September 2004
Volume 45, Issue 9
Free
Immunology and Microbiology  |   September 2004
Epigenetic Silencing of the CIITA Gene and Posttranscriptional Regulation of Class II MHC Genes in Ocular Melanoma Cells
Author Affiliations
  • Michael Radosevich
    From the Department of Immunology, University College London, University of London, Institutes of Ophthalmology and Child Health, and Department of Ocular Immunology, Moorfields Eye Hospital, London, United Kingdom; and the
  • Zhimin Song
    From the Department of Immunology, University College London, University of London, Institutes of Ophthalmology and Child Health, and Department of Ocular Immunology, Moorfields Eye Hospital, London, United Kingdom; and the
  • Joan C. Gorga
    From the Department of Immunology, University College London, University of London, Institutes of Ophthalmology and Child Health, and Department of Ocular Immunology, Moorfields Eye Hospital, London, United Kingdom; and the
  • Bruce Ksander
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts.
  • Santa Jeremy Ono
    From the Department of Immunology, University College London, University of London, Institutes of Ophthalmology and Child Health, and Department of Ocular Immunology, Moorfields Eye Hospital, London, United Kingdom; and the
Investigative Ophthalmology & Visual Science September 2004, Vol.45, 3185-3195. doi:https://doi.org/10.1167/iovs.04-0111
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      Michael Radosevich, Zhimin Song, Joan C. Gorga, Bruce Ksander, Santa Jeremy Ono; Epigenetic Silencing of the CIITA Gene and Posttranscriptional Regulation of Class II MHC Genes in Ocular Melanoma Cells. Invest. Ophthalmol. Vis. Sci. 2004;45(9):3185-3195. https://doi.org/10.1167/iovs.04-0111.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Primary uveal melanocytes and many ocular melanoma cells are resistant to interferon (IFN)-γ–mediated induction of major histocompatibility complex (MHC) class II molecule expression. This suppression of class II MHC induction is considered to be one of the ways in which the eye is able to inhibit inflammatory responses. However, the mechanism(s) of this suppression is unknown. In this study, we have probed the molecular basis of this phenotype and report two distinct mechanisms underlying this phenotype.

methods. Primary ocular melanocytes and ocular melanoma cell lines (retaining this IFN-γ–resistant class II MHC phenotype) were examined for the expression of class II MHC molecules on the cell surface by flow cytometry. Class II MHC gene expression was further examined using Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses.

results. The IFN-γ signal-transduction pathway was found to be intact by electrophoretic mobility shift assay (EMSA) and transfection of reporter constructs. The lack of class II MHC gene expression appears to result from at least two mechanisms: (1) a specific inhibition of CIITA (class II transactivator) gene expression (reminiscent of trophoblasts), and (2) posttranscriptional regulation of class II MHC genes.

conclusions. The inability of primary uveal melanocytes and ocular melanoma cells to express class II MHC molecules after treatment with IFN-γ has been found to map to two distinct points in the class II MHC biosynthetic pathway. The predominant mechanism appears to involve the silencing of the endogenous gene encoding the class II transactivator (CIITA). Here, the blockade does not involve signal transduction from the IFN-γ receptor, but rather involves a specific silencing of the CIITA gene. A second mechanism involves the posttranscriptional regulation of class II MHC genes.

We have studied both primary uveal melanocytes and ocular melanoma cells to understand how ocular melanoma cells often evade immune surveillance. 1 These studies were begun with a study of the status of class I and II major histocompatibility complex (MHC) expression on the surface of these cells and were extended to both biochemical and molecular studies on the transcriptional control of class II MHC genes within these cells. Because molecular studies (e.g., promoter studies and electrophoretic mobility shift assays [EMSAs]) depend on the availability of a large number of cells, we performed our detailed studies on the regulation of class II MHC genes using ocular melanoma cell lines that accurately mimic the class II MHC phenotype of uveal melanocytes. 2  
We and others have found that primary ocular melanocytes do not constitutively express class II MHC molecules on the cell surface. 3 In the current study, these cells could not be induced to express class II MHC molecules on the cell surface by stimulation with interferon (IFN)-γ. (This is in contrast to the phenotype on most nucleated cells.) Our analysis of a large panel of ocular melanoma cell lines (Ono SJ, manuscript in preparation) indicates that most ocular melanoma cells, like primary ocular melanocytes, cannot express class II MHC molecules on the cell surface when stimulated with IFN-γ. We present one mechanism responsible for this blockade, illustrated by the situation in a representative human ocular melanoma cell line (Melanoma 202). 
