NKG2D ligand tumor expression and association with clinical outcome in early breast cancer patients: an observational study.

BACKGROUND: Cell surface NKG2D ligands (NKG2DL) bind to the activating NKG2D receptor present on NK cells and subsets of T cells, thus playing a role in initiating an immune response. We examined tumor expression and prognostic effect of NKG2DL in breast cancer patients. METHODS: Our study population (n = 677) consisted of all breast cancer patients primarily treated with surgery in our center between 1985 and 1994. Formalin-fixed paraffin-embedded tumor tissue was immunohistochemically stained with antibodies directed against MIC-A/MIC-B (MIC-AB), ULBP-1, ULBP-2, ULBP-3, ULBP-4, and ULBP-5. RESULTS: NKG2DL were frequently expressed by tumors (MIC-AB, 50% of the cases; ULBP-1, 90%; ULBP-2, 99%; ULBP-3, 100%; ULBP-4, 26%; ULBP-5, 90%) and often showed co-expression: MIC-AB and ULBP-4 (p = 0.043), ULBP-1 and ULBP-5 (p = 0.006), ULBP-4 and ULBP-5 (p < 0.001). MIC-AB (p = 0.001) and ULBP-2 (p = 0.006) expression resulted in a statistically significant longer relapse free period (RFP). Combined expression of these ligands showed to be an independent prognostic parameter for RFP (p < 0.001, HR 0.41). Combined expression of all ligands showed no associations with clinical outcome. CONCLUSIONS: We demonstrated for the first time that NKG2DL are frequently expressed and often co-expressed in breast cancer. Expression of MIC-AB and ULBP-2 resulted in a statistically significant beneficial outcome concerning RFP with high discriminative power. Combination of all NKG2DL showed no additive or interactive effect of ligands on each other, suggesting that similar and co-operative functioning of all NKG2DL can not be assumed. Our observations suggest that among driving forces in breast cancer outcome are immune activation on one site and tumor immune escape on the other site.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Cellular expression, trafficking, and function of two isoforms of human ULBP5/RAET1G

Background: The activating immunoreceptor NKG2D is expressed on Natural Killer (NK) cells and subsets of T cells. NKG2D contributes to anti-tumour and anti-viral immune responses in vitro and in vivo. The ligands for NKG2D in humans are diverse proteins of the MIC and ULBP/RAET families that are upregulated on the surface of virally infected cells and tumours. Two splicing variants of ULBP5/RAET1G have been cloned previously, but not extensively characterised. Methodology/Principal Findings: We pursue a number of approaches to characterise the expression, trafficking, and function of the two isoforms of ULBP5/RAET1G. We show that both transcripts are frequently expressed in cell lines derived from epithelial cancers, and in primary breast cancers. The full-length transcript, RAET1G1, is predicted to encode a molecule with transmembrane and cytoplasmic domains that are unique amongst NKG2D ligands. Using specific anti-RAET1G1 antiserum to stain tissue microarrays we show that RAET1G1 expression is highly restricted in normal tissues. RAET1G1 was expressed at a low level in normal gastrointestinal epithelial cells in a similar pattern to MICA. Both RAET1G1 and MICA showed increased expression in the gut of patients with celiac disease. In contrast to healthy tissues the RAET1G1 antiserum stained a wide variety or different primary tumour sections. Both endogenously expressed and transfected RAET1G1 was mainly found inside the cell, with a minority of the protein reaching the cell surface. Conversely the truncated splicing variant of RAET1G2 was shown to encode a soluble molecule that could be secreted from cells. Secreted RAET1G2 was shown to downregulate NKG2D receptor expression on NK cells and hence may represent a novel tumour immune evasion strategy. Conclusions/Significance: We demonstrate that the expression patterns of ULBP5RAET1G are very similar to the well-characterised NKG2D ligand, MICA. However the two isoforms of ULBP5/RAET1G have very different cellular localisations that are likely to reflect unique functionality

Contemporary review of risk-stratified management in acute uncomplicated and complicated diverticulitis

BACKGROUND: Acute colonic diverticulitis is a common clinical condition. Severity of the disease is based on clinical, laboratory, and radiological investigations and dictates the need for medical or surgical intervention. Recent clinical trials have improved the understanding of the natural history of the disease resulting in new approaches to and better evidence for the management of acute diverticulitis. METHODS: We searched the Cochrane Library (years 2004-2015), MEDLINE (years 2004-2015), and EMBASE (years 2004-2015) databases. We used the search terms "diverticulitis, colonic" or "acute diverticulitis" or "divertic*" in combination with the terms "management," "antibiotics," "non-operative," or "surgery." Registers for clinical trials (such as the WHO registry and the https://clinicaltrials.gov/) were searched for ongoing, recruiting, or closed trials not yet published. RESULTS: Antibiotic treatment can be avoided in simple, non-complicated diverticulitis and outpatient management is safe. The management of complicated disease, ranging from a localized abscess to perforation with diffuse peritonitis, has changed towards either percutaneous or minimally invasive approaches in selected cases. The role of laparoscopic lavage without resection in perforated non-fecal diverticulitis is still debated; however, recent evidence from two randomised controlled trials has found a higher re-intervention in this group of patients. CONCLUSIONS: A shift in management has occurred towards conservative management in acute uncomplicated disease. Those with uncomplicated acute diverticulitis may be treated without antibiotics. For complicated diverticulitis with purulent peritonitis, the use of peritoneal lavage appears to be non-superior to resection

