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

Article Title: A soluble form of IL-27Rα is a natural IL-27 antagonist.

doi: 10.4049/jimmunol.1303435

Figure Lengend Snippet: FIGURE 1. sIL-27Ra is released from differ- ent primary human cell types. (A) Purified CD4+

Article Snippet: To investigate whether sIL27Ra could form complexes with IL-27 in the sera, we developed an ELISA by using mouse anti-human IL-27Ra mAb or a control isotype mAb as coating Abs, and goat biotinylated polyclonal anti-human IL-27 Ab (R&D Systems) as detection Ab.

Techniques:

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

Article Title: A soluble form of IL-27Rα is a natural IL-27 antagonist.

doi: 10.4049/jimmunol.1303435

Figure Lengend Snippet: FIGURE 2. ELISA analysis of sIL-27Ra in the sera of healthy indi- viduals. (A) Levels of sIL-27Ra were measured by ELISA in the sera from healthy individuals. (B) The correlation between sIL-27Ra and IL-27 se- rum levels is shown. Although a logarithmic scale is used, linear regression was calculated using the actual values. (C) Sera from six pregnant women, collected at various times during pregnancy (6–10 sera per time point), were tested by sIL-27Ra ELISA. The mean values (6SEM) are represented. (D) Sera from four nonpregnant healthy individuals (Da–d) and four pregnant women (De–h) were tested in a sandwich ELISA using the indicated Abs as capture Abs and biotinylated anti-human IL-27 Ab as detection Ab.

Article Snippet: To investigate whether sIL27Ra could form complexes with IL-27 in the sera, we developed an ELISA by using mouse anti-human IL-27Ra mAb or a control isotype mAb as coating Abs, and goat biotinylated polyclonal anti-human IL-27 Ab (R&D Systems) as detection Ab.

Techniques: Enzyme-linked Immunosorbent Assay, Sandwich ELISA

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

Article Title: A soluble form of IL-27Rα is a natural IL-27 antagonist.

doi: 10.4049/jimmunol.1303435

Figure Lengend Snippet: FIGURE 3. Biochemical characterization of sIL- 27Ra. Lysates from COS7 cells transiently transfected with vector control or vector expressing IL-27RaV5His (A), and anti–IL-27Ra immunoprecipitates from the lysate of IL-27RaV5His–transfected COS7 cells before or after N-glycanase treatment (B), were analyzed by immunoblot using anti–IL-27Ra Ab. (C) Concentrated culture supernatant from KMH2 cells (left) or human sera (right) was submitted to immunoprecipitation with goat or mouse anti–IL-27Ra Abs, followed by protein G beads, or with anti–IL-27Ra beads, as indicated, and analyzed by anti–IL-27Ra immunoblot. (D) Treatment of anti–IL-27Ra immunoprecipitate from KMH2 cul- ture supernatant with N-glycanase causes a shift in the apparent molecular mass of sIL-27Ra from 90/70 to 60 kDa. Cross-reacting IgH is indicated by an asterisk. Kilodaltons of molecular size markers are reported on the left.

Article Snippet: To investigate whether sIL27Ra could form complexes with IL-27 in the sera, we developed an ELISA by using mouse anti-human IL-27Ra mAb or a control isotype mAb as coating Abs, and goat biotinylated polyclonal anti-human IL-27 Ab (R&D Systems) as detection Ab.

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Western Blot, Immunoprecipitation

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

Article Title: A soluble form of IL-27Rα is a natural IL-27 antagonist.

