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: All samples were supplemented with protease inhibitors (1 mM PMSF, 1 mg/ml pepstatin, and 1 mg/ml leupeptin) and precleared with protein G–Sepharose beads (GE Healthcare) before incubation with Abs. sIL-27Ra was immunoprecipitated using either mouse mAb or goat polyclonal anti–IL-27Ra Ab (R&D Systems), in parallel to control Abs (mouse MOPC141 mAb or purified goat IgG; Sigma Aldrich), followed by incubation with protein G–Sepharose beads, or mouse anti-human IL-27Ra mAb covalently linked to agarose beads using AminoLink Plus immobilization kit (Thermo Scientific).

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: All samples were supplemented with protease inhibitors (1 mM PMSF, 1 mg/ml pepstatin, and 1 mg/ml leupeptin) and precleared with protein G–Sepharose beads (GE Healthcare) before incubation with Abs. sIL-27Ra was immunoprecipitated using either mouse mAb or goat polyclonal anti–IL-27Ra Ab (R&D Systems), in parallel to control Abs (mouse MOPC141 mAb or purified goat IgG; Sigma Aldrich), followed by incubation with protein G–Sepharose beads, or mouse anti-human IL-27Ra mAb covalently linked to agarose beads using AminoLink Plus immobilization kit (Thermo Scientific).

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: All samples were supplemented with protease inhibitors (1 mM PMSF, 1 mg/ml pepstatin, and 1 mg/ml leupeptin) and precleared with protein G–Sepharose beads (GE Healthcare) before incubation with Abs. sIL-27Ra was immunoprecipitated using either mouse mAb or goat polyclonal anti–IL-27Ra Ab (R&D Systems), in parallel to control Abs (mouse MOPC141 mAb or purified goat IgG; Sigma Aldrich), followed by incubation with protein G–Sepharose beads, or mouse anti-human IL-27Ra mAb covalently linked to agarose beads using AminoLink Plus immobilization kit (Thermo Scientific).

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: All samples were supplemented with protease inhibitors (1 mM PMSF, 1 mg/ml pepstatin, and 1 mg/ml leupeptin) and precleared with protein G–Sepharose beads (GE Healthcare) before incubation with Abs. sIL-27Ra was immunoprecipitated using either mouse mAb or goat polyclonal anti–IL-27Ra Ab (R&D Systems), in parallel to control Abs (mouse MOPC141 mAb or purified goat IgG; Sigma Aldrich), followed by incubation with protein G–Sepharose beads, or mouse anti-human IL-27Ra mAb covalently linked to agarose beads using AminoLink Plus immobilization kit (Thermo Scientific).

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: All samples were supplemented with protease inhibitors (1 mM PMSF, 1 mg/ml pepstatin, and 1 mg/ml leupeptin) and precleared with protein G–Sepharose beads (GE Healthcare) before incubation with Abs. sIL-27Ra was immunoprecipitated using either mouse mAb or goat polyclonal anti–IL-27Ra Ab (R&D Systems), in parallel to control Abs (mouse MOPC141 mAb or purified goat IgG; Sigma Aldrich), followed by incubation with protein G–Sepharose beads, or mouse anti-human IL-27Ra mAb covalently linked to agarose beads using AminoLink Plus immobilization kit (Thermo Scientific).

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: All samples were supplemented with protease inhibitors (1 mM PMSF, 1 mg/ml pepstatin, and 1 mg/ml leupeptin) and precleared with protein G–Sepharose beads (GE Healthcare) before incubation with Abs. sIL-27Ra was immunoprecipitated using either mouse mAb or goat polyclonal anti–IL-27Ra Ab (R&D Systems), in parallel to control Abs (mouse MOPC141 mAb or purified goat IgG; Sigma Aldrich), followed by incubation with protein G–Sepharose beads, or mouse anti-human IL-27Ra mAb covalently linked to agarose beads using AminoLink Plus immobilization kit (Thermo Scientific).

Techniques: Enzyme-linked Immunosorbent Assay

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

Journal: Cancer Discovery

Article Title: IL27 Signaling Serves as an Immunologic Checkpoint for Innate Cytotoxic Cells to Promote Hepatocellular Carcinoma

doi: 10.1158/2159-8290.cd-20-1628

Figure Lengend Snippet: Figure 2. IL27R signaling regulates NK-cell accumulation and function in HCC. A–C, Differential gene expression analysis of HCC tumors from DEN- treated Il27ra+/− (n = 6) and Il27ra−/− (n = 6) mice as determined by NanoString (Cancer Immunopanel; P < 0.05). A, The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes. HTLV-I, human T-cell lymphotropic virus, type I. B, Gene set enrichment analysis enrichment plots for NK gene signatures in tumor of DEN-treated Il27ra−/− mice compared with Il27ra+/− controls. NES, normalized enrichment score. C, Violin plot for the enrichment of subset-specific gene expression in tumors from Il27ra−/− mice (see Methods for details). D, Il15 gene expression in nontumor (NT) and tumor (T) tissue as determined by qRT-PCR. Gene expression was first normalized to Rpl32 and then to gene expression in nontumor tissue from Il27ra+/− mice. **, P < 0.01, unpaired Student t test (two-tailed). Single-cell suspensions of nontumor and tumor tissues from Il27ra+/− (n = 5) and Il27ra−/− (n = 5). DEN-injected 10-month-old mice were stained for Live/Dead, CD45, TCRβ, NK1.1, CD49a, and CD49b analyzed by FACS. E–H, Represen tative FACS plots (E) and quantified percentage of NK1.1+TCRβ− cells in CD45+ gate (F) as well as CD49a+NK1.1+ and CD49b+NK1.1+ cells in NK1.1+TCRβ− gate (G and H) in NT and T tissues of DEN-treated Il27ra+/− (n = 5) and Il27ra−/− (n = 5) mice. Data are mean ± SEM from at least two independent experiments. I, Correlation between presence of NK cells in tumors and IL27RA, IL27EBI3, and IL27P28 expression in the TCGA HCC cohort. Expression of IL27RA or IL27EBI3 in tumors with a “high” abundance of NK cells (NK+; n = 100) or low abundance of NK cells (NK−; n = 269) as determined by CIBERSORT. FPKM, fragments per kilobase of transcript per million mapped fragments. J, Correlation of activated NK-cell signature with IL27RA expression in scRNA-seq of human HCC (GSE151530; ref. 53). For F and H–J, *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Slides were blocked with 5% goat serum in 1% BSA–PBS for 20 minutes, and then they were incubated with primary antibodies for Ki-67 (1:100; BioLegend, 151202, RRID:AB_2566621), p-ERK1/2 (1:400; Cell Signaling, 4370, RRID: AB_2315112), IL27R (34N4G11; Novus Biologicals, NBP2-19015, RRID:AB_2916313), α-SMA (1:500; Abcam, 124964, RRID:AB_11129103), and RAE-1 (R&D Systems, AF1136, RRID:AB_2238016) overnight at 4°C.

Techniques: Gene Expression, Virus, Quantitative RT-PCR, Two Tailed Test, Injection, Staining, Expressing

Journal: Cancer Discovery

Article Title: IL27 Signaling Serves as an Immunologic Checkpoint for Innate Cytotoxic Cells to Promote Hepatocellular Carcinoma

doi: 10.1158/2159-8290.cd-20-1628

Figure Lengend Snippet: Figure 5. IL27R signaling promotes tumor growth in NASH-driven HCC. Eight-week-old MUP-uPA+Il27ra−/− and MUP-uPA+Il27ra+/− control mice were fed with a WD for 8 months. A, Representative macroscopic and microscopic [hematoxylin and eosin (H&E) staining] images of livers with tumors. NT, non tumor; T, tumor. B, Tumor load and tumor number of MUP-uPA+Il27ra+/− (n = 7) and MUP-uPA+Il27ra−/− (n = 7) male mice. C and D, qRT-PCR analysis of relative gene expression of Ccnd1 (C) and Lcn2 (D) in NT and T tissue from MUP-uPA+ l27ra+/− (n = 6) and MUP-uPA+Il27ra−/− (n = 6) male mice. E and F, Histologic analysis and quantification of collagen content determined by Van Gieson (E) or Trichrome (F) staining of liver sections from MUP-uPA+Il27ra+/− and MUP-uPA+Il27ra−/− male mice. G–I, FACS analysis of CD45+ immune cells in NT and T tissue of MUP-uPA+Il27ra+/− and MUP-uPA+Il27ra−/− mice. G, Percent age of CD8α+TCRβ+ and CD4+TCRβ+ cells among CD45+ populations. H and I, Percentage of NK1.1+TCR β+ (H) and CD49a+ NK1.1+ and CD49b+ NK1.1+ (I) cells among CD45+ populations in NT and T tissues of MUP-uPA+Il27ra+/− and MUP-uPA+Il27ra−/− mice (n = 4–6). J and K, qRT-PCR analysis of relative gene expression of Cxcr6 and Gzmb (J) as well as Raet1 and H60b (K) in NT and T tissues from MUP-uPA+Il27ra+/− and MUP-uPA+Il27ra−/− mice (n = 6–13). L, FACS analysis of MHC-I expression on CD45−CD31−TER119− tumor cells from MUP-uPA+Il27ra+/− and MUP-uPA+Il27ra−/− mice (n = 4). Representative histogram and mean fluorescence intensity (MFI) of expression are presented. M, qRT-PCR analysis of relative gene expression of Tap1 in tumors from MUP-uPA+Il27ra+/− and MUP-uPA+Il27ra−/− female and male mice (n = 6). C and M, Gene expression was first normalized to Rpl32 and then to that in T tissue from MUP-uPA+Il27ra+/− mice. D, J, and K, Gene expression was first normalized to Rpl32 and then to gene expression in NT tissue of MUP-uPA+Il27ra+/− mice. Data are mean ± SEM from at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, unpaired Student t test (two-tailed); Tukey multiple comparisons test. ns, not significant.

Article Snippet: Slides were blocked with 5% goat serum in 1% BSA–PBS for 20 minutes, and then they were incubated with primary antibodies for Ki-67 (1:100; BioLegend, 151202, RRID:AB_2566621), p-ERK1/2 (1:400; Cell Signaling, 4370, RRID: AB_2315112), IL27R (34N4G11; Novus Biologicals, NBP2-19015, RRID:AB_2916313), α-SMA (1:500; Abcam, 124964, RRID:AB_11129103), and RAE-1 (R&D Systems, AF1136, RRID:AB_2238016) overnight at 4°C.

Techniques: Control, Staining, Quantitative RT-PCR, Gene Expression, Expressing, Fluorescence, Two Tailed Test

Journal: Cancer Discovery

Article Title: IL27 Signaling Serves as an Immunologic Checkpoint for Innate Cytotoxic Cells to Promote Hepatocellular Carcinoma

doi: 10.1158/2159-8290.cd-20-1628

Figure Lengend Snippet: Figure 6. IL27R signaling exerts its action via innate cytotoxic lymphocytes. DEN-treated Il27ra+/− and Il27ra−/− mice were administered anti-NK1.1 or IgG isotype control antibodies for 5.5 months prior to tumor development analysis at 10 months. A, Representative images of macroscopic and microscopic view of tumor-bearing livers. H&E, hematoxylin and eosin; NT, nontumor; T, tumor. B, Tumor load and tumor number in the IgG treatment group: Il27ra+/− (n = 6), Il27ra−/− (n = 6) and anti-NK1.1 treatment group: Il27ra+/− (n = 10) and Il27ra−/−(n = 7) mice. C, Representative images and quantification of α-SMA staining of HCC sections from DEN-treated Il27ra+/− (n = 3) and Il27ra−/− (n = 3) mice injected with anti-NK1.1 antibody or isotype control. D, Mean fluorescence intensity (MFI) of NKp46 surface expression on NK1.1+TCRβ− cells from livers of Il27ra+/− and Il27ra−/− mice as determined by FACS (n = 4). E, qRT-PCR analysis of Ncr1 (NKp46) gene expression in CD49b+ NK cells purified from spleens of wild-type naive mice and stimulated in vitro with rIL27 (n = 3). Gene expression was first normalized to Rpl32 and then to gene expression in untreated condition. F, Representative images of macroscopic view of tumor- bearing livers from Ncr1+/gfpIl27ra+/− and Ncr1+/gfpIl27ra−/− DEN-treated mice analyzed at 10 months of age. G, Tumor load and tumor number among Ncr1+/gfpIl27ra+/− (n = 7) and Ncr1+/gfpIl27ra−/− (n = 6) mice compared with Il27ra+/− (n = 15) and Il27ra−/− (n = 12) mice from the cohorts shown in Fig. 1K. Data are mean ± SEM from at least three independent experiments. *, P < 0.05; **, P < 0.01, unpaired Student t test (two-tailed). ns, not significant.

