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Bio X Cell anti human cd3

(A) Gene expression levels of glucose transporters (Glut1 and Glut3) and the key enzymes in glycolysis (HK2, GPI, PFK1, TPI, ENO1, PKM2 and LDHα) in different T cell subsets. Th1, Th2 and Th17 cells were polarized from naïve T cells purified from healthy donors in the presence of related polarization cytokine conditions. nTreg cells were directly purified from PBMCs of healthy donors. Total RNA was isolated from each cell type and analyzed by real-time PCR. Expression levels of each gene were normalized to β-actin expression level and adjusted to the levels in naïve CD4+ T cells (served as 1). Data shown are mean ± SD from four independent donors. (B) Gene expression levels of key enzymes in cholesterol synthesis (HMGCR, HMGCS1, SQLE, and IDI1), as well as fatty acid oxidation (CPT-1) and synthesis (ACC1 and FASN) in different T cell subsets. Cell preparations and assays were identical to (A). (C) and (D) Tumor-derived CD4+ Treg and γδ Treg cells had higher gene expression levels of glucose transporters and the key enzymes in glycolysis (in C) and lipid metabolism (in D) than those of naïve CD4+ and Th1 cells. Tumor-derived Treg cells: CD4 TregE1 is a melanoma-specific Treg cells and γδ Treg31 & 76 cells are derived from TILs of breast cancer patients. Relative mRNA expression level of each gene was determined by real-time PCR, normalized to β-actin expression and then adjusted to the level in naïve CD4+ T cells. (E) nTreg cells produced higher amounts of the key metabolites involved in the glycolysis and tricarboxylic acid cycle than other T cell subsets. Th1, Th2, Th17 and nTreg cells were prepared as (A). The cell lysates from different T cell subsets were extracted and analyzed using a LCtriple quadruple mass spectrometry for determination of cellular glucose metabolites. Metabolite levels are normalized to naïve CD4 cell group. Relative levels of intermediate metabolites in the glycolysis and TCA-cycle pathways are shown as mean ± SD from representative of three independent T cell subsets with similar results. (F) and (G) Inhibition of glycolysis and lipid metabolism dramatically blocked Treg cell suppressive capacity on T cell proliferation (in F) and prevented Treg-induced responder T cell senescence (in G). nTreg cells were pretreated with pharmacological glucose transporter, glycolysis and lipid metabolism inhibitors for 48 hours, including phloretin (2 μM), 2-DG (1 mM), LND (125 μM), and 3BrPA (30 μM), etomoxir (100 μM), C75 (5 μM), orlistat (10 μM), 25-HC (0.25 μg/ml), simvastatin (2 μM), respectively. Naïve CD4+ T cells were then co-cultured with inhibitor-pretreated or untreated Treg cells for 3 days. Proliferation of co-cultured naïve T cells stimulated by <t>anti-CD3</t> antibody was determined by [3H]-thymidine incorporation assays, and SA-β-Gal expression in treated T cells was also determined. Data shown are mean ± SD from representative of three independent experiments with similar results.* p<0.05 and ** p<0.01, compared with the medium only group.

Anti Human Cd3, supplied by Bio X Cell, used in various techniques. Bioz Stars score: 95/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti human cd3 - by Bioz Stars, 2026-09

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1) Product Images from "TLR8-mediated metabolic control of human Treg function: a mechanistic target for cancer immunotherapy"

Article Title: TLR8-mediated metabolic control of human Treg function: a mechanistic target for cancer immunotherapy

Journal: Cell metabolism

doi: 10.1016/j.cmet.2018.09.020

Figure Legend Snippet: (A) Gene expression levels of glucose transporters (Glut1 and Glut3) and the key enzymes in glycolysis (HK2, GPI, PFK1, TPI, ENO1, PKM2 and LDHα) in different T cell subsets. Th1, Th2 and Th17 cells were polarized from naïve T cells purified from healthy donors in the presence of related polarization cytokine conditions. nTreg cells were directly purified from PBMCs of healthy donors. Total RNA was isolated from each cell type and analyzed by real-time PCR. Expression levels of each gene were normalized to β-actin expression level and adjusted to the levels in naïve CD4+ T cells (served as 1). Data shown are mean ± SD from four independent donors. (B) Gene expression levels of key enzymes in cholesterol synthesis (HMGCR, HMGCS1, SQLE, and IDI1), as well as fatty acid oxidation (CPT-1) and synthesis (ACC1 and FASN) in different T cell subsets. Cell preparations and assays were identical to (A). (C) and (D) Tumor-derived CD4+ Treg and γδ Treg cells had higher gene expression levels of glucose transporters and the key enzymes in glycolysis (in C) and lipid metabolism (in D) than those of naïve CD4+ and Th1 cells. Tumor-derived Treg cells: CD4 TregE1 is a melanoma-specific Treg cells and γδ Treg31 & 76 cells are derived from TILs of breast cancer patients. Relative mRNA expression level of each gene was determined by real-time PCR, normalized to β-actin expression and then adjusted to the level in naïve CD4+ T cells. (E) nTreg cells produced higher amounts of the key metabolites involved in the glycolysis and tricarboxylic acid cycle than other T cell subsets. Th1, Th2, Th17 and nTreg cells were prepared as (A). The cell lysates from different T cell subsets were extracted and analyzed using a LCtriple quadruple mass spectrometry for determination of cellular glucose metabolites. Metabolite levels are normalized to naïve CD4 cell group. Relative levels of intermediate metabolites in the glycolysis and TCA-cycle pathways are shown as mean ± SD from representative of three independent T cell subsets with similar results. (F) and (G) Inhibition of glycolysis and lipid metabolism dramatically blocked Treg cell suppressive capacity on T cell proliferation (in F) and prevented Treg-induced responder T cell senescence (in G). nTreg cells were pretreated with pharmacological glucose transporter, glycolysis and lipid metabolism inhibitors for 48 hours, including phloretin (2 μM), 2-DG (1 mM), LND (125 μM), and 3BrPA (30 μM), etomoxir (100 μM), C75 (5 μM), orlistat (10 μM), 25-HC (0.25 μg/ml), simvastatin (2 μM), respectively. Naïve CD4+ T cells were then co-cultured with inhibitor-pretreated or untreated Treg cells for 3 days. Proliferation of co-cultured naïve T cells stimulated by anti-CD3 antibody was determined by [3H]-thymidine incorporation assays, and SA-β-Gal expression in treated T cells was also determined. Data shown are mean ± SD from representative of three independent experiments with similar results.* p<0.05 and ** p<0.01, compared with the medium only group.

