF-beta and IL-10 mRNA in resting PBMCs but to a lesser extent in activated cells. Moreover, they significantly increased the percent of CD4+CD25+FoxP3+CD127− Tregs in activated and naïve PBMCs. Functionally, both compounds caused a significant reduction in the stimulation index in allogeneic MLR. Together, our data demonstrate for the first time that SS and Harp can induce human Tregs in vitro and therefore have great potential as anti-inflammatory agents.

High levels of thyroid hormone impair regulatory T cells function via reduced PD-1 expression

2021 ◽  
Author(s):  
Yi Zhong ◽  
Ting-Ting Lu ◽  
Xiao-Mei Liu ◽  
Bing-Li Liu ◽  
Yun Hu ◽  
...  
Keyword(s):  
T Cells ◽  
Thyroid Hormone ◽  
Regulatory T Cells ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Dependent Manner ◽  
Peripheral Blood Mononuclear

Abstract Context Regulatory T cells (Tregs) dysfunction plays an important role in the development and progression of Graves’ disease (GD). Programmed cell death 1 (PD-1) prompts FoxP3 in Tregs expression and enhances the suppressive activity of Tregs. Whether abnormal expression of PD-1 contributes to the breakdown of Tregs and the role of thyroid hormone in the PD-1 expression of Tregs in GD remain substantially undefined. Objective To evaluate the role of PD-1 in Tregs function and triiodothyronine (T3) in PD-1 expression in patients with GD and mice treated with T3. Methods We recruited 30 patients with GD and 30 healthy donors. PD-1 expression in Tregs and Tregs function were determined. To evaluate the effects of thyroid hormone on PD-1 expression in Tregs, we used T3 for the treatment of human peripheral blood mononuclear cells (PBMCs). We then treated mice with T3 to confirm the effect of thyroid hormone on PD-1 expression in Tregs and Tregs function in vivo. Results PD-1 expression in Tregs and the suppressive function of Tregs significantly decreased in patients with GD. T3 reduced PD-1 expression in human Tregs in a concentration- and time-dependent manner in vitro. High levels of circulating T3 reduced PD-1 expression in Tregs, impaired Tregs function, and disrupted T-helper cell (Th1 and Th2) balance in mice treated with T3. Conclusions Tregs dysfunction in GD patients might be due to down-regulation of PD-1 expression in Tregs induced by high levels of serum T3.

scholarly journals Rabbit ATG but not horse ATG promotes expansion of functional CD4+CD25highFOXP3+ regulatory T cells in vitro

Blood ◽  
2008 ◽  
Vol 111 (7) ◽  
pp. 3675-3683 ◽  
Author(s):  
Xingmin Feng ◽  
Sachiko Kajigaya ◽  
Elena E. Solomou ◽  
Keyvan Keyvanfar ◽  
Xiuli Xu ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Expression Patterns ◽  
Cell Number ◽  
Therapeutic Effects ◽  
Peripheral Blood Mononuclear ◽  
Activation Markers

Abstract Regulatory T cells (Treg) play important roles in suppressing immune responses and maintaining tolerance. Rabbit antithymocyte globulin (rATG) and horse ATG (hATG) are widely used in the treatment of immune-mediated syndromes, but their effects on Treg are unknown. We show here that in vitro culture of normal human peripheral blood mononuclear cells (PBMCs) with a low-dose rATG resulted in marked expansion of functional Treg by converting CD4+CD25− T cells to CD4+CD25+ T cells. hATG did not expand but rather decreased Treg. Immuno-blot showed increased expression of FOXP3 and NFAT1 in CD4+CD25− and CD4+CD25+ T cells exposed to rATG. PBMCs treated with rATG displayed increased interleukin-10 in culture supernatants than those treated with hATG. Furthermore, rATG and hATG showed differences in their potential to stimulate CD4+ T cells as examined using different activation markers. Microarray revealed that rATG induced markedly different gene-expression patterns in PBMCs, compared with hATG-treated or untreated PBMCs. Our findings indicate that rATG expanded Treg, probably through transcriptional regulation by enhanced NFAT1 expression, in turn conferring CD4+CD25− T cell FOXP3 expression and regulatory activity. The therapeutic effects of rATG may occur not only because of lymphocyte depletion but also enhanced Treg cell number and function.

