Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Mar;63(3):217-27.
doi: 10.1369/0022155414563800. Epub 2014 Dec 3.

Immunohistochemical validation and expression profiling of NKG2D ligands in a wide spectrum of human epithelial neoplasms

Hiromi Fujita  1   2 , Yutaka Hatanaka  1 , Yoichi Sutoh  1   2 , Yuta Suzuki  1 , Koji Oba  3 , Kanako C Hatanaka  1 , Tomoko Mitsuhashi  1 , Noriyuki Otsuka  2 , Kazunori Fugo  2 , Masanori Kasahara  2 , Yoshihiro Matsuno  1
Affiliations

Immunohistochemical validation and expression profiling of NKG2D ligands in a wide spectrum of human epithelial neoplasms

Hiromi Fujita et al. J Histochem Cytochem. 2015 Mar.

Abstract

The MHC class I-chain-related proteins (MICs) and the UL16-binding proteins (ULBPs) are inducible stress response molecules that work as activators of a specific receptor, NKG2D, which is expressed on effector cells, such as NK cells and subsets of T cells. In this study, we sought to explore the biological significance of NKG2D ligands in human neoplasms by comprehensively examining the immunohistochemical expression profile of NKG2D ligands in a variety of human epithelial neoplasms. Following careful validation of the immunohistochemical specificity and availability of anti-human ULBP antibodies for formalin-fixed paraffin-embedded (FFPE) materials, the expression of NKG2D ligands was analyzed in FFPE tissue microarrays comprising 22 types of epithelial neoplastic tissue with their non-neoplastic counterpart from various organs. Hierarchical cluster analysis demonstrated a positive relationship among ULBP2/6, ULBP3, ULBP1, and ULBP5, whose expression patterns were similar across all of the neoplastic tissues examined. In contrast, MICA/B, as well as ULBP4, did not appear to be related to any other ligand. These expression profiles of NKG2D ligands in human neoplasms based on well-validated specific antibodies, followed by hierarchical cluster analysis, should help to clarify some functional aspects of these molecules in cancer biology, and also provide a path to the development of novel tumor-type-specific treatment strategies.

Keywords: NKG2D ligands; epithelial neoplasms; immunohistochemistry.

PubMed Disclaimer

Conflict of interest statement

Declaration of Conflicting Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Representative immunohistochemical patterns of NKG2D ligands. The intensity of staining, as the proportion of positive cases among all cases, was assessed using a semi-quantitative system as: no (A; score 0 in MICA/B), low (B, score 1 in ULBP3) or high (C; score 2 in ULBP5) by two observers. Scores of 0 and 1 were defined as negative, and a score of 2 was defined as positive. Scale, 100 μm.
Figure 2.
Figure 2.
Validation of antibodies using ULBP-transfected COS7 cells. We confirmed the specificity of all antibodies against UL16-binding proteins (ULBP) using western blotting with cell lysates (A) and immunohistochemistry with a FFPE cell block (B) prepared from each ULBP transfectant, respectively. The anti-ULBP2 antibody cross-reacted with ULBP5 and ULBP6 in western blotting but only with ULBP6 in FFPE. Therefore, this antibody was used to mark ULBP2 and ULBP6. Scale, 50 μm.
Figure 3.
Figure 3.
Expression profiles for NKG2D ligands in non-neoplastic epithelial tissues. Hierarchical cluster analysis based on the expression profiles of NKG2D ligands demonstrated two distinct ligand-based clusters and three distinct tissue-based clusters: white, N-null type; blue, N-variable type; pink, N-complete type (right side).
Figure 4.
Figure 4.
Diverse expression of NKG2D ligands in non-neoplastic prostate tissue. The upper panels show immunohistochemistry for ULBP1 (A), ULBP2/6 (B), and ULBP4 (C) as positive, and the lower panels for MICA/B (D), ULBP3 (E), and ULBP5 (F) as negative. Scale, 100 μm.
Figure 5.
Figure 5.
Expression profiles of NKG2D ligands in neoplastic epithelial tissues. Hierarchical cluster analysis based on the expression profiles of NKG2D ligands demonstrated five distinct tissue-based clusters: white, T-null type; blue, T-variable A type; yellow, T-variable B type; green, T-variable C type; pink, T-complete type. A dendrogram constructed from the expression profiles obtained in the ligand-based cluster analysis (top) was similar to the phylogenetic tree based on the DNA promoter sequences of the ligands reported previously by Eagle et al. (2006).
Figure 6.
Figure 6.
Variable expression pattern of NKG2D ligands in uterine endometrial carcinoma. Uterine endometrial carcinoma was placed in the T-variable B group (Figure 5). Only MICA/B showed a negative pattern (A), whereas the other ligands, including ULBP3 (B) and ULBP4 (C), were positive. Scale, 100 μm.
Figure 7.
Figure 7.
Differential expression of NKG2D ligands between the two histological types of thyroid carcinoma. Thyroid follicular carcinoma was divided into the T-variable C type (A–C; B was Score 0), and papillary carcinoma into the T-complete type (D–F). (F) Inset shows non-neoplastic follicular epithelium. Scale, 50 μm.
See this image and copyright information in PMC

References

    1. Bacon L, Eagle RA, Meyer M, Easom N, Young NT, Trowsdale J. (2004). Two human ULBP/RAET1 molecules with transmembrane regions are ligands for NKG2D. J Immunol 173:1078-1084. - PubMed
    1. Bahram S, Bresnahan M, Geraghty DE, Spies T. (1994). A second lineage of mammalian major histocompatibility complex class I genes. Proc Natl Acad Sci U S A 91:6259-6263. - PMC - PubMed
    1. Bauer S, Groh V, Wu J, Steinle A, Phillips JH, Lanier LL, Spies T. (1999). Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285:727-729. - PubMed
    1. Castriconi R, Dondero A, Negri F, Bellora F, Nozza P, Carnemolla B, Raso A, Moretta L, Bottino C. (2007). Both CD133(+) and CD133(-) medulloblastoma cell lines express ligands for triggering NK receptors and are susceptible to NK-mediated cytotoxicity. Eur J Immunol 37:3190-196. - PubMed
    1. Cerwenka A, Bakker AB, McClanahan T, Wagner J, Wu J, Phillips JH, Lanier LL. (2000). Retinoic acid early inducible genes define a ligand family for the activating NKG2D receptor in mice. Immunity 12:721-727. - PubMed

Publication types

MeSH terms

Substances