Techniques Used: Gene Expression, Purification, Isolation, Real-time Polymerase Chain Reaction, Expressing, Derivative Assay, Produced, Mass Spectrometry, Inhibition, Cell Culture

(A) Gene expression levels of glucose transporters and the key enzymes in glycolysis in different T cell subsets after stimulation with anti-CD3 antibody. Cell subset preparations and assays were identical to Figure 1. Total RNA was isolated from each cell type and analyzed by real-time PCR. Expression levels of each gene were normalized to β-actin expression levels and adjusted to the levels in naïve CD4 cells (served as 1). Data shown are mean ± SD from four independent healthy donors. (B) Comparisons of gene expression levels of key enzymes involved in glucose metabolism in different T cell subsets before and after anti-CD3 stimulations. Anti-CD3 activated Treg cells displayed strong desire for glucose metabolism compared with effector T cells. T cells were stimulated with or without anti-CD3 for 8 hours and total RNA was isolated from each cell type and analyzed by real-time PCR. Expression levels of each gene were normalized to β-actin expression levels and adjusted to the levels in naïve CD4+ T cells (served as 1). Data shown are mean ± SD from four independent healthy donors. *p<0.05 and **p<0.01, compared with the Treg results before anti-CD3 activation. #p<0.01, compared with the other T cell subsets with anti-CD3 activation. (C) Treg cells had higher glucose uptake than other T cell subsets no matter activation status. Cell subset preparations were identical to Figure 1. T cell subsets were stimulated with/without anti-CD3 antibody for 24 hours, and glucose uptake was determined by the flow cytometry after addition of 2-NBDG for 15 min. Results shown are a representative from three independent experiments. (D) Treg cells produced more L-lactate than other T cell subsets. Cell subset preparations and stimulations were identical to Figure 1. The L-lactate levels in the culture supernatants were determined by the Glycolysis cell-based assay kit. Results shown are mean ± SD from the summary of three independent experiments. **p<0.01, compared with the levels in naïve CD4 T cells. #p<0.01, compared with the other T cell subsets. (E) Activated tumor-derived CD4 Treg and γδ Treg cells also have high gene expression levels of glucose transporters and the key enzymes in glycolysis compared with those of activated Th1 cells. Cell preparations and assays are identical to (B). Data shown are mean ± SD from three independent experiments with similar results.

Figure Legend Snippet: (A) Gene expression levels of glucose transporters and the key enzymes in glycolysis in different T cell subsets after stimulation with anti-CD3 antibody. Cell subset preparations and assays were identical to Figure 1. Total RNA was isolated from each cell type and analyzed by real-time PCR. Expression levels of each gene were normalized to β-actin expression levels and adjusted to the levels in naïve CD4 cells (served as 1). Data shown are mean ± SD from four independent healthy donors. (B) Comparisons of gene expression levels of key enzymes involved in glucose metabolism in different T cell subsets before and after anti-CD3 stimulations. Anti-CD3 activated Treg cells displayed strong desire for glucose metabolism compared with effector T cells. T cells were stimulated with or without anti-CD3 for 8 hours and total RNA was isolated from each cell type and analyzed by real-time PCR. Expression levels of each gene were normalized to β-actin expression levels and adjusted to the levels in naïve CD4+ T cells (served as 1). Data shown are mean ± SD from four independent healthy donors. *p<0.05 and **p<0.01, compared with the Treg results before anti-CD3 activation. #p<0.01, compared with the other T cell subsets with anti-CD3 activation. (C) Treg cells had higher glucose uptake than other T cell subsets no matter activation status. Cell subset preparations were identical to Figure 1. T cell subsets were stimulated with/without anti-CD3 antibody for 24 hours, and glucose uptake was determined by the flow cytometry after addition of 2-NBDG for 15 min. Results shown are a representative from three independent experiments. (D) Treg cells produced more L-lactate than other T cell subsets. Cell subset preparations and stimulations were identical to Figure 1. The L-lactate levels in the culture supernatants were determined by the Glycolysis cell-based assay kit. Results shown are mean ± SD from the summary of three independent experiments. **p<0.01, compared with the levels in naïve CD4 T cells. #p<0.01, compared with the other T cell subsets. (E) Activated tumor-derived CD4 Treg and γδ Treg cells also have high gene expression levels of glucose transporters and the key enzymes in glycolysis compared with those of activated Th1 cells. Cell preparations and assays are identical to (B). Data shown are mean ± SD from three independent experiments with similar results.