Establishing a method for the expansion of human regulatory T cells with a high-efficiency in vitro

2020 ◽  
Vol 10 (5) ◽  
pp. 694-700
Author(s):  
Shuo Tao ◽  
Sujuan Feng ◽  
Bengfang Fan ◽  
Lihua Zhao ◽  
Poxuan Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Magnetic Beads ◽  
High Efficiency ◽  
Mixed Lymphocyte Reaction ◽  
Mononuclear Cells ◽  
Cultured Cells ◽  
Peripheral Blood Mononuclear ◽  
Expansion Ability

The objective of this study is to establish a method to isolate and culture regulatory T cells with a high-efficiency in vitro. The CD4+, CD25+, and CD127low T cells were isolated through fluorescence activated cells sorting and cultured in vitro with anti-CD3-anti-CD28-coated microbeads and IL-2 for two weeks. The number of cells was determined to evaluate the expansion ability, the phenotype of the expanded cells was identified with flow cytometry, and the suppressive function was examined by mixed lymphocyte reaction. The CD4+CD25+CD127low T cells were efficiently amplified in vitro using magnetic beads (Fe3O4). A mean expansion of (755.5 ± 213.5) fold was obtained after the cells were cultured for two weeks. In addition, the expressions of CD4, CD19, CD8, and CD25 in the expanded cells were estimated at (98.3 ± 1.04)%, (0.039 ± 0.021)%, (0.443 ± 0.239)%, and (97.6 ± 1.35)%, respectively. FOXP3+ cells accounted for (87.7 ± 5.5)% of the expanded cells, while Helios+ cells accounted for (73.3 ± 2.9)%. The cultured cells demonstrated the highest suppressive capacity with (84.39 ± 1.98)% suppression of the activated peripheral blood mononuclear cells at a responder: regulatory T cells (Treg) ratio of 1:1. This research suggests a method to obtain a large quantity of Tregs with a high-efficiency in vitro. The expanded cells remain in their original phenotype and demonstrate the ability of immune suppression, which plays enormous significance in the cell therapy for treating autoimmune diseases and transplant rejections.

scholarly journals Precision engineering of an anti-HLA-A2 chimeric antigen receptor in regulatory T cells for transplant immune tolerance

2021 ◽  
Author(s):  
Yannick D. Muller ◽  
Leonardo M.R. Ferreira ◽  
Emilie Ronin ◽  
Patrick Ho ◽  
Vinh Nguyen ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Chimeric Antigen Receptor ◽  
Mononuclear Cells ◽  
Antigen Receptor ◽  
Single Chain ◽  
Transplant Tolerance ◽  
Peripheral Blood Mononuclear

Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-zeta signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR+TCRdeficient human Tregs maintained both Treg phenotype and function in vitro. Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR+TCRdeficient Tregs did not impair the function of these HLA-A2+ islets, whereas similarly engineered A2-CAR+TCRdeficientCD4+ conventional T cells rejected the islets in less than 2 weeks. A2-CAR+TCRdeficient Tregs delayed graft-versus-host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance.

scholarly journals Anti-lymphocyte globulin stimulates normal human T cells to proliferate and to release lymphokines in vitro. A study at the clonal level

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 956-963
Author(s):  
GC Barbano ◽  
A Schenone ◽  
S Roncella ◽  
R Ghio ◽  
A Corcione ◽  
...  
Keyword(s):  
T Cells ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Interleukin 2 ◽  
Suppressor Cells ◽  
Peripheral Blood Mononuclear ◽  
Gm Csf ◽  
Recombinant Interleukin 2 ◽  
Macrophage Colony

