Journal: Cancer research

Article Title: IL15RA drives antagonistic mechanisms of cancer development and immune control in lymphocyte-enriched triple-negative breast cancers.

doi: 10.1158/0008-5472.CAN-14-0637

Figure Lengend Snippet: Figure 4. IL15 is expressed by TNBCs and basal BCCLs and its silencing specifically impairs growth in high IL15RA–expressing cell lines. Correlation of gene- expression levels between IL15RA and IL15 across A, 579 TNBC (GSE31519) and B, 51 BCCLs. The Pearson r values and P values for significance are shown. C, relative gene expression of IL15 in 51 BCCL dichotomized into the basal- like (triangles) and luminal-like (dots) phenotypes, whereby each symbol represents a cell line. D, ELISA analysis of IL15 levels in a subset of basal or luminal BCCLs. HaCat cells were included as a positive control. IL15 levels were assessed in whole cells (lysate). E to G, cell population growth assays of MDA-MB-231 (E), HCC1143 (F), (high levels of both IL15 and IL15RA), and SKBR3 (G) cells (low levels of both IL15 and IL15RA) following IL15 knockdown by two different shRNA species (IL15 sh26 and IL15 sh27). H, caspase-3/7 activation was assessed in HCC1143 cells transduced with a control shRNA (nontargeting) or shRNAs targeting IL15, 24 hours after seeding the cells for the cell population growth assay, equivalent to 7 days posttransduction. I, quantification of shRNA-mediated IL15 knockdown in MDA-MB-231 cells by qPCR. Data representative of at least two independent experiments; values are average SD; sample size, 3; , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: When indicated, a blocking anti-IL15 monoclonal mouse antibody (MAB2471; R&D Systems) was added at 10 mg/mL to BCCLs 30 minutes before coculture with PBMC.

Techniques: Expressing, Gene Expression, Enzyme-linked Immunosorbent Assay, Positive Control, Knockdown, shRNA, Activation Assay, Transduction, Control, Growth Assay

Journal: Cancer research

Article Title: IL15RA drives antagonistic mechanisms of cancer development and immune control in lymphocyte-enriched triple-negative breast cancers.

doi: 10.1158/0008-5472.CAN-14-0637

Figure Lengend Snippet: Figure 5. IL15 signals through IL15RA to promote phosphorylation of ERK1/2, JAK1, STAT1, STAT2, AKT, and PRAS40 in BCCLs. A, HCC1143 cells (high IL15RA), were stimulated with 100 ng/mL of IL15 for 15 minutes, then lysed and protein phosphorylation assessed using a human phospho- protein array. Phospho-protein patterns were consistent in two independent experiments; for a full description of the phospho- antibodies included in the array please refer to Supplementary Fig. S1. B, IL15 induced increased ERK1/2, AKT, STAT2, PRAS40, and STAT1 phosphorylation as assessed by densitometry in one representative experiment; error bars, SD of the densitometry readings from the experimental replicates. C, phospho-protein array results were validated by Western blotting using a different set of phospho-antibodies and a wider set of BCCLs: either high IL15RA, expressing such as HCC1954 and HCC1143, or low IL15RA, expressing such as SKBR3. D, Western blot analysis of JAK1 and JAK2 phosphorylation in response to IL15 stimulation in HCC1954, HCC1143, and MDA- MB-231 (high IL15RA) or SKBR3 (low IL15RA) cell lines. E, Western blot analysis of STAT3, STAT5, and STAT6 phosphorylation in response to IL15 stimulation in HCC1143 cells. F, Western blot analysis of JAK1 and ERK1/2 phosphorylation in response to IL15 stimulation following siRNA- mediated IL15RA knockdown (IL15R siRNA #0, #1, and #2) in HCC1954 cells; nontargeting siRNAs used as negative control.

Article Snippet: When indicated, a blocking anti-IL15 monoclonal mouse antibody (MAB2471; R&D Systems) was added at 10 mg/mL to BCCLs 30 minutes before coculture with PBMC.

Techniques: Phospho-proteomics, Protein Array, Western Blot, Expressing, Knockdown, Negative Control

Journal: Cancer research

Article Title: IL15RA drives antagonistic mechanisms of cancer development and immune control in lymphocyte-enriched triple-negative breast cancers.

doi: 10.1158/0008-5472.CAN-14-0637

Figure Lengend Snippet: Figure 6. IL15RA knockdown impairs motility of basal BCCLs by a JAK1/STAT1–dependent, ERK1/ 2–independent mechanism. A, MDA-MB-231 and HCC1954 cell migration was investigated using a standard Transwell assay in cells transfected with a control siRNA (nontargeting) and siRNAs targeting IL15RA (IL15R siRNA#0, IL15R siRNA#2). B, MDA-MB-231 cells were seeded on a thin layer of collagen type I and adhesion assessed following transfection with a control siRNA (nontargeting) and siRNAs targeting IL15RA (IL15R siRNA#0, IL15R siRNA#2). C, Western blot analysis showing efficacy of JAK1 knockdown 72 hours posttransfection. D, MDA- MB-231 and HCC1954 cell migration was investigated using a standard Transwell assay in cells transfected with a control siRNA (nontargeting) and siRNAs targeting JAK1 (JAK1 siRNA#47, JAK1 siRNA48). E, HCC1954 cell population growth was assessed in cells transfected with a control siRNA (nontargeting) and siRNAs targeting JAK1 (JAK1 siRNA#47, JAK1 siRNA48). F, the impact of JAK1 knockdown on IL15-induced phosphorylation of STAT1 and ERK1/2 was assessed in HCC1954 cells by Western blot analysis. G, quantitation by densitometry of the IL15-induced phosphorylation of STAT1 as showed in F. H, quantitation by densitometry of the IL15-induced phosphorylation of ERK1/2 as showed in F. Data are representative of at least two independent experiments; values are average SD; sample size, 3; , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: When indicated, a blocking anti-IL15 monoclonal mouse antibody (MAB2471; R&D Systems) was added at 10 mg/mL to BCCLs 30 minutes before coculture with PBMC.