Techniques Used: Gene Expression, Isolation, Real-time Polymerase Chain Reaction, Expressing, Activation Assay, Flow Cytometry, Produced, Cell Based Assay, Derivative Assay

(A) Significantly increased SA-β-Gal+ T cell populations were induced in anti-CD3-activated naïve CD4+ and CD8+ T cells cultured in the medium with different concentrations of glucose for 3 days. Data shown are mean ± SD of T cells from three individual healthy donors. Normal medium with 11 mM glucose served as a control. (B) Addition of high concentration of glucose markedly rescued responder T cell senescence induced by nTreg cells and tumor-derived Treg cells. Anti-CD3 activated CD4+ T cells were co-cultured with Treg cells for 3 days with different concentrations of glucose. SA-β-Gal expression in responder CD4+ T cells was determined. Data shown are mean ± SD from three independent experiments. **p<0.01, compared with the naïve CD4 only group. #p<0.01, compared with the Tregtreated with normal concentration of glucose (11mM) group. (C) and (D) TLR8 ligand Poly-G3 treatment significantly reversed Treg suppressive capacity on T cell proliferation (in C) and prevented Treg-induced responder T cell senescence (in D). Different types of Treg cells were co-cultured with naïve CD4+ T cells in the presence or absence of Poly-G3 (3 μg/ml) or Poly-T3 (control) for 3 days. Proliferation of cocultured naïve T cells stimulated by anti-CD3 antibody was determined by [3H]-thymidine incorporation assays (in C), and SA-β-Gal expression in treated naïve T cells was determined (in D). Data shown are mean ± SD from representative of three independent experiments with similar results. **p<0.01, compared with the respective medium only and Poly-T3 treatment groups. (E) Alterations of genes involved in glucose metabolism were identified and ranked in nTreg cells after treatment with Poly-G3 at 24 hours. Gene alterations were normalized to log2 expression level. Human nTreg cells were isolated from PBMCs of two healthy donors and treated with Poly-G3 for different time points. Total RNA was purified and pooled, and transcriptome analyses of Treg cells were performed using the Illumina wholegenome Human HT-12 BeadChips. (F) Poly-G3 treatment increased glucose levels in the culture medium of both nTreg and tumor-derived Treg cells. Different types of Treg cells and naïve CD4+ T cells were cultured in the presence or absence of Poly-G3 or Poly-T3 (Control) for 3 days, and glucose levels in the culture medium were determined. **p<0.01, compared with the None and Poly-T3 treatment groups. (G) and (H) Poly-G3 treatment significantly decreased glucose uptake by nTreg and tumorderived Treg cells. Glucose uptake was determined by the flow cytometry with addition of 2-NBDG for 15 min after 3 day culture. Results shown in histogram (H) are mean ± SD from four independent experiments. *p<0.05 and **p<0.01, compared with the respective medium only and control Poly-T3 treatment groups.

Figure Legend Snippet: (A) Significantly increased SA-β-Gal+ T cell populations were induced in anti-CD3-activated naïve CD4+ and CD8+ T cells cultured in the medium with different concentrations of glucose for 3 days. Data shown are mean ± SD of T cells from three individual healthy donors. Normal medium with 11 mM glucose served as a control. (B) Addition of high concentration of glucose markedly rescued responder T cell senescence induced by nTreg cells and tumor-derived Treg cells. Anti-CD3 activated CD4+ T cells were co-cultured with Treg cells for 3 days with different concentrations of glucose. SA-β-Gal expression in responder CD4+ T cells was determined. Data shown are mean ± SD from three independent experiments. **p<0.01, compared with the naïve CD4 only group. #p<0.01, compared with the Tregtreated with normal concentration of glucose (11mM) group. (C) and (D) TLR8 ligand Poly-G3 treatment significantly reversed Treg suppressive capacity on T cell proliferation (in C) and prevented Treg-induced responder T cell senescence (in D). Different types of Treg cells were co-cultured with naïve CD4+ T cells in the presence or absence of Poly-G3 (3 μg/ml) or Poly-T3 (control) for 3 days. Proliferation of cocultured naïve T cells stimulated by anti-CD3 antibody was determined by [3H]-thymidine incorporation assays (in C), and SA-β-Gal expression in treated naïve T cells was determined (in D). Data shown are mean ± SD from representative of three independent experiments with similar results. **p<0.01, compared with the respective medium only and Poly-T3 treatment groups. (E) Alterations of genes involved in glucose metabolism were identified and ranked in nTreg cells after treatment with Poly-G3 at 24 hours. Gene alterations were normalized to log2 expression level. Human nTreg cells were isolated from PBMCs of two healthy donors and treated with Poly-G3 for different time points. Total RNA was purified and pooled, and transcriptome analyses of Treg cells were performed using the Illumina wholegenome Human HT-12 BeadChips. (F) Poly-G3 treatment increased glucose levels in the culture medium of both nTreg and tumor-derived Treg cells. Different types of Treg cells and naïve CD4+ T cells were cultured in the presence or absence of Poly-G3 or Poly-T3 (Control) for 3 days, and glucose levels in the culture medium were determined. **p<0.01, compared with the None and Poly-T3 treatment groups. (G) and (H) Poly-G3 treatment significantly decreased glucose uptake by nTreg and tumorderived Treg cells. Glucose uptake was determined by the flow cytometry with addition of 2-NBDG for 15 min after 3 day culture. Results shown in histogram (H) are mean ± SD from four independent experiments. *p<0.05 and **p<0.01, compared with the respective medium only and control Poly-T3 treatment groups.