The data presented in this article suggest that the lack of class II MHC expression in ocular Melanoma 202 cells (representative of most ocular melanoma cell lines we have examined) is due to a specific block in the expression of the class II MHC transactivator (CIITA). These data indicate that CIITA blockade is critical in preventing class II MHC expression on at least a subset of ocular melanoma cells. Because the phenotype of Melanoma 202 cells mirrors that of primary uveal melanocytes, the data also suggest that this mechanism may explain the class II MHC blockade observed in these cells. As such, the data may also add to our understanding of immune privilege. 
Materials and Methods
Cells and Cell Lines
Uveal melanocytes were purified from human biopsy specimens, as previously described. 4 Melanoma 202 and Melanoma 285 cells were derived at the Schepens Eye Research Institute in the Ksander laboratory. The cells used are early-passage cells (less than five passages from stored frozen stock). Tumor tissue was dissected from surrounding normal uveal tissue and enzymatically digested to yield a single-cell suspension in a Petri dish containing 10 mL collagenase at 150 U/mL (Sigma-Aldrich, St. Louis, MO) in complete RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 2.0 mM glutamine (BioWhittaker), 100 U/mL penicillin, 100 U/mL streptomycin, 0.1% amphotericin B (Fungizone; BioWhittaker), 0.01 M HEPES (BioWhittaker), and 0.5% 2-β-mercaptoethanol (Sigma-Aldrich). Tissue was incubated for 90 minutes at 37°C, after which the released cells were removed and the debris allowed to settle. Cells in the culture supernatant were recovered, washed three times, and examined microscopically for the presence of viable tumor cells. Ocular melanoma cells were maintained in complete RPMI 1640 medium. Cells were incubated at 37°C with 5% CO2 and passaged when they reached a confluent monolayer by treatment with trypsin-EDTA (BioWhittaker). Tumor cell lines were demonstrated to be ocular melanoma cells by the expression of melanoma antigen genes (MAGEs) that are transcriptionally active only in melanoma cells and are not active in normal nonmalignant cells. 5 HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), and 100 U/mL penicillin-streptomycin. Priess cells, a B lymphoblastoid cell line that expresses both class I MHC and all class II isotypes (used as a positive control in Western blot experiments) were maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS and 100 U/mL penicillin-streptomycin. 
Flow Cytometry
HeLa cells and Melanoma 202 cells (5 × 105) were plated in six-well plates and were allowed to attach to the plates for 12 hours. IFN-γ was either added or not added at a concentration of 500 U/mL, and the cells were incubated for 48 hours. The cells were then incubated with an anti-HLA-DR (clone L243; BD Biosciences, San Jose, CA) monoclonal antibody, which is directly conjugated to phycoerythrin (PE). MHC class II expression on HeLa and melanoma cells was determined by flow cytometry (Epics XL fluorescence-activated cell sorter [FACS]; Beckman-Coulter, Fullerton, CA) and analyzed with the accompanying software (Coulter system 2; Beckman-Coulter). 
Immunofluorescent Staining
Two tenths of a million each of the melanoma cells 285 or 202 were passed into double-chambered slides (Nunc, Inc., Naperville, IL) and incubated at 37°C with 5% CO2 overnight. IFN-γ was added in the culture medium to a final concentration of 200 U/mL and incubated for 24 hours. The medium was then removed, and the cells were immersed in methanol for 15 minutes and washed by quickly dipping the chamber slide in and out of water 10 times. The chamber was then air dried. To stain the cells, L243 supernatant (anti-DRA) was added to the slide and incubated in a dark, humidified chamber at room temperature. The second incubation was with FITC-conjugated anti-mouse IgG (Sigma-Aldrich). The coverslips were mounted with 1 drop of antifade mounting medium (VectaShield; Vector Laboratories, Inc., Burlington, CA). 
