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Since HLA-E heavy chains accumulate free of their light β2-microglobulin (β2m) subunit, raising mAbs to folded HLA-E heterodimers has been difficult, and mAb characterization has been controversial. Herein, mAb W6/32 and 5 HLA-E-restricted mAbs (MEM-E/02, MEM-E/07, MEM-E/08, DT9, and 3D12) were tested on denatured, acid-treated, and natively folded (both β2m-associated and β2m-free) HLA-E molecules. Four distinct conformations were detected, including unusual, partially folded (and yet β2m-free) heavy chains reactive with mAb DT9. In contrast with previous studies, epitope mapping and substitution scan on thousands of overlapping peptides printed on microchips revealed that mAbs MEM-E/02, MEM-E/07, and MEM-E/08 bind three distinct α1 and α2 domain epitopes. All three epitopes are linear since they span just 4-6 residues and are 'hidden' in folded HLA-E heterodimers. They contain at least one HLA-E-specific residue that cannot be replaced by single substitutions with polymorphic HLA-A, HLA-B, HLA-C, HLA-F, and HLA-G residues. Finally, also the MEM-E/02 and 3D12 epitopes are spatially distinct. In summary, HLA-E-specific residues are dominantly immunogenic, but only when heavy chains are locally unfolded. Consequently, the available mAbs fail to selectively bind conformed HLA-E heterodimers, and HLA-E expression may have been inaccurately assessed in some previous oncology, reproductive immunology, virology, and transplantation studies. [ABSTRACT FROM AUTHOR]

Paired cultures of early-passage melanoma cells and melanocytes were established from metastatic lesions and the uninvolved skin of five patients. In this stringent autologous setting, cDNA profiling was used to analyze a subset of 1477 genes selected by the Gene Ontology term 'immune response'. Human Leukocyte Antigen E ( HLA-E) was ranked 19th among melanoma-overexpressed genes and was embedded in a transformation signature including its preferred peptide ligand donors HLA-A, HLA-B, HLA-C, and HLA-G. Mostly undetectable in normal skin and 39 nevi (including rare and atypical lesions), HLA-E was detected by immunohistochemistry in 17/30 (57%) and 32/48 (67%) primary and metastatic lesions, respectively. Accordingly, surface HLA-E was higher on melanoma cells than on melanocytes and protected the former (6/6 cell lines) from lysis by natural killer ( NK) cells, functionally counteracting co-expressed triggering ligands. Although lacking HLA-E, melanocytes (4/4 cultures) were nevertheless (and surprisingly) fully protected from NK cell lysis. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) aminopeptidases ERAP1 and ERAP2 contribute to generate HLA class I binding peptides. Recently, we have shown that the expression of these enzymes is high and coordinated (with each other and with HLA class I molecules) in immortalized B cells, but variable and imbalanced in human tumour cell lines of various non-lymphoid lineages. Herein, this issue was investigated in vivo by testing ERAP1 and ERAP2 expression in normal non-lymphoid tissues and their malignant counterparts. ERAP1 and ERAP2 were detected exclusively in the epithelial cells of over half of the tested normal tissues. Four ERAP1/ERAP2 phenotypes (+/+, -/-, +/- and -/+) were detected, and the presence of either or both enzymes was not necessarily associated with HLA class I expression. In more than 160 neoplastic lesions, the expression of either or both aminopeptidases was retained, lost (most frequently, particularly ERAP1) or acquired as compared to the normal counterparts, depending on the tumour histotype. The double-negative (-/-) phenotype was the most frequent, and significantly (P = 0.013) associated with a lack of detectable HLA class I antigens. In selected neoplastic lesions, ERAP1 and ERAP2 were also tested for their enzymatic (peptide-trimming) activities. Expression and function were found to correlate, indicating that immunohistochemistry detects active enzymes in vivo. Thus, dissociation in the expression of ERAP1, ERAP2 and HLA class I may already be present in some normal tissues, but malignant transformation causes additional losses, gains and imbalances in specific tumour histotypes, and these alter the peptide-trimming ability of tumour cells in vivo. J. Cell. Physiol. 