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

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: International Immunopharmacology

Article Title: IL-35 interferes with splenic T cells in a clinical and experimental model of acute respiratory distress syndrome

doi: 10.1016/j.intimp.2018.12.024

Figure Lengend Snippet: (A, E) Pretreatment with IL-35 neutralizing antibodies (anti-P35 or anti-Ebi3) aggravated LPS- and CLP-induced ARDS. Lungs from each experimental group were processed for histological examination after H&E staining. Compared with the LPS + IgG group, thickened alveolar wall, alveolar hemorrhage and collapse, inflammatory cells in filtration were more severe and pretreated with IL-35 neutralizing antibodies prior to LPS challenge. (B, D) Lung injury scores were estimated by the method of Mikawa, which is from the following four indicators of lung injury score: alveolar congestion; bleeding; gap or vascular wall neutrophil infiltration or aggregation; alveolar septal thickening or transparent membrane formation. 0 marks: no or very slight damage, 1 marks: mild injury, 2 marks: moderate injury, 3 marks: severe injury, 4 marks: very severe damage, the cumulative increase in the number of lesions of the total score is the pathological score of the ARDS. Pretreatment with IL-35 neutralizing antibodies lungs injury were more severe than LPS + IgG group. **p < 0.01, ***p < 0.001, by the two-way ANOVA followed by LSD multiple comparisons test, compared with the LPS + IgG group.

Article Snippet: Neutralization assays were performed by giving 50 μg anti-mouse IL-35 EBI3 (Rockland Immunochemicals) or anti-mouse IL-12A p35 (Abcam Systems) 30 min after CLP or LPS.

Techniques: Staining, Filtration, Membrane

Journal: International Immunopharmacology

Article Title: IL-35 interferes with splenic T cells in a clinical and experimental model of acute respiratory distress syndrome

doi: 10.1016/j.intimp.2018.12.024

Figure Lengend Snippet: Interleukin 35 (IL-35) blockade upregulated the production of proinflammatory cytokines and downregulated anti-inflammatory cytokines during ARDS models. Cytokine and chemokine concentrations in BALF and blood specimens from five mice treated with or without anti–Ebi3 or anti–P35 blocking antibodies were determined by mice cytokine/chemokine magnetic bead panel kit assays 24 h after onset of ARDS. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by the one-way ANOVA followed by LSD multiple comparisons test, compared with mice treated with isotypical IgG control. ARDS featured increased CXCL-1, TNF-α, IL-6, and IL-17A and reduced IFN-γ, IL-10, IL-2, and IL-13 after neutralizing antibody treatment (anti-IL-35 Ebi3 or anti-IL-35 P35) in BALF and serum.

Article Snippet: Neutralization assays were performed by giving 50 μg anti-mouse IL-35 EBI3 (Rockland Immunochemicals) or anti-mouse IL-12A p35 (Abcam Systems) 30 min after CLP or LPS.

Techniques: Blocking Assay, Control

Journal: Cancer Research

Article Title: Interleukin-30/IL27p28 Shapes Prostate Cancer Stem-like Cell Behavior and Is Critical for Tumor Onset and Metastasization

doi: 10.1158/0008-5472.can-17-3117

Figure Lengend Snippet: Figure 1. Expression of IL30 and IL30R by PCSLCs, murine, and human prostate tissues. A, Cytofluorimetric analyses of gp130-(CD130) and IL6Ra- (CD126) expression in PIN-SCs. B, Relative expression SD of IL30 mRNA. CTRL, PIN-SCs. ANOVA, P < 0.0001. , P < 0.01, Tukey HSD test compared with CTRL, EV, or EV-IL30shRNA (clones D and B). C, Western blot analyses of IL30 protein expression. D, ELISA assay of IL30 release by CTRL (182.82 6.9 pg/mL), EV-PIN-SCs (168.21 10.82 pg/mL), IL30PIN-SCs (2424.46 83.9 pg/mL), EV-IL30shPIN-SCs (170.44 13.09 pg/mL), and IL30shPIN-SCs (clone D, 6.76 1.87 pg/mL; clone B, 7.53 1.38 pg/mL). ANOVA, P < 0.0001. , P < 0.01, Tukey HSD test compared with CTRL, EV, or EV-IL30shRNA (clones D and B). E, IL30 immunostaining in normal prostate, PIN (11 weeks), and in poorly differentiated tumor (28 weeks) of TRAMP mice. IL30 (brown) colocalizes with Sca-1 (red) in PIN; scale bars, 50 mm (left two); 30 mm (right two and inset). F, Expression of IL6Ra and gp130 in PIN and in poorly differentiated adenocarcinoma (AC) of TRAMP mice; scale bars, 50 mm (left two); 30 mm (right two). G, IL30 immunostaining in normal prostate, PIN (IL30/CD133 colocalization), and in poorly differentiated adenocarcinoma; scale bars, 20 mm (left two and inset); 30 mm (right two).

