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 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: iScience

Article Title: Interferon-γ and IL-27 positively regulate type 1 regulatory T cell development during adaptive tolerance

doi: 10.1016/j.isci.2025.112308

Figure Lengend Snippet:

Article Snippet: Mouse Anti-Mouse IL-27 p28 (clone MM27.7B1) , BioXCell , Cat #BE0326; RRID:AB_2819053.

Techniques: Recombinant, Saline, Staining, Red Blood Cell Lysis, Sequencing, Software

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

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 1. DNA vaccination-based anti-IL-27 Abs are highly specific. The Western blot shows that our DNA vaccination-based anti-IL-27 Ab binds mouse IL-27 p28 (lane 1; 27 kDa), but not recombinant mouse IL-18, IL-12, or TNF- (lanes 2, 3, and 4, respectively). A, Coomassie Blue staining verifies the appearance of each cytokine on the loaded gel. B, Western blot showing that of these cytokines, our DNA vaccination-based anti-IL-27 Ab binds only mouse IL-27 p28. These Ab also bound natural mouse IL-27 (verified by sequencing) from supernatant of activated MOGp35–55-specific cultured primary draining lymph node cells (not shown).

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: Western Blot, Recombinant, Staining, Sequencing, Cell Culture

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

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 2. Anti-p28 Abs suppresses ongoing severe EAE. A, Four groups of 10 mice each were subjected to MOGp35–55-induced EAE. Beginning at the onset of disease (day 17), these mice were repeatedly (every other day) administered 100 g/mouse of anti-p28 Ab (f), IgG obtained from naive Lewis rats (Œ), or PBS (E). An observer blind to the experimental procedure scored EAE daily. The experiment summarized in Fig. 2 shows the results of one of three experiments performed under similar experimental conditions, with similar results. The mean maximal score SE represents six mice per group. The other four mice were killed on day 30 and subjected to histological evaluation (see Fig. 3). B, Five groups of six mice each were subjected to MOGp35–55-induced EAE. Beginning at the onset of disease (day 17), these mice were repeatedly (every other day) administered 100 g of anti-p28 Ab/mouse (f), anti-IL-18 Ab (F), anti-IL-1 Ab (), IgG obtained from Lewis rats previously subjected to an empty plasmid administration (Œ), or PBS (E). An observer blind to the experimental procedure scored EAE daily. Results are shown as the mean maximal score SE of six mice per group. C, Three groups of six mice each were subjected to induction of transferred EAE. Beginning at the onset of disease (day 5), these mice were repeatedly (every other day) administered 100 g of anti-p28 Ab/mouse (f), IgG obtained from naive Lewis rats (Œ), or PBS (E). An observer blind to the experimental procedure scored EAE daily. Results are shown as mean maximal score SE of six mice per group.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: Plasmid Preparation

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

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 4. The beneficial effect of anti-IL-27 is dependent on the con- tinuing administration of protective Abs. Three groups of six mice each were subjected to active induction of EAE. Beginning 1 day after the onset of disease (day 17), these mice were treated with either a single dose of anti-p28 Ab (100 g/mouse; E) or with repeated administration (every other day) of this Ab (Œ) or PBS (f). An observer blind to the experi- mental procedure scored EAE daily. Results are shown as the mean max- imal score SE of six mice per group.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques:

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

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 3. Anti-IL-27 therapy reduces the histological score of EAE. Histological evaluation was conducted 30 days after disease induction. Lumbar spinal cord samples from naive mice or from EAE mice treated with PBS, IgG from naive mice, or anti-IL-27 p28 Abs were subjected to histological analysis (nine sections each group). The arrowheads point to the parenchymal mononuclear cell infiltration. The scale for mononuclear cell infiltration used was: 0, no mononuclear cell infiltration; 1, one to five perivascular lesions per section with minimal parenchymal infiltration; 2, five to 10 perivascular lesions per section with parenchymal infiltration; and 3, 10 perivascular lesions per section with extensive parenchymal infiltration. The mean histological score SE was calculated for each group.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques:

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

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 5. Protective administration of anti-IL-27 Abs decreases in vivo polarization of CD4 T cells into Th1 and suppresses IFN- production by Ag-specific T cells. C57BL/6 mice (three per group) were subjected to active induction of EAE and then to repeated administration (days 12, 14, and 16) of anti-IL-27 p28 Abs (100 g), PBS, or normal rat IgG. On day 17 cervical lymph node cells (that drain the autoimmune site) were subjected to intracellular staining of IL-4 and IFN-. A, FACS analysis of CD4 T cells in this experiment. This experiment represents results obtained in three different independent experiments with very similar data. Subsequently, cervical lymph node T cells from these mice were cultured in the presence of 100 M MOGp35–55. After 72 h of stimulation, supernatants were assayed for the protein level of IFN- (B) and IL-4 (not shown). This experiment represents results obtained in three different independent experiments with very similar data.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: In Vivo, Staining, Cell Culture

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

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 6. Neutralizing the function of IL-27 reduces IFN- produc- tion by IFN--producing T cells. A, C57BL/6 mice (three per group) were subjected to active induction of EAE and then to repeated administration (days 3 and 6) of 100 g of anti-IL-27 p28 Abs (group 3), PBS (group 2), or normal rat IgG (group 1). On day 9, spleen cells were subjected to spot ELISA as previously described (38). A, Relative number of positive spots per 107 cultured cells. The average size of positive spots was analyzed. B, The MOGp33–55-specific CD4 T cell line was cultured with or without 100 M MOGp33–55. Cultured cells were supplemented with anti-IL-27 Abs at a final concentration of 10 g/ml (), normal rat IgG (f), or PBS (E). After 60 h of incubation, cells were plates in spot ELISA plates for an additional 24 h for the detection of IFN--positive spots (38). Number of positive spots (y-axis) and spot sizes (x-axis; logarithmic scale) determined as previously described (46).

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Concentration Assay, Incubation