Techniques Used: Cell Culture, Control, Concentration Assay, Derivative Assay, Expressing, Isolation, Purification, Flow Cytometry

(A) and (B) Poly-G3 treatment down-regulated gene (in A) and protein (in B) expressions of Glut1 and Glut3 in human Treg cells. Treg and control CD4+CD25− effector cells were treated with Poly-G3 (3 μg/ml) for 48 hours. Total RNA was isolated from the T cells and analyzed by real-time PCR. The expression levels of each gene were normalized to β-actin expression levels and adjusted to the levels in untreated T cells (in A). Treated nTreg cells were also determined for Glut1 and Glut3 protein expression using the flow cytometry analysis (in B). Data shown in histograms are representative of average of three independent experiments ± SD. *p<0.05 and **p<0.01, compared with the medium only group. (C) Decreased Glut1 and Glut3 protein expression was induced by Poly-G3 treatment in nTreg cells but not in control CD4+ T cells after 3-day culture. Glut1 and Glut3 (green) expression was determined by an indirect immunofluorescence assay with a confocal microscopy. Scale bar, 50 μm. Results shown in the right histograms are mean ± SD of fluorescence intensity (MFI) quantifications of glucose transporters from three independent experiments. *p<0.05 and **p<0.01, compared with the medium only group. (D) Poly-G3 treatment down-regulated Glut1 and Glut3 membrane expression and promoted its intracellular translocation in nTreg cells. Cell treatment and procedure were identical to (C). Percentages of glucose transporter expression in cell membrane or intracellular were counted and shown in the right histograms. Scale bar, 25 μm. Results are mean ± SD of positive cells from three independent experiments. **p<0.01, compared with the medium only group. (E) and (F) Inhibition of glucose transport significantly promoted the Poly-G3-mediated reversal of Treg suppression on responder T cell proliferation (in E) and induction of cell senescence (in F). nTreg cells were pretreated with or without glucose transporter inhibitor phloretin (2 μM) for 2 days, and then co-cultured with naive CD4+ T cells in the presence or absence of Poly-G3 (3 μg/ml) for 3 days. Proliferation of co-cultured naïve T cells stimulated with anti-CD3 antibody was determined by [3H]-thymidine incorporation assays (in E), and SA-β-Gal expression in treated T cells was determined (in F). Data shown are mean ± SD from three independent experiments with similar results. **p<0.01 between the comparison groups.

Figure Legend Snippet: (A) and (B) Poly-G3 treatment down-regulated gene (in A) and protein (in B) expressions of Glut1 and Glut3 in human Treg cells. Treg and control CD4+CD25− effector cells were treated with Poly-G3 (3 μg/ml) for 48 hours. Total RNA was isolated from the T cells and analyzed by real-time PCR. The expression levels of each gene were normalized to β-actin expression levels and adjusted to the levels in untreated T cells (in A). Treated nTreg cells were also determined for Glut1 and Glut3 protein expression using the flow cytometry analysis (in B). Data shown in histograms are representative of average of three independent experiments ± SD. *p<0.05 and **p<0.01, compared with the medium only group. (C) Decreased Glut1 and Glut3 protein expression was induced by Poly-G3 treatment in nTreg cells but not in control CD4+ T cells after 3-day culture. Glut1 and Glut3 (green) expression was determined by an indirect immunofluorescence assay with a confocal microscopy. Scale bar, 50 μm. Results shown in the right histograms are mean ± SD of fluorescence intensity (MFI) quantifications of glucose transporters from three independent experiments. *p<0.05 and **p<0.01, compared with the medium only group. (D) Poly-G3 treatment down-regulated Glut1 and Glut3 membrane expression and promoted its intracellular translocation in nTreg cells. Cell treatment and procedure were identical to (C). Percentages of glucose transporter expression in cell membrane or intracellular were counted and shown in the right histograms. Scale bar, 25 μm. Results are mean ± SD of positive cells from three independent experiments. **p<0.01, compared with the medium only group. (E) and (F) Inhibition of glucose transport significantly promoted the Poly-G3-mediated reversal of Treg suppression on responder T cell proliferation (in E) and induction of cell senescence (in F). nTreg cells were pretreated with or without glucose transporter inhibitor phloretin (2 μM) for 2 days, and then co-cultured with naive CD4+ T cells in the presence or absence of Poly-G3 (3 μg/ml) for 3 days. Proliferation of co-cultured naïve T cells stimulated with anti-CD3 antibody was determined by [3H]-thymidine incorporation assays (in E), and SA-β-Gal expression in treated T cells was determined (in F). Data shown are mean ± SD from three independent experiments with similar results. **p<0.01 between the comparison groups.

