Journal: Oncoimmunology

Article Title: CD4 + T cells potently induce epithelial-mesenchymal-transition in premalignant and malignant pancreatic ductal epithelial cells–novel implications of CD4 + T cells in pancreatic cancer development

doi: 10.1080/2162402X.2014.1000083

Figure Lengend Snippet: H6c7 cells acquire a spindle-shaped morphology in the presence of activated CD4+ T-effs and T-regs. Representative images (n = 7 experiments with T cells isolated from seven individual donors) showing cell morphology of H6c7 cells monocultured (mono) or directly co-cultured with T-effs (co T-effs) or T-regs (co T-regs) for 72 h, either in the absence (w/o) or presence of activation beads (+ beads). Magnification x 400.

Article Snippet: After 24 h, medium was removed and 1×10 4 freshly isolated T-regs or T-effs, diluted in 125 μL X-Vivo medium/well (Lonza, BE04-418F) were added, either in the absence or presence of 5 × 10 3 T cell activation/expansion beads (Miltenyi Biotec, 130-091-441) which had been prepared according to the manufacturer's instructions.

Techniques: Isolation, Cell Culture, Activation Assay

Journal: Oncoimmunology

Article Title: CD4 + T cells potently induce epithelial-mesenchymal-transition in premalignant and malignant pancreatic ductal epithelial cells–novel implications of CD4 + T cells in pancreatic cancer development

doi: 10.1080/2162402X.2014.1000083

Figure Lengend Snippet: An altered EMT marker expression predominates in H6c7 cells when co-cultured with activated CD4+ T-effs. H6c7 cells were monocultured (mono) or directly co-cultured with T-effs (co T-effs) or T-regs (co T-regs) for 72 h, either in the absence (w/o) or presence of activation beads (+ beads). E-cadherin, L1CAM, vimentin, ZEB-1, Snail and Slug expression were analyzed by RT-qPCR. TBP was used as house-keeping gene for control. Data represent the median values with quartiles (Q0,75 as upper, Q0.25 as lower deviation) of 5–7 independent experiments with T cells isolated from seven individual donors. * = p < 0.05.

Article Snippet: After 24 h, medium was removed and 1×10 4 freshly isolated T-regs or T-effs, diluted in 125 μL X-Vivo medium/well (Lonza, BE04-418F) were added, either in the absence or presence of 5 × 10 3 T cell activation/expansion beads (Miltenyi Biotec, 130-091-441) which had been prepared according to the manufacturer's instructions.

Techniques: Marker, Expressing, Cell Culture, Activation Assay, Quantitative RT-PCR, Control, Isolation

Journal: Oncoimmunology

Article Title: CD4 + T cells potently induce epithelial-mesenchymal-transition in premalignant and malignant pancreatic ductal epithelial cells–novel implications of CD4 + T cells in pancreatic cancer development

doi: 10.1080/2162402X.2014.1000083

Figure Lengend Snippet: An altered EMT marker expression predominates in H6c7 cells when co-cultured with activated CD4+ T-effs. H6c7 cells were monocultured (mono) or directly co-cultured with T-effs (co T-effs) or T-regs (co T-regs) for 72 h, either in the absence (w/o) or presence of activation beads (+ beads). Flow cytometry analysis of E-cadherin, L1CAM and vimentin expression in detached mono- and co-cultured H6c7 cells. Data are presented as median values with quartiles quartiles (Q0,75 as upper, Q0.25 as lower deviation) of 3–6 independent experiments with T cells isolated from 3–6 individual donors. * = p < 0.05.

Article Snippet: After 24 h, medium was removed and 1×10 4 freshly isolated T-regs or T-effs, diluted in 125 μL X-Vivo medium/well (Lonza, BE04-418F) were added, either in the absence or presence of 5 × 10 3 T cell activation/expansion beads (Miltenyi Biotec, 130-091-441) which had been prepared according to the manufacturer's instructions.

Techniques: Marker, Expressing, Cell Culture, Activation Assay, Flow Cytometry, Isolation

Journal: Oncoimmunology

Article Title: CD4 + T cells potently induce epithelial-mesenchymal-transition in premalignant and malignant pancreatic ductal epithelial cells–novel implications of CD4 + T cells in pancreatic cancer development

doi: 10.1080/2162402X.2014.1000083

Figure Lengend Snippet: An altered EMT marker expression predominates in H6c7 cells when co-cultured with activated CD4+ T-effs. H6c7 cells were monocultured (mono) or directly co-cultured with T-effs (co T-effs) or T-regs (co T-regs) for 72 h, either in the absence (w/o) or presence of activation beads (+ beads). Immunocytochemistry were used to detect (A) E-cadherin, (B) L1CAM and (C) vimentin in mono- and co-cultured H6c7 cells still adherent in 96-well-plates. Representative stainings of three independent experiments with T cells isolated from three individual donors are shown at 580-fold magnification.

Article Snippet: After 24 h, medium was removed and 1×10 4 freshly isolated T-regs or T-effs, diluted in 125 μL X-Vivo medium/well (Lonza, BE04-418F) were added, either in the absence or presence of 5 × 10 3 T cell activation/expansion beads (Miltenyi Biotec, 130-091-441) which had been prepared according to the manufacturer's instructions.

Techniques: Marker, Expressing, Cell Culture, Activation Assay, Immunocytochemistry, Isolation

Journal: Oncoimmunology

Article Title: CD4 + T cells potently induce epithelial-mesenchymal-transition in premalignant and malignant pancreatic ductal epithelial cells–novel implications of CD4 + T cells in pancreatic cancer development

doi: 10.1080/2162402X.2014.1000083

Figure Lengend Snippet: Greater invasiveness of H6c7 cells after co-culture with activated CD4+ T-effs. H6c7 cells were mono-cultured (mono) or directly co-cultured with T-effs (co T-effs) or T-regs (co T-regs) for 72 h, either in the absence (w/o) or presence of activation beads (+ beads). Cell invasion of H6c7 cells was determined in a modified boyden chamber on collagen-I coated transwells after 24 h. Median values with quartiles quartiles (Q0,75 as upper, Q0.25 as lower deviation) of % invaded cells from three independent experiments with T cells isolated from three individual donors are shown. * = p < 0.05.

Article Snippet: After 24 h, medium was removed and 1×10 4 freshly isolated T-regs or T-effs, diluted in 125 μL X-Vivo medium/well (Lonza, BE04-418F) were added, either in the absence or presence of 5 × 10 3 T cell activation/expansion beads (Miltenyi Biotec, 130-091-441) which had been prepared according to the manufacturer's instructions.

Techniques: Co-Culture Assay, Cell Culture, Activation Assay, Modification, Isolation

Journal: Oncoimmunology

Article Title: CD4 + T cells potently induce epithelial-mesenchymal-transition in premalignant and malignant pancreatic ductal epithelial cells–novel implications of CD4 + T cells in pancreatic cancer development

doi: 10.1080/2162402X.2014.1000083

Figure Lengend Snippet: (See previous page). Activated CD4+ T-effs induce EMT and increase cell invasion in T3M4 cells. (A) Representative images (n = 3 experiments with T cells isolated from three individual donors) showing cell morphology of T3M4 cells monocultured (mono) or directly co-cultured with T-effs (co T-effs) or T-regs (co T-regs) for 72 h, either in the absence (w/o) or presence of activation beads (beads). Magnification x 280. (B) E-cadherin, L1CAM, vimentin, ZEB-1, Snail and Slug expression were analyzed by RT-qPCR in the differentially cultured T3M4 cells. TBP was used as house-keeping gene for control. Data represent the median values with quartiles (Q0,75 as upper, Q0.25 as lower deviation) of four independent experiments with T cells isolated from four individual donors. * = p < 0.05. (C) Cell invasion of T3M4 cells was determined in an xCelligence Real time Analyzer with collagen-I coated transwells after 24 h. Since maximum cell invasion was reached after 16 h, cell invasion of mono- and co-cultured T3M4 cells presented as cell indices determined over a time period of 16 h. One representative invasion assay from three independent experiments with T cells isolated from three individual donors is shown.

Article Snippet: After 24 h, medium was removed and 1×10 4 freshly isolated T-regs or T-effs, diluted in 125 μL X-Vivo medium/well (Lonza, BE04-418F) were added, either in the absence or presence of 5 × 10 3 T cell activation/expansion beads (Miltenyi Biotec, 130-091-441) which had been prepared according to the manufacturer's instructions.

Techniques: Isolation, Cell Culture, Activation Assay, Expressing, Quantitative RT-PCR, Control, Invasion Assay

Journal: Oncoimmunology

Article Title: CD4 + T cells potently induce epithelial-mesenchymal-transition in premalignant and malignant pancreatic ductal epithelial cells–novel implications of CD4 + T cells in pancreatic cancer development

doi: 10.1080/2162402X.2014.1000083

Figure Lengend Snippet: Elevated levels of IL-8, IL-6 and TNF-α in co-cultures with activated CD4+ T-effs. Supernatants from monocultured (mono) or directly T-eff co-cultured (co T-effs) H6c7 cells, either in the absence (w/o) or presence of activation beads (+ beads), were analyzed after 72 h for the presence of (A) IL-8, (B) IL-6 and (C) TNF-α. Data are expressed as concentrations (pg/mL) in supernatants and represent median values with quartiles quartiles (Q0,75 as upper, Q0.25 as lower deviation) of three independent experiments with T cells isolated from 3 individual donors.

Article Snippet: After 24 h, medium was removed and 1×10 4 freshly isolated T-regs or T-effs, diluted in 125 μL X-Vivo medium/well (Lonza, BE04-418F) were added, either in the absence or presence of 5 × 10 3 T cell activation/expansion beads (Miltenyi Biotec, 130-091-441) which had been prepared according to the manufacturer's instructions.

Techniques: Cell Culture, Activation Assay, Isolation

Journal: Oncoimmunology

Article Title: CD4 + T cells potently induce epithelial-mesenchymal-transition in premalignant and malignant pancreatic ductal epithelial cells–novel implications of CD4 + T cells in pancreatic cancer development

doi: 10.1080/2162402X.2014.1000083

Figure Lengend Snippet: TNF-α and IL-6 account for the acquisition of the mesenchymal and invasive phenotype of H6c7 cells during co-culture with activated CD4+ T-effs. H6c7 cells were directly co-cultured with T-effs (co T-effs) and activation beads (+ beads) for 72 h. As indicated, 10 μg/mL of Rituximab (control blocking) or blocking agents specific for IL-6 (Tocilizumab) or TNF-α (Etanercept) were added during co-culture. (A) Representative images of three independent experiments showing cell morphologies of differentially treated H6c7 cells during T-eff co-culture. Magnification x 280 (upper panel) and x 560 (lower panel). (B) E-cadherin, L1CAM, vimentin and ZEB-1 expression were analyzed by RT-qPCR. TBP was used as house-keeping gene for control. Data represent the median values with quartiles quartiles (Q0,75 as upper, Q0.25 as lower deviation) of 3–4 independent experiments. (C) Cell invasion of differentially treated H6c7 cells was determined in a modified boyden chamber on collagen-I coated transwells after 24 h. Median values with quartiles (Q0,75 as upper, Q0.25 as lower deviation) of % invaded cells from three independent experiments with T cells isolated from three individual donors are shown. * = p < 0.05.

Article Snippet: After 24 h, medium was removed and 1×10 4 freshly isolated T-regs or T-effs, diluted in 125 μL X-Vivo medium/well (Lonza, BE04-418F) were added, either in the absence or presence of 5 × 10 3 T cell activation/expansion beads (Miltenyi Biotec, 130-091-441) which had been prepared according to the manufacturer's instructions.

Techniques: Co-Culture Assay, Cell Culture, Activation Assay, Control, Blocking Assay, Expressing, Quantitative RT-PCR, Modification, Isolation

Journal: Oncoimmunology

Article Title: CD4 + T cells potently induce epithelial-mesenchymal-transition in premalignant and malignant pancreatic ductal epithelial cells–novel implications of CD4 + T cells in pancreatic cancer development

doi: 10.1080/2162402X.2014.1000083

Figure Lengend Snippet: Distribution of CD4+ T cells and EMT markers in PanINs within CP tissues. Representative E-cadherin, L1CAM and vimentin stainings of CP tissues demonstrate partially reduced E-cadherin expression (indicated by the arrow) and enhanced L1CAM and vimentin expression in PanINs surrounded by a dense stroma enriched with CD4+ T cells (magnification x 400). Note, that besides vimentin expression in PanINs surrounded by a CD4+ T cells containing stroma, the stromal compartment itself was characterized by strong vimentin expression, too.

Article Snippet: After 24 h, medium was removed and 1×10 4 freshly isolated T-regs or T-effs, diluted in 125 μL X-Vivo medium/well (Lonza, BE04-418F) were added, either in the absence or presence of 5 × 10 3 T cell activation/expansion beads (Miltenyi Biotec, 130-091-441) which had been prepared according to the manufacturer's instructions.

Techniques: Expressing

Journal: Oncoimmunology

Article Title: CD4 + T cells potently induce epithelial-mesenchymal-transition in premalignant and malignant pancreatic ductal epithelial cells–novel implications of CD4 + T cells in pancreatic cancer development

doi: 10.1080/2162402X.2014.1000083

Figure Lengend Snippet: Simplified overview on the mutual epithelia-stroma-interaction promoting EMT and malignancy during inflammation-associated PDAC development. (A) Under physiological conditions, pancreatic ducts are embedded in a homeostatic stromal microenvironment comprising fibroblasts, T cells, and macrophages and exhibit an epithelial and polarized phenotype. (B) Upon chronic inflammation, predominantly CD4+ T effs and macrophages enrich in the pancreatic tissue leading to increased level of inflammatory mediators such as TNF-α and IL-6. This may either directly result from the infiltrated inflammatory cells or from an enhanced secretion by stromal and/or the epithelial cells upon their reciprocal interaction. As a result, elevated TNF-α and IL-6 levels promote EMT in pancreatic ducts leading to loss of E-cadherin expression, upregulation of mesenchymal proteins such as vimentin and L1CAM. This probably paves the way for an early dissemination of epithelial cells.21,22 Elevated epithelial L1CAM expression, in turn, impairs the proliferation of CD4+ T-effs and promotes infiltration of CD4+CD25+CD127-CD49d- T-regs into the pancreas as well as the generation of immunosuppressive CD4+CD25-CD69+ T cells. These various subsets of regulatory T cells contribute to the manifestation of an immunosuppressive microenvironment, e.g., through TGF-β1. Besides its immunoregulatory function, TGF-β1 can directly induce EMT in epithelial/carcinoma cells but may also indirectly contribute to EMT by promoting transdifferentiation of pancreatic stellate cells into myofibroblasts and modulating the phenotype of macrophages. (C) Manifestation of these mechanisms shapes the stromal compartment of PDAC diminishing the percentage of CD4+ T-effs while increasing the percentages of T-reg subsets as well as myofibroblasts. The predominance of these stromal cell types results in a further elevation of TGF-β1 which augments an immunosuppressive microenvironment and EMT induction in carcinoma cells ultimately supporting malignant progression of PDAC.

Article Snippet: After 24 h, medium was removed and 1×10 4 freshly isolated T-regs or T-effs, diluted in 125 μL X-Vivo medium/well (Lonza, BE04-418F) were added, either in the absence or presence of 5 × 10 3 T cell activation/expansion beads (Miltenyi Biotec, 130-091-441) which had been prepared according to the manufacturer's instructions.

Techniques: Expressing