Journal: Molecular Medicine Reports

Article Title: Cytotoxic effect of CLL-1 CAR-T cell immunotherapy with PD-1 silencing on relapsed/refractory acute myeloid leukemia

doi: 10.3892/mmr.2021.11847

Figure Lengend Snippet: Construction of CLL-1 CAR, shRNA PD-1/CLL-1 CAR and CLL-1-overexpressing K562 cells. (A) CLL-1 CAR vector. The third-generation CAR was composed of the variable-light domain, followed and variable-heavy domain of the CLL-1 scFv, the CD8 hinge and TM region, CD28, the OX40 co-stimulatory domains and the CD3 + signaling domain. (B) shRNA PD-1/CLL-1 CAR vector. The third-generation shRNA PD-1/CLL-1 CAR was composed of the variable-light domain, followed and variable-heavy domain of the CLL-1 scFv, the CD8 hinge and TM region, CD28, the OX40 co-stimulatory domains, the CD3 + signaling domain and shRNA PD-1. (C) PSE2970 vector. The PSE2970 lentiviral vector, was constructed to express the CLL-1 antigen in the K562 cell line (stK562). (D) Flow cytometry plots exhibiting the phenotype of CAR-T cell subsets. The frequency of CD4 + and CD8 + T cells was assessed in primary T cells from patients with AML or healthy donors transduced with CLL-1 CAR or shRNA PD-1/CLL-1 CAR. (E) Bar graph of flow cytometry data illustrating the immune phenotype of CLL-1 and shRNA PD-1/CLL-1 CAR-T cells. NS, the proportion of CD4 + vs. CD8 + CAR-T cells from patients; *P<0.05, the proportion of CD4 + vs. CD8 + CAR-T cells from healthy donors. (F) Representative flow cytometry dot plots demonstrating transduction efficiency. (G) Transfection efficiency was evaluated in CLL-1 and shRNA PD-1/CLL-1 CAR-T cells. (H) Representative flow cytometry dot plots for PD-1 expression on the CLL-1 CAR-T cells and shRNA PD-1/CLL-1 CAR-T cells. (I) PD-1 expression was evaluated in CLL-1 CAR-T and shRNA PD-1/CLL-1 CAR-T cells using flow cytometry. *P<0.05. (J) PD-1 mRNA expression was measured in CLL-1 and shRNA PD-1/CLL-1 CAR-T cells using reverse transcription-quantitative polymerase chain reaction. *P<0.05. AML, acute myeloid leukemia; CLL-1, C-type lectin-like molecule-1; scFv, single-chain variable fragment; TM, transmembrane; PD-1, programmed cell death-1; CAR, chimeric antigen receptor; APC, allophycocyanin; PerCP, peridinin chlorophyll; NS, not significant.

Article Snippet: The T cells were cultured in AIM-V T cell medium (Gibco; Thermo Fisher Scientific, Inc.) containing 100 IU/ml recombinant human IL-2 (cat. no. AF-200-02; PeproTech, Inc.), 5 ng/ml recombinant human IL-7 (cat. no. AF-200-07; PeproTech, Inc.) and 5 ng/ml recombinant human IL-15 (cat. no. AF-200-15; PeproTech, Inc.), and co-stimulated with anti-CD3 (cat. no. 170-076-116; Miltenyi Biotec, Inc.) and -CD28 antibodies (cat. no. 170-076-117; Miltenyi Biotec, Inc.) for 18–24 h in a 5% CO 2 atmosphere at 37°C.

Techniques: shRNA, Plasmid Preparation, Construct, Flow Cytometry, Transduction, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cutting edge: immune cells as sources and targets of the IL-10 family members?

doi: 10.4049/jimmunol.168.11.5397

Figure Lengend Snippet: FIGURE 3. Modulation of the T cell expression of novel IL-10 family members by costimulation and functional polarization. T cells were stimulated with anti-CD3 and anti-CD28 mAbs in the presence of IL-12/anti-IL-4 mAb (polarization toward T1), IL-4/anti-IFN- mAb (polarization toward T2), IL-10/TGF-1.2 (polarization toward regulatory T cells), or isotypic control mAbs, or cultured without stimulation in the presence of isotypic control mAbs for 6, 18, 42, and 66 h. Gene expression of IL-10 homologs was analyzed by real-time RT-PCR. A, Expression in nonstimulated and stimulated nonpolarized cells is shown as relative to that of the housekeeping gene. B, Expression in stimulated polarized cells relative to stimulated nonpolarized cells is shown. Data from three independent assays are given as mean SEM.

Article Snippet: To study the effect of T cell costimulation and functional polarization (Fig. 3), T cells were cultured either in the presence of 5 g/ml IgG1 and 5 g/ml IgG2a (controls) or stimulated with anti-CD3 (Cilag) and anti-CD28 mAbs (R&D Systems) coated on culture vessel (1 g/cm2 each), in the presence of 5 g/ml IgG1 and 5 g/ml IgG2a, 10 ng/ml IL-12 and 5 g/ml anti-IL-4 mAb, 10 ng/ml IL-4 and 5 g/ml anti-IFN- mAb (all from R&D Systems), or 10 ng/ml IL-10 (PeproTech, Rock Hill, SC) and 10 ng/ml TGF- 1.2 (R&D Systems) for 6, 18, 42, and 66 h. Human EBV-transformed B cells were provided by Dr. N. Babel (Charite, Berlin, Germany).

Techniques: Expressing, Functional Assay, Control, Cell Culture, Gene Expression, Quantitative RT-PCR

Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 1 Coadministration of CD3/CD28 and CpG induce robust amounts of IL-10 expression in splenocytes when compared to either signal alone. (A) Splenocytes were treated with CpG, CD3/CD28, CD3/CD28/CpG, or left untreated for 72 hours and analyzed for IL0 production by ELISA. (B) Splenocytes were treated with CD3/CD28/CpG for 0, 48, or 72 hours, and IL-10 levels were measured by ELISA. (C) Supernatants of splenocytes treated with either CpG or control ODN (ctrl CpG) in the absence or presence of CD3/CD28 for 72 hours were analyzed for IL-10 production by ELISA. (D) Splenocytes were treated with CpG in the presence or absence of various T cell-activating antibodies (CD28, CD3, or CD3/CD28) for 72 hours, and IL-10 expression in the supernatants was measured via ELISA. (E) Supernatants from splenocytes treated as indicated for 72 hours were analyzed for IL-10 production by ELISA. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 2 Coordination between T cells and macrophages induces the highest expression of IL-10 in a cell-contact dependent manner. (A, B) Supernatants from wild type and nude (A, Balb/c background) or wild type and SCID (B, C3H background) splenocytes were treated with CD3/CD28/CpG for 72 hours and then analyzed for IL-10 production by ELISA. (C) Non-depleted splenocytes or the ones depleted for CD3, CD4, CD8, DC, and NK cells were treated with CD3/CD28/CpG for 72 hours, and the supernatants were analyzed for IL-10 expression via ELISA. (D) Purified splenic B cells, DC, CD4+ T cells, or peritoneal macrophages were coincubated in the presence or absence of purified CD4+ T cells, treated with CD3/CD28/CpG for 72 hours and analyzed for IL-10 expression in the supernatants by ELISA. (E) Peritoneal macrophages were coincubated in the presence or absence of purified NK, whole T (CD3+ T), CD4+ T, or a combination of CD4+ T and NK cells; treated with CD3/CD28/CpG for 72 hours; and analyzed for IL-10 expression in the supernatants via ELISA. (F) Peritoneal macrophages were coincubated with purified CD4+ cells in the presence or absence of a transwell barrier, treated with CD3/CD28/CpG for 72 hours, and analyzed for IL-10 expression in the supernatant via ELISA. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Purification

Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 3 CD3/28/CpG induces IL-10 production in both macrophages and CD4+ T cells. (A) Diagram of FACS analysis. (B, C) Splenocytes left untreated or treated with CpG, CD3/CD28, CD3/CD28/CpG for 72 hours and number of CD4 (B) or F4/80 (C) were measured via FACS. (C, D) Median fluorescence intensity of IL10 in the CD4+ (D) or F4/80+ (E) gated cells. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Fluorescence

Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 4 CD3/CD28/CpG regulates IL-10 via activation of NF-κB1,pERK, and STAT3. (A) Splenocytes were treated as indicated for 72 hours and IL-10 levels were measured by ELISA. (B) Supernatants from wild type or NF-κB1−/−splenocytes treated with CD3/CD28, CpG, or TX (CD3/CD28/CpG) for 72 hours were analyzed for IL-10 production by ELISA. (C) Peritoneal macrophages from wild type or NF-κB1−/−mice were coincubated with purified CD4+ T cells from either wild type or NF-κB1−/−mice, treated with CD3/CD28/CpG for 72 hours and the supernatants were measured for IL-10 expression by ELISA. (D) Splenocytes treated with CD3/CD28, CpG, or TX (CD3/CD28/CpG) for 72 hours were stained for CD4 and pERK or CD4 and pSTAT3. Histograms show pERK or pSTAT3 levels from gated T cells. (E) Quantification of median fluorescence intensity of pERK levels in CD4 cells (N = 3). (F) Splenocytes were treated with ERK inhibitor U0126 at various doses for 72 hours in presence of CD3/CD28/CpG, and IL-10 levels were measure by ELISA. (G) Splenocytes were treated with STAT3 inhibitor NSC 74859 at various doses for 72 hours in presence of CD3/ CD28/CpG, and IL-10 levels were measure by ELISA. (H) Wild type or NF-κB1−/−splenocytes in presence or absence of STAT3 inhibitor (STAT3i, 50 μM) were treated with CD3/CD28/CpG for 72 hours and analyzed for IL-10 production by ELISA. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Purification, Expressing, Staining, Fluorescence

Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 5 Activation or inhibition of CD40/CD154 pathway in the presence of CD3/CD28/CpG acts as a switch in the expression of IL-10 or IL-30. (A) Supernatants from wildtype or CD40−/−splenocytes were treated with CD3/CD28/CpG for 72 hours were analyzed for IL-10 production by ELISA. (B) Supernatants from wild type or CD154−/−splenocytes treated with CD3/CD28/CpG for 72 hours were analyzed for IL-10 production by ELISA. (C, D) Splenocytes from wild type mice were treated with the indicated treatments in the presence of control or anti-CD40 antibodies for 72 hours, and IL-10 (C) or IL-30 (D) expression was measured in the supernatants using ELISA. (E, F) Splenocytes from wild type mice were treated with the indicated treatments in the presence of control or anti-CD40 antibodies for 24, 48, or 72 hours, and IL-10 (E) or IL-30 (F) expression was measured in the supernatants using ELISA. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Activation Assay, Inhibition, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 6 Regulation of IL-10 and IL-30 via activation of CD40 signaling. (A) Supernatants from splenocytes treated with CpG, CD3/CD28, TX (CD3/CD28/CpG), in the presence or absence of rIL-10 for 72 hours were analyzed for IL-30 production by ELISA. (B) Wild type or IL-10−/−splenocytes were treated as indicated for 72 hours, and IL-30 levels were measured by ELISA. (C) Splenocytes were treated with CD3/CD28/CpG in the presence of recombinant IL30 at 50 and 100 ng/ml for 72 hours, and IL-10 levels were measured by ELISA. (D) Wild type or IL-10−/−splenocytes were treated with CD3/CD28/CpG for 72 hours in the presence of control or anti-CD40 antibodies, and IL-30 levels were measured by ELISA. (E) Harvested pellets of splenocytes treated with anti-CD40 or control antibody for the indicated time points were probed for pSTAT3, p-p65, p50/105, and actin. (F) Splenocytes were treated with CD3/CD28/CpG in the presence or absence of anti-CD40 activating antibody and the levels of p-p65 and p-STAT3 in the CD4+ T cells or macrophages were measured via flow cytometry. (G) Supernatants from wild type or NF-κB1−/−splenocytes treated with CpG, CD3/CD28, TX (CD3/CD28/CpG), or CD3/CD28/CpG in the presence of activating anti-CD40 for 72 hours were analyzed for IL-30 production by ELISA. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Control, Flow Cytometry

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Antagonism of the A 2A R enhances TCR-stimulated IL-2 mRNA increases in primary human CD4 + T cells and Jurkat T cells. (A) Box plots (top) and difference plots (bottom) show data from naïve and memory CD4 + T cells isolated from the peripheral blood of 20 healthy donors, stimulated with plate-bound anti-CD3 and soluble anti-CD28, and grown in conditions promoting TH1 or TH2 differentiation for three days in the presence or absence of ZM-241385 (ZM). IL-2 mRNA levels were determined by qPCR. In the box plots (top), the height of the box plots equals the interquartile range (IQR) and the horizontal line within the box indicates the median value. The whiskers extend to the lowest and highest data points within 1.5 X IQR and the open circles indicate the outliers, which lie above or below the whiskers. In the difference plots (bottom), open circles show pairwise differences in IL-2 mRNA for each sample when treated with ZM-241385 (ZM) or not (Con). To the right of the open circles are the median values (closed circles) and 95% confidence intervals. (B) Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the absence or presence of ZM-241385 (ZM) for three days. IL-2 mRNA levels were determined by qPCR and normalized to the amount produced by the TCR-stimulated control. Data represent the mean ± SE from 8 experiments. * , p < 0.05; *** , p < 0.001; **** , p < 0.0001.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Isolation, Produced, Control

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: A dominant negative Gα s construct, Gα s DN3, which blocks signaling from G s -coupled receptors, enhances TCR-stimulated IL-2 mRNA increases. Jurkat cells were nucleofected with Gα s DN3 or empty vector (pcDNAI/Amp) and then stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 3 days. IL-2 mRNA levels were determined by qPCR and normalized to the amount produced by the TCR-stimulated control. Data represent the mean ± SE from 8 experiments. * , p < 0.05.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Dominant Negative Mutation, Construct, Plasmid Preparation, Produced, Control

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Gα s siRNA and adenylyl cyclase inhibition with ddA decrease TCR-stimulated IL-2 mRNA levels. Jurkat cells were nucleofected with Gα s siRNA or NT siRNA as described in Methods (A-C) and stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 3 days (A, C). Gα s siRNA significantly decreased levels of Gα s mRNA (A), Gα s protein (B), and IL-2 mRNA (C). Data for (A) and (C) represent the mean ± SE from 8 experiments. (B) Left, each immunoblot is representative of three immunoblots. Right, quantification of protein expression levels in the presence of Gα s siRNA relative to NT siRNA. Data represent mean ± SE from 3 experiments. (D) Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 3 days in the presence or absence of ddA. Data represent the mean ± SE from 17 experiments. mRNA levels were determined by qPCR. * , p < 0.05; ** , p < 0.01; **** , p < 0.0001.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Inhibition, Western Blot, Expressing

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Inhibiting cAMP production decreases activity of the IL-2 promoter without affecting IL-2 mRNA stability. (A) ddA does not decrease stability of IL-2 mRNA. After 3 days of TCR stimulation with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of ddA, Jurkat cells were incubated for the indicated times with Actinomycin D to inhibit transcription, and the rate of IL-2 mRNA degradation was measured. In both cases, the rates of IL-2 mRNA degradation fit a single exponential. Data represent means ± SD from triplicate determinations from a single experiment representative of 4 experiments. (B) ddA decreases IL-2 promoter activity in a luciferase reporter assay. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of ddA for 3 days following nucleofection with the indicated plasmids. (B) Data represent means ± SD from triplicate determinations from a single assay representative of 6 assays. (C) Data represent the means ± SE of values from stimulated cells expressing IL2/pGL3 from the 6 assays. ** , p < 0.01.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Activity Assay, Incubation, Luciferase, Reporter Assay, Expressing

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Gα s siRNA, but not Gα s DN3, decreases TCR-stimulated cAMP. Jurkat cells were nucleofected with the indicated siRNA or plasmids and then assayed for cAMP accumulation as described in Methods. The TCR was stimulated with 2.5 µg/ml plate-bound anti-CD3 and 2.5 µg/ml soluble anti-CD28 (A and B), and the A 2A R was stimulated with 300 µM CGS-21680 (C). Data in (A) represent the mean ± SE from 3 experiments and data in (B and C) represent the mean ± SE from 9 experiments. * , p < 0.05.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques:

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Evidence for an inhibitory effect of cAMP on TCR-stimulated IL-2 mRNA levels after at least 2 days of TCR stimulation. (A) The potentiating effect of A 2A R antagonism was only observed after at least two days of TCR stimulation. IL-2 levels peaked within 24 hours of TCR stimulation and then decreased over the next 48 hours. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies in the presence or absence of ZM-241385 (ZM) and IL-2 mRNA levels were determined by qPCR at the indicated times. Data represent the means ± SD from a single experiment that is representative of three such experiments. (B) Stimulation of the TCR for three days followed by one hour of ddA treatment leads to potentiation of TCR-stimulated IL-2 mRNA levels by ddA. After three days of TCR stimulation with plate-bound anti-CD3 and soluble anti-CD28, Jurkat cells were treated with ddA for one hour before determination of IL-2 mRNA levels by qPCR. Data represent the mean ± SE from 14 experiments. *** , p < 0.001.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques:

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Model of how the source and context of activated Gα s and cAMP may determine whether they enhance or inhibit TCR-stimulated IL-2 transcription. Interactions between the TCR and peptide-major histocompatibility complex (MHC) lead to recruitment of CD4 and its associated kinase, p56-Lck, which phosphorylates tyrosine residues in the cytoplasmic tails of the TCR subunits, leading to recruitment and phosphorylation of the tyrosine kinase, ZAP-70. CD28 co-stimulation provides an additional signal that is needed for complete T cell activation and regulation of IL-2 production . ZAP-70 and p56-Lck then phosphorylate and activate numerous downstream target proteins, including PLC-γ, leading to Ca 2+ increases and activation of a variety of downstream pathways including translocation of NFAT to the nucleus and activation of IL-2 transcription (black and white pathway). Gα s stimulated by a mechanism that does not involve G s PCRs, but which could potentially involve the TCR, enhances TCR-stimulated IL-2 transcription by a mechanism that may involve binding of pCREB to the CRE site of the IL-2 promoter [ ] during the initial stages of TCR stimulation (green pathway, Stimulatory Step 1). In contrast G s PCRs decrease TCR-stimulated IL-2 transcription, potentially by utilizing both Gα s and Gβγ signaling in cells that have been exposed to at least two days of TCR stimulation (red pathway, Inhibitory Step 2). The inhibitory G s PCR/Gα s /cAMP pathway may involve binding of CREM, which gradually replaces pCREB, to the CRE site of the IL-2 promoter or the formation of NFAT/ICER complexes on NFAT/AP-1 composite sites in the IL-2 promoter , leading to repression of transcription (Inhibitory Step 2). Previous studies suggest that cAMP increases stimulated by the TCR are smaller and more transient than those stimulated by G s PCRs, as depicted by the relative sizes of the cAMP symbols, and this may contribute to the opposite effects on IL-2 transcription. Simultaneously, Gβγ may inhibit TCR-stimulated IL-2 transcription by decreasing TCR-stimulated Ca 2+ increases through Ca v 1 channels (Inhibitory Step 2), which are activated by the TCR by an unknown mechanism . Ca 2+ -calmodulin-activated calcineurin dephosphorylates NFAT, exposing a nuclear localization sequence (NLS) and leading to nuclear translocation.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Immunopeptidomics, Phospho-proteomics, Activation Assay, Translocation Assay, Binding Assay, Sequencing

Journal: Cell reports

Article Title: T Cell-Intrinsic IRF5 Regulates T Cell Signaling, Migration, and Differentiation and Promotes Intestinal Inflammation

doi: 10.1016/j.celrep.2020.107820

Figure Lengend Snippet: (A) CD4+ T cells were activated with anti-CD3/CD28 over 72 h. IRF5 expression by western blot. IRF5−/−CD4+ T cells served as a negative control (representative of 2 independent experiments).

Article Snippet: InVivom Ab anti-human CD28 (9.3) , Bio X Cell , Cat# BE0248.

Techniques: Expressing, Western Blot, Negative Control

Journal: Cell reports

Article Title: T Cell-Intrinsic IRF5 Regulates T Cell Signaling, Migration, and Differentiation and Promotes Intestinal Inflammation

doi: 10.1016/j.celrep.2020.107820

Figure Lengend Snippet: (A and B) IRF5+/+, IRF5+/−, or IRF5−/−CD4+ T cells were activated in vitro with anti-CD3/CD28.

Article Snippet: InVivom Ab anti-human CD28 (9.3) , Bio X Cell , Cat# BE0248.

Techniques: In Vitro

Journal: Cell reports

Article Title: T Cell-Intrinsic IRF5 Regulates T Cell Signaling, Migration, and Differentiation and Promotes Intestinal Inflammation

doi: 10.1016/j.celrep.2020.107820

Figure Lengend Snippet: (A–C) IRF5+/+, IRF5+/−, or IRF5−/−CD4+ T cells were activated with anti-CD3/CD28.

Article Snippet: InVivom Ab anti-human CD28 (9.3) , Bio X Cell , Cat# BE0248.

Techniques:

Journal: Cell reports

Article Title: T Cell-Intrinsic IRF5 Regulates T Cell Signaling, Migration, and Differentiation and Promotes Intestinal Inflammation

doi: 10.1016/j.celrep.2020.107820

Figure Lengend Snippet: (A–C) CD4+ T cells from IRF5+/+ and IRF5−/−mice were stimulated in vitro with anti-CD3/CD28 at the indicated times and phosphorylated proteins were assessed by (A and B) western blot. Representative of 3–4 independent experiments. (C) Flow cytometry. Top: representative flow cytometry with MFI indicated. Bottom: summary phospho-proteins (3 replicates/group).

Article Snippet: InVivom Ab anti-human CD28 (9.3) , Bio X Cell , Cat# BE0248.

Techniques: In Vitro, Western Blot, Flow Cytometry

Journal: Cell reports

Article Title: T Cell-Intrinsic IRF5 Regulates T Cell Signaling, Migration, and Differentiation and Promotes Intestinal Inflammation

doi: 10.1016/j.celrep.2020.107820

Figure Lengend Snippet: CD45RA+ CD4+ T cells were isolated from rs2004640/rs2280714 TT/TT or GG/CC carriers (n = 14/genotype). (A and B) Cells were treated with anti-CD3/CD28 for 72 h.

Article Snippet: InVivom Ab anti-human CD28 (9.3) , Bio X Cell , Cat# BE0248.

Techniques: Isolation

Journal: Cell reports

Article Title: T Cell-Intrinsic IRF5 Regulates T Cell Signaling, Migration, and Differentiation and Promotes Intestinal Inflammation

doi: 10.1016/j.celrep.2020.107820

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: InVivom Ab anti-human CD28 (9.3) , Bio X Cell , Cat# BE0248.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Sonication, DNA Library Preparation, Purification, Activation Assay, cDNA Synthesis, Software