Increased percentage of T_reg cells in <i>Il27ra</i>-deficient mice.

<p>(A & B) tumors induced by injection of 25 µg of MCA into <i>Il27ra</i>^+/+ and <i>Il27ra</i>^−/− were harvested at 14 weeks post-induction and 5 µm sections analyzed by immunohistochemistry to detect FoxP3⁺ cells. (A) Representative photographs (magnification = 100×) of tumor sections stained with FoxP3 (brown). (B) The average Foxp3⁺ cells per field of view, over 5 fields of view per mouse, is indicated (n = 7 per group). Bars indicate the average of 7/8 mice per genotype. (C-E) 14 or 28 days after PyMT tumor transplant (as indicated), lymphocytes from <i>Il27ra</i>^+/+ (filled grey circles) and <i>Il27ra</i>^−/− (open black squares) host spleen, TDLN and NDLN were harvested. Single cell preparations were stained with antibodies against CD4, CD25 and Foxp3 for flow cytometry. The percentage of FoxP3⁺CD25⁺ cells in the CD4⁺ compartment is shown for (C) TDLN, (D) spleen and (E) non-tumor draining lymph node. n = 5 per group *p<.05, **p<.01, ***p<.001 (unpaired t test).</p

Interleukin-27 Signaling Promotes Immunity against Endogenously Arising Murine Tumors

<div><p>Interleukin-27 (IL-27) is a pleiotropic cytokine but its immunosuppressive effects predominate during many <i>in vivo</i> immunological challenges. Despite this, evidence from tumor cell line transfer models suggested that IL-27 could promote immune responses in the tumor context. However, the role of IL-27 in immunity against tumors that develop <i>in situ</i> and in tumor immunosurveillance remain undefined. In this study, we demonstrate that tumor development and growth are accelerated in IL-27 receptor α <i>(Il27ra)</i>-deficient mice. Enhanced tumor growth in both carcinogen-induced fibrosarcoma and oncogene-driven mammary carcinoma was associated with decreased interferon-γ production by CD4 and CD8 T cells and increased numbers of regulatory T-cells (T_reg). This is the first study to show that IL-27 promotes protective immune responses against endogenous tumors, which is critical as the basis for future development of an IL-27 based therapeutic agent.</p> </div

Accelerated development and growth of PyMT-induced mammary tumors in <i>Il27ra</i>-deficient mice.

<p>(A-D) WT mouse mammary epithelial cells (MMECs), retrovirally transduced with the pMIG-PyMT vector were transplanted to <i>Il27ra</i>^+/+ (filled grey circles) and <i>Il27ra</i>^−/− (open black squares) hosts (n = 9/group). Tumor development and growth was monitored by <i>in vivo</i> GFP imaging and measurement of palpable tumors using Vernier calipers for 280 days. (A & B) tumor growth, as measured by calipers, of individual <i>Il27ra</i>^+/+ mice (A) and <i>Il27ra</i>^−/− mice (B) with palpable mammary tumor. (C) cumulative % of mice with a detectable GFP signal in the mammary gland. (D) Average days to tumor detection (GFP) for mice that developed tumors for each genotype. (E) Primary mammary tumors that arose after implantation of pMIG-PyMT transduced tissue were excised and dissected into pieces (∼1 mm³) and surgically transplanted into groups of <i>Il27ra</i>^+/+ and <i>Il27ra</i>^−/− mice (n = 5/group/time point). Mean tumor diameter (mm) at harvest, 14 and 28 days p.t., is shown. * p<.05 (unpaired t tests). Data presented are from one of three independent studies.</p

Reduced IFN-γ production by activated CD4⁺ T cells in tumor bearing <i>Il27ra</i>-deficient mice.

<p>(A, B, D, F–H) Fibrosarcomas were induced in <i>Il27ra</i>^+/+ (grey bars) and <i>Il27ra</i>^−/− (black bars) by injecting 25 µg of MCA s.c.. 14 wk after tumor induction, lymphocytes from spleen, tumor draining LN (TDLN; inguinal) and non tumor draining LN (NDLN; contralateral inguinal) of tumor bearing mice were harvested (n = 6 per genotype). (C, E) PyMT-driven mammary carcinoma cells were transplanted into <i>Il27ra</i>^+/+ (grey bars) and <i>Il27ra</i>^−/− (black bars) mice. 32 days after tumor transplantation, lymphocytes from spleen, TDLN and NDLN of tumor bearing mice were harvested (n = 3 per genotype). Cells were re-stimulated with PMA and ionomycin for 5 hours in the presence of a protein transport inhibitor then IFNγ, IL-4, IL-10 and IL-17 production was determined by flow cytometry. (A) Representative cytometry plots illustrating IFNγ versus CD44 expression. (B-H) The percentage of cytokine (as indicated) producing cells in the CD4⁺ CD44^hi gate (B, C, F-H) or the CD8⁺ CD44^hi gate (D, E) is shown. Error bars indicate SEM. * p<.05, ** p<.01, *** p<.001 (unpaired t tests). Each dataset is representative of two independent studies.</p

Rapid development of MCA-induced fibrosarcoma in <i>Il27ra</i>-deficient mice.

