Journal: PLoS ONE

Article Title: Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

doi: 10.1371/journal.pone.0008747

Figure Lengend Snippet: A) A549 subpopulations collected from different cell growth stages (as indicated) were stained for CEACAM1 with mAb clone 283340, for CEACAM5 with Col-1, for CEACAM6 with mAb 9A6 and for CEACAM7 with BAC2 (thick line). The background fluorescence was determined by incubating the cells with control IgG antibody instead of primary anti-CEACAM antibody (thin line). Subsequently, samples were analyzed by flow cytometry revealing CEACAM1 expression in confluent A549-NT and A549-T cells. Additionally, a minor fraction of confluent A549-T cells expressed CEACAM6. The data shown are representative of three independent experiments. B) Determination of different CEACAMs in whole cell lysates of A549 cells cultured in the non-confluent log phase, the confluent phase and the spheroidal unanchored growing cells by immunoblotting with mAb specific for CEACAM1, CEACAM5 and CEACAM6. The detection of beta-actin served as a loading control. The data shown are representative of three different experiments. C) Soluble CEACAM1, CEACAM5 and CEACAM6 forms are released in the cell culture supernatant of confluent A549 cells. The released CEACAM1, CEACAM5 and CEACAM6 molecules were quantified by specific sandwich ELISA as described in the section. The mean values ± SD (*p≤0.005) were determined from triplicates. The experiment was repeated twice.

Article Snippet: The mAb Col-1 was purchased from Invitrogen (Carlsbad, CA) and the anti-human CEACAM1 mAb clone 283340 from R&D systems (Minneapolis, MN).

Techniques: Staining, Fluorescence, Control, Flow Cytometry, Expressing, Cell Culture, Western Blot, Sandwich ELISA

Journal: PLoS ONE

Article Title: Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

doi: 10.1371/journal.pone.0008747

Figure Lengend Snippet: A) For quantification of CEACAM1 cell surface expression in confluent and log phase cultured A549 cells, samples were analyzed by flow cytometry utilizing the QuiFiKit approach as described in . Briefly, cells were stained for CEACAM1 with mAb clone 283340. Fluorescence was quantitated using QuiFiKit calibration-beads as described in . The data shown are means ± SD (*p≤0.007) of three different experiments. B) Immunoblot analyses of confluent and proliferating A549 cell lysates were done as described in using monospecific mAbs directed against CEACAM1 (clone 283340), CEACAM5 (Col-1), and CEACAM6 (9A6), respectively and visualized by HRP-coupled secondary antibody and ECL detection. Beta-actin served as a loading control. The blots shown are representative of three separate experiments. C) The fraction of CEACAM1 expressing A549 cells increased over time as cells were kept in the confluent state. To analyze the CEACAM1 expression in A549 cells grown to confluence (day 0), plus 1 day, plus 3 days, plus 5 days and plus 7 days, cells were stained with mAb clone 283340 (thick line) and isotype matched control antibody (thin line) followed by FITC-conjugated secondary antibody. Subsequently, samples were measured by flow cytometry. The data shown are representative of three different experiments.

Article Snippet: The mAb Col-1 was purchased from Invitrogen (Carlsbad, CA) and the anti-human CEACAM1 mAb clone 283340 from R&D systems (Minneapolis, MN).

Techniques: Expressing, Cell Culture, Flow Cytometry, Staining, Fluorescence, Western Blot, Control

Journal: PLoS ONE

Article Title: Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

doi: 10.1371/journal.pone.0008747

Figure Lengend Snippet: RT of RNA isolated from confluent A549 cells were applied to four different PCR reactions using primer pairs specific four each of the four CEACAM1 isoforms. Products corresponding to CEACAM1-4L (266 bp), CEACAM1-4S (245 bp), CEACAM1-3L (177 bp), and CEACAM1-3S (145 bp), respectively, were amplified. The size of oligonucleotide markers is shown on the left.

Article Snippet: The mAb Col-1 was purchased from Invitrogen (Carlsbad, CA) and the anti-human CEACAM1 mAb clone 283340 from R&D systems (Minneapolis, MN).