Ectopic Expression of CIITA
Melanoma 202 and 285 cells (5 × 105) were passed into chambered slides and rested for 24 hours at 37°C in a 5% CO2 incubator. The cells were washed extensively with serum-free medium before transfection. The plasmid DNAs PCDNA/CIITA and PCDNA/INOnk2 were purified with a plasmid (Qiagen, Inc., Valencia, CA) and then added, at 1.5 μg each to 1 mL serum-free RPMI-1640 medium containing 4 μL lipophilic transfection agent (Lipofectamine; Invitrogen-Life Technologies, Gaithersburg, MD). Cells were incubated for 25 hours at 37°C until 1.5 mL complete medium (RPMI1640 containing 10% FCS and 1× penicillin-streptomycin) was added. 
Western Blot Analysis
The samples were applied to 12% acrylamide gels in reducing Laemmli sample buffer. After electrophoresis, the samples were transferred overnight at 250 mA to nitrocellulose membranes (Immobilon-P; Millipore, Bedford, MA) in a transfer unit (Hoeffer Mighty Small Transfer Unit; Amersham Biosciences, Amersham, UK). After transfer, the membranes were washed for 30 minutes in 0.1% (vol/vol) Tween-20 in 100 mM Tris and 0.9% sodium chloride (pH 7.5; TTBS: Tris, Tween-buffered saline) and then incubated for 30 minutes in a 1:200 dilution of a rabbit anti-class I, -DR, -DQ, or -DP heteroserum (all obtained from Hidde Ploegh, Department of Pathology, Harvard Medical School, Boston, MA) in TTBS. The membranes were washed and stained (Vectastain ABC [avidin-biotinylated enzyme complex]; Vector Laboratories), with diaminobenzidine plus nickel chloride as the substrate. The approximate positions of the class Iα chains and the class IIα and -β chains are indicated in Figure 3 with arrows. 
RNA Extraction and RT-PCR
Melanoma 285 and 202 cells (1.5 × 106) were incubated for 24 hours and treated with IFN-γ (200 U/mL) for 24 hours, and RNA was extracted (TRIzol; Invitrogen-Life Technologies). M-MLV reverse transcriptase and random primer (Invitrogen-Gibco, Grand Island, NY) were applied for reverse transcription, and PCR was performed in the same tube under conditions of 94°C for 1 minute, 55°C for 2 minutes, and 72°C for 3 minutes for 30 cycles. The primers used for PCR were: DRA-sense (S): 5′-GAGTTCTATCTGAATCCTG-3′; DRA-antisense (AS): 5′-GTTCTGCTGCATTGCTTTTGC-3′; DRB-S: 5′-GGCCTGATCCAGAATGGAGAT-3′; DRB-AS: 5′-ATGAGGCGCTGTCATCAATGC-3′; DQA-S: 5′-TGGACTTGCTACATGACCTAG-3′; DQA-AS: 5′-GCAGAGGGTATGCATTAACTT-3′; DQB-S: 5′-GAGCCCACAGTGACCATC-3′; DQB-AS: 5′-TGATGGGGTTCTGGAGG-3′; DMA-S: 5′-CTATCGCTGAAGTGTTCACGC-3′; DMA-AS: 5′-AGATTTATTGCCTTGTGGGGG-3′; DMB-S: 5′-AGCTTGTCATGCCTCACAGCA-3′; DMB-AS: 5′-AACAATCACCAGTTGCTGTCC-3′; Invariant Chain S: 5′-GCAGAGGCGGTCTTCAACATC-3′; Invariant Chain AS: 5′-TGATAACAGCTTGGCTGAGC-3′; NFX-S: 5′-CCCGAATTCTCATCTCAGGCTGATCCGTGA-3′; NFX-AS: 5′-GGGGAATTCATGCGTTGCCTGGCTCCACG-3′; GAPDH-S: 5′-TGATGACATCAAGAAGGTGGT-3′; and GAPDH-AS: 5′-CAGTGAGGGTCTCTCTCTTCC-3′. The CIITA-specific primers 5 and HMG I–specific primers 6 have been described previously. 
