Status report from 'double agent HLA': health and disease.

The human major histocompatibility complex is a multi-gene ~4 Mb of DNA including the highly polymorphic HLA genes which are fundamental in effecting a healthy immune response. Detailed knowledge of the structure and function of HLA genes and their alleles and HLA proteins and their allotypes has rapidly evolved because of their role in clinical transplantation. A significant additional finding is the association or linkage of a wide range of diseases with HLA. We review the function of HLA and the history of disease association studies and focus on specific informative examples in the context of recent genome-wide screening analyses.

ULBP2 and RAET1E NKG2D ligands are independent predictors of poor prognosis in ovarian cancer patients.

The human activating immune receptor, NKG2D, binds to a diverse array of cellular ligands of the MIC and unique long 16 (UL16)-binding protein (ULBP)/retinoic acid early transcript (RAET) family. NKG2D is thought to participate in anticancer immune responses. By using tissue microarrays representing over 300 patients with defined clinicopathological factors, we present the first comprehensive screen of the expression of all NKG2D ligands in primary ovarian cancers. NKG2D ligands were expressed by the majority of tumors; however, the level of expression varied considerably. By categorizing each tumor as having negative, low or high expression, it was shown that high expression of several NKG2D ligands is inversely correlated with disease survival. Patients whose tumors had high expression of RAET1E (p = 0.037), ULBP1 (p = 0.036) and ULBP3 (p = 0.004) surviving a median of 11, 14 and 11 months, respectively, compared with disease-specific survival of 29, 30 and 25 months in patients whose tumors showed no expression of these ligands. These results contrast with previous findings showing that high level NKG2D ligand expression is associated with good prognosis in colorectal cancer and suggest a fundamental difference in the involvement of NKG2D-mediated immunity in these two types of cancer. By using multivariate analysis, the factors retaining independent prognostic significance were International Federation of Gynecologists and Obstetricians stage (p < 0.001), presence of residual disease (p = 0.003), ULBP2 (p = 0.042) and RAET1E (p = 0.030).

Maintenance of pluripotency in human embryonic stem cells cultured on a synthetic substrate in conditioned medium.

Realizing the potential clinical and industrial applications of human embryonic stem cells (hESCs) is limited by the need for costly, labile, or undefined growth substrates. Here we demonstrate that trypsin passaging of the hESC lines, HUES7 and NOTT1, on oxygen plasma etched tissue culture polystyrene (PE-TCPS) in conditioned medium is compatible with pluripotency. This synthetic culture surface is stable at room temperature for at least a year and is readily prepared by placing polystyrene substrates in a radio frequency oxygen plasma generator for 5 min. Modification of the polystyrene surface chemistry by plasma etching was confirmed by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), which identified elemental and molecular changes as a result of the treatment. Pluripotency of hESCs cultured on PE-TCPS was gauged by consistent proliferation during serial passage, expression of stem cell markers (OCT4, TRA1-60, and SSEA-4), stable karyotype and multi-germlayer differentiation in vitro, including to pharmacologically responsive cardiomyocytes. Generation of cost-effective, easy-to-handle synthetic, defined, stable surfaces for hESC culture will expedite stem cell use in biomedical applications.

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

PURPOSE: NKG2D (natural killer group 2, member D) 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 its prognostic effect has not been investigated in such a large cohort. EXPERIMENTAL DESIGN: In our study, 462 primary colorectal tumors were screened for the 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) showed 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 tumor-node-metastasis stage I tumors, but became progressively less frequent in stages II, III, and IV 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. The combination of MIC and tumor-node-metastasis stage was found to be the strongest predictor of survival, splitting patients into eight groups and suggesting prognostic value in clinical assessment. Of particular interest were stage I patients with low expression of MIC who had a similar survival to stage III patients, and may be candidates for adjuvant therapy.

UXT interacts with the transcriptional repressor protein EVI1 and suppresses cell transformation.