Techniques: Isolation, Amplification

Journal: PLoS ONE

Article Title: Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

doi: 10.1371/journal.pone.0008747

Figure Lengend Snippet: Characterization of the CEACAM1 isoform pattern as determined by quantitative RT-PCR of the two A549 subpopulations.

Article Snippet: The mAb Col-1 was purchased from Invitrogen (Carlsbad, CA) and the anti-human CEACAM1 mAb clone 283340 from R&D systems (Minneapolis, MN).

Techniques: Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

doi: 10.1371/journal.pone.0008747

Figure Lengend Snippet: A) Analyses of the cell surface expression of different CEACAMs in the non-confluent log phase cultured parental A549 cells stably transfected with an empty vector (a), CEACAM1-4L (b), the mutant form of the intracellular ITIM motif CEACAM1-4L-Y459F/Y486F (c) and CEACAM5 (d). Samples were analyzed by flow cytometry using mAbs that specifically bind the different CEACAMs (thick line) or isotype matched control antibody (thin line) followed by FITC-conjugated secondary antibody. Data show one of three different, representative stably transfected A549 clones. B) Representative phase contrast images demonstrating the morphology of A549 cells stably transfected with empty vector (a), plasmids encoding for CEACAM1-4L (b), the ITIM mutant form CEACAM1-4L(Y459F/Y486F) (c), CEACAM5 (d) and CEACAM1-4S (e). Bar, 50 µm. C) Percentage of viable cells determined by the flow cytometry based annexin V-FITC/PI approach as described in using cell spheroidals and aggregates harvested from the culture supernatant of wild type (wt) A549 cells and A549 cells transfected with control vector, CEACAM1-4L (Y459F/Y486F) or A549-CEACAM5. Data are shown as the percentage of viable cells as the mean of three independent experiments +/− Standard deviation. D) Growth properties of control vector transfected and CEACAM1-4L transfected A549 cells as determined by the MTS based method as described in . A549- vector (white circles) and A549-CEACAM1 transfected cells (black circles) were seeded into in 96-well cell culture plates at a density of 25,000 cells/well. Following standard cell culture for 1, 2, 3, 4 and 5 days, respectively, the tetrazolium compound MTS was added and the samples were incubated at 37°C in a humidified, 5% CO2 atmosphere for 4 h. Experiments were performed in triplicate and results presented are expressed as means of OD 490 nm ± SD (*p≤0.005). The data show one representative result of three independent repeats of the experiment.

Article Snippet: The mAb Col-1 was purchased from Invitrogen (Carlsbad, CA) and the anti-human CEACAM1 mAb clone 283340 from R&D systems (Minneapolis, MN).

Techniques: Expressing, Cell Culture, Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, Flow Cytometry, Control, Clone Assay, Standard Deviation, Incubation

Journal: PLoS ONE

Article Title: Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

doi: 10.1371/journal.pone.0008747

Figure Lengend Snippet: A) The expression of the proliferation marker Ki67 is limited to A549-T cells that also expressed CEACAM6 on their cell surface. Confluent CEACAM6-negative and CEACAM6-positive A549-T cells were separated by mAb 9A6 loaded magnetic protein G microbeads and μMAS magnetic sorting columns (Miltenyi Biotec) as described in . Immunoblot analysis of confluent CEACAM6-negative and CEACAM6-positive A549-T cell lysates was performed applying a Ki67 specific antibody followed by HRP-coupled secondary antibody and ECL detection. Beta-actin served as a loading control. The data shown are representative of three separate experiments. B) Cell cycle analysis of CEACAM6 negative and CEACAM6 positive A549-T cells. CEACAM6-negative and CEACAM6-positive A549 cells were fixed in 80% ethanol and stained with propidium iodide, and analyzed by flow cytometry as described in “ .” The DNA content in the different cell fractions is given in arbitrary units on the X-axis. Cells in the G2-M phase (second peak) contained twice as much DNA as cells in the G0-G1 phase (first peak). Cells between the peaks represent cells in the S-phase. The relative proportions of cells in the various phases are shown above the DNA profiles. Filled curve, CEACAM6 negative A549 cells; thick curve, CEACAM6 positive A549 cells. C) Confluent control sh-plasmid transfected A549-T (A549-shControl) and shCEACAM6 transfected A549-T cells (A549-shCC6) were stained for CEACAM1 with mAb clone 283340 and for CEACAM6 with mAb 9A6 (thick lines). The background fluorescence was determined by incubating the cells with control IgG antibody (thin lines). Samples were analyzed by flow cytometry. Compared to A549-shControl cells, A549-shCC6 cells completely lacked CEACAM6 expression, but continued to express CEACAM1. D) Phase contrast images of (a) control sh-plasmid transfected A549-T and (b) shCEACAM6 transfected A549-T cells. Confluent control sh-plasmid transfected A549-T piled up and formed unanchored spheroidal cell aggregates on top of the monolayer revealing insufficient contact inhibition. (b) In contrast, A549-shCC6 cells formed well spread monolayers without detection of unanchored, spheroidal cell growth indicative tight contact inhibition. Bar, 50 µm.

Article Snippet: The mAb Col-1 was purchased from Invitrogen (Carlsbad, CA) and the anti-human CEACAM1 mAb clone 283340 from R&D systems (Minneapolis, MN).

Techniques: Expressing, Marker, Western Blot, Control, Cell Cycle Assay, Staining, Flow Cytometry, Plasmid Preparation, Transfection, Fluorescence, Inhibition

Journal: PLoS ONE

Article Title: Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

doi: 10.1371/journal.pone.0008747

Figure Lengend Snippet: Primer sequences used in RT-PCR.

Article Snippet: The mAb Col-1 was purchased from Invitrogen (Carlsbad, CA) and the anti-human CEACAM1 mAb clone 283340 from R&D systems (Minneapolis, MN).

Techniques:

Journal: The Journal of investigative dermatology

Article Title: Cytokine-induced CEACAM1 expression on keratinocytes is characteristic for psoriatic skin and contributes to a prolonged lifespan of neutrophils.

doi: 10.1038/jid.2008.303

Figure Lengend Snippet: Figure 1. Keratinocytes in the outer epidermal layer of psoriatic lesions specifically express CEACAM1. CEACAM1 expression on cutaneous biopsies from individuals with (a) melanoma, from (b) healthy individuals, or patients with (c) psoriasis, (d) atopic dermatitis, or (e) nummular dermatitis was determined by immunohistochemistry analysis as described in ‘Materials and Methods’. The presence of the Munro-Saboureau microabscess in the outer layer of psoriatic skin (c) is indicated by *. Arrows indicate CEACAM1 expression in the dermis of (b) healthy individuals or patients with (d) atopic or (e) nummular dermatitis. (f) CD3 expression in biopsies from patients with psoriasis. Controls are inserted in the relevant figures. Scale bars ¼ 100 mm.

Article Snippet: Soluble CEACAM1 measurements were carried out according to a similar procedure, using the anti-CEACAM1 mAb 19.6 as the capture antibody (2mg ml 1) and a biotinylated goat anti-recombinant CEACAM1 Ab (R&D Systems; BAF2244) as the detection antibody (0.1 mg ml 1).

Techniques: Expressing, Immunohistochemistry

Journal: The Journal of investigative dermatology

Article Title: Cytokine-induced CEACAM1 expression on keratinocytes is characteristic for psoriatic skin and contributes to a prolonged lifespan of neutrophils.

doi: 10.1038/jid.2008.303

Figure Lengend Snippet: Figure 2. Culture supernatants of activated skin-infiltrating T cells induce expression of CEACAM1 transcripts and protein by NHEK in an IFN-c-dependent manner. (a) NHEK were cultured with different dilutions of supernatants of in vitro-activated skin-infiltrating T cells, depleted or not for the presence of IFN-g, and the expression of CEACAM1 transcripts was analyzed by real-time RT-PCR following 24 hours of incubation. (b) NHEK were cultured in medium only (Cont.) or in the presence of 30 or 5% culture supernatant, derived from psoriatic skin-infiltrating T cells, that had been activated for 24 hours in vitro with anti- CD3 and CD28 mAbs (30% ND and 5% ND). In addition, supernatants depleted for the presence of IFN-g used at a concentration of 30% (30% D) or 5% (5% D) were added in parallel. As a control, NHEK were stimulated with anti-CD3 and CD28 mAbs (CD3CD28). Expression of CEACAM1 was determined by immunohistochemistry analysis after 48 hours of incubation. Scale bar ¼ 100 mm.