Electrophoretic Mobility Shift Assay
Nuclear extracts and EMSAs were performed as previously described. 7 8 9  
Transfections and Luciferase Analysis
Transient transfections of HeLa and Melanoma 202 cells where performed by with a commercial reagent (Superfect Transfection Reagent; Qiagen, Inc.). For luciferase activity measurement, cells were washed twice in PBS and then lysed in the six-well plates with 150 μL 1× reporter lysis buffer (Promega, Madison, WI) for 15 minutes at room temperature. To access the luciferase activity, 30 μL lysate was added to 50 μL luciferase assay reagent, as indicated in the manufacturer’s protocol (Promega). PIII.Luc and PIV.Luc were generously provided by Jenny P. Y. Ting (University of North Carolina at Chapel Hill). 10 The empty pLG2-basic vector (Promega) was used as the negative control. 
Results
Extracellular Expression of HLA-DR on Primary Uveal Melanocytes, Melanoma 202 Cells, and HeLa Cells after Stimulation with IFN-γ
Unlike primary skin melanocytes, human uveal melanocytes do not express class II MHC molecules on stimulation with IFN-γ (Fig. 1) . To understand the molecular basis of this phenomenon, we also analyzed a large panel of ocular melanoma cell lines (12 cell lines in total), to permit molecular studies such as gene transfection experiments and signal-transduction studies. In this screening, we found that most human ocular melanoma cell lines (10/12) retained the class II MHC–negative phenotype of primary uveal melanocytes (Ono SJ, manuscript submitted). We chose a representative human cell line from this panel (Melanoma 202) for the molecular studies presented in this article. Another ocular melanoma cell line 285, which expresses class II MHC genes in response to IFN-γ, but not class II proteins (due to a posttranscriptional block; Ono SJ, manuscript in preparation), represents an alternative pathway for the class II negative phenotype observed in the panel of ocular melanoma cells. 
We initially examined the expression of extracellular HLA-DR in Melanoma 202 and HeLa cells after stimulating the cells with IFN-γ. HeLa cells were used as a positive control, because many studies have demonstrated that class II molecules are expressed on the cell surface of these cells. After treatment with IFN-γ for 24 hours, there was substantial expression of HLA-DR on the surface of HeLa cells (Fig. 2A) . However, there was no expression of HLA-DR molecules on the surface of Melanoma 202 cells (Fig. 2B) , reminiscent of what we observed in primary uveal melanocytes (Fig. 1) and in many other ocular melanoma cell lines (data not shown). 
Lack of MHC Class II Protein in Melanoma 202 and 285 Cells
IFN-γ–treated ocular melanoma cells were then analyzed to determine whether they express any of the class II MHC isotypes (HLA-DR, -DP, or -DQ). The B-cell line Priess was used as a positive control for the Western blot analyses, as it expresses class I MHC and all three human class II MHC molecules constitutively. Melanoma 285 cells are an additional ocular melanoma cell line that expresses both the CIITA transactivator and class II MHC genes on IFN-γ treatment, but does not express class II MHC polypeptides because of a posttranscriptional block (manuscript in preparation). 
Melanoma 202 and Melanoma 285 cells were cultured in the absence or presence of IFN-γ and then examined at several time points after addition of IFN-γ or vehicle alone (0, 16, 24, 36, and 48 hours) for the expression of class II by Western blot. In Figure 3A , lane 1 contains 1 μg of purified HLA-DR from Priess cells, lane 2 contains Priess cells, and lanes 3 to 7 contain Melanoma 285 cells stimulated with IFN-γ for 0, 16, 24, 36, and 48 hours, respectively. Lanes 8 to 12 contain Melanoma 202 cells stimulated with IFN-γ for 0, 16, 24, 36, and 48 hours, respectively. 