Techniques Used: Control, Isolation, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Immunofluorescence, Confocal Microscopy, Fluorescence, Membrane, Translocation Assay, Inhibition, Cell Culture, Comparison

(A) Poly-G3 treatment significantly down-regulated gene expression levels of key glycolytic enzymes in both nTreg and tumor-derived Treg cells. Different types of human Treg cells and control effector CD4+ T cells were treated with or without Poly-G3 or Poly-T3 for 48 hours. Total RNA was isolated from the T cells and analyzed by real-time PCR. The expression levels of each gene were normalized to β-actin expression levels and adjusted to the levels in untreated T cells (medium). Data shown in nTreg and control CD4+ T cells are mean ± SD from four independent donors. Data for CD4 TregE1 and γδ Treg31 are averages of three independent experiments. *p<0.05 and **p<0.01, compared with the medium only group. (B) and (C) Blockage of glycolysis in nTreg cells using specific pharmacological inhibitors dramatically enhanced the effects of Poly-G3-mediated reversal of Treg suppression on responder T cell proliferation (in B) and induction of cell senescence (in C). nTreg cells were pretreated with glycolysis inhibitors, including 2-DG (1 mM), LND (125 μM), and 3-BrPA (30 μM), respectively for 48 hours. Naïve CD4+ T cells were then co-cultured with inhibitor-pretreated or untreated Treg cells for 3 days in the presence or absence of Poly-G3. Proliferation of co-cultured naïve T cells stimulated with anti-CD3 antibody was determined by [3H]-thymidine incorporation assays (in B), and SA-β-Gal expression in treated T cells was determined (in C). Data shown are mean ± SD from representative of three independent experiments with similar results. **p<0.01 and #p<0.01, compared with the respective medium only group. (D) TLR8 signaling activation decreased the key metabolites involved in glycolysis and TCA in nTreg cells. nTreg cells were cultured in T cell medium in the presence of Poly-G3 or Poly-T3 for 72 hours. Glucose metabolites from the nTreg cell lysates were analyzed using a LC-triple quadruple mass spectrometry, and metabolite levels are normalized to medium group. Relative levels of intermediate metabolites in the glycolysis and TCA-cycle pathways are shown. Data shown are mean ± SD from representative of three independent nTreg cells with similar results. *p<0.05 and **p<0.01, compared with the medium only group.

Figure Legend Snippet: (A) Poly-G3 treatment significantly down-regulated gene expression levels of key glycolytic enzymes in both nTreg and tumor-derived Treg cells. Different types of human Treg cells and control effector CD4+ T cells were treated with or without Poly-G3 or Poly-T3 for 48 hours. Total RNA was isolated from the T cells and analyzed by real-time PCR. The expression levels of each gene were normalized to β-actin expression levels and adjusted to the levels in untreated T cells (medium). Data shown in nTreg and control CD4+ T cells are mean ± SD from four independent donors. Data for CD4 TregE1 and γδ Treg31 are averages of three independent experiments. *p<0.05 and **p<0.01, compared with the medium only group. (B) and (C) Blockage of glycolysis in nTreg cells using specific pharmacological inhibitors dramatically enhanced the effects of Poly-G3-mediated reversal of Treg suppression on responder T cell proliferation (in B) and induction of cell senescence (in C). nTreg cells were pretreated with glycolysis inhibitors, including 2-DG (1 mM), LND (125 μM), and 3-BrPA (30 μM), respectively for 48 hours. Naïve CD4+ T cells were then co-cultured with inhibitor-pretreated or untreated Treg cells for 3 days in the presence or absence of Poly-G3. Proliferation of co-cultured naïve T cells stimulated with anti-CD3 antibody was determined by [3H]-thymidine incorporation assays (in B), and SA-β-Gal expression in treated T cells was determined (in C). Data shown are mean ± SD from representative of three independent experiments with similar results. **p<0.01 and #p<0.01, compared with the respective medium only group. (D) TLR8 signaling activation decreased the key metabolites involved in glycolysis and TCA in nTreg cells. nTreg cells were cultured in T cell medium in the presence of Poly-G3 or Poly-T3 for 72 hours. Glucose metabolites from the nTreg cell lysates were analyzed using a LC-triple quadruple mass spectrometry, and metabolite levels are normalized to medium group. Relative levels of intermediate metabolites in the glycolysis and TCA-cycle pathways are shown. Data shown are mean ± SD from representative of three independent nTreg cells with similar results. *p<0.05 and **p<0.01, compared with the medium only group.

Techniques Used: Gene Expression, Derivative Assay, Control, Isolation, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Activation Assay, Mass Spectrometry