Article Snippet: Soluble CEACAM1 measurements were carried out according to a similar procedure, using the anti-CEACAM1 mAb 19.6 as the capture antibody (2mg ml 1) and a biotinylated goat anti-recombinant CEACAM1 Ab (R&D Systems; BAF2244) as the detection antibody (0.1 mg ml 1).

Techniques: Expressing, Cell Culture, In Vitro, Quantitative RT-PCR, Incubation, Derivative Assay, Concentration Assay, Control, Immunohistochemistry

Journal: The Journal of investigative dermatology

Article Title: Cytokine-induced CEACAM1 expression on keratinocytes is characteristic for psoriatic skin and contributes to a prolonged lifespan of neutrophils.

doi: 10.1038/jid.2008.303

Figure Lengend Snippet: Figure 3. IFN-c induces cell-surface expression of CEACAM1 by NHEK. NHEK were cultured for 48 hours in the (a) absence or (b) presence of IFN-g. Cell- surface expression of CEACAM1 was determined by immunohistochemistry (a, b) or western blotting analysis (c). Negative control for immunohistochemical analysis is inserted in (b). Scale bar ¼ 100 mm.

Article Snippet: Soluble CEACAM1 measurements were carried out according to a similar procedure, using the anti-CEACAM1 mAb 19.6 as the capture antibody (2mg ml 1) and a biotinylated goat anti-recombinant CEACAM1 Ab (R&D Systems; BAF2244) as the detection antibody (0.1 mg ml 1).

Techniques: Expressing, Cell Culture, Immunohistochemistry, Western Blot, Negative Control, Immunohistochemical staining

Journal: The Journal of investigative dermatology

Article Title: Cytokine-induced CEACAM1 expression on keratinocytes is characteristic for psoriatic skin and contributes to a prolonged lifespan of neutrophils.

doi: 10.1038/jid.2008.303

Figure Lengend Snippet: Figure 4. OSM induces cell-surface expression of CEACAM1 on human reconstituted epidermis. Human reconstituted epidermis was cultured for 48 hours in (a) the absence or (b) the presence of 30% T-cell-derived culture supernatant, with (c) OSM or (d) IL-17, both at a concentration of 10 ng ml1. The expression of CEACAM1 was determined by immunohistochemistry analysis. Scale bar ¼ 100 mm.

Article Snippet: Soluble CEACAM1 measurements were carried out according to a similar procedure, using the anti-CEACAM1 mAb 19.6 as the capture antibody (2mg ml 1) and a biotinylated goat anti-recombinant CEACAM1 Ab (R&D Systems; BAF2244) as the detection antibody (0.1 mg ml 1).

Techniques: Expressing, Cell Culture, Derivative Assay, Concentration Assay, Immunohistochemistry

Journal: The Journal of investigative dermatology

Article Title: Cytokine-induced CEACAM1 expression on keratinocytes is characteristic for psoriatic skin and contributes to a prolonged lifespan of neutrophils.

doi: 10.1038/jid.2008.303

Figure Lengend Snippet: Figure 5. IL-17 does not induce CEACAM1 expression on NHEK. NHEK were cultured in medium only (Cont.) or in the presence of either a culture supernatant (30%, T-cell SN) of in vitro-activated psoriatic skin-infiltrating T cells, IFN-g (10 ng ml1), or IL-17 and the expression of CEACAM1 was analyzed by immunohistochemistry after 48 hours of incubation. Negative controls for the two conditions that induce CEACAM1 expression are inserted in the figures. Scale bars ¼ 100 mm.

Article Snippet: Soluble CEACAM1 measurements were carried out according to a similar procedure, using the anti-CEACAM1 mAb 19.6 as the capture antibody (2mg ml 1) and a biotinylated goat anti-recombinant CEACAM1 Ab (R&D Systems; BAF2244) as the detection antibody (0.1 mg ml 1).

Techniques: Expressing, Cell Culture, In Vitro, Immunohistochemistry, Incubation

Journal: The Journal of investigative dermatology

Article Title: Cytokine-induced CEACAM1 expression on keratinocytes is characteristic for psoriatic skin and contributes to a prolonged lifespan of neutrophils.

doi: 10.1038/jid.2008.303

Figure Lengend Snippet: Figure 6. Cytokines produced by activated skin-infiltrating T cells induce the expression of CEACAM1-S and -L isoform transcripts on primary keratinocytes. (a) NHEK were cultured for 24 hours with a combinations of cytokines, each used at a concentration of 10 ng ml1 or with a culture supernatant (30%) of in vitro- activated psoriatic skin-infiltrating T cells, in the presence or absence of neutralizing mAbs specific for IFN-g or IL-17 and the expression of transcripts for the short (3S and 4S) and long (3L and 4L) isoforms of CEACAM1 was analyzed by real-time RT-PCR. (b) The specificity of the primers for the different isoforms was validated by classic PCR on NHEK, cultured in the presence of T-cell culture supernatant.

Article Snippet: Soluble CEACAM1 measurements were carried out according to a similar procedure, using the anti-CEACAM1 mAb 19.6 as the capture antibody (2mg ml 1) and a biotinylated goat anti-recombinant CEACAM1 Ab (R&D Systems; BAF2244) as the detection antibody (0.1 mg ml 1).

Techniques: Produced, Expressing, Cell Culture, Concentration Assay, In Vitro, Quantitative RT-PCR

Journal: The Journal of investigative dermatology

Article Title: Cytokine-induced CEACAM1 expression on keratinocytes is characteristic for psoriatic skin and contributes to a prolonged lifespan of neutrophils.

doi: 10.1038/jid.2008.303

Figure Lengend Snippet: Figure 7. IFN-c-induced expression of CEACAM1 on keratinocytes protects neutrophils from spontaneous apoptosis. (a) One million of freshly isolated peripheral blood neutrophils were cultured for 24 hours in medium alone or (b) 200 ng ml1 GM-CSF or were cocultured with either NHEK that had been preincubated for 48 hours (c) in medium, (d) with 10 ng ml1 IFN-g, or with (e) wild type CHO cells or with CHO cells expressing (f) the long or (g) the short isoform of CEACAM1. Frequencies of viable and early or late apoptotic cells were determined by measuring Annexin-V binding (x axis) and PI incorporation (y axis) using flow cytometry. Results shown in (a) to (g) are representative of three independent experiments with neutrophils from different donors and the percentages of cells in the quadrants are indicated in each graph. (h) Percentages of viable cells are represented as the mean±SD.

Article Snippet: Soluble CEACAM1 measurements were carried out according to a similar procedure, using the anti-CEACAM1 mAb 19.6 as the capture antibody (2mg ml 1) and a biotinylated goat anti-recombinant CEACAM1 Ab (R&D Systems; BAF2244) as the detection antibody (0.1 mg ml 1).

Techniques: Expressing, Isolation, Cell Culture, Binding Assay, Flow Cytometry

Journal: The Journal of investigative dermatology

Article Title: Cytokine-induced CEACAM1 expression on keratinocytes is characteristic for psoriatic skin and contributes to a prolonged lifespan of neutrophils.

doi: 10.1038/jid.2008.303

Figure Lengend Snippet: Figure 8. CEACAM1 engagement delays neutrophil apoptosis. One million of freshly isolated peripheral blood neutrophils were either cultured (a) in medium only, (b) with 200 ng ml1 GM-CSF, (c) with 30 mg ml1 of the anti- CEACAM1 mAb 19.6, or (d) with 30 mg ml1 of an isotype control mAb for various periods of time. Frequencies of viable and early or late apoptotic cells were determined by measuring Annexin-V binding and PI incorporation using flow cytometry. Representative results from four independent experiments with neutrophils from different donors. Percentages of cells in the quadrants are indicated. (e) Kinetics of viable cell frequencies are expressed as the mean±SD of the values obtained in the four experiments.

Article Snippet: Soluble CEACAM1 measurements were carried out according to a similar procedure, using the anti-CEACAM1 mAb 19.6 as the capture antibody (2mg ml 1) and a biotinylated goat anti-recombinant CEACAM1 Ab (R&D Systems; BAF2244) as the detection antibody (0.1 mg ml 1).

