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

Recovery and analysis of DNA from fixed tissue

This research was undertaken to: 1) Develop techniques for the recovery of DNA from fixed and paraffin wax embedded tissues. 2) Assess the potential for use of this DNA for the diagnosis of lymphoma. 3) Contribute to the understanding of the effects of fixation on DNA.;Initial investigations used pure DNA and lymphocytes to determine the effect of five fixatives on the integrity, recovery and restriction endonuclease digestion of the nucleic acid. Two fixatives, Bouin and formol sublimate, proved unsuitable for further analysis. DNA recovery and Southern analysis was then attempted using Carnoy, formol saline and neutral buffered formalin fixed and paraffin wax embedded tissue. From this an optimised method featuring prolonged incubation of tissue with Proteinase K and SDS at 37°C followed by purification was developed. Within limits imposed by fixation this DNA was suitable for Southern analysis and amplification by PCR. A simplified DNA recovery method involving overnight incubation of paraffin wax sections in Proteinase K at 55°C without purification was also evaluated. This gave satisfactory results by PCR but the maximum product size obtained was 500 bp compared with 1250 bp using the optimised method.;DNA was better preserved after Carnoy than following formalin fixation and this was reflected in consistently superior Southern and PCR results. Formalin fixation induced degradation of DNA, which increased as fixation time was extended. This made Southern analysis for the identification of B and T cell rearrangements in lymphomas unreliable. However, the t(14:18) translocation was demonstrated successfully by PCR in formalin fixed follicular lymphomas.;The results of these investigations show the importance of the pH of fixatives on the preservation of DNA. They also suggest that chemical interactions with DNA and associated proteins occur using fixatives containing formalin and mercury.

Molecular diagnostics

Oxford, UKxv, 412 hlm.: bibl., ref., index; 25 c

Application of phage display to high throughput antibody generation and characterization.

We have created a high quality phage display library containing over 1010 human antibodies and describe its use in the generation of antibodies on an unprecedented scale. We have selected, screened and sequenced over 38,000 recombinant antibodies to 292 antigens, yielding over 7,200 unique clones. 4,400 antibodies were characterized by specificity testing and detailed sequence analysis and the data/clones are available online. Sensitive detection was demonstrated in a bead based flow cytometry assay. Furthermore, positive staining by immunohistochemistry on tissue microarrays was found for 37% (143/381) of antibodies. Thus, we have demonstrated the potential of and illuminated the issues associated with genome-wide monoclonal antibody generation.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

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

Subcellular localisation of RAET1G1.

<p>(A) By confocal microscopy, little RAET1G1 co-localised with MHC Class I (W6/32 antibody) at the cell surface, suggesting that the majority of RAET1G protein is found inside the cell. RAET1G1 does co-localise with calreticulin, a protein expressed predominately in the endoplasmic reticulum (ER). Co-localisation was also seen with the golgi apparatus marker GM130, as highlighted in inset. (C) Antibody internalisation experiment to prove that some RAET1G1 protein does reach the cell surface where it can be internalised into the endocytic pathway. Live cells were incubated with an anti-myc tag antibody prior to fixing and staining. Red represents myc tag staining, purple is staining with an antibody to the early endosome marker EEA1, and green is RAET1G1. Only RAET1G1 positive cells showed staining with the myc antibody; RAET1G1 negative cells only stained with anti-EEA1. In a magnified image EEA1, myc and RAET1G1 co-localised in vesicles (white, marked by arrows) showing that RAET1G1 is being internalised into early endosomes. Also, EEA1 negative, RAET1G1 positive, myc positive vesicles could be seen (yellow), and probably represent other compartments in the endocytic pathway. These data indicate that the vast majority of RAET1G1 protein is present in the ER and early golgi apparatus, and hence does not acquire Endo H resistance. A small minority of RAET1G1 can reach the cell surface, where it can be internalised into the endocytic pathway.</p

Generation of specific anti-RAET1G1 antiserum.

<p>(A) A rabbit polyclonal antiserum was raised against the cytoplasmic domain of RAET1G. By western blot a protein of approximately 70 kDa was recognised in Cos7 cells transfected with a construct encoding an RAET1G-GFP fusion protein, but not in untransfected Cos7 cells. (B) By confocal microscopy the anti-RAET1G antiserum specifically stains (red) Cos7 cells transfected with RAET1G-GFP (green), but not the highly related molecule ULBP2-GFP.</p