NKG2D ligand expression in human colorectal cancer reveals associations with prognosis and evidence for immunoediting

PURPOSE: NKG2D binds to cellular ligands of the MIC and ULBP/RAET family. These ligands have restricted expression in normal tissue, but are frequently expressed on primary tumors. The role of NKG2D ligands is thought to be important in carcinogenesis but prognostic impact has not been investigated in such a large cohort. EXPERIMENTAL DESIGN: In our study 462 primary colorectal tumors were screened for expression of all MIC / ULBP / RAET proteins and NK cell infiltration. Tumor microarray technology was used for the purpose of this investigation. RESULTS: NKG2D ligands were expressed by the majority of colorectal tumors; however the level of expression varied considerably. High expression of MIC (68 versus 56 months) or RAET1G (74 versus 62 months) demonstrated improved patient survival. Tumors expressing high levels of MIC and RAET1G showed improved survival of 77 months over tumors that expressed high levels of one ligand or low levels of both. High-level expression of all ligands was frequent in TNM stage 1 tumors, but became progressively less frequent in stage 2, 3 and 4 tumors. Expression of MIC was correlated with NK cellular infiltration. CONCLUSION: The observations presented are consistent with an immunoediting mechanism that selects tumor cells that have lost or reduced their expression of NKG2D ligands. Combination of MIC and TNM stage was found to be the strongest predictor of survival splitting patients into 8 groups and suggesting prognostic value in clinical assessment. Of particular interest were stage 1 patients with low expression of MIC who had a similar survival to stage 3 patients, and may be candidates for adjuvant therapy

RAET1G1 protein is poorly expressed at the cell surface.

<p>(A) Stable cell lines expressing N-terminal GFP fusion proteins of ULBP2, and RAET1G1, were created in HT1080 cells. The transfectants expressed equivalent levels of transgene, as evident by total GFP fluorescence (open histogram), plotted versus untransfected HT1080 cells (gray shaded histogram). Cell surface expression was assessed by staining with an anti-GFP monoclonal antibody followed by an Alexa-Fluor 633 nm (far red) secondary antibody. The RAET1G1 transfectant had only a modest level of staining over untransfected cells, whereas the ULBP2 transfectant stained at a high level. This indicates that a much smaller percentage of RAET1G1-GFP transgene is present at the cell surface when compared to the closely related molecule ULBP2. (B) This observation was confirmed by confocal microscopy. The ULBP2-GFP transfectant showed clearly defined cell surface fluorescence in all cells, whereas cell surface RAET1G1-GFP could not be observed. (C) Stable transfectants were radiolabelled and chased for 180 minutes. Radiolabelled RAET1G1 and ULBP2 were then immunoprecipitated with an anti-GFP antibody. Endo H digests reveal that a substantial proportion of ULBP2 has acquired Endo H resistance after 180 minutes, and hence has trafficked to the cell surface. In contrast RAET1G1 remains Endo H sensitive.</p

The truncated isoform, RAET1G2, can be secreted by cells and can downregulate NKG2D expression on NK cells.

<p>(A) RAET1G2 transcript encodes a protein that is secreted from the cell. C-terminally V5 tagged RAET1G1 and RAET1G2 were transfected into Cos7 cells, RAET1G2 protein was readily detected in the tissue culture media of transfected cells by western blot with an anti-V5 antibody, whereas RAET1G1 was not demonstrated. (B) Soluble RAET1G2 down regulates NKG2D expression on natural killer lymphocytes. Isolated peripheral blood CD3− CD56+ NK cells were incubated for 24 hours at 37°C with culture supernatant from RAET1G1 transfected COS cells (solid black profile) or RAET1G2 transfected COS cells (open dashed profile) and examined for NKG2D expression levels using a PE-labelled anti-NKG2D antibody and flow cytometry. Supernatant from the RAET1G2 transfectant caused a substantial downregulation of NKG2D expression (mean fluorescence intensity = 41.75) compared with supernatant from the RAET1G1 transfectant (mean fluorescence intensity = 144.7). The solid white profile shows background staining with a PE-labelled mouse isotype control (mean fluorescence intensity = 8.1). (C) Soluble RAET1G2 binding to NK cells occurs rapidly but is lost within 24 hours of culture. V5 epitope tagged RAET1G2 was incubated at 37°C with isolated peripheral blood natural killer cells and assayed for binding after 1 hour (solid black profile) and 24 hours (open dashed profile) of incubation. RAET1G2 binding was detected by staining with anti-V5 antibody and PE-labelled goat anti-mouse second stage antibody followed by flow cytometric analysis. The solid white profile depicts background staining with the second stage antibody alone. (D) Coomassie stained SDS-PAGE gel of soluble recombinant RAET1G (rRAET1G). (E) rRAET1G down regulates NKG2D expression. Isolated peripheral blood CD3− CD56+ NK cells were incubated for 24 hours at 37°C with a range of concentrations of rRAET1G. Downregulation of NKG2D was observed at concentrations of rRAET1G as low as 100 ng/ml, but not in the presence of an irrelevant control his-tagged protein.</p