Techniques: Isolation, Cell Culture, Control, Binding Assay, Flow Cytometry

Journal: European Journal of Immunology

Article Title: TIM‐3 and CEACAM1 do not interact in cis and in trans

doi: 10.1002/eji.201948400

Figure Lengend Snippet: Expression analysis of TIM‐3 and CEACAM1. Flow cytometry analysis of TIM‐3 expression on freshly isolated PBMCs derived from healthy donors. CD19 + cells (A), CD56 + cells (B), and CD14 + cells (C) were analyzed for TIM‐3 expression. (D) Immature (iDC) and mature DCs were stained with TIM‐3 mAb (black histograms) or isotype control (open histograms). (A–D) Left: one representative donor; right: each dot represents one donor. Median is shown. (E and F) CD4 + and CD8 + T cells in freshly isolated PBMCs and PBMCs stimulated in vitro with staphylococcal enterotoxin E (SEE) (E) or immobilized aCD3/aCD28 mAb (F) for 3, 6, and 10 days were analyzed for TIM‐3, CEACAM1, and CD25 expression, respectively. In the stimulated samples, the gate was set on proliferated (CFSE low ) cells. Left panels: dot plots from one representative donor are shown. For better visibility larger dots were used in dot plots depicting TIM‐3 and CEACAM1 expression in CD8 + T cells upon stimulation with SEE. Middle‐right panels: histogram overlay shows gMFI of CD25 expression on day 10 of the indicated populations. Right panels: summarized data of CD25 expression in the TIM3 + versus TIM3 + /CD66a + subset upon SEE (17 donors/8 experiments with 1–3 donors each) or aCD3/aCD28‐stimulation (11 donors/6 experiments with 1–3 donors each). (G) Bar diagrams from five representative donors (from three experiments with one or two donors) show percentages of CD66a + ‐expressing cells within the TIM3 + population. For statistical evaluation, paired t ‐tests (A–C; E and F) and one‐way ANOVA followed by Tukey's multiple comparison test (D) were performed (*** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05; ns, p > 0.05).

Article Snippet: For ELISA‐based binding studies, recombinant human Gal‐9 expressed in E. coli or in HEK293T cells and recombinant human CEACAM1 expressed in the murine myeloma cell line NSO cells were purchased from R&D systems.

Techniques: Expressing, Flow Cytometry, Isolation, Derivative Assay, Staining, Control, In Vitro, Comparison

Journal: European Journal of Immunology

Article Title: TIM‐3 and CEACAM1 do not interact in cis and in trans

doi: 10.1002/eji.201948400

Figure Lengend Snippet: Evaluation of CEACAM1 function in a reporter cell system. (A) Schematic of CEACAM1‐4L and CEACAM1‐4S proteins. (B) Flow cytometry analysis of triple parameter reporter cells (TPR) and T cell stimulator cells (TCS). Open histograms: control cells; filled histograms: expression of indicated molecules on TPR and TCS. (C) Gating strategy and one representative stimulation experiment of control TPR and TPR‐expressing CEACAM1‐4S and CEACAM1‐4L with control TCS or TCS‐expressing CEACAM1 is shown. eGFP, eCFP, and mCherry expression was measured via flow cytometry. The histograms of unstimulated cells are also depicted. The geometric MFI (gMFI) value is shown for each histogram. (D) The indicated TPRs were stimulated with control TCS or TCS‐expressing CEACAM1. Reporter activation is shown as fold induction (gMFI of TCS‐stimulated cells/gMFI of unstimulated cells). Results are from ten independent experiments performed in triplicates. Note that some data points overlap. For statistical evaluation, paired t ‐tests were performed (**** p ≤ 0.0001; ** p ≤ 0.01; * p ≤ 0.05; ns, p > 0.05).

Article Snippet: For ELISA‐based binding studies, recombinant human Gal‐9 expressed in E. coli or in HEK293T cells and recombinant human CEACAM1 expressed in the murine myeloma cell line NSO cells were purchased from R&D systems.

Techniques: Flow Cytometry, Control, Expressing, Activation Assay

Journal: European Journal of Immunology

Article Title: TIM‐3 and CEACAM1 do not interact in cis and in trans

doi: 10.1002/eji.201948400

Figure Lengend Snippet: TIM‐3 does not modulate CEACAM1 function. (A) Flow cytometry analysis of TPR reporter cells coexpressing CEACAM1‐4L and TIM‐3 (gray histograms). Reactivity of antibodies to CEACAM1 and TIM‐3 to control TPR is shown as open histograms. (B) CEACAM1‐4L reporter cells coexpressing TIM‐3 were stimulated with control TCS or TCS‐expressing CEACAM1. Results are from seven independent experiments performed in triplicates. (C) Expression of membrane‐bound anti‐CD3 Ab fragment and TIM‐3 on TCS‐TIM‐3 (gray histograms). Reactivity of the used antibodies to control cells is shown as open histograms. (D) The indicated TPRs were stimulated with control TCS or TCS‐expressing TIM‐3. Results are from five independent experiments performed in triplicates. (B and D) eGFP, eCFP, and mCherry expression was measured via flow cytometry. Reporter activation is shown as fold induction (gMFI of TCS‐stimulated cells/gMFI of unstimulated cells). Note that some data points overlap. For statistical evaluation, paired t ‐tests were performed (**** p ≤ 0.0001; ** p ≤ 0.01; ns, p > 0.05).

Article Snippet: For ELISA‐based binding studies, recombinant human Gal‐9 expressed in E. coli or in HEK293T cells and recombinant human CEACAM1 expressed in the murine myeloma cell line NSO cells were purchased from R&D systems.

Techniques: Flow Cytometry, Control, Expressing, Membrane, Activation Assay

Journal: European Journal of Immunology

Article Title: TIM‐3 and CEACAM1 do not interact in cis and in trans

doi: 10.1002/eji.201948400

Figure Lengend Snippet: CEACAM1 does not mediate TIM‐3 signaling. (A) Schematic of WT and mutated TIM‐3 molecules (left) and flow cytometric analysis of TPR reporter cells expressing the indicated TIM‐3 molecules (gray histograms) and control TPR (open histogram). (B) The indicated TPR were stimulated with control TCS or TCS‐expressing CEACAM1. eGFP, eCFP, and mCherry expression was measured via flow cytometry. Results are shown for five independent experiments performed in triplicates. Reporter activation is shown as fold induction (gMFI of TCS‐stimulated cells/gMFI of unstimulated cells). For statistical evaluation, unpaired t ‐tests were performed (ns, p > 0.05).

Article Snippet: For ELISA‐based binding studies, recombinant human Gal‐9 expressed in E. coli or in HEK293T cells and recombinant human CEACAM1 expressed in the murine myeloma cell line NSO cells were purchased from R&D systems.

Techniques: Expressing, Control, Flow Cytometry, Activation Assay

Journal: European Journal of Immunology

Article Title: TIM‐3 and CEACAM1 do not interact in cis and in trans

doi: 10.1002/eji.201948400

Figure Lengend Snippet: TIM‐3 and CEACAM1 do not interact in trans . (A) Binding of indicated Ig fusion proteins to immobilized recombinant galectin‐9 (Gal‐9) and CEACAM1 proteins was analyzed by ELISA. Results are representative for four independently performed experiments. Information on the used proteins is provided in the material and method section. (B) Jurkat cells transduced to express ICOS, TIM‐3, or CEACAM1 were probed with the respective antibodies and analyzed by flow cytometry (gray histograms). Open histograms show the reactivity of control Jurkat cells. Bottom: results of a representative binding experiment of indicated Ig fusion proteins used at a final concentration of 10 μg/mL to control Jurkat cells or Jurkat cells expressing ICOS, TIM‐3, or CEACAM1. Binding was detected via flow cytometry by an APC‐conjugated goat‐anti‐human IgG (Fc‐specific) Ab. (C) Binding of indicated Ig fusion proteins (final concentrations: 31.6, 10, 3.16, 1, and 0.316 μg/mL) to control Jurkat cells and Jurkat cells expressing ICOS, TIM‐3, or CEACAM1. Binding was detected as described in (B). Results are shown from three different experiments performed in duplicates. Binding signals (gMFI) were normalized to background binding (gMFI values obtained with secondary reagent only). (D) Binding of CEACAM1‐Ig (final concentration: 10 μg/mL) to control Jurkat cells and CEACAM1‐expressing Jurkat cells in absence or presence of a blocking CEACAM1 mAb. Results are shown from four different experiments performed in duplicates. (E) Cells coexpressing eCFP or eGFP and the indicated cell surface molecules were preincubated for 1 h and conjugate formation between cells expressing different fluorescent proteins was assessed via flow cytometry. (F) Results of three independently performed cell–cell binding assays are summarized. (A, C, D, and F) Standard deviation is shown.