(A) Significant alterations in 24 genes involved in the mTOR signaling pathway were identified and ranked in nTreg cells after treatment with or without TLR8 ligand Poly-G3 at 24 hours. Gene alterations were normalized to log2 expression level. Human nTreg cells were isolated from PBMCs of two healthy donors and treated with Poly-G3 for different time points. Total RNA was purified and transcriptome analyses of Treg cells were performed using the Illumina whole-genome Human HT-12 BeadChips. (B) Suppression of phosphorylation and subsequent activation of mTOR signaling in Treg cells treated with Poly-G3. Treg cells were treated with or without Poly-G3 (3 μg/ml) for 30 minutes and then phosphorylated mTOR, p70S6K, and 4E-BP1 in Treg cells were determined by the flow cytometry. Protein levels shown in the right histograms are summarized as the mean ± SD from three independent experiments. **p<0.01 compared with the medium only group. (C) Blockage of p38 signaling prevented TLR8-mediated inhibition of mTOR signaling. nTreg cells were treated with p38 inhibitor SB203580 (10 μM) in the presence or absence of Poly-G3 (3 μg/ml) for 30 minutes and then phosphorylated mTOR and p70S6K in Treg cells were determined by the flow cytometry. Results shown in the right histogram are summarized as the mean ± SD from three independent experiments. **p<0.01 compared with the other treatment groups. (D) and (E) Blockage of mTOR signaling with mTOR inhibitor rapamycin partially reversed Treg suppression and promoted the Poly-G3-mediated reversal of Treg suppressive activities on responder T cell proliferation (in D) and induction of cell senescence (in E). nTreg cells were pretreated with or without rapamycin (300 nM) for 1 day, and then co-cultured with naive CD4+ T cells in the presence or absence of Poly-G3 (3 μg/ml) for 3 days. Proliferation of co-cultured naïve T cells stimulated by anti-CD3 antibody was determined by [3H]-thymidine incorporation assays (in D), and SAβ-Gal expression in treated T cells was determined (in E). Data shown are mean ± SD from representative of three independent experiments. **p<0.01 between the comparison groups. (F) and (G) Activation of mTOR signaling with Retro-RHEB transfection promoted Treg suppression and prevented the Poly-G3-mediated reversal of Treg suppressive activities on responder T cell proliferation (in F) and induction of cell senescence (in G). Activated nTreg cells were infected with retrovirus carrying RHEB gene or control vector for 48 hours. Infected Treg cells were then co-cultured with naive CD4+ T cells in the presence or absence of Poly-G3 (3 μg/ml) for 3 days. Proliferation and SA-β-Gal expression of cocultured naïve T cells were determined as above. Data shown are mean ± SD from three independent experiments with similar results. *p<0.05 and **p<0.01 between the comparison groups. (H) Relative expression levels of HIF1α in different T cell subsets before and after anti-CD3 stimulations. TCR-activated Treg cells showed significantly elevated HIF1α expression compared with that of effector T cells. T cells were stimulated with or without anti-CD3 for 8 hours and total RNA was isolated from each cell type and analyzed by real-time PCR. Expression level of HIF1α was normalized to β-actin expression and adjusted to the level in naïve CD4+ T cells (served as 1). Data shown are mean ± SD from four independent healthy donors. (I) Activated tumor-derived CD4+ Treg and γδ Treg cells also had higher HIF1α gene expression than activated Th1 cells. Cell treatment and assays were the same as in (H). Data shown are mean ± SD from three independent experiments with similar results. (J) and (K) Poly-G3 treatment down-regulated HIF1α mRNA expression in human Treg cells. nTreg and tumorderived Treg cells, and control CD4+ T cells were treated with Poly-G3 (3 μg/ml) or Poly-T3 for 48 hours. Total RNA was isolated from the T cells and analyzed by real-time PCR. The gene expression levels of HIF1α were normalized to β-actin expression levels and adjusted to the levels in untreated T cells. Data shown in histograms are representative of mean ± SD from three independent experiments. *p<0.05 and **p<0.01, compared with the medium only group. (L) and (M) Inhibition of HIF1α signaling alleviated Treg suppression and significantly promoted the Poly-G3-mediated reversal of Treg suppressive activities on responder T cell proliferation (in L) and induction of cell senescence (in M). nTreg cells were pretreated with or without HIF1α inhibitors YC-1 (5 μM) or 2-ME (10 μM) for 1 day, and then co-cultured with naive CD4+ T cells in the presence or absence of Poly-G3 (3 μg/ml) for 3 days. Proliferation and SA-β-Gal expression of co-cultured naïve T cells were determined as above. Data shown are mean ± SD from three independent experiments with similar results. *p<0.05 and #p<0.01, compared with the respective medium only group. (N) and (O) Activation of HIF1α signaling in nTreg cells dramatically augmented Treg suppression and blocked the effects of Poly-G3-mediated reversal of Treg suppression on responder T cell proliferation (in N) and induction of cell senescence (in O). nTreg cells were pretreated with or without HIF1α activator DMOG (0.1mM) for 1 day and then co-cultured with naive CD4+ T cells in the presence or absence of Poly-G3 (3 μg/ml) for 3 days. Proliferation and SA-β-Gal expression of cocultured naïve T cells were determined. Data shown are mean ± SD from representative of three independent experiments with similar results. *p<0.05 and **p<0.01, between the comparison groups.

Figure Legend Snippet: (A) Significant alterations in 24 genes involved in the mTOR signaling pathway were identified and ranked in nTreg cells after treatment with or without TLR8 ligand Poly-G3 at 24 hours. Gene alterations were normalized to log2 expression level. Human nTreg cells were isolated from PBMCs of two healthy donors and treated with Poly-G3 for different time points. Total RNA was purified and transcriptome analyses of Treg cells were performed using the Illumina whole-genome Human HT-12 BeadChips. (B) Suppression of phosphorylation and subsequent activation of mTOR signaling in Treg cells treated with Poly-G3. Treg cells were treated with or without Poly-G3 (3 μg/ml) for 30 minutes and then phosphorylated mTOR, p70S6K, and 4E-BP1 in Treg cells were determined by the flow cytometry. Protein levels shown in the right histograms are summarized as the mean ± SD from three independent experiments. **p<0.01 compared with the medium only group. (C) Blockage of p38 signaling prevented TLR8-mediated inhibition of mTOR signaling. nTreg cells were treated with p38 inhibitor SB203580 (10 μM) in the presence or absence of Poly-G3 (3 μg/ml) for 30 minutes and then phosphorylated mTOR and p70S6K in Treg cells were determined by the flow cytometry. Results shown in the right histogram are summarized as the mean ± SD from three independent experiments. **p<0.01 compared with the other treatment groups. (D) and (E) Blockage of mTOR signaling with mTOR inhibitor rapamycin partially reversed Treg suppression and promoted the Poly-G3-mediated reversal of Treg suppressive activities on responder T cell proliferation (in D) and induction of cell senescence (in E). nTreg cells were pretreated with or without rapamycin (300 nM) for 1 day, and then co-cultured with naive CD4+ T cells in the presence or absence of Poly-G3 (3 μg/ml) for 3 days. Proliferation of co-cultured naïve T cells stimulated by anti-CD3 antibody was determined by [3H]-thymidine incorporation assays (in D), and SAβ-Gal expression in treated T cells was determined (in E). Data shown are mean ± SD from representative of three independent experiments. **p<0.01 between the comparison groups. (F) and (G) Activation of mTOR signaling with Retro-RHEB transfection promoted Treg suppression and prevented the Poly-G3-mediated reversal of Treg suppressive activities on responder T cell proliferation (in F) and induction of cell senescence (in G). Activated nTreg cells were infected with retrovirus carrying RHEB gene or control vector for 48 hours. Infected Treg cells were then co-cultured with naive CD4+ T cells in the presence or absence of Poly-G3 (3 μg/ml) for 3 days. Proliferation and SA-β-Gal expression of cocultured naïve T cells were determined as above. Data shown are mean ± SD from three independent experiments with similar results. *p<0.05 and **p<0.01 between the comparison groups. (H) Relative expression levels of HIF1α in different T cell subsets before and after anti-CD3 stimulations. TCR-activated Treg cells showed significantly elevated HIF1α expression compared with that of effector T cells. T cells were stimulated with or without anti-CD3 for 8 hours and total RNA was isolated from each cell type and analyzed by real-time PCR. Expression level of HIF1α was normalized to β-actin expression and adjusted to the level in naïve CD4+ T cells (served as 1). Data shown are mean ± SD from four independent healthy donors. (I) Activated tumor-derived CD4+ Treg and γδ Treg cells also had higher HIF1α gene expression than activated Th1 cells. Cell treatment and assays were the same as in (H). Data shown are mean ± SD from three independent experiments with similar results. (J) and (K) Poly-G3 treatment down-regulated HIF1α mRNA expression in human Treg cells. nTreg and tumorderived Treg cells, and control CD4+ T cells were treated with Poly-G3 (3 μg/ml) or Poly-T3 for 48 hours. Total RNA was isolated from the T cells and analyzed by real-time PCR. The gene expression levels of HIF1α were normalized to β-actin expression levels and adjusted to the levels in untreated T cells. Data shown in histograms are representative of mean ± SD from three independent experiments. *p<0.05 and **p<0.01, compared with the medium only group. (L) and (M) Inhibition of HIF1α signaling alleviated Treg suppression and significantly promoted the Poly-G3-mediated reversal of Treg suppressive activities on responder T cell proliferation (in L) and induction of cell senescence (in M). nTreg cells were pretreated with or without HIF1α inhibitors YC-1 (5 μM) or 2-ME (10 μM) for 1 day, and then co-cultured with naive CD4+ T cells in the presence or absence of Poly-G3 (3 μg/ml) for 3 days. Proliferation and SA-β-Gal expression of co-cultured naïve T cells were determined as above. Data shown are mean ± SD from three independent experiments with similar results. *p<0.05 and #p<0.01, compared with the respective medium only group. (N) and (O) Activation of HIF1α signaling in nTreg cells dramatically augmented Treg suppression and blocked the effects of Poly-G3-mediated reversal of Treg suppression on responder T cell proliferation (in N) and induction of cell senescence (in O). nTreg cells were pretreated with or without HIF1α activator DMOG (0.1mM) for 1 day and then co-cultured with naive CD4+ T cells in the presence or absence of Poly-G3 (3 μg/ml) for 3 days. Proliferation and SA-β-Gal expression of cocultured naïve T cells were determined. Data shown are mean ± SD from representative of three independent experiments with similar results. *p<0.05 and **p<0.01, between the comparison groups.

Techniques Used: Expressing, Isolation, Purification, Phospho-proteomics, Activation Assay, Flow Cytometry, Inhibition, Cell Culture, Comparison, Transfection, Infection, Control, Plasmid Preparation, Real-time Polymerase Chain Reaction, Derivative Assay, Gene Expression

Figure Legend Snippet: KEY RESOURCES TABLE

Techniques Used: Derivative Assay, Recombinant, Reverse Transcription, Selection, Cell Based Assay, Software

Incubation:

Article Title: Dextran-based T-cell expansion nanoparticles for manufacturing CAR T cells with augmented efficacy.
Article Snippet: .. First, the anti-CD3 (clone OKT-3, BioXCell) and anti-CD28(clone 9.3, BioXCell) antibodies were dissolved in PBS buffer (0.5 mg/ml, 200 μl), Next, DBCO-Sulfo-NHS (3.32 μl, 10 mm) and Cy5-NHS (8 μl, 1.5mM in DMSO, Cytiva, PA15101) was added into anti-CD3 solution, DBCO-Sulfo-NHS (3.32 μl, 10 mm) and Alexa Fluor (AF) 488-NHS (8 μl, 1.5mM in DMSO, Thermo Fisher Scientific) was added into anti-CD28 solution and further incubated for 3 hours at room temperature. ..