Techniques: Knockdown, Migration, Transwell Assay, Transfection, Control, Western Blot, Phospho-proteomics, Quantitation Assay

Journal: Cancer research

Article Title: IL15RA drives antagonistic mechanisms of cancer development and immune control in lymphocyte-enriched triple-negative breast cancers.

doi: 10.1158/0008-5472.CAN-14-0637

Figure Lengend Snippet: Figure 7. IL15RA/IL15–expressing BCCLs elicit STAT5 phosphorylation in PBMC upon cocultivation. Flow-cytometry analysis of STAT5 phosphorylation in PBMC cocultured 1:1 with BCCLs or stimulated with recombinant IL15 (rIL15; 100 pg/mL) for 30 minutes. IL15-blocking antibody (aIL15) effects were assessed by adding 30 minutes before coculture. The effect of IL15RA knockdown was also assessed following transduction and selection of MDA-MB-231 cells with shRNAs targeting IL15RA (IL15R sh3) or IL15 (IL15 sh26 and IL15 sh27) as well as a control shRNA (nontargeting). Data are representative of at least two independent experiments; values are average SD; sample size, 3; , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: When indicated, a blocking anti-IL15 monoclonal mouse antibody (MAB2471; R&D Systems) was added at 10 mg/mL to BCCLs 30 minutes before coculture with PBMC.

Techniques: Expressing, Phospho-proteomics, Flow Cytometry, Recombinant, Blocking Assay, Knockdown, Transduction, Selection, Control, shRNA

Journal: eLife

Article Title: Clearance of senescent decidual cells by uterine natural killer cells in cycling human endometrium

doi: 10.7554/eLife.31274

Figure Lengend Snippet: ( A ) Left panel: uNK cell density in the subluminal stroma was quantified using a standardized immunohistochemistry protocol in LH timed endometrial biopsies (n = 1,997). Right panel: corresponding centile graph. Color code on the left. ( B ) Left panel: example of the tissue distribution of CD56 + uNK cells (brown staining) at LH + 10. Scale bar = 250 μm. Right panel: Pearson’s correlation analysis of stromal cell and uNK cell densities. A total of 80 randomly selected images from 20 biopsies were analyzed. ( C ) Representative images of an eosin stained primary culture decidualized for 8 days incubated for 18 hr with or without uNK cells isolated from luteal phase endometrium. Scale bar = 100 μm. ( D ) SAβG activity in undifferentiated or day eight decidualized EnSCs co-cultured with or without uNK cells in the presence or absence of the apoptosis inhibitor Z-VAD-FMK (Z-VAD, 10 μM) or the granzyme activity inhibitor 3,4-DCI (25 μM). ( E ) Secretion of IL-15 secretion was measured every 48 hr in the supernatant of primary EnSCs decidualized for the indicated time-points following pretreatment with vehicle, dasatinib (250 nM) or palbociclib (1 μM). ( F ) SAβG activity in undifferentiated or day eight decidualized EnSCs co-cultured with or without uNK cells in the presence or absence of an IL-15 blocking antibody (1 μg/ml). Data are mean ±SEM of 3 biological replicates unless stated otherwise. Different letters above the error bars indicate that those groups are significantly different from each other at p<0.05. 10.7554/eLife.31274.015 Figure 5—source data 1. uNK cell mediated immune surveillance and clearance of senescent cells.

Article Snippet: Inhibitors were pre-incubated with EnSCs for 1 hr prior to and during co-cultures as follows: Z-VAD-FMK (apoptosis inhibitor) (10 μM), 3,4-DCI (25 μM) (granzyme activity inhibitor), anti-IL-15 inhibitory antibody (1 μg/ml, MAB247, Bio-Techne, Abingdon, UK) and inhibitory anti-NKG2D antibody (10 μg/ml, MAB139, Bio-Techne).

Techniques: Immunohistochemistry, Staining, Incubation, Isolation, Activity Assay, Cell Culture, Blocking Assay

Journal: eLife

Article Title: Clearance of senescent decidual cells by uterine natural killer cells in cycling human endometrium

doi: 10.7554/eLife.31274

Figure Lengend Snippet: We propose that rapid endometrial growth during the proliferative phase is important for implantation as it imparts replication stress in a subpopulation of EnSCs. Upon cell cycle exit at G0/G1, this subpopulation of stressed EnSCs do not differentiate into specialist decidual cells but undergo acute cellular senescence and secrete a host of inflammatory mediators (senescence associated secretory phenotype; SASP) involved in endometrial receptivity. In parallel, Il-15 secreted by differentiated decidual cells activates uNK cells, which then target and eliminate senescent cells through granule exocytosis. Systematic clearance of acutely senescent decidual cells by uNK cells not only remodels but also rejuvenates the endometrium at the time of embryo implantation.

Article Snippet: Inhibitors were pre-incubated with EnSCs for 1 hr prior to and during co-cultures as follows: Z-VAD-FMK (apoptosis inhibitor) (10 μM), 3,4-DCI (25 μM) (granzyme activity inhibitor), anti-IL-15 inhibitory antibody (1 μg/ml, MAB247, Bio-Techne, Abingdon, UK) and inhibitory anti-NKG2D antibody (10 μg/ml, MAB139, Bio-Techne).

Techniques: