Immunohistochemical Validation and Expression Profiling of NKG2D Ligands in a Wide Spectrum of Human Epithelial Neoplasms

It is widely accepted that the natural history of human neoplasms is influenced by a variety of microenvironmental factors, such as blood supply, hypoxia, and immunological surveillance. Natural killer (NK) cells are one of the key players in the immunological response to neoplastic cells, and their function is regulated by a delicate balance of signals initiated from a variety of activating and inhibitory receptors on NK cells. The activating receptor NKG2D (natural-killer group 2, member D) belongs to the family of C-type lectin-like type II transmembrane proteins and is expressed by a range of effector cells, such as NK cells, NKT cells, γδT cells (Wu et al. 1999; Jamieson et al. 2002) and CD8⁺ T cells (Ehrlich et al. 2005). One of the characteristics of the NKG2D system is that there are multiple ligands for the receptor. The NKG2D receptor ligands are distant homologs of major histocompatibility complex (MHC) class I molecules (Bauer et al. 1999; Cerwenka et al. 2000; Diefenbach et al. 2000), and include two families in humans: the MHC class I-chain-related proteins (MIC) A and B (Bauer et al. 1999), and the UL16-binding proteins (ULBPs) 1-6 (Cosman et al. 2001; Radosavljevic et al. 2002; Chalupny et al. 2003; Bacon et al. 2004; Eagle et al. 2009b). The amino acid sequences and domain structures of NKG2D ligands are variable. MICA and MICB consist of α1, α2 and α3 domains (Bauer et al. 2000; Bahram et al. 1994), whereas each ULBP consists of only two Ig-like domains (α1 and α2). Moreover, two members (ULBPs 4, -5) of the ULBP family are anchored to the cell membrane by a transmembrane region, whereas other members are linked to the cell surface via glycosyl-phosphatidylinositol anchors (Bacon et al. 2004; Eagle et al. 2009b). Although NKG2D, as a single receptor, combines with these several distinct ligands, it is still unclear why multiple ligands exist for this one invariant receptor.

The cellular expression of these ligands can be up-regulated in response to a variety of stimuli, such as viral infection, tissue ischemia, heat shock and malignant transformation (Groh et al. 1996; Gasser et al. 2005; Groh et al. 2001). In humans, the expression of NKG2D ligands is known to be rare in normal tissues, but frequent in both primary tumors and tumor-derived cell lines (Groh et al. 1996; Raffaghello et al. 2004; Pende et al. 2002; Coudert et al. 2006). Many reports have already described the expression of MICA/B in a broad range of normal and tumor tissues in humans, including various carcinomas (breast, lung, colon, kidney, ovary and prostate), leukemias, gliomas, neuroblastomas and melanomas (Groh et al. 1999; Vetter et al. 2002; Friese et al. 2003; Salih et al. 2003; Watson et al. 2006; Castriconi et al. 2007). Similarly, it has been reported that ULBPs are expressed in several types of human tumors, and that up-regulation of NKG2D ligands in cancer is associated with patient survival (McGilvray et al. 2010; McGilvray et al. 2009). Moreover, recent studies have strongly suggested that the expression levels of these ligands are associated with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity (Inagaki et al. 2009), which is one of the key mechanisms responsible for the antitumor effect of antibody therapeutics. This topic is of considerable interest because the potential to manipulate NKG2D ligand expression could offer promise in the treatment of tumors. However, as comprehensive details of NKG2D ligand expression patterns in human tissues are still largely lacking, the significance of NKG2D ligands in the pathobiological behavior of human neoplasms remains speculative. In previous immunohistochemical studies, the specificity of the antibodies employed was unclear, and both the analyzed neoplasms and ligands were considerably limited.

This study identified the specificity of the antibodies using transfected cells, and clarified differences in ligand expression between non-neoplastic and neoplastic tissues in the same individual. This is the first reported study to have clarified the expression patterns of eight NKG2D ligands during malignant transformation using six validated and specific antibodies.

In this study, we attempted to obtain an accurate overall picture of the expression patterns of NKG2D ligands in a variety of human tissues, both neoplastic and non-neoplastic, by employing well-validated specific antibodies. A critical issue affecting the reliabilityof immunohistochemistry is the specificity and applicability of the antibodies used. Because of this point, the results of previous immunohistochemical studies of human NKG2D ligands need to be validated. In fact, our pilot study using western blotting revealed that several commercially available antibodies against ULBP2 cross-reacted with ULBP5 and ULBP6 (data not shown), and existing commercially available antibodies against ULBP5 were not applicable for FFPE immunohistochemistry. Therefore, we first validated the specificity and FFPE applicability of the antibodies very carefully, including commercially available antibodies and an antibody we had raised ourselves. As a result, we succeeded in distinguishing six different NKG2D ligands reliably on the basis of FFPE tissue immunohistochemistry: ULBP1, ULBP2/6, ULBP3, ULBP4, ULBP5, and MICA/B.