<p>Groups of <i>Il27ra</i>^+/+ (filled grey circles) and <i>Il27ra</i>^−/− (open black squares) mice were treated with a single dose of 10 µg (A, B & C) or 25 µg (D) of MCA and tumor development was monitored weekly for 20 weeks. Tumors >3 mm in mean diameter and progressively growing were scored positive. (A & B) tumor growth curves of individual <i>Il27ra</i>^+/+ (A) and <i>Il27ra</i>^−/− (B) mice with sarcoma. (C & D) cumulative incidence (%) in groups of mice injected with 10 μg (C; <i>Il27ra</i>^+/+ n = 10, <i>Il27ra </i>^−/− n = 11, data are representative of 2 independent experiments) and 25 μg (D; data from 2 experiments are combined to give n = 19 per group) of MCA respectively. The overall tumor incidence is indicated. * p<.05 (Log-rank test).</p

<i>ELF5</i> expression in normal breast and breast cancer.

<p><i>ELF5</i> expression in the UNC337 breast cancer cohort according to subtype. Top panel shows combined results with statistical analysis (thick black line, median;, box, interquartile range 25%–75% of points; whiskers, 1.5× interquartile range; crosses, individual tumors). Bottom panel <i>ELF5</i> expression in all individual tumor and normal samples. Basal and normal had significantly higher expression of ELF5.</p

ELF5 specifies breast cancer subtype.

<p>(A) Sub network of breast cancer subtype gene sets derived from forced ELF5 expression in MCF7 luminal breast cancer cells (inner node color) and knockdown of ELF5 expression in HCC1937 basal breast cancer cells. Node size is proportional to gene set size; thicker green lines indicate greater gene set overlap. Nodes are positioned according to similarity in gene sets. Labels in bold type indicate the functional significance of the four clusters generated, label is plain type is the gene set name. The full network is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001461#pbio.1001461.s016" target="_blank">Figure S16</a>. (B–D) expression signature analysis of the ELF5-induced changes in molecular subtype produced by ELF5 knockdown in HCC1937 cells (B), or forced ELF5 expression in MCF7 cells (C), or T47D cells (D). Bars show the indicated comparisons that produce the associated <i>p</i>-values. BS, borderline significance; NS, not significant.</p

ELF5 suppresses the estrogen-sensitive phenotype.

<p>(A) Western blot showing reduced expression of key genes involved in the response to estrogens following induction of ELF5 expression. (B) Reduced transcriptional activity of reporters of <i>ER</i> and <i>FOXA1</i> (<i>UGT2B17</i> promoter) transcriptional activity following induction of ELF5 in MCF7 cells. Black bars, -DOX, grey bars +DOX 72 h for <i>ERE</i> and 24 h and 48 h for <i>FOXA1</i>. (C) Cell accumulation in MCF7-V5 cell cultures with (+E) or without (−E) 10 nM estrogen treatment, or following expression of ER (+ER) and 10 nM E in the context of induced ELF5. Black bars, -DOX; grey bars +DOX, 72 h and 144 h, respectively. (D) interaction of ELF5-regulated gene sets with estrogen-regulated gene sets. <i>p</i>-Values and odds ratios derived from hypergeometric tests. Number of genes in brackets. (E) Enrichment of gene sets in ELF5 ChIP targets either down (Dn) or Up in response to forced ELF5-V5 expression in T47D cells with DOX. P-Values for hypergeometric tests from GSEA (upper case) or Oncomine (lower case).</p

Elf5 modulates the adhesion of breast cancer cells.

<p>(A) Quantification of detached cells in cultures treated with DOX (+D) compared to no induction (−D). (B) Ability of DOX-treated cells to replate 4 h after trypsin destruction of attachment proteins, compared to untreated cells. Data are expressed as a percentage of replated untreated cells. (C) Proportion of apoptotic cells in DOX treated (grey bars) compared to untreated (black bars) T47D-ELF5-V5 cells, measured by flow cytometry using the M30 antibody. (D) Expression and activation of key cell adhesion proteins following DOX induction of ELF5-V5 expression.</p

Visualization of the transcriptional functions of ELF5 in breast cancer.

<p>Affymetrix transcript profiling following induction of ELF5 in T47D and MCF7 luminal breast cancer cells, analysed by LIMMA and GSEA. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001461#s2" target="_blank">Results</a> are visualized using the enrichment map plug-in for Cytoscape. Each circular node is a gene set with diameter proportional to the number of genes. The outer node color represents the magnitude and direction of enrichment (see scale) in T47D cells, inner node color enrichment in MCF7 cells. Thickness of the edges (green lines) is proportional to the similarity of gene sets between linked nodes. The most related clusters are placed nearest to each other. The functions of prominent clusters are shown. The network can be examined in detail using the scalable PDF in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001461#pbio.1001461.s004" target="_blank">Figure S4</a>.</p