doi: 10.4049/jimmunol.1303435

Figure Lengend Snippet: FIGURE 4. Role of metalloproteases in the production of sIL-27Ra. (A) COS7 cells were cotransfected with GFP vector and pcDNA3 control vector or pcDNA3 encoding IL-27RaV5His. Two days after transfection, cells were stained with isotype control (filled gray histogram) or anti–IL-27Ra (bold line) Abs and analyzed by FACS. Staining observed in GFP-positive cells is shown. (B) Cell culture supernatants from transfected COS7 cells were tested by Western blot for detection of sIL-27R. Only Abs recognizing the extracellular domain of IL-27Ra, but not its intracellular C terminus (V5 mAb), detected sIL-27Ra. (C) Cell culture supernatants from IL-27RaV5His–transfected COS7 cells, incubated for the indicated times with GM6001, TAPI-0 (both at 50 mM), or DMSO control, were tested by sIL-27Ra ELISA. No sIL-27Ra was detected in the culture supernatant from vector control-transfected COS7 cells. (D) Cell lysates from COS7 cells collected 48 h after transfection were analyzed by immunoblot with anti-V5 Ab to monitor IL-27RaV5His expression levels. On a longer exposure, a smaller band that was weaker in the presence of metalloprotease inhibitors and might correspond to the transmembrane and intracellular domains was observed (data not shown). One representative experiment of two to three is shown in (A)–(D). (E) KMH2 cells were incubated for 3 d with various con- centrations of GM6001, TAPI-0, or DMSO. Levels of sIL-27Ra measured by ELISA in the culture supernatants and cell numbers determined by trypan blue exclusion are shown as the mean 6 SEM of relative levels of three independent experiments. (F) Purified CD4+ T cells were stimulated for 5 d with CD2/CD3/ CD28 beads and IL-2 and various concentrations of GM6001, TAPI-0, or DMSO. Relative levels of sIL-27Ra and cell numbers observed in four independent experiments performed with different donors are shown. Inhibitor doses .25 mM were not used in CD4+ T cells because they resulted in cell toxicity. (G) Cell membrane IL-27Ra expression (mIL-27Ra) was analyzed by FACS in activated CD4+ T cells cultured for 5 d in the presence of GM6001, TAPI-0 (both at 25 mM), or DMSO. The mean fluorescence intensity of mIL-27Ra staining is indicated. (H) The mean fluorescence intensity of mIL-27Ra staining observed in GM6001 or TAPI-0–treated CD4+ T cells relative to that observed in the DMSO control is shown as mean 6 SEM of four independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: To investigate whether sIL27Ra could form complexes with IL-27 in the sera, we developed an ELISA by using mouse anti-human IL-27Ra mAb or a control isotype mAb as coating Abs, and goat biotinylated polyclonal anti-human IL-27 Ab (R&D Systems) as detection Ab.

Techniques: Plasmid Preparation, Control, Transfection, Staining, Cell Culture, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Membrane

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

Article Title: A soluble form of IL-27Rα is a natural IL-27 antagonist.

doi: 10.4049/jimmunol.1303435

Figure Lengend Snippet: FIGURE 5. Natural sIL-27Ra antagonizes IL-27 signaling. (A) Concentrated cell culture supernatant of KMH2 cells was submitted to immuno- precipitation with control or anti–IL-27Ra Abs, in the absence or presence of 100 ng rIL-27. Immunoprecipitates (lanes 1–4) and a fraction of su- pernatant before immunoprecipitation (lane 5) were analyzed by anti–IL-27Ra and anti-EBI3 immunoblots. (B and C) rIL-27 was preincubated for 15 min at 37˚C with various concentrations of rIL-27R-Fc or gp130-Fc fusion proteins, or purified natural sIL-27Ra, before addition to BL2 cells for 15 min. Cell lysates were analyzed for STAT1 activation with anti–phospho-STAT1 Ab and subsequently with STAT1 Ab to monitor STAT1 levels. (D) Similar experiment was conducted using IFN-g in place of IL-27. (E) Inhibition of IL-27 binding to BL2 cells. rIL-27 (2 ng) was preincubated or not with purified natural sIL-27Ra (2 ng) before incubation with BL2 cells. IL-27 binding was measured by FACS. One representative experiment of two to three is shown.

Article Snippet: To investigate whether sIL27Ra could form complexes with IL-27 in the sera, we developed an ELISA by using mouse anti-human IL-27Ra mAb or a control isotype mAb as coating Abs, and goat biotinylated polyclonal anti-human IL-27 Ab (R&D Systems) as detection Ab.

Techniques: Cell Culture, Immunoprecipitation, Control, Western Blot, Activation Assay, Inhibition, Binding Assay, Incubation

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

Article Title: A soluble form of IL-27Rα is a natural IL-27 antagonist.

doi: 10.4049/jimmunol.1303435

Figure Lengend Snippet: FIGURE 6. Analysis of sIL-27Ra and IL-27 levels in the sera of CD patients. Serum levels of sIL-27Ra (A) and IL-27 (B) were determined by ELISA in 52 CD patients and 28 healthy individuals. The correlation between sIL-27Ra and IL-27 serum levels (C) and the molar ratio between IL-27 and sIL-27Ra serum values (D) are shown. Although a logarithmic scale is used in (C), linear regression was calculated using the actual values.

Article Snippet: To investigate whether sIL27Ra could form complexes with IL-27 in the sera, we developed an ELISA by using mouse anti-human IL-27Ra mAb or a control isotype mAb as coating Abs, and goat biotinylated polyclonal anti-human IL-27 Ab (R&D Systems) as detection Ab.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Biochemical Journal

Article Title: Mutations in the transmembrane and juxtamembrane domains enhance IL27R transforming activity

doi: 10.1042/bj20110351

Figure Lengend Snippet: Figure 1 Mutations of IL27R that enhance the transforming activity of IL27R

Article Snippet: Anti-human IL27R (TCCR/WSX-1) antibody (R&D Systems) was conjugated to Alexa Fluor® 647 using an Alexa Fluor® 647 antibody labelling kit (Molecular Probes).

Techniques: Activity Assay

Journal: Biochemical Journal

Article Title: Mutations in the transmembrane and juxtamembrane domains enhance IL27R transforming activity

doi: 10.1042/bj20110351

Figure Lengend Snippet: Figure 2 Mutant IL27R proteins exhibit enhanced transforming activity compared with IL27R-WT

Article Snippet: Anti-human IL27R (TCCR/WSX-1) antibody (R&D Systems) was conjugated to Alexa Fluor® 647 using an Alexa Fluor® 647 antibody labelling kit (Molecular Probes).

Techniques: Mutagenesis, Activity Assay

Journal: Biochemical Journal

Article Title: Mutations in the transmembrane and juxtamembrane domains enhance IL27R transforming activity

doi: 10.1042/bj20110351

Figure Lengend Snippet: Figure 3 Cells expressing transforming IL27R mutants display increased activation of signalling proteins

Article Snippet: Anti-human IL27R (TCCR/WSX-1) antibody (R&D Systems) was conjugated to Alexa Fluor® 647 using an Alexa Fluor® 647 antibody labelling kit (Molecular Probes).

Techniques: Expressing, Activation Assay

Journal: Biochemical Journal

Article Title: Mutations in the transmembrane and juxtamembrane domains enhance IL27R transforming activity

doi: 10.1042/bj20110351

Figure Lengend Snippet: Figure 4 F523S and F523A mutations of IL27R do not enhance the transforming activity of IL27R-WT

Article Snippet: Anti-human IL27R (TCCR/WSX-1) antibody (R&D Systems) was conjugated to Alexa Fluor® 647 using an Alexa Fluor® 647 antibody labelling kit (Molecular Probes).

Techniques: Activity Assay

Journal: Biochemical Journal

Article Title: Mutations in the transmembrane and juxtamembrane domains enhance IL27R transforming activity

doi: 10.1042/bj20110351

Figure Lengend Snippet: Figure 5 The IL27R- mutant exhibits enhanced homodimeric complex formation

Article Snippet: Anti-human IL27R (TCCR/WSX-1) antibody (R&D Systems) was conjugated to Alexa Fluor® 647 using an Alexa Fluor® 647 antibody labelling kit (Molecular Probes).

Techniques: Mutagenesis

Journal: Biochemical Journal

Article Title: Mutations in the transmembrane and juxtamembrane domains enhance IL27R transforming activity

doi: 10.1042/bj20110351

Figure Lengend Snippet: Figure 6 Comparison of the transforming properties of IL27R proteins to known transforming haemopoietic mutations

Article Snippet: Anti-human IL27R (TCCR/WSX-1) antibody (R&D Systems) was conjugated to Alexa Fluor® 647 using an Alexa Fluor® 647 antibody labelling kit (Molecular Probes).

Techniques: Comparison

Journal: Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research

Article Title: IL-27 Promotes Proliferation of Human Leukemic Cell Lines Through the MAPK/ERK Signaling Pathway and Suppresses Sensitivity to Chemotherapeutic Drugs.

doi: 10.1089/jir.2015.0091

Figure Lengend Snippet: FIG. 1. Expression of the IL-27R complex on human tumor cell lines. (A) mRNA expression of IL-27Ra and gp130 in 9 human tumor cell lines as indicated was analyzed by RT-PCR using specific primers. NC represents a negative control in which cDNA was replaced by H2O. The PCR products were separated by 2% agarose gel electrophoresis and the molecular weights were determined using the 100-bp DNA ladder. (B) Cell surface expression of IL-27Ra and gp130 on 9 human tumor cell lines as indicated was detected by flow cytometry using PE-conjugated specific antibodies. Shaded histograms are isotype-matched mAb staining. Unshaded histograms are IL-27Ra and gp130 staining, respectively. RT-PCR, reverse transcription polymerase chain reaction.

Article Snippet: Cell surface expression of IL-27Ra or gp130 was detected using PE-conjugated anti-IL-27Ra monoclonal antibody or PE-conjugated anti-gp130 monoclonal antibody (R&D Systems).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Agarose Gel Electrophoresis, Cytometry, Staining, Reverse Transcription, Polymerase Chain Reaction

Journal: Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research

Article Title: IL-27 Promotes Proliferation of Human Leukemic Cell Lines Through the MAPK/ERK Signaling Pathway and Suppresses Sensitivity to Chemotherapeutic Drugs.

doi: 10.1089/jir.2015.0091

Figure Lengend Snippet: FIG. 4. IL-27-activated STAT pathway was mediated through the receptor complex. (A) Intracellular phosphorylation of STAT1 and STAT3 activated by IL-27 was detected by flow cytometry on leukemic cell lines as indicated. Shaded histograms are untreated controls. Unshaded histograms are STAT1-pTyr701 (top) and STAT3-pTyr705 (bottom) staining, respectively. (B, C) IL-27 was preincubated for 15 min with either IL-27Ra-Fc or gp130-Fc before addition to OCI-AML5 (B) and TF-1 (C) cells for 15-min treatment. OCI-AML5 (B) and TF-1 (C) cells were pretreated with anti-gp130 Ab for 15 min, followed by IL-27 stimulation. Intracellular phosphorylation of STAT1 and STAT3 was analyzed by flow cy- tometry. (D) TF-1 cells were deprived of GM-CSF in 5% serum assay medium containing IL-27 in the absence or presence of either IL-27Ra-Fc or anti-gp130 Ab at indicated concentrations. Cell proliferation was measured by 3H-thymidine incorporation after 2-day incubation. *P < 0.05 for IL-27 plus IL-27Ra-Fc versus IL-27 alone. ***P < 0.001 for IL-27 plus both IL-27Ra-Fc and anti-gp130 Ab versus IL-27 alone. (continued/)

Article Snippet: Cell surface expression of IL-27Ra or gp130 was detected using PE-conjugated anti-IL-27Ra monoclonal antibody or PE-conjugated anti-gp130 monoclonal antibody (R&D Systems).

Techniques: Phospho-proteomics, Cytometry, Staining, Serum Assay, Incubation

Journal: Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research

Article Title: IL-27 Promotes Proliferation of Human Leukemic Cell Lines Through the MAPK/ERK Signaling Pathway and Suppresses Sensitivity to Chemotherapeutic Drugs.

doi: 10.1089/jir.2015.0091

Figure Lengend Snippet: FIG. 5. Kinetic binding interactions of IL-27 with each receptor chain and of anti-gp130 Ab with gp130. SPR sensorgrams were ob- tained from injections of IL- 27 at concentrations of 0.25, 0.5, 1, 2, 4, and 8 nM over IL-27Ra-Fc captured surface (A), human IL-27 at concen- trations of 2, 4, 8, 16, and 32 nM over gp130-Fc cap- tured surface (B), and of anti- gp130 antibody at concen- trations of 6.25, 12.5, 25, 50, 100, and 200 nM over gp130-Fc captured surface (C). Experimental data were globally fitted with a 1:1 Langmuir binding model for the determination of kinetic constants. Each sensorgram shown is representative of 3 independent experiments. Color images available on- line at www.liebertpub.com/ jir

Article Snippet: Cell surface expression of IL-27Ra or gp130 was detected using PE-conjugated anti-IL-27Ra monoclonal antibody or PE-conjugated anti-gp130 monoclonal antibody (R&D Systems).

Techniques: Binding Assay