Journal: Cells

Article Title: IL-6 Blockade Enhances the Efficacy of CDK4/6 Inhibitor in BRCA1 -Mutant Triple-Negative Breast Cancer Cells

doi: 10.3390/cells14201602

Figure Lengend Snippet: Abemaciclib inhibited BRCA1 -mutant TNBC cell growth. SUM149, HCC1937, and MDA-MB-436 cells were treated with abemaciclib for 72 h. ( A ) Cell viability was determined by MTT assay. IC50 of abemaciclib was calculated and the IC50 ± SE values were listed at the bottom. ( B ) Cell proliferation was measured by BrdU cell proliferation assay. The drug concentrations used in the BrdU assay were selected based on the IC50 values determined in the MTT assay ( A ) to assess proliferation at biologically relevant doses. Data represents SD from three independent experiments (n = 3). ** p < 0.01, **** p < 0.0001.

Article Snippet: The human BRCA1 -mutant TNBC cell line MDA-MB-436 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA; cat. no. HTB-13), SUM149 and HCC1937 were obtained from the University of Maryland Greenebaum Comprehensive Cancer Center Translational Laboratory (Baltimore, MD, USA).

Techniques: Mutagenesis, MTT Assay, BrdU Cell Proliferation Assay, BrdU Staining

Journal: Cells

Article Title: IL-6 Blockade Enhances the Efficacy of CDK4/6 Inhibitor in BRCA1 -Mutant Triple-Negative Breast Cancer Cells

doi: 10.3390/cells14201602

Figure Lengend Snippet: Bazedoxifene synergizes with abemaciclib in BRCA1 -mutant TNBC cells. Cell viability was measured by MTT assay in SUM149 ( A ), HCC1937 ( B ), and MDA-MB-436 ( C ) cells after treatment with bazedoxifene alone, abemaciclib alone, and the combination for 24 h (performed in triplicate assays). The CI values were reported in the bottom panel. *** p < 0.001, and **** p < 0.0001.

Article Snippet: The human BRCA1 -mutant TNBC cell line MDA-MB-436 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA; cat. no. HTB-13), SUM149 and HCC1937 were obtained from the University of Maryland Greenebaum Comprehensive Cancer Center Translational Laboratory (Baltimore, MD, USA).

Techniques: Mutagenesis, MTT Assay

Journal: Cells

Article Title: IL-6 Blockade Enhances the Efficacy of CDK4/6 Inhibitor in BRCA1 -Mutant Triple-Negative Breast Cancer Cells

doi: 10.3390/cells14201602

Figure Lengend Snippet: GP130 knockdown enhances the sensitivity of abemaciclib in BRCA1 -mutant TNBC cells. ( A ) The knockdown efficiency of GP130 and its downstream P-STAT3 (Y705) and STAT3 were detected by western blot. GAPDH is used as a loading control. Western blot analysis was performed once due to sample limitations. ( B ) Cell viability of GP130 knockdown alone, abemaciclib alone, and the combination treatment was measured by MTT assay (performed in triplicate assays). * p < 0.05, ** p < 0.01, and *** p < 0.001. Original western blot images can be found in the .

Article Snippet: The human BRCA1 -mutant TNBC cell line MDA-MB-436 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA; cat. no. HTB-13), SUM149 and HCC1937 were obtained from the University of Maryland Greenebaum Comprehensive Cancer Center Translational Laboratory (Baltimore, MD, USA).

Techniques: Knockdown, Mutagenesis, Western Blot, Control, MTT Assay

Journal: Cells

Article Title: IL-6 Blockade Enhances the Efficacy of CDK4/6 Inhibitor in BRCA1 -Mutant Triple-Negative Breast Cancer Cells

doi: 10.3390/cells14201602

Figure Lengend Snippet: Significant inhibitory effects of bazedoxifene and abemaciclib combination on the migration of BRCA1 -mutant TNBC cells. Wound healing assay was performed to evaluate cell migration in SUM149 ( A ), HCC1937 ( B ), and MDA-MB-436 ( C ) cells with bazedoxifene alone, abemaciclib alone, and the combination treatment for 24 h. Representative images of scratches with bazedoxifene alone (B15: 15 μM bazedoxifene; B20: 20 μM bazedoxifene), abemaciclib alone (A2.5: 2.5 μM abemaciclib), and the bazedoxifene and abemaciclib (B+A) combination treatment. The scale bar is 20 µM. The statistical analysis of cell invasion was shown in the right panel. Relative migration was quantified by measuring the wound area and statistical analysis was performed on data from triplicate assays. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The human BRCA1 -mutant TNBC cell line MDA-MB-436 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA; cat. no. HTB-13), SUM149 and HCC1937 were obtained from the University of Maryland Greenebaum Comprehensive Cancer Center Translational Laboratory (Baltimore, MD, USA).

Techniques: Migration, Mutagenesis, Wound Healing Assay

Journal: Cells

Article Title: IL-6 Blockade Enhances the Efficacy of CDK4/6 Inhibitor in BRCA1 -Mutant Triple-Negative Breast Cancer Cells

doi: 10.3390/cells14201602

Figure Lengend Snippet: Significant inhibitory effects of bazedoxifene and abemaciclib combination on the invasion of BRCA1 -mutant TNBC cells. Transwell invasion assay was performed in triplicate to evaluate cell invasion. Representative images of invasive cells of SUM149 ( A ), HCC1937 ( B ), and MDA-MB-436 ( C ) with bazedoxifene alone, abemaciclib alone, and the combination treatment for 24 h (B15: 15 μM bazedoxifene; B20: 20 μM bazedoxifene; A1: 1 μM abemaciclib; A2.5: 2.5 μM abemaciclib and bazedoxifene and abemaciclib (B+A) combination treatment). The scale bar is 10 µM. The statistical analysis of cell invasion was shown in the right panel. * p < 0.05, and **** p < 0.0001.

Article Snippet: The human BRCA1 -mutant TNBC cell line MDA-MB-436 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA; cat. no. HTB-13), SUM149 and HCC1937 were obtained from the University of Maryland Greenebaum Comprehensive Cancer Center Translational Laboratory (Baltimore, MD, USA).

Techniques: Mutagenesis, Transwell Invasion Assay

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: eLife

Article Title: Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function

doi: 10.7554/eLife.91392

Figure Lengend Snippet: Immunophenotyping of thymi from Ikzf1 -fl-Foxp3-YFP-Cre or Foxp3-YFP-Cre control mice ( a ). Bisulfite DNA methylation analysis of the Foxp3 CNS2/TSDR region in peripheral regulatory T cells (Treg) and Tconv from Ikzf1 -fl-Foxp3-YFP-Cre or Foxp3-YFP-Cre control mice ( b ). Quantification of total peripheral Treg, CD44 lo cTreg, and CD44 hi eTreg in Ikzf1 -fl-Foxp3-YFP-Cre and Foxp3-YFP-Cre mice ( c ). Bisulfite DNA methylation analysis of the IFNg promoter ( d ) and IFNg intronic enhancer ( e ) regions in peripheral Treg and Tconv from Ikzf1 -fl-Foxp3-YFP-Cre or Foxp3-YFP-Cre control mice.

Article Snippet: Conventional CD4+CD25-negative and CD4+CD25+ Tregs cells were purified from the lymphocytes of wild-type and Ikzf1 -cko mutant mice using Miltenyi Treg isolation kit (cat # 130-091-041).

Techniques: Control, DNA Methylation Assay

Journal: eLife

Article Title: Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function

doi: 10.7554/eLife.91392

Figure Lengend Snippet: Example histograms ( a ) and quantified expression ( b ) of Ikaros protein by peripheral Treg and Tconv from Foxp3-YFP-Cre (green) and Ikzf1 -fl-Foxp3-YFP-Cre (red) mice (n=6 mice per group). ( c ) Expression of CD25, ICOS, GITR, and PD1 by wild-type (WT) (green) and Ikzf1 -cko (red) Treg (n=6 mice per group). ( d ) Flow cytometric measurement of Foxp3, Aiolos, Eos, and Helios protein expression in WT and Ikzf1 -cko Treg (n=6 mice per group). ( e ) Transcriptomic analysis of WT vs. Ikzf1 -cko Treg gene expression. Top differentially expressed genes (FDR <0.05) organized into eight clusters in ex vivo or in vitro stimulated WT and Ikzf1 -cko Treg. The heatmap represents scaled transcripts per million (tpm, n=3 replicates per group).

Article Snippet: Conventional CD4+CD25-negative and CD4+CD25+ Tregs cells were purified from the lymphocytes of wild-type and Ikzf1 -cko mutant mice using Miltenyi Treg isolation kit (cat # 130-091-041).

Techniques: Expressing, Gene Expression, Ex Vivo, In Vitro

Journal: eLife

Article Title: Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function

doi: 10.7554/eLife.91392

Figure Lengend Snippet: ( a ) Known negative regulators of Treg function up-regulated in ex vivo Ikzf1-cko Treg, ( b ) positive regulators of Treg function down-regulated in ex vivo Ikzf1-cko Treg, ( c ) negative regulators of Treg function up-regulated in in vitro stimulated Ikzf1-cko Treg, ( d ) positive regulators of Treg function down-regulated in in vitro stimulated Ikzf1-cko Treg. See for all differential genes and for a larger list of functionally relevant differentially expressed genes.

Article Snippet: Conventional CD4+CD25-negative and CD4+CD25+ Tregs cells were purified from the lymphocytes of wild-type and Ikzf1 -cko mutant mice using Miltenyi Treg isolation kit (cat # 130-091-041).

Techniques: Ex Vivo, In Vitro

Journal: eLife

Article Title: Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function

doi: 10.7554/eLife.91392

Figure Lengend Snippet: ( a ) Secretion of IL-2 and IFNg protein by WT and Ikzf1 -cko Treg measured by ELISA (n=3). ( b ) IL-2, IFNg, and TNFa production by Treg from WT mice vs. mice with a dominant-negative form of Ikaros (IkDN) measured by ELISA (n=3). ( c ) IL-2-induced phosphorylation of STAT5 in WT and Ikzf1 -cko Treg measured by flow cytometry in vitro. Mean fluorescence intensity (MFI) and individual histograms (inset, n=3) are depicted. ( d ) Activation-induced proliferation of WT (closed) and Ikzf1 -cko (open) Treg measured by dye dilution (n=3). ( e ) Gene ontology analysis of genes down-regulated (top panel) and up-regulated (bottom panel) in Ikzf1 -cko compared to WT Treg. The x-axis is fold enrichment and numbers to the right are unique genes in each pathway. ( f ) Differential expression of core Treg, nTreg, aTreg, cTreg, and eTreg genes in ex vivo (left panel) and in vitro stimulated (right panel) WT and Ikzf1 -cko Treg. The heatmap represents z-score and genes significantly differentially expressed are shown in red.

Article Snippet: Conventional CD4+CD25-negative and CD4+CD25+ Tregs cells were purified from the lymphocytes of wild-type and Ikzf1 -cko mutant mice using Miltenyi Treg isolation kit (cat # 130-091-041).

Techniques: Enzyme-linked Immunosorbent Assay, Dominant Negative Mutation, Phospho-proteomics, Flow Cytometry, In Vitro, Fluorescence, Activation Assay, Quantitative Proteomics, Ex Vivo

Journal: eLife

Article Title: Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function

doi: 10.7554/eLife.91392

Figure Lengend Snippet: ( a ) Expression of B-catenin by wild-type (WT) (left histogram) vs. Ikzf1 -cko (right histogram) Treg measured by flow cytometry (plot depicts B-catenin mean fluorescence intensity (MFI) from n=3 experiments). ( b ) IFNg secretion by WT (top panels) vs. Ikzf1 -cko (bottom panels) Treg activated with (right panels) or without (left panels) the Wnt pathway inhibitor PKF. ( c ) IFNg secretion (measured by ELISA) by Ikzf1-cko Treg, but not by conventional T cells, is inhibited in a dose-dependent manner by PKF (n=3).

Article Snippet: Conventional CD4+CD25-negative and CD4+CD25+ Tregs cells were purified from the lymphocytes of wild-type and Ikzf1 -cko mutant mice using Miltenyi Treg isolation kit (cat # 130-091-041).

Techniques: Expressing, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function

doi: 10.7554/eLife.91392

Figure Lengend Snippet: Assessment of wild-type (WT) and Ikzf1 -cko ex vivo and stimulated regulatory T cells (Treg) library reproducibility by principal component ( a ) and spearman correlation ( b ) analysis. ( c ) Hierarchical clustering of 12,030 differentially accessible regions (DAR, FDR <0.05, abs[logFC]>1) into seven groups from the root. Red vs. blue indicate increased vs. decreased accessibility. ( d ) Enrichment of GO terms for genes nearest to differential accessibility (DAR) (FDR <0.05,>5 genes). ( e ) Association of DAR accessibility changes with changes in expression of the nearest gene. Enrichment is over the mean increase or decrease in expression, indicated by red vs. blue.

Article Snippet: Conventional CD4+CD25-negative and CD4+CD25+ Tregs cells were purified from the lymphocytes of wild-type and Ikzf1 -cko mutant mice using Miltenyi Treg isolation kit (cat # 130-091-041).

Techniques: Ex Vivo, Expressing

Journal: eLife

Article Title: Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function

doi: 10.7554/eLife.91392

Figure Lengend Snippet: Assessment of WT and Ikzf1 -cko H3K27ac ChIP-seq library reproducibility by principal component ( a ) and pairwise spearman correlation ( b ) analysis. Overlap ( c ) and Pearson correlation between ATAC-seq and H3K27ac ChIP-seq peaks and H3K27ac and gene expression ( d ). Identification of super-enhancer regions based on H3K27ac density ( e ). The Venn diagram (inset) depicts the number of super-enhancers shared between WT and Ikzf1 -cko Treg (orange), unique to WT Treg (green), and unique to Ikzf1 -cko Treg (red).

Article Snippet: Conventional CD4+CD25-negative and CD4+CD25+ Tregs cells were purified from the lymphocytes of wild-type and Ikzf1 -cko mutant mice using Miltenyi Treg isolation kit (cat # 130-091-041).

Techniques: ChIP-sequencing, Gene Expression

Journal: eLife

Article Title: Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function

doi: 10.7554/eLife.91392

Figure Lengend Snippet: Differential analysis of open chromatin ( a ) and H3K27ac ( b ) in WT vs. Ikzf1 -cko Treg (FDR <0.05, n=3). Peaks with FC >2 are depicted in red. ( c ) Ikaros ChIP-seq signal (read density) at genomic regions shared (panel 2) in Tconv (blue) vs. Treg (red), unique to Tconv (panel 3), or unique to Treg (panel 4). Panel 1 depicts read densities at the same regions in control input libraries. ( d ) Unique vs. shared Ikaros binding sites in Treg (red) vs. Tconv (green) open chromatin. ( e ) Enrichment of Ikaros ChIP-seq signal (footprint, solid lines) or input background (transparent lines) at accessible Ikaros motifs (AGGAA) in WT Treg (blue) and Tconv (red). ( f ) Expression (tpm) of genes with open chromatin enriched for the Ikaros consensus binding motif in WT vs. Ikzf1 -cko Treg. ( g ) Enrichment of TF consensus binding motifs in genomic regions that are more accessible in Ikzf1 -cko Treg. Inset depicts motifs for Ikaros/Ikzf1, Stat, Nfat, Rbpj, Rela, and AP-1. ( h ) Open chromatin (top tracks) and H3K27ac (bottom tracks) in WT (blue) and Ikzf1 -cko (red) Treg, Ikaros binding sites (purple marks) and Foxp3 binding sites (red marks) in WT Treg, and Foxp3 binding sites in Ikzf1 -cko Treg (green marks) at the Bcl6 locus. ( i ) Enrichment of TF consensus binding motifs in genomic regions that are less accessible in Ikzf1 -cko Treg. Inset depicts motifs for forkhead family members. In ( g ) and ( i ), factors with roles in Treg function are colored green and purple, forkhead family members are colored red, and factors differentially expressed in Ikzf1 -cko Treg are indicated with an asterisk. ATAC-seq was performed on Treg purified directly ex vivo, while ChIP-seq analyses were performed on Treg expanded in vivo using IL-2/anti-IL-2 complexes.

Article Snippet: Conventional CD4+CD25-negative and CD4+CD25+ Tregs cells were purified from the lymphocytes of wild-type and Ikzf1 -cko mutant mice using Miltenyi Treg isolation kit (cat # 130-091-041).

Techniques: ChIP-sequencing, Control, Binding Assay, Expressing, Purification, Ex Vivo, In Vivo

Journal: eLife

Article Title: Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function

doi: 10.7554/eLife.91392

Figure Lengend Snippet: ( a–c ) Ikaros-dependent changes in accessibility, enhancer signatures, and Foxp3 occupancy at genes differentially expressed in Ikzf1 -cko regulatory T cells (Treg). Open chromatin (top tracks) and H3K27ac (bottom tracks) in wild-type (WT) (blue) and Ikzf1 -cko (red) Treg, Ikaros binding sites (purple marks) and Foxp3 binding sites (red marks) in WT Treg, and Foxp3 binding sites in Ikzf1 -cko Treg (green marks) at the Ifng ( a ) and Notch2 ( b ) and Irf4 ( c ) loci. ( d ) Overlap between Ikaros ChIP-seq peaks and differentially acetylated regions is indicated in red in the pie charts. ( e ) Differential H3K27ac (z-score) at top Ikaros-bound DEG in ex vivo and stimulated WT and Ikzf1 -cko Treg.

Article Snippet: Conventional CD4+CD25-negative and CD4+CD25+ Tregs cells were purified from the lymphocytes of wild-type and Ikzf1 -cko mutant mice using Miltenyi Treg isolation kit (cat # 130-091-041).

Techniques: Binding Assay, ChIP-sequencing, Ex Vivo

Journal: eLife

Article Title: Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function

doi: 10.7554/eLife.91392

Figure Lengend Snippet: ( a ) Input vs. Foxp3 ChIP-seq at genomic regions shared or unique in wild-type (WT) (blue) vs. Ikzf1 -cko (red) Treg (n=3 per group). ( b ) Foxp3- (green), Ikaros- (red), and Foxp3-Ikaros co-bound (orange) open chromatin regions (OCR). Inset depicts Foxp3-Ikaros co-bound regions in WT (green) vs. Ikzf1 -cko (red) Treg. ( c ) Foxp3 binding sites in WT (green) vs. Ikzf1 -cko (red) Treg. ( d ) Expression (tpm) of genes enriched for accessible Foxp3 consensus motifs in WT vs. Ikzf1 -cko Treg. ( e ) 293T cells transfected with FLAG-tagged full-length Ikaros (Ik1), DNA-binding mutant Ikaros (Ik7), or Runx1 alone (lanes 1–4) or in combination with untagged Foxp3 (lanes 5–7). Whole extracts (panels 1–2) or Foxp3-immunoprecipitated extracts (panels 3–4) probed for Foxp3 or FLAG. ( f ) IL-2 or IFNg production (left panel) and Foxp3 ChIP-qPCR at Il2 promoter (right panel) in Tconv transduced with vector, Foxp3, Ik7/DN, or Foxp3 + Ik7/DN. ( g ) Concordant vs. discordant genes co-regulated in Ikzf1 -cko vs. IkDN Treg (odds ratio = 5.19, P -value = 2.2 × 10 –16 ). ( h, i ) Open chromatin (top) and H3K27ac (bottom) in WT (blue) and Ikzf1 -cko (red) Treg, Ikaros binding (purple marks), and Foxp3 binding (red marks) in WT Treg, and Foxp3 binding in Ikzf1 -cko Treg (green marks) at Il2 ( h ) and Tcf7 ( i ). Figure 6—source data 1. Immunoblot analysis of Foxp3-Ikaros co-precipitation in transfected HEK293T cells.

Article Snippet: Conventional CD4+CD25-negative and CD4+CD25+ Tregs cells were purified from the lymphocytes of wild-type and Ikzf1 -cko mutant mice using Miltenyi Treg isolation kit (cat # 130-091-041).

Techniques: ChIP-sequencing, Binding Assay, Expressing, Transfection, Mutagenesis, Immunoprecipitation, ChIP-qPCR, Transduction, Plasmid Preparation, Western Blot

Journal: eLife

Article Title: Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function

doi: 10.7554/eLife.91392

Figure Lengend Snippet: Assessment of wild-type (WT) and Ikzf1 -cko library reproducibility by principal component ( a ) and pairwise spearman correlation ( b ) analysis. ( c ) Strand cross-correlation analysis of FoxP3 ChIP-seq signal vs. noise corresponding to the broad peak (red) and phantom peak (blue; mappability bias). ( d ) Differential H3K27ac (z-score) at top Foxp3-bound differential expression gene (DEG) in ex vivo and stimulated WT and Ikzf1 -cko Treg. ( e ) Ikaros-dependent changes in accessibility, enhancer signatures, and Foxp3 occupancy at the Il2ra, Rbpj, and Maml3 genes differentially expressed in Ikzf1 -cko Treg. Open chromatin (top tracks) and H3K27ac (bottom tracks) in WT (blue) and Ikzf1 -cko (red) Treg, Ikaros binding sites (purple marks) and Foxp3 binding sites (red marks) in WT Treg, and Foxp3 binding sites in Ikzf1 -cko Treg (green marks).

Article Snippet: Conventional CD4+CD25-negative and CD4+CD25+ Tregs cells were purified from the lymphocytes of wild-type and Ikzf1 -cko mutant mice using Miltenyi Treg isolation kit (cat # 130-091-041).

Techniques: ChIP-sequencing, Quantitative Proteomics, Ex Vivo, Binding Assay

Journal: eLife

Article Title: Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function

doi: 10.7554/eLife.91392

Figure Lengend Snippet: Frequencies of total ( a ), memory ( b, c ), and naïve ( d ) phenotype Tconv in secondary lymphoid tissues of 6–8 week-old Ikzf1 -fl-Foxp3-YFP-Cre (purple) and Foxp3-YFP-Cre (green) mice (n=7). ( e ) In vitro suppressive activity of wild-type (WT) (blue) vs. Ikzf1 -deficient (orange) Treg against Tconv from WT mice. ( f ) In vitro suppressive activity of WT Treg against Tconv from WT mice (blue) vs. Ikzf1 -deficient Treg against Tconv from Ikzf1 -cko mice (orange). ( g ) In vitro suppressive activity of WT Treg against Tconv from WT (blue) vs. Ikzf1 -deficient mice (orange). Tconv proliferation was measured by dye dilution in all cultures (n=3). ( h ) Frequencies of Foxp3+PD1 hi CXCR5 hi follicular regulatory T cells (Tfr) and PD1 hi CXCR5 hi follicular helper T cells (Tfh) in secondary lymphoid tissues of 6-month-old Ikzf1 -fl-Foxp3-YFP-Cre (purple) and Foxp3-YFP-Cre (green) mice (n=7). ( i ) Total serum levels of IgM, IgG, and IgA from Ikzf1 -fl-Foxp3-YFP-Cre vs. Foxp3-YFP-Cre mice (n=4). Frequency of IgA-positive B cells and surface density (MFI) of IgA-positive B cells in spleen ( j ) and mesenteric lymph nodes ( k ) from 9-month-old WT (green) vs. Ikzf1 -deficient (red) mice (N=3). p-values are indicated for significant differences.

Article Snippet: Conventional CD4+CD25-negative and CD4+CD25+ Tregs cells were purified from the lymphocytes of wild-type and Ikzf1 -cko mutant mice using Miltenyi Treg isolation kit (cat # 130-091-041).

Techniques: In Vitro, Activity Assay

Journal: eLife

Article Title: Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function

doi: 10.7554/eLife.91392

Figure Lengend Snippet: Wild-type (WT) CD4 +CD25- Tconv were transferred alone (red), or together with WT (green) or Ikzf1 -cko (blue) CD4 +CD25+Treg into RAG1ko mice (n=5). Animal weight was monitored for 40 days ( a ), and intestines were scored for pathology at the gross and histologic levels ( b ). Example histopathology of colons from RAG1ko recipients of WT Tconv ( c ), WT Tconv+WT Treg ( d ), and WT Tconv+ Ikzf1 cko Treg ( e ). Hematoxylin and eosin (H&E) (top row), CD4 (middle row), and Foxp3 (bottom row) staining are shown at 200x. Scale bar = 100 µm. Mean Foxp3 + cells per 200 X field from n=3 animals is 3.4 in ( d ) and 22 in ( e ), p <0.05.

Article Snippet: Conventional CD4+CD25-negative and CD4+CD25+ Tregs cells were purified from the lymphocytes of wild-type and Ikzf1 -cko mutant mice using Miltenyi Treg isolation kit (cat # 130-091-041).

Techniques: Histopathology, Staining

Journal: eLife

Article Title: Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function

doi: 10.7554/eLife.91392

Figure Lengend Snippet: Spleens and colons from Rag1ko recipients of wild-type (WT) Tconv ( a ), WT Tconv plus WT Treg ( b ) and WT Tconv plus Ikzf1 -cko Treg ( c ) harvested at day 40 post-transfer. Quantitation of total CD4 + T cells ( d, e ), CD4+CD44+ effector Tconv ( f, g ), and CD4+Foxp3+ Treg ( h, i ) in the spleens ( d, f, h ) and mesenteric lymph nodes ( e, g, i ) from Rag1ko recipients of WT Tconv (orange), WT Tconv plus WT Treg (blue) and WT Tconv plus Ikzf1 -cko Treg (green) harvested at day 40 post-transfer. Role of Ikaros in Treg-dependent acquired cardiac transplant tolerance. ( j ) B6 Ikzf1 -fl-Foxp3-YFP-Cre (red) or Foxp3-YFP-Cre (black) mice received BALB/c cardiac allografts, donor-specific transfusion (DST), and anti-CD40L. Graft survival was monitored for 100 days. ( k ) Histopathological analysis of cardiac grafts harvested at day 14 post-transplant from Foxp3-YFP-Cre and Ikzf1 -fl-Foxp3-YFP-Cre recipients (n=3, scale = 200 x, scale bar = 100 µm).

Article Snippet: Conventional CD4+CD25-negative and CD4+CD25+ Tregs cells were purified from the lymphocytes of wild-type and Ikzf1 -cko mutant mice using Miltenyi Treg isolation kit (cat # 130-091-041).

Techniques: Quantitation Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Armored TGFβRIIDN ROR1-CAR T cells reject solid tumors and resist suppression by constitutively-expressed and treatment-induced TGFβ1

doi: 10.1136/jitc-2023-008261

Figure Lengend Snippet: CAR-1, but not CAR-2, suppressed tumor progression in the orthotopic OVCAR-3 mouse xenograft model of ovarian cancer, and exhibited higher cytokine response to solid tumor lines in vitro. (A) Quantification of ROR1 expression on the surface of various solid tumor cancer cell lines; the experiment was performed in duplicates employing anti-ROR1 Ab from BD Biosciences; a separate experiment was also performed in duplicates using anti-ROR-1 Abs from Miltenyi Biotec and R&D Systems with similar results. (B) Representative killing curves of CAR T cells against various solid cancer cell lines (OVCAR-3, Capan-2, and NCI-H226) in an 18 hours co-culture. (C) Cytokine production from the experiments in (B) was quantified by ELISA, pooled results from three independent donors are shown, mean±SEM. Experimental groups were compared by mixed effect model (D–I): Efficacy of CAR T cells in in vivo ovarian cancer OVCAR-3 xenograft model: NSG mice (five mice/group) were implanted (i.p.) with OVCAR-3 cell line (10e6 cells/mouse) at day −7, followed by staging at day −1; CAR T cells (5e6 CAR + T cells/mouse) were administered (i.v.) at day 0 (D); tumor progression was quantified by bioluminescence imaging; groups were compared by mixed effect analysis with Tukey’s post hoc test. (E, F) Body weight was monitored (G); blood was sampled at the indicated time points to quantify CAR + T cells in both CD8 + and CD4 + subpopulations (H) as well as memory T cells (I). Groups were compared by two way analysis of variance with Sidak’s post hoc test (H) or Student’s t-test (I). All results are presented as mean±SEM; statistical significance is denoted as *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, ns-non-significant. Ab, antibody; BLI, bioluminescence; CAR, chimeric antigen receptor; IFN, interferon; IL, interleukin; i.p., intraperitoneal; i.v., intravenous; ROR1, receptor tyrosine kinase-like orphan receptor 1; T EM , efector memeory T cells; T CM - central memory T cells; TNF, tumor necrosis factor; UTD, un-transduced T cells.

Article Snippet: The human CD4 + and CD8 + T cells were purified from buffy coats via positive selection using a 1:1 ratio of CD4-MicroBeads and CD8-MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol resulting in a mixture of enriched CD4 + and CD8 + T cells.

Techniques: In Vitro, Expressing, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, In Vivo, Imaging

Journal: Journal for Immunotherapy of Cancer

Article Title: Armored TGFβRIIDN ROR1-CAR T cells reject solid tumors and resist suppression by constitutively-expressed and treatment-induced TGFβ1

doi: 10.1136/jitc-2023-008261

Figure Lengend Snippet: Dominant negative TGFβRII (DN) obstructed TGFβ1 signaling in T cells transduced with CAR-1 and reduced the inhibitory effect of TGFβ1 on CAR T cells’ cytotoxic activity against pancreatic cancer cell line AsPC-1 in vitro. (A) schematic diagram of constructs of CAR-1 alone and CAR-1 armored with DN. (B) At day 8 of transduction, CAR expression (left: flow plots, center: graph from the flow plots) and memory phenotype (right) of both CD8 + and CD4 + T cells transduced with CAR-1 or CAR-1+DN were analyzed by flow cytometry; three independent experiments were performed, employing three donors, with similar results. (C) Expression of TGFβRII in T cells transduced with CAR-1 or CAR-1+DN was assessed by flow cytometry; three independent experiments were performed, employing three donors, with similar results. (D) CAR T cells were IL-2 starved for 22 hours to synchronize the cells followed by treatment with TGFβ1 (10 ng/mL) for 5, 15, or 30 min; cells were then stained with pSmad2/3 and subject to flow cytometry analysis; left panel: flow plots: right panel: graph from the plots in the left panel (results are the mean±SE of three donors). (E) Expression of ROR1 on AsPC-1 cell line was assessed by flow cytometry. (F) AsPC-1 was co-cultured with CAR T cells without or with TGFβ1 (1 or 10 ng/mL); tumor cell lysis was measured by xCELLigence; left: % cytolysis (results are representative of three donors); right: cytotoxic relative potency of CAR T cells treated with TGFβ1 versus non-treatment (results are the mean±SE of three donors). Mean±SE of three donors. Two-way analysis of variance with Sidak’s post hoc test *p<0.05; ***p<0.001; **p<0.01; ***p<0.001. (G) Cytokine production from the experiments in (F) was quantified by ELISA; results are the mean±SE of three donors. (H,I) Production of TGFβ1 either in active or latent form by various solid tumor cell lines (H) or by AsPC-1 ectopically overexpressing TGFβ1 (I) was assessed by ELISA; data are representative of two independent experiments with similar results. (J) AsPC-1 overexpressing TGFβ1 (AsPC-1/TGFβ1) or AsPC-1 control was co-cultured with CAR T cells, % cytolysis of tumor cells was shown, results are the representative of three donors. (K) Cytokine production from the experiments in (J) was quantified by ELISA. Mean±SE of three donors; Student’s t-tests; statistical significance is denoted as *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, ns-non-significant. CAR, chimeric antigen receptor; IFN, interferon; IL, interleukin; TGF, transforming growth factor; TNF, tumor necrosis factor; UTD, un-transduced T cells.

Article Snippet: The human CD4 + and CD8 + T cells were purified from buffy coats via positive selection using a 1:1 ratio of CD4-MicroBeads and CD8-MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol resulting in a mixture of enriched CD4 + and CD8 + T cells.

Techniques: Dominant Negative Mutation, Transduction, Activity Assay, In Vitro, Construct, Expressing, Flow Cytometry, Staining, Cell Culture, Lysis, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Armored TGFβRIIDN ROR1-CAR T cells reject solid tumors and resist suppression by constitutively-expressed and treatment-induced TGFβ1

doi: 10.1136/jitc-2023-008261

Figure Lengend Snippet: TGFβRIIDN attenuated the inhibitory effect of TGFβ1 in AsPC-1 xenograft model of pancreatic cancer overexpressing TGFβ1. NSG mice (five mice/group) were implanted subcutaneously with AsPC-1/TGFβ1 cells (1e6 cells/mouse) at day −15, followed by staging and CAR T-cell infusion (i.v., 5e6 CAR + T cells/mouse) at day 0 (A) and tumor volumes were monitored (B–C). Statistical significance determined by Mann-Whitney test (B) and Wilcoxon matched—pairs signed-rank test (C) T cells isolated from peripheral blood at the indicated time points were quantified by flow cytometry for total cell number (D) or CAR + components in both CD8 + and CD4 + subpopulations (E) results were analyzed by Mann-Whitney test. Blood from mice sampled at day 5 and day 15 post T-cell infusion was quantified for TGFβ1, groups were compared by Student’s t-test (F). Tumor tissues collected at day 7 post T-cell infusion and at study termination were subjected to immunohistochemistry staining for TGFβ1, CD3, and IgG control (G); the number of ROR1 + cells was quantified by MACSima imaging cycling staining high content immunofluorescent microscopy (H–J); tissues were probed for CD8a (T cells), ROR1 (CAR-targeted tumor antigen), TGFβ and alpha-smooth muscle actin (myofibroblasts, stroma) with DAPI counterstaining for nuclei (H); T cell-rich, T cell-intermediate and T cell-low regions of tumors were evaluated; an example of higher magnification of these regions is shown in (I); and the quantification of ROR1 + cells from each of these regions (n=3 each) is shown in violin plots representing cell number distribution in each group (J); statistical analysis was performed by two-way analysis of variance with Sidak’s post hoc test. All results are presented as mean±SEM; statistical significance is denoted as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. BLI, bioluminescene; CAR, chimeric antigen receptor; DAPI, 4',6-diamidino-2-phenylindole; DN, dominant negative; i.v., intravenous; ROR1, receptor tyrosine kinase-like orphan receptor 1; s.c., subcutaneous; SMA, smooth muscle actin; TGF, transforming growth factor; UTD, un-transduced T cells.

Article Snippet: The human CD4 + and CD8 + T cells were purified from buffy coats via positive selection using a 1:1 ratio of CD4-MicroBeads and CD8-MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol resulting in a mixture of enriched CD4 + and CD8 + T cells.

Techniques: MANN-WHITNEY, Isolation, Flow Cytometry, Immunohistochemistry, Staining, Control, Imaging, Microscopy, Dominant Negative Mutation