The EVI1 transcriptional repressor is critical to the normal development of a variety of tissues and participates in the progression of acute myeloid leukaemias. The repressor domain (Rp) was used to screen an adult human kidney yeast two-hybrid library and a novel binding partner designated ubiquitously expressed transcript (UXT) was isolated. Enforced expression of UXT in Evi1-expressing Rat1 fibroblasts suppresses cell transformation and UXT may therefore be a negative regulator of Evi1 biological activity. The Rp-binding site for UXT was determined and non-UXT-binding Evi1 mutants (Evi1Delta706-707) were developed which retain the ability to bind the corepressor mCtBP2. Evi1Delta706-707 transforms Rat1 fibroblasts, showing that the interaction is not essential for Evi1-mediated cell transformation. However, Evi1Delta706-707 produces an increased proportion of large colonies relative to wild-type, showing that endogenous UXT has an inhibitory effect on Evi1 biological activity. Exogenous UXT still suppresses Evi1Delta706-707-mediated cell transformation, indicating that it inhibits cell proliferation and/or survival by both Evi1-dependent and Evi1-independent mechanisms. These observations are consistent with the growth-suppressive function attributed to UXT in human prostate cancer. Our results show that UXT suppresses cell transformation and might mediate this function by interaction and inhibition of the biological activity of cell proliferation and survival stimulatory factors like Evi1.

Conservation and expression of a novel alternatively spliced Evi1 exon.

The Evi1 transcriptional repressor protein is expressed in a developmentally regulated manner, is essential for normal development, participates in regulating cell proliferation and differentiation of cells of haemopoietic and neuronal origin and contributes to the progression of leukaemia. In this report we describe a new murine Evi1 gene transcript (Delta105) that contains two alternatively spliced regions encoding a 9 amino acid insertion (Rp+9) within the repressor domain (Rp) and a 105 amino acid C-terminal truncation. Abundant levels of the 105 amino acid truncated protein are observed in murine leukaemia cells. The combined primary sequence alterations do not affect the DNA binding, transcriptional repressor or CtBP2 protein binding properties of Evi1 but they do reduce its transforming and cell proliferation stimulating activities. Reduced transforming activity is most likely due to the C-terminal truncation as the activity of Evi1 containing either Rp or Rp+9 is indistinguishable. Both isoforms exist in all murine tissues and cell lines examined. However, only the Rp+9 alternative splice variant is also found in humans and other vertebrates. Murine and human forms of Evi1 with Rp or Rp+9 exist. The additional 9 amino acids are encoded by a conserved 27 nucleotide exon, the overall structural organisation of the gene being preserved in the two species. The function of the Rp+9 and Delta105 splice variants is unknown although the conservation of Rp+9 throughout evolution in vertebrate species suggests it is essential to the broad spectrum of biological activities attributed to this developmentally essential protein.

Expression of the stress-related MHC class I chain-related protein MICA is an indicator of good prognosis in colorectal cancer patients.

The realization of targeted cancer therapy has driven the need to improve selection of patients with colorectal cancer for adjuvant therapy, leading to a search for potential new prognostic markers. There is accumulating evidence that immunosurveillance acts as an extrinsic tumor suppressor. As genetic instability is an early event in colorectal cancer, this can lead to altered expression of molecules conferring resistance to immune attack. Hence, molecules up or downregulated in this process may impact on patient survival. In our study, 449 colorectal tumors were screened for expression of the stress-related protein MICA, which functions as a ligand for the NKG2D receptor and whose expression confers susceptibility to both T- and NK-cell attack. Intensity of MICA expression was quantified using automated image analysis and MICA expression showed no correlation with conventional clinicopathological variables. In contrast, survival analysis showed a significant correlation between higher levels of MICA expression and improved disease-specific survival, with independent prognostic significance in multivariate analysis. Thus, patients with low levels of MICA and a poor prognosis may be good candidates for aggressive chemotherapy. In contrast, patients with high expression of MICA may be candidates for the antibody therapies, as they should be susceptible to NK killing by antibody dependent cellular cytotoxicity.