Abstract Human peripheral blood mononuclear cells (PBMC) were stimulated in vitro with anti-lymphocyte globulin (ALG), and the phenotypic and functional properties of the blasts obtained were investigated. When stained with monoclonal antibodies (MoAbs), all of the blasts were identified as T cells that expressed predominantly the CD4 phenotype (70% of the cells). The remaining blasts were CD8+. These findings demonstrate that ALG stimulates both helper-inducer and cytotoxic- suppressor cells at random since the CD4 to CD8 ratio in the stimulated blasts was the same as in resting PBMC. This ratio is different from that observed in short-term cultures of T cells stimulated with phytohemagglutinin (PHA) under the same conditions (CD4 to CD8 ratio less than 1). ALG-stimulated T cells were cloned by limiting dilution in the presence of recombinant Interleukin-2 (rIL-2). The clones obtained were expanded and maintained in long term cultures with rIL-2. Thirty-two clones were tested for their capacity of producing colony stimulating activity (CSA) or burst promoting activity (BPA). Twenty- eight of them produced CSA and 12 produced BPA. No correlation was found between the surface phenotype and the ability of the clones to produce CSA or BPA (ie, both the CD4+ and CD8+ clones released the cytokines). When 16 of the same clones were tested for II-2 and gamma interferon (gamma IFN) production, 12 were found to be gamma INF and IL- 2 producers. All of the gamma IFN producers also released IL-2, whereas in the single clones no correlation was found with the capacity of releasing BPA and CSA. Supernatants from selected T-cell clones were also tested for hematopoietic growth factor activities in the presence of neutralizing antisera to human granulocyte-macrophage colony stimulating factor (GM-CSF) or to Interleukin-3 (IL-3). It was found that most CSA was attributable to GM-CSF, whereas BPA was mainly related to the presence of IL-3.

scholarly journals Efficient Therapeutic Function and Mechanisms of Human Polyclonal CD8+CD103+Foxp3+ Regulatory T Cells on Collagen-Induced Arthritis in Mice

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Juan Sun ◽  
Yiming Yang ◽  
Xiaona Huo ◽  
Beibei Zhu ◽  
Zhenhua Li ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
Regulatory T Cells ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Therapeutic Effects ◽  
Igg Antibody ◽  
Th1 Cell ◽  
Inflammatory Microenvironment

Objective. To investigate the potential therapeutic effect in a rheumatoid arthritis model of stable human CD8+ regulatory T cells (hCD8+Tregs) induced by TGF-β1 and rapamycin (RAPA) in vitro. Methods. Human CD8+T cells were isolated from human peripheral blood mononuclear cells and induced/expanded with TGF-β1 and RAPA along with anti-CD3/28 beads and IL-2 in vitro and harvested as hCD8+Tregs. The phenotypes, suppressive characteristics, and stability of the hCD8+Tregs in an inflammatory microenvironment were examined in vitro. Human CD8+Tregs were transfused into an acollagen-induced arthritis (CIA) mouse model, and their therapeutic effects and related mechanisms were investigated. Results. Human CD8+Tregs induced by TGF-β1/RAPA showed high expression of Foxp3 and CD103, exhibited vigorous suppression ability, and were stable in inflammatory microenvironments. In CIA mice, the clinical scores, levels of anti-collagen IgG antibody, and cartilage destruction were significantly reduced after adoptive transfusion with hCD8+Tregs. Moreover, hCD8+Treg treatment significantly reduced the number of Th17 cells, increased the number of CD4+IFN-γ+T cells, and produced self CD4+Foxp3+Tregs in vivo. In an in vitro cell coculture assay, hCD8+Tregs significantly inhibited mouse CD4+ effector T cell proliferation, induced mouse CD4+Foxp3+Treg and CD4+IFN-γ+Th1 cell production, reduced Th17 cell development, and downregulated CD80/86 expression on mature DCs (mDCs). Conclusion. TGF-β1/RAPA can induce hCD8+Tregs with stable suppressive characteristics, which could significantly alleviate the severity of CIA based on their stable suppressive ability in an inflammatory microenvironment and further influence the function of other downstream cell subtypes. Human CD8+Tregs might be a therapeutic strategy for rheumatoid arthritis.

scholarly journals Anti-lymphocyte globulin stimulates normal human T cells to proliferate and to release lymphokines in vitro. A study at the clonal level

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 956-963 ◽  
Author(s):  
GC Barbano ◽  
A Schenone ◽  
S Roncella ◽  
R Ghio ◽  
A Corcione ◽  
...  
Keyword(s):  
T Cells ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Interleukin 2 ◽  
Suppressor Cells ◽  
Peripheral Blood Mononuclear ◽  
Gm Csf ◽  
Recombinant Interleukin 2 ◽  
Macrophage Colony

Human peripheral blood mononuclear cells (PBMC) were stimulated in vitro with anti-lymphocyte globulin (ALG), and the phenotypic and functional properties of the blasts obtained were investigated. When stained with monoclonal antibodies (MoAbs), all of the blasts were identified as T cells that expressed predominantly the CD4 phenotype (70% of the cells). The remaining blasts were CD8+. These findings demonstrate that ALG stimulates both helper-inducer and cytotoxic- suppressor cells at random since the CD4 to CD8 ratio in the stimulated blasts was the same as in resting PBMC. This ratio is different from that observed in short-term cultures of T cells stimulated with phytohemagglutinin (PHA) under the same conditions (CD4 to CD8 ratio less than 1). ALG-stimulated T cells were cloned by limiting dilution in the presence of recombinant Interleukin-2 (rIL-2). The clones obtained were expanded and maintained in long term cultures with rIL-2. Thirty-two clones were tested for their capacity of producing colony stimulating activity (CSA) or burst promoting activity (BPA). Twenty- eight of them produced CSA and 12 produced BPA. No correlation was found between the surface phenotype and the ability of the clones to produce CSA or BPA (ie, both the CD4+ and CD8+ clones released the cytokines). When 16 of the same clones were tested for II-2 and gamma interferon (gamma IFN) production, 12 were found to be gamma INF and IL- 2 producers. All of the gamma IFN producers also released IL-2, whereas in the single clones no correlation was found with the capacity of releasing BPA and CSA. Supernatants from selected T-cell clones were also tested for hematopoietic growth factor activities in the presence of neutralizing antisera to human granulocyte-macrophage colony stimulating factor (GM-CSF) or to Interleukin-3 (IL-3). It was found that most CSA was attributable to GM-CSF, whereas BPA was mainly related to the presence of IL-3.

scholarly journals TLR7 and RIG-I dual-adjuvant loaded nanoparticles drive broadened and synergistic responses in dendritic cells in vitro and generate unique cellular immune responses in influenza vaccination

2020 ◽  
Author(s):  
Randall Toy ◽  
M. Cole Keenum ◽  
Pallab Pradhan ◽  
Katelynn Phang ◽  
Patrick Chen ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Immune Responses ◽  
Cellular Immunity ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Mouse Bone Marrow ◽  
Peripheral Blood Mononuclear ◽  
Flu Vaccination

ABSTRACTAlthough the existing flu vaccines elicit strong antigen-specific antibody responses, they fail to provide effective, long term protection – partly due to the absence of robust cellular memory immunity. We hypothesized that co-administration of combination adjuvants, mirroring the flu-virus related innate signaling pathways, could elicit strong cellular immunity. Here, we show that the small molecule adjuvant R848 and the RNA adjuvant PUUC, targeting endosomal TLR7s and cytoplasmic RLRs respectively, when delivered together in polymer nanoparticles (NP), elicits a broadened immune responses in mouse bone marrow-derived dendritic cells (mBMDCs) and a synergistic response in both mouse and human plasmacytoid dendritic cells (pDCs). In mBMDCs, NP-R848-PUUC induced both NF-κB and interferon signaling. Interferon responses to co-delivered R848 and PUUC were additive in human peripheral blood mononuclear cells (PBMCs) and synergistic in human FLT3-differentiated mBMDCs and CAL-1 pDCs. Vaccination with NPs loaded with H1N1 Flu antigen, R848, and PUUC increased percentage of CD8+ T-cells in the lungs, percentage of antigen-specific CD4+T-cells in the spleen, and enhanced overall cytokine-secreting T cell percentages upon antigen restimulation. Also in the spleen, T lymphopenia, especially after in vitro restimulation, was observed. Our results demonstrate that simultaneous engagement of TLR7 and RIG-I pathways using particulate carriers is a potential approach to improve cellular immunity in flu vaccination.GRAPHICAL ABSTRACT

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