216: 742–749, 2008, © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Three paired (from the same donor) sets of melanoma cells and normal melanocytes, established as early-passage cultures from metastatic lesions and the uninvolved skin of three patients, were comparatively cDNA profiled by macroarrays (approximately 1,200 genes) and reverse transcription (RT)-PCR. While 145 gene products were significantly (at least twofold) upregulated or downregulated in at least 1 pair, and 23 were in at least 2 pairs, only 3 (the signal transducer and activator of transcription STAT2, collagen type VI, and CD9) were concordantly modulated (downregulation) in all 3 pairs. Array results were validated by RT-PCR on a small panel of surgically removed nevocellular nevi and metastatic melanoma lesions, and by immunohistochemistry on a large panel of benign and malignant lesions of the nevomelanocytic lineage. The three gene products were downregulated at different stages of melanoma progression. STAT2 was detectable in nevi (5/5) and most primary melanomas (11/12), but was lost in 10/15 metastatic lesions. Collagen type VI was expressed in nevi (5/5) and primary melanomas below a Breslow thickness of 1 mm (3/3), but was lost in 24/24 primary melanomas above this threshold, and in metastatic melanomas (10/10). The tetraspanin CD9 molecule was expressed in 18/18 nevi, but was lost in 20/28 primary melanomas (including thin lesions), and in 24/52 metastatic lesions. These data provide the proof of principle that cDNA profiling of paired melanocyte/melanoma cultures sorts out novel, early signatures of melanocyte transformation that could contribute to the clinical management of patients at high risk of metastatic disease. J. Cell. Physiol. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Class II MHC genes (for example, the human HLA-DRA gene) are expressed at the cell surface in many professional and nonprofessional antigen-presenting cells in a variety of anatomical locations. Here, we report about 13 mouse transgenic lines (11 of which have not been previously described) generated with four distinct sets of DRA transgenes carrying progressive, informative 5′ and 3′ deletions. DRA expression was assessed in B lymphocytes, dendritic cells, macrophages, and extra-hematopoietic cells (particularly kidney epithelial cells). A compact transcriptional unit was identified that efficiently directs DRA expression [both constitutive and interferon (IFN)-γ induced] in extra-hematopoietic tissues and dendritic cells. It extends from position−266 upstream of the transcription initiation site to position +119 downstream of the last DRA exon. The same fragment, however, did not efficiently direct IFN-γ-induced DRA expression in macrophages, that required additional 5′ sequences. Thus, IFN-γ uses distinct promoter segments and mechanisms to up-regulate class II in different cell lineages. In contrast to previous results in transgenic mice expressing murine class II transgenes, we were unable to generate reproducible patterns of HLA-DRA expression in B cells. [ABSTRACT FROM AUTHOR]

A consistent lack of DNA methylation at one or both of two GCGC (Hha I) restriction sites in the 5′ region of the HLA-DRA gene has been previously documented by the use of methyl-sensitive restriction enzymes in human cells and tissues, irrespectively of their expression of DRα products. Evidence presently available, however, does not exclude that a lack of methylation in this region, although not sufficient, might be necessary for gene expression. In this report, we show that only one of the 5′-GCGC sites is protected, although less efficiently than in man, from CG→ mCG modifications in tissues and cells of transgenic mice carrying an expressed single copy of the HLA-DRA gene/diploid genome. We demonstrate that the two 5′ GCGC sites of the HLA-DRA transgene are fully methylated in DRα splenocytes (more than 80% T-lymphocytes), while one of them (the most 5′ site) is not methylated in a fraction of DRα splenocytes (more than 80% T-lymphocytes), while one of them (the most 5′ site) is note methylated in a fraction of DRα+ splenocytes (more than 95% B-lymphocytes). These results provide evidence that absence of DNA methylation in the 5′ region is not necessary for, but might be associated with and possibly secondary to the expression of the DRA gene. [ABSTRACT FROM AUTHOR]