Article Snippet: For ELISA‐based binding studies, recombinant human Gal‐9 expressed in E. coli or in HEK293T cells and recombinant human CEACAM1 expressed in the murine myeloma cell line NSO cells were purchased from R&D systems.

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Control, Concentration Assay, Expressing, Blocking Assay, Standard Deviation

Journal: European Journal of Immunology

Article Title: TIM‐3 and CEACAM1 do not interact in cis and in trans

doi: 10.1002/eji.201948400

Figure Lengend Snippet: TIM‐3 and CEACAM1 do not interact in cis . (A) HEK293T cells were cotransfected with indicated molecules. Flow cytometry analysis was performed 2 days after transfection. Open histograms: staining with an isotype control; filled histograms: staining of indicated molecules. Bar diagrams show results of three independent experiments. (B) Analysis of in cis association of indicated interaction partners via FRET. mRuby3 (561 nm excitation; PE filter) and mNeonGreen (488 nm excitation; FITC filter) fusion constructs were cotransfected in HEK293T cells and analyzed by flow cytometry. For FRET detection, 488 nm laser light was used for fluorophore excitation, and mRuby3 emission (600 nm longpass filter) was measured. (C) Fluorescence images of HEK293T cells coexpressing the indicated molecules. FRET yield was determined by donor recovery after acceptor photobleaching and calculated pixelwise. Representative images are shown. Scale bar = 5 μm. (D) Quantification of average FRET yield per cell ( n = 46; 35; 57). For statistical evaluation, one‐way ANOVA followed by Tukey's test was performed (ns, p > 0.05; **** p ≤ 0.0001). (A and D) Standard error of the mean is shown.

Article Snippet: For ELISA‐based binding studies, recombinant human Gal‐9 expressed in E. coli or in HEK293T cells and recombinant human CEACAM1 expressed in the murine myeloma cell line NSO cells were purchased from R&D systems.

Techniques: Flow Cytometry, Transfection, Staining, Control, Construct, Fluorescence

Journal: European Journal of Immunology

Article Title: TIM‐3 and CEACAM1 do not interact in cis and in trans

doi: 10.1002/eji.201948400

Figure Lengend Snippet: Cytoplasmic sequences of TIM‐3 and CEACAM1 induce inhibitory signals. (A) Schematic of mICOS chimera; (B) Left: surface expression of mICOS::Δcyt, mICOS::PD‐1, mICOS::TIM‐3, mICOS::TIM‐3_mut, mICOS::CEACAM1‐L, and mICOS::CEACAM1‐S on TPR cells (dark gray histograms). Reactivity of mICOS Ab with control TPR is shown as an open histogram. Right: Flow cytometry analysis of TCS‐mICOS‐L. Open histograms: control cells; filled histograms: expression of indicated molecules. (C) mICOS::Δcyt, mICOS::PD‐1, mICOS::TIM‐3, mICOS::TIM‐3_mut, mICOS::CEACAM1‐L, and mICOS::CEACAM1‐S reporter cells were stimulated with control TCS or TCS‐expressing mICOS‐L. For statistical evaluation, one‐way ANOVA followed by Dunnett's test was performed (**** p ≤ 0.0001; ns, p > 0.05). eGFP, eCFP, and mCherry expression was measured via flow cytometry. Results are shown for four independent experiments performed in triplicates. Reporter activation is shown as fold induction (gMFI of mICOS‐L‐stimulated cells/gMFI of control‐stimulated cells). It should be noted that some data pointes overlap. Control stimulation is shown as a dotted line.

Article Snippet: For ELISA‐based binding studies, recombinant human Gal‐9 expressed in E. coli or in HEK293T cells and recombinant human CEACAM1 expressed in the murine myeloma cell line NSO cells were purchased from R&D systems.

Techniques: Expressing, Control, Flow Cytometry, Activation Assay