Article Snippet: Recombinant (r) murine (m) IL30 (#7430-ML) was purchased from R&D Systems.

Techniques: Expressing, Clone Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Immunostaining

Journal: Cancer Research

Article Title: Interleukin-30/IL27p28 Shapes Prostate Cancer Stem-like Cell Behavior and Is Critical for Tumor Onset and Metastasization

doi: 10.1158/0008-5472.can-17-3117

Figure Lengend Snippet: Figure 3. Effects of IL30 overproduction or silencing on subcutaneous PCSLC-derived tumors. A, Histology and immunohistochemistry of IL30PIN-SC and IL30shPIN-SC tumors versus controls; scale bars, 50 mm (bottom); 30 mm (other and inset). B, Immunohistochemical features of IL30PIN-SC and EV-IL30PIN-SC tumors; scale bars, 30 mm; 20 mm (granulocytes). C and D, Immune cells in IL30–overexpressing (C) or IL30-silenced (D) PIN-SC tumors. Results are expressed as mean SD of positive cells/field evaluated at 400 (0.180 mm2 field) by immunohistochemistry. , values significantly (P < 0.05) different from controls. H&E, hematoxylin and eosin.

Article Snippet: Recombinant (r) murine (m) IL30 (#7430-ML) was purchased from R&D Systems.

Techniques: Derivative Assay, Immunohistochemistry, Immunohistochemical staining

Journal: Cancer Research

Article Title: Interleukin-30/IL27p28 Shapes Prostate Cancer Stem-like Cell Behavior and Is Critical for Tumor Onset and Metastasization

doi: 10.1158/0008-5472.can-17-3117

Figure Lengend Snippet: Figure 4. IL30's ability to regulate gene expression of PCSLCs and PCSLC-derived tumors. A, Fold differences of mRNAs between rIL30–treated and untreated PIN-SCs. A significant threshold of 2-fold change in gene expression corresponded to P < 0.001. B, Fold differences of mRNAs between IL30shPIN-SCs and EV- IL30shPIN-SCs. Results from the latter are comparable to those from untransfected cells. A significant threshold of 2-fold change in gene expression corresponded to P < 0.001. C–E, Immunohistochemical features of prostate draining LNs, IL30PIN-SC, and EV-IL30PIN-SC orthotopic tumors; scale bars, 30 mm. F, Silencing of STAT1 and STAT3 in PIN-SCs, as confirmed by Western blot. G, Fold differences of mRNAs between Stat1 siRNA- or Stat3 siRNA- or CTRL siRNA-transfected PIN-SCs cultured with rIL30 and untreated CTRL siRNA-transfected PIN-SCs. Results from the latter are comparable with those from untreated and untransfected cells. A significant threshold of 2-fold change in gene expression corresponded to P < 0.001. , P < 0.05 by Student t test compared with rIL30–treated CTRL siRNA-transfected PIN-SCs. H, Fold differences of mRNAs between Stat1 siRNA- or Stat3 siRNA-transfected PIN-SCs cultured with rIL30 and rIL30–treated CTRL siRNA-transfected PIN-SCs. A significant threshold of 2-fold change in gene expression corresponded to P < 0.001.

Article Snippet: Recombinant (r) murine (m) IL30 (#7430-ML) was purchased from R&D Systems.

Techniques: Gene Expression, Derivative Assay, Immunohistochemical staining, Western Blot, Transfection, Cell Culture

Journal: Cancer Research

Article Title: Interleukin-30/IL27p28 Shapes Prostate Cancer Stem-like Cell Behavior and Is Critical for Tumor Onset and Metastasization

doi: 10.1158/0008-5472.can-17-3117

Figure Lengend Snippet: Figure 5. IL30 favors PCSLC metastasis to the lungs involving CXCR4/CXCL12 axis. A, Histology and immunohistochemistry of lung metastasis in IL30PIN-SC and EV-IL30PIN-SC tumor bearing mice. Scale bars, 50 mm (bottom); 30 mm (other). B, Expression of CXCR4 and CXCL12 in lung metastasis developed in IL30PIN-SC and EV-IL30PIN-SC tumor-bearing mice; scale bars, 30 mm. C, Migration of IL30–treated PIN-SCs toward CXCL12. Results are expressed as mean SD. ANOVA, P < 0.0001. , Tukey HSD test compared with CTRL and rIL30þAb-CXCR4 (P < 0.05) or EVsup and IL30LV-DNAsupþAb-CXCR4 (P < 0.01). , P < 0.01, Tukey HSD test compared with CTRL, rIL30þAb-CXCR4, EVsup, and IL30LV-DNAsupþAb-CXCR4. , Tukey HSD test compared with CTRL, rIL30þAb-CXCR4, EVsup, and IL30LV- DNAsupþAb-CXCR4 (P < 0.01) or IL30LV-DNAsup (P < 0.05). D, Histology and immunohistochemistry of lung metastasis in mice bearing orthotopic IL30PIN-SC and EV-PIN-SC tumors; scale bars, 50 mm; 30 mm (bottom and insets). E, Immune cells in lung metastasis of mice bearing orthotopic IL30PIN-SC tumors versus controls. Results are expressed as mean SD of positive cells/field (400) evaluated by immunohistochemistry. , values significantly (P < 0.05) different from values in EV-PIN-SC and PIN-SC tumors. H&E, hematoxylin and eosin.

Article Snippet: Recombinant (r) murine (m) IL30 (#7430-ML) was purchased from R&D Systems.

Techniques: Immunohistochemistry, Expressing, Migration

Journal: Cancer Research

Article Title: Interleukin-30/IL27p28 Shapes Prostate Cancer Stem-like Cell Behavior and Is Critical for Tumor Onset and Metastasization

doi: 10.1158/0008-5472.can-17-3117

Figure Lengend Snippet: Figure 6. IL30 promotes PCSLC dissemination in the LNs and bone marrow involving CXCR5/CXCL13 upregulation. A, Histology and immunohistochemistry of IL30PIN-SC orthotopic tumors. Isolated Sca-1þ/IL30þ cells (red arrow) inside the blood vessels; scale bars, 50 mm (left); 20 mm (right). B, Immunohistochemistry of LNs draining IL30PIN-SC or EV-IL30PIN-SC orthotopic tumors. The inset shows CK5/6 staining of the primary tumor. In LNs, CK5/6þ colocalizes with Sca-1; scale bars, 30 mm; 20 mm (bottom). C, Immunohistochemistry of bone marrow from mice bearing IL30PIN-SC and PIN-SC orthotopic tumors; scale bars, 50 mm (top); 30 mm (bottom). D, Migration of IL30–treated PIN-SCs toward CXCL13. Results are expressed as mean SD. ANOVA, P < 0.0001. , Tukey HSD test compared with CTRL and rIL30þAb-CXCR5 (P < 0.05) or EVsup and IL30LV-DNAsupþAb-CXCR5 (P < 0.01). , Tukey HSD test compared with CTRL, rIL30þAb-CXCR5, EVsup, and IL30LV-DNAsupþAb-CXCR5 (P < 0.01) or IL30LV-DNAsup (P < 0.05).

Article Snippet: Recombinant (r) murine (m) IL30 (#7430-ML) was purchased from R&D Systems.

Techniques: Immunohistochemistry, Isolation, Staining, Migration