il27r  (Novus Biologicals)

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