As described above, previous reports have indicated that there was almost no expression of NKG2D ligands in normal tissues, whereas the present study demonstrated diverse expression of NKG2D in non-neoplastic tissues with an apparently normal histology. This difference may be attributable to the fact that, in the present study, clinical samples were obtained from cancer patients, but not from healthy individuals, and tumor-associated changes, such as inflammation or immunological reaction, may have occurred in the otherwise apparently normal cells and tissues from these patients. Therefore, in order to minimize the impact of this potential limitation on our evaluation of normal tissues, we focused on differences in the expression levels of each NKG2D ligand between neoplastic and corresponding non-neoplastic lesions in the same individual.

Tissue-based cluster analysis divided non-neoplastic tissues into the N-null type, which consisted mainly of common stratified squamous epithelium (tongue, larynx, esophagus, uterine cervix, and skin) and simple flattened epithelium (alveolar epithelium of the lung); the N-variable and N-complete types consisted mainly of non-squamous epithelia, such as ductal and glandular epithelial cells. Accordingly, it appears that, in general, NKG2D ligands are of less importance for maintaining the function of squamous epithelial cells unless they are subjected to severe pathological disturbance. Although most squamous epithelia showed a low level of expression, characteristic expression of ULBP4 was observed in the skin, in agreement with a recent study (Chalupny et al. 2003). In contrast, in ductal or glandular epithelial cells, at least one of the NKG2D ligands was frequently expressed, though any significant relationship was not found with specific ligands. This implies that local NK cell-mediated immunological defense may differ between squamous- and glandular epithelium-lined tissues, and may be variably regulated among tissues and organs. In addition, groups of ligands shared a similar expression pattern in non-neoplastic tissues, and cluster analysis revealed two distinct clusters: the ULBP5-ULBP3-MICA/B cluster and the ULBP2/6-ULBP1-ULBP4 cluster (Fig. 3). Thus, it seems possible to assume that ligand expression in normal cells might be regulated in a ligand-set manner, at least in some tissues. More interestingly, on the basis of a comparative analysis of the gene promoters of the six NKG2D ligands reported by Eagle et al. (2006), ligands were divisible into three groups using specific antibodies: ULBP2/6 and ULBP5; ULBP1 and ULBP3; and MICA/B and ULBP4. In fact, each of the two clusters presented in this study includes either one of the ligands in each group.

With regard to neoplastic tissues, recent studies have revealed that MICs and ULBPs are variably expressed, and often co-expressed in ovarian (McGilvray et al. 2010) and colorectal (McGilvary et al. 2009) cancers, and that their expression is associated with prognosis. However, whether there are many correlations of ligand expression pattern among various tissues and organs remains an interesting question. In the present study, ligand-based cluster analysis of neoplastic tissues indicated that the dendrogram (based on immunohistochemical expression profiles) was closely similar to the phylogenetic tree (constructed on the basis of the ligand promoter DNA sequences; Fig. 5, inset shows the phylogenic tree presented by Eagle et al. (2009)). This similarity between these two molecular features—the protein expression profile in neoplastic tissues and DNA phylogeny in normal cells—may imply that genetic control of NKG2D ligand expression is basically conserved even during cellular stress resulting from malignant transformation, without marked epigenetic alteration.

Histologically, most squamous cell carcinomas, especially those of the upper aerodigestive tract (tongue, larynx, esophagus and lung) and skin, were included in the T-null type. The only exception was squamous cell carcinoma of the uterine cervix, because it showed a relatively high rate of ligand positivity (T-variable B). Textor et al. (2008) also investigated the differential expression of NKG2D ligands in cervical carcinogenesis, and demonstrated an increased rate of positivity for MICA in 20% of cervical carcinomas, being similar to the rate we observed in the present study. Close association with HPV infection as a carcinogenetic factor may be one of the explanations for this unique characteristic of squamous cell carcinoma of the uterine cervix. On the other hand, tissue-based cluster analysis of neoplastic tissues demonstrated that null-type squamous cell carcinomas, except for those of the lung, specifically expressed ULBP4, unlike other tissues in the null-type cluster. This expression pattern of ULBP4 is similar to that of a murine homologue H60c (Takada et al. 2008), as reported by Whang et al. (2009). However, no expression of ULBP4 was observed in squamous cell carcinoma of the lung. This difference may be attributable to a non-squamous origin of squamous cell carcinoma in the lung; i.e., de novo squamous carcinogenesis from bronchial columnar epithelium.

In this study, using validated and specific antibodies, we analyzed the immunohistochemical expression of NKG2D ligands in neoplastic lesions and their normal counterpart tissues. The results indicated that epithelial neoplasms show a characteristic pattern of NKG2D ligand expression, suggesting that expression of the ligand proteins may be controlled by promoter-dependent transcriptional regulation. The data presented should serve as a useful reference for other investigators in future studies of NKG2D ligand functions. For instance, neoplasms expressing NKG2D ligands might be potential targets for antibody-based therapy, and NKG2D ligand expression might be useful as a surrogate marker reflecting ADCC activity. Our present study has attempted to clarify why multiple NKG2D ligands exist, and also the patterns of expression of NKG2D ligands in various neoplasms, as we considered that these molecules could have potential therapeutic applications as direct targets and modulators of ADCC activity in a variety of neoplasms through antibody treatment. In addition, these IHC evaluations may yield some predictive markers. We also considered that future studies focusing on correlations with clinical factors might lead to the identification of prognostic markers.

We thank Ms Kyoko Fujii for her excellent technical assistance in the antibody validation.