The results clearly show that the positive control Priess cells expressed both the α and β chains of class II but neither of the two melanoma lines expressed both chains any time point after addition of IFN-γ. In Figures 3B and 3C similar results were obtained for the α and β chains of HLA-DP and -DQ. In Figure 3D a similar experiment was performed, except that the antibody was against the class I heavy chain. In addition to the positive control showing presence of MHC class I, both Melanoma 285 and 202 cells expressed enhanced levels of class I MHC molecules in response to IFN-γ treatment. A notable finding was that Melanoma 285 cells expressed a twofold higher level of class I heavy chain than Melanoma 202 cells at all time points after IFN-γ treatment. These Western blots demonstrate that in addition to the class II MHC molecule’s not being expressed on the cell surface, it is also not translated and expressed in the cytoplasm of Melanoma 202 (or 285) cells. 
Transfection of Ocular Melanoma Cells with a CIITA Expression Vector
Melanoma 202 tumor cells did not express class II molecules on the cell surface or in the cytoplasm when stimulated with IFN-γ. To determine whether expression of class II MHC molecules could be achieved on ectopic expression of CIITA, tumor cells were transfected with either the PcDNA I/INOnk2 (empty) expression vector or the same vector with the CIITA cDNA. All cells were examined by immunohistochemical staining for HLA-DR. The data shown in Figure 4B indicate that when Melanoma 202 cells were transfected with a CIITA expression vector, HLA-DR molecules were detected on the surface of the cells. These data indicate that all the components necessary for cell surface expression of HLA-DR are present in Melanoma 202 tumor cells and indicate that a lack of CIITA may account for the failure of these cells to express class II MHC genes and proteins. 
Presence of a Functional CIITA Protein in Melanoma 202 Cells
The previous experiments showed that the block in class II MHC expression on IFN-γ–treated uveal melanocytes is not due to a defective IFN-γ receptor (class I MHC molecules are induced) or class II MHC genes. (Class II MHC molecules are expressed on ectopic expression of CIITA.) Rather, the MHC blockade appeared to map to the level of either CIITA expression or the expression of wild-type–functional CIITA. To differentiate between the two possibilities of (1) a defective CIITA protein and (2) a block to CIITA gene transcription, we next performed a series of transfection and expression studies to map the CIITA defect further. We transfected HeLa cells (known to be able to express class II MHC genes and molecules on IFN-γ stimulation) and Melanoma 202 cells with an HLA-DRA promoter-driven reporter construct (DRA-LUC; nucleotides −260 to +11; Fig. 5A ) that we constructed. The transfected cells were then stimulated with recombinant IFN-γ and DRA promoter luciferase activity measured 48 hours after cytokine addition. Whereas transcription from the DRA-LUC reporter was enhanced in HeLa cells, the DRA promoter was not activated in Melanoma 202 cells (Fig. 5B) . These data suggest that either no CIITA protein is being made in these cells or that it is defective. 
Transcription of Class II Genes in Ocular Melanoma Cells
We next determined whether the α and β chain genes of HLA-DR and -DQ are transcribed in IFN-γ–treated ocular Melanoma 202 cells. As mentioned previously, Melanoma 285 cells express class II MHC genes on IFN-γ stimulation, but (as shown in Fig. 3 ) they do not express class II antigens because of a posttranscriptional block. Data on IFN-γ–inducible class II MHC gene transcription in Melanoma 285 cells are included to illustrate that the class II MHC expression defect observed in uveal melanocytes and ocular melanomas can manifest in at least two molecular pathways. 
The RT-PCR conditions described in the Methods section were used to reverse transcribe and amplify tumor cell RNA for evaluation of class II gene transcription. Total RNA was recovered from the Melanoma 202 and 285 cell lines cultured in either the absence or presence of 200 U/mL of IFN-γ and class II MHC gene transcription assessed at three time points. The results are displayed in Figure 6 . GAPDH was amplified from each sample, indicating that intact RNA was present in each sample, and that the starting levels of cDNA were similar in the RT-PCR reactions. Care was also taken to assure that the RT-PCR was performed at the linear phase of the amplification (i.e., semiquantitative PCR conditions). In the absence of IFN-γ, neither Melanoma 202 nor 285 cells expressed significant levels of HLA-DRA or -DRB or HLA-DQA or -DQB transcripts. Although transcripts of all four class II MHC genes were detected in both IFN-γ–treated melanoma lines, this transcription was significantly weaker in the Melanoma 202 cell line than in the 285 cell line or in other IFN-γ–inducible cells (Fig. 6 and data not shown). 
The data in Figure 6 indicate that there was a quantitative difference in the transcription of class II MHC genes in Melanoma 202 and 285 cells. To compare more carefully the magnitude of difference in class II MHC gene transcription in these cell lines, we performed extensive RT-PCR analyses of cDNAs generated from RNAs extracted from these cell lines. These experiments were performed in the linear phases of the amplifications, and input cDNAs were also serially diluted (after normalization by total RNA and GAPDH levels). Densitometric and Scatchard analysis of the levels of HLA-DRA transcripts detected in Figure 7 (and similar experiments) revealed that the levels of class II MHC transcripts induced in Melanoma 202 cells are only 4% of that induced in Melanoma 285 or HeLa cells. Thus, although a low level of class II MHC transcripts can be induced in Melanoma 202 cells, it is significantly below that observed in other cell lines. 
Transcription of Class II Accessory Molecules
Because several genes involved in antigen presentation are coexpressed with the structural class II MHC genes, we next asked whether their expression is also impaired in IFN-γ–treated ocular melanoma cells. As in the experiments described earlier, the 202 and 285 melanoma cells were cultured in the absence or presence of IFN-γ, and total RNA was recovered and analyzed by semiquantitative RT-PCR for invariant chain (Ii), DM α and β chain transcripts. The results shown in Figure 8 indicate that Melanoma 285 tumor cells constitutively transcribed DM and Ii, and the level of transcription was increased after treatment with IFN-γ. By contrast, neither DM nor Ii was constitutively transcribed in Melanoma 202 tumor cells, and treatment with IFN-γ only weakly enhanced the abundance of DMA and -B transcripts. As was determined for the transcription of class II MHC genes, densitometric/Scatchard analysis indicated that the number of DMA and -B transcripts in IFN-γ–treated Melanoma 202 cells was less than 4% of that found in IFN-γ–treated Melanoma 285 cells. In contrast, although there were no Ii transcripts detected in untreated Melanoma 202 cells, there was no defect in the IFN-γ induction of the invariant chain gene when compared with Melanoma 285 or HeLa cells. This suggests that the defect in Melanoma 202 cells that strongly impairs the IFN-γ inducibility of class II MHC and DMA/DMB genes does not affect IFN-γ induction of the invariant chain gene. We have provided our interpretation of this result in the Discussion section. 
Transcription of CIITA in Ocular Melanoma Cells
Because ectopic expression of CIITA was able to induce the expression of class II MHC molecules on the surface of Melanoma 202 (but not 285) cells (Fig. 4 and data not shown), we hypothesized that the defect in Melanoma 202 cells either mapped at the level of CIITA transcription or was due to a structural defect in the CIITA polypeptide. Transcription of CIITA and two other factors implicated in class II MHC gene expression (HMGI/Y, also known as HMGA1 and NFX.1) was examined to determine whether the impaired IFN-γ–induced transcription of class II MHC and accessory molecules was attributable to the inappropriate expression of these regulatory proteins. As in the previous experiments, ocular melanoma cells were cultured in the absence or presence of IFN-γ, and RT-PCR was used to examine transcription (Fig. 9) . In the absence of IFN-γ, Melanoma 202 and 285 tumors expressed little or no CIITA mRNA. Of note, whereas IFN-γ treatment of Melanoma 202 tumor cells failed to induce transcription of the CIITA gene, there was a robust induction of the CIITA gene in Melanoma 285 cells (comparable to that seen in other IFN-γ–responsive cells, such as HeLa). Thus, the blockade in class II MHC gene induction observed in Melanoma 202 cells appeared to map upstream of CIITA gene induction, whereas the blockade in Melanoma 285 cells mapped downstream of both CIITA and class II MHC gene transcription. 
Nuclear Translocation of Stat1 and IRF-1
Because the CIITA gene in Melanoma 202 cells is resistant to IFN-γ–mediated activation, we next investigated the integrity of the IFN-γ signal-transduction pathway in these cells. EMSA was performed to see whether two key signal-transduction molecules, Stat1 and IRF-1, would translocate to the nucleus and bind to DNA-binding elements. Jurkat cells, which are known to have a functional IFN-γ signal-transduction pathway, were used as a positive control. Jurkat and Melanoma 202 cells were stimulated with IFN-γ for 16 hours, and then nuclear extracts were prepared. The extracts where then incubated with a radiolabeled double-stranded γ-activating sequence (GAS) oligonucleotide and then run on a polyacrylamide gel. It is clear that in both the Jurkat and Melanoma 202 cells Stat1 translocated to the nucleus and bound to a GAS (Fig. 10) . A similar experiment was performed with the results shown in Figure 11 , except that an IRE (IRF-1 response element) was used as the radiolabeled probe. In both the Jurkat and Melanoma 202 cells. IRF-1 translocated to the nucleus and bound to an IRE. These two signaling pathways for CIITA gene activation are therefore intact in Melanoma 202 cells. 
Presence of Transcription Factors for CIITA Expression in Melanoma 202 Cells
Given the EMSA data, it is still possible that Stat1 and IRF-1 translocate to the nucleus and have functional DNA binding domains but have mutant activation domains. To determine whether Stat1 and IRF-1 (and all other factors required for IFN-γ induction of CIITA) are functional proteins, promoter–reporter constructs with the IFN-γ–inducible portions of the CIITA promoter were transfected into Melanoma 202 cells. Transfection of the plasmid PIVCIITA.Luc (Fig. 12) , which contains both the GAS- and IRF-1 binding sites, showed that this promoter was strongly IFN-γ inducible in Melanoma 202 cells. Because it has also been demonstrated that promoter III of CIITA is inducible by IFN-γ (although its role is minimal when compared with promoter IV), we transfected PIIICIITA.Luc (Fig. 13) into HeLa and Melanoma 202 cells. Promoter III was IFN-γ inducible in both cell lines, although to a lesser extent. Taken together, these results demonstrate that the IFN-γ signal–transduction pathway is intact in Melanoma 202 cells and that all necessary transcription factors are present and functional, to achieve activation of heterologous CIITA reporter constructs. 
Discussion
The expression of MHC class II molecules is essential for the development of inflammation mediated by CD4+ T cells. 11 The inhibition of inflammatory responses within immune-privileged sites such as the eye coincides with a lack of class II MHC–positive antigen-presenting cells and a failure to induce class II MHC molecules in surrounding tissues after stimulation with IFN-γ. 12 To gain a better understanding of the regulation of class II MHC gene expression in ocular immune-privileged sites, we examined class II MHC gene expression in uveal melanocytes and two ocular melanoma cell lines. Our analysis of a large panel of ocular melanoma cell lines indicates that the majority of ocular melanomas do not express class II on the cell surface (Ono SJ, manuscript in preparation). We have presented herein data on two cell lines that exhibit two different points of blockade in class II MHC gene expression. 
In this study, MHC class II molecules were not expressed on the surface of primary uveal melanocytes and two human ocular melanoma cell lines, and class II MHC protein was not present in the cytoplasm of the two melanoma cell lines (as determined by Western blot). Also, there was no block in the class II biosynthetic pathway (in Melanoma 202 cells), because ectopic expression of CIITA delivered class II MHC molecules to the surface of Melanoma 202 cells. Ectopic expression of CIITA did not deliver class II MHC molecules to the surface of Melanoma 285 cells, however, indicating that the block to MHC antigen expression in those cells is downstream of class II MHC gene transcription (data not shown). 
In the case of Melanoma 202 cells, the impairment in class II MHC gene transcription was due to the inability of IFN-γ to induce strong activation of the endogenous CIITA gene. The integrity of the IFN-γ signal-transduction pathway responsible for activating CIITA expression was intact, as assessed in a series of experiments. Stat1 and IRF-1 translocated to the nucleus after IFN-γ receptor engagement. All transcription factors required for the IFN-γ–mediated activation of the CIITA gene were found to be present and functional, since both promoters III and IV of CIITA were activated in transiently transfected Melanoma 202 cells. Ectopic expression of CIITA delivered HLA-DR molecules to the cell surface, indicating that no other defects in class II MHC expression exist within Melanoma 202 cells. 
Given these results, there are several possible explanations for the suppression of CIITA expression in ocular melanoma cells. It is possible that there is a deletion or a mutation in the CIITA gene, since only a minimal amount of CIITA mRNA is produced in Melanoma 202 cells. This mutation would probably be in the regulatory region of the gene in the promoter or enhancer sequences. This type of mutation could have been produced during the malignant transformation of these cells. However, we think this is unlikely because we have to detect any SNIPs in the Melanoma 202 CIITA gene or promoter (data not shown), and primary ocular melanocytes also exhibited a class II MHC–negative phenotype on addition of IFN-γ. We interpret these data to suggest that the class II–unresponsive phenotype of IFN-γ–treated ocular melanoma cell lines is indicative of the situation in primary uveal melanocytes. Indeed, our analysis of other ocular melanoma cell lines indicates that the Melanoma 202 phenotype is observed in most human ocular melanoma cells (Ono SJ, manuscript in preparation). 
It is also possible that a transacting suppressor is working on the endogenous CIITA gene. There is evidence to suggest that this is one way to control class II expression. Transforming growth factor (TGF)-β has been shown to inhibit IFN-γ–induced class II gene expression in several cell types, 13 14 15 and its effects appear to be mediated through suppression of synthesis of CIITA mRNA. 16 17  
Another example occurs in B-cell differentiation, during which class II MHC gene transcription is silenced. 18 19 20 This downregulation of class II MHC gene expression during B-cell differentiation into plasmocytes has been demonstrated to correlate with a decrease in CIITA expression. A CIITA expression vector restores class II MHC gene expression in plasmocytes. 20 These results, along with the evidence that class II expression is not detected on stable heterokaryons derived from fusions between human B cells and mouseplasmocytes, suggests that plasmocytes express a potential repressor molecule that inhibits CIITA gene expression. 18 19 We think it unlikely that the CIITA gene is actively suppressed in Melanoma 202 cells in view of our transfection study results. Both promoters III and IV of CIITA were readily induced by IFN-γ, and it appears that a transrepressor does not exist in these cells. 
We think it is most likely that the CIITA gene silencing observed in Melanoma 202 cells is due to epigenetic mechanisms, such as alterations in chromatin structure or methylation that affects promoter accessibility to transcription factors. 21 The silencing of transcription of certain genes has been shown to be mediated by the deacetylation of histones. 22 The other distinct possibility is direct methylation of the CIITA gene. The placenta, like the eye, is an immune-privileged site, and it has been clearly demonstrated that trophoblast cells do not express class II MHC molecules on the cell surface. 23 24 25 26 This stems from the unresponsiveness of class II MHC genes to IFN-γ stimulation, reminiscent of that observed in the Melanoma 202 cell line. 27 28 This inhibition of MHC class II expression on fetal trophoblast cells is one of the various mechanisms that have been proposed to explain the lack of a vigorous immune response at the maternal–fetal interface. 29 30 31 32 33 This repression of the CIITA gene is due to methylation of promoter IV of CIITA. 28 34 35 36 It is therefore possible that methylation is responsible for the lack of CIITA expression in ocular melanoma cells. