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

Article Title: Naïve CD4 + T cells carrying a TLR2 agonist overcome TGF–β-mediated tumor immune evasion

doi: 10.4049/jimmunol.1700396

Figure Lengend Snippet: (A) Indicated naïve untreated CFSE labeled CD4+ T cells were co-cultured with 0.1 μg/ml CD3ε Abs and TLR2−/− APCs in the absence (I) or presence (II) of Th1 polarization or (III) with Th1 polarization and 10 μg/ml of Pam3Cys4 for 96 hours. (IV) Indicated naïve CD4+ TPam3 cells were stimulated as in (II). The corresponding CFSE dilution and IFN-γ accumulation are shown as a percent value in the inset. (B) Naïve untreated OT2 were co-cultured with OVA peptide (OVAp) bearing TLR2−/− APCs in the absence (I) or presence (II) of Th1 polarization or (III) with Th1 polarization and 10 μg/ml of Pam3Cys4 for 96 hours and assessed for IFN-γ expression. (IV) Naïve OT2Pam3 cells were stimulated as in (II). (C) Naïve human CD4+ T cells with or without Pam3Cys4 cargo were stimulated under Th1 polarizing conditions with CD3ε Ab-conjugated beads for 96 hours and assessed for IFN-γ expression. (A–C) are representative results from at least three independent experiments.

Article Snippet: T h 1 polarization for human CD4 + T cells was accomplished with CellXVivo Human Th1 Cell Differentiation Kit (R&D Systems) in accordance with manufacturers recommendations.

Techniques: Labeling, Cell Culture, Expressing

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

Article Title: Naïve CD4 + T cells carrying a TLR2 agonist overcome TGF–β-mediated tumor immune evasion

doi: 10.4049/jimmunol.1700396

Figure Lengend Snippet: (A) Naïve B6 CD4+ T, TLR2−/− CD4+ T and OT2 cells with or without Pam3Cys4 cargo were stimulated with TLR2-deficient splenocyte-derived APCs bearing CD3ε Abs (B6 or TLR2−/− CD4+ T cells) or OVAp (OT2 cells) in the presence of IL-12 and indicated amounts of TGF-β1. After 72 hours culture supernatants were assessed for IFN-γ by ELISA. Data are representative of at least four independent experiments. (B) Naïve untreated OT2 and OT2Pam3 cells were stimulated with TLR2−/− APCs bearing OVAp cultured under Th1 polarization, 0.5 ng/ml TGF-β1 or Th1 polarization + 0.5 ng/ml TGF-β1. After 5 days of culture OT2 cells were evaluated for the percent abundance of Foxp3, T-bet and IFN-γ expression. Data shown is a representative result of three independent experiments. (C) Naïve untreated OT2 and OT2Pam3 cells were stimulated as in (A) and then fractionated from 72 hrs later, mixed at indicated ratios with responder CFSE labeled responder OT1 cells and re-cultured in triplicate wells with TLR2−/− APCs bearing peptides specific for OT2 and OT1. (Upper panel) Representative CFSE dilution plots of OT2 regulators were mixed with OT1 responders at a 1:1 ratio. (Lower panel) Mean CFSE dilution ± S.E of responders over indicated regulator to responder ratios (**p< 0.01). The result shown is representative of three independent experiments.

Article Snippet: T h 1 polarization for human CD4 + T cells was accomplished with CellXVivo Human Th1 Cell Differentiation Kit (R&D Systems) in accordance with manufacturers recommendations.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing, Labeling

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

Article Title: Naïve CD4 + T cells carrying a TLR2 agonist overcome TGF–β-mediated tumor immune evasion

doi: 10.4049/jimmunol.1700396

Figure Lengend Snippet: (A) Indicated naïve CD4+ T cells unactivated (black line) or activated for 10 mins with plate bound 0.1μg/ml CD3ε Ab and 1 μg/ml soluble CD28 Ab with (red line) or without (blue line) Pam3Cys4 cargo were evaluated for p-AktSer473. Left histograms are a representative result from 5 experiments. In the right panel data is shown as a individual and mean relative MFI calculated as the ratio of the MFI with Pam3Csy4 cargo to the MFI of untreated CD4+ cells ± S.D. where **p < 0.01. (B) B6 CD4+ TPam3 left untreated and unstimulated (black line) or pretreated for 30 mins with either indicated concentrations of Ly2924002 (green lines) or DMSO vehicle (red line) and then TCR stimulated as in (A) and assessed for p-AktSer473. The result is representative of 2 independent experiments. (C) B6 CD4+ T and B6 CD4+ TPam3 cells were pretreated Ly2924002 as in (B) and activated as in (A) under Th1 polarization & 0.5 ng/ml TGF-β1 and assessed for indicated transcripts 36 hrs after culture by semiquantitative real time RT-PCR. Data is normalized to transcript levels in freshly isolated untreated naïve B6 CD4+ T cells and the result shown is representative of 3 independent experiments. (D) Indicated naïve CD4+ T cells were pretreated with DMSO vehicle or 0.3 μM of Ly2924002 for 30 mins and stimulated as in (C) for 72 hours and assessed for percent abundance of Foxp3 and T-bet expression. Data are representative of 3 independent experiments.

Article Snippet: T h 1 polarization for human CD4 + T cells was accomplished with CellXVivo Human Th1 Cell Differentiation Kit (R&D Systems) in accordance with manufacturers recommendations.

Techniques: Quantitative RT-PCR, Isolation, Expressing

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

Article Title: Naïve CD4 + T cells carrying a TLR2 agonist overcome TGF–β-mediated tumor immune evasion

doi: 10.4049/jimmunol.1700396

Figure Lengend Snippet: Both TCR/CD28 engagement and TLR2-MyD88 signaling stimulates PI3 kinase-mediated Akt activation as measured by phosphorylation at serine 473. Akt activation induces mTORC1 to promote T-bet activation to stimulate Th1 lineage determination. TGF-β–signaling leads to SMAD2/3 phosphorylation, which in turn drives Foxo1/Foxo3a mediated transcription of Foxp3 to specify iTreg development. SMAD3 and Foxo1/Foxo3a activity are antagonized by Akt activation. TCR/CD28 activated CD4+ TPam3 cells exposed to a TGF-β + Th1 cytokine environment (e.g., IL-12) favor Th1 over iTreg lineage determination due to additional Akt activation contributed by TLR2-MyD88 dependent PI3 kinase activation.

Article Snippet: T h 1 polarization for human CD4 + T cells was accomplished with CellXVivo Human Th1 Cell Differentiation Kit (R&D Systems) in accordance with manufacturers recommendations.

Techniques: Activation Assay, Phospho-proteomics, Activity Assay

Journal: International journal of molecular sciences

Article Title: Effect of Airborne Particulate Matter on the Immunologic Characteristics of Chronic Rhinosinusitis with Nasal Polyps.

doi: 10.3390/ijms23031018

Figure Lengend Snippet: Figure 5. The pivotal effect of PM10-induced IL-33 for Th2 differentiation effects of conditioned media on PM10-induced human nasal polyp derived fibroblast (NPDFs-CM) on the inflammatory cytokine production in CD4+ T cells. (A) Th2 differentiation was determined by the expression of GATA3 and ST2 (IL-33 receptor). (B,C) The protein levels of IFN-γ, IL-17A, IL-4, IL-13, and IL-10 in CD4+ T cell populations were determined by flow cytometry according to the addition of ST2. The graphic data represents the means ± SEM (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.0001 as compared to the PM10-treated group.

Article Snippet: TMCs (1 × 106 cells) were cultured with 1 μg/mL of phytohemagglutinin (PHA) in NPDF-CM for 72 h. For Th2 differentiation, CD4+ T cells from TMCs were purified using a CD4+ T Cell Isolation Kit MicroBeads (Miltenyi Biotech, Bisley, Surrey, UK), and purified CD4+ T cells were differentiated using a CellXVivo Human Th2 Cell Differentiation Kit (RnDSystems, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Derivative Assay, Expressing, Cytometry