Article Snippet: Slides were blocked with 5% goat serum in 1% BSA–PBS for 20 minutes, and then they were incubated with primary antibodies for Ki-67 (1:100; BioLegend, 151202, RRID:AB_2566621), p-ERK1/2 (1:400; Cell Signaling, 4370, RRID: AB_2315112), IL27R (34N4G11; Novus Biologicals, NBP2-19015, RRID:AB_2916313), α-SMA (1:500; Abcam, 124964, RRID:AB_11129103), and RAE-1 (R&D Systems, AF1136, RRID:AB_2238016) overnight at 4°C.

Techniques: Control, Staining, Injection, Fluorescence, Expressing, Quantitative RT-PCR, Gene Expression, Purification, In Vitro, Two Tailed Test

Journal: Cancer Discovery

Article Title: IL27 Signaling Serves as an Immunologic Checkpoint for Innate Cytotoxic Cells to Promote Hepatocellular Carcinoma

doi: 10.1158/2159-8290.cd-20-1628

Figure Lengend Snippet: Figure 7. Pharmacologic blockade of IL27 or IL27R suppresses HCC tumor growth in the NASH model. A, Eight-week-old MUP-uPA+ mice were fed a WD for 8 months and received IgG or anti-IL27 (SRF381) for 3.5 months prior to the assessment of tumor development at 10 months. B, Representative images of macroscopic and microscopic views of tumor-bearing livers. H&E, hematoxylin and eosin. C, Tumor load and tumor number of IgG isotype control (n = 10)– and anti-IL27 (n = 11)–treated mice. Representative images and quantification of fibrosis (collagen content) as determined by Trichrome staining (D) and α-SMA staining (E) of liver sections from MUP-uPA+ mice that received IgG isotype control (n = 4) or anti-IL27 (n = 4) treatment. Single- cell suspensions of livers from mice that received IgG isotype control (n = 6–7) or anti-IL27 (n = 6–7) treatment were analyzed by FACS. Percentage of CD4+TCRβ+ and CD8α+TCRβ+ cells (F) and NK1.1+TCRβ− cells (G) among CD45+ populations is presented. H, qRT-PCR analysis of relative gene expression of Gzmb, Tnfsf10, Klrk1, Cxcr6, and Ncr1 in nontumor (NT) and tumor (T) tissue from IgG (n = 10–15) and anti-IL27 (n = 10–17) mice. I, Eight-week-old MUP-uPA+ mice were fed a WD for 8 months and received IgG or anti-IL27R for 4 months prior to the assessment of tumor development at 10 months. J, Representative macroscopic images of livers with developed tumors. K, Tumor load and tumor number of IgG group (n = 8) and anti-IL27R (n = 10) mice. Data are mean ± SEM from at least three independent experiments. *, P < 0.05; **, P < 0.01, unpaired Student t test (two-tailed).

Article Snippet: Slides were blocked with 5% goat serum in 1% BSA–PBS for 20 minutes, and then they were incubated with primary antibodies for Ki-67 (1:100; BioLegend, 151202, RRID:AB_2566621), p-ERK1/2 (1:400; Cell Signaling, 4370, RRID: AB_2315112), IL27R (34N4G11; Novus Biologicals, NBP2-19015, RRID:AB_2916313), α-SMA (1:500; Abcam, 124964, RRID:AB_11129103), and RAE-1 (R&D Systems, AF1136, RRID:AB_2238016) overnight at 4°C.

Techniques: Control, Staining, Quantitative RT-PCR, Gene Expression, Two Tailed Test