Article Title: Dextran-based T-cell expansion nanoparticles for manufacturing CAR T cells with augmented efficacy
Article Snippet: .. First, the anti-CD3 (clone OKT-3, BioXCell) and anti-CD28(clone 9.3, BioXCell) antibodies were dissolved in PBS buffer (0.5 mg/ml, 200 μl), Next, DBCO-Sulfo-NHS (3.32 μl, 10 mm) and Cy5-NHS (8 μl, 1.5 mM in DMSO, Cytiva, PA15101) was added into anti-CD3 solution, DBCO-Sulfo-NHS (3.32 μl, 10 mm) and Alexa Fluor (AF) 488-NHS (8 μl, 1.5 mM in DMSO, Thermo Fisher Scientific) was added into anti-CD28 solution and further incubated for 3 h at room temperature. ..

Concentration Assay:

Article Title: Dextran-based T-cell expansion nanoparticles for manufacturing CAR T cells with augmented efficacy
Article Snippet: The NPs were obtained by centrifugation (10,600 × g , 10 min, Heraeus fresco 21, Thermo Fisher Scientific) and washed with PBS 3 times. .. First, the anti-CD3 (clone OKT-3, BioXCell) and anti-CD28(clone 9.3, BioXCell) antibodies were dissolved in PBS buffer (0.5 mg/ml, 200 μl), and the DBCO-Sulfo-NHS (CCT-A124, Vector Laboratories) was dissolved in DMSO to final concentration at 10 mM. .. Next, 3.32 μL of the above DBCO-Sulfo-NHS Ester was added to each antibody solution and incubated at room temperature for 30 min. Then, the reaction was quenched with 18 μl of Tris-buffer (1 M, pH 8, Thermo Fisher Scientific) for 15 min.

Article Title: Dextran-based T-cell expansion nanoparticles for manufacturing CAR T cells with augmented efficacy.
Article Snippet: The NPs were obtained by centrifugation (10600 g, 10 min, Heraeus fresco 21, Thermo Fisher Scientific) and washed with PBS 3 times. .. First, the anti-CD3 (clone OKT-3, BioXCell) and anti-CD28(clone 9.3, BioXCell) antibodies were dissolved in PBS buffer (0.5 mg/ml, 200 μl), and the DBCO-Sulfo-NHS (CCT-A124, Vector Laboratories) was dissolved in DMSO to final concentration at 10 mM. ..

Isolation:

Article Title: An imbalance between CD80 and CD86 levels and CD4 regulatory T cell number and transendocytosis function exists in the liver in autoimmune hepatitis.
Article Snippet: .. 2X105 freshly isolated CD4 T cells from peripheral blood or liver tissue were co-cultured in 5 complete RPMI with 1 μg/mL anti-CD3 (OKT3; BioXCell) for 16 hours alongside Chinese 6 Hamster Ovary (CHO) cells either transduced to express human CD80 labelled with a C-7 terminal Green Fluorescent Protein (GFP) tag (CHO-CD80) or not (CHO-Blank) at 1:1 ratio. .. 8 Anti-CTLA4 (Ticilimumab a gift from Pfizer) was added at 20 μg/mL, recombinant human 9 interleukin (IL) 2 or IL10 (both Peprotech) were added at stated concentrations with or 10 without 20 μg/mL anti-CD210 (anti-IL10RA) (clone 3F9, BD Biosciences), as stated.

Article Title: An imbalance between CD80 and CD86 levels and CD4 regulatory T cell number and transendocytosis function exists in the liver in autoimmune hepatitis
Article Snippet: .. In total, 2 × 10 5 freshly isolated CD4 T cells from peripheral blood or liver tissue were co-cultured in complete RPMI with 1 μg/ml anti-CD3 (OKT3; BioXCell) for 16 hours alongside Chinese Hamster Ovary (CHO) cells either transduced to express human CD80 labelled with a C-terminal Green Fluorescent Protein (GFP) tag (CHO-CD80) or not (CHO-Blank) at 1:1 ratio. .. Anti-CTLA4 (ticilimumab was a gift from Pfizer) was added at 20 μg/ml, and recombinant human interleukin (IL) 2 or IL10 (both Peprotech) was added at stated concentrations with or without 20 μg/ml anti-CD210 (anti-IL10RA) (clone 3F9, BD Biosciences), as stated.

Activation Assay:

Article Title: Rewiring STAT signaling from the cell surface with Trikine immunotherapeutics
Article Snippet: .. For human T cell activation, cells were activated with plate-bound anti-human CD3ε (1 μg ml −1 , clone OKT-3, BioXCell) and soluble anti-human CD28 (5 μg ml −1 , clone 9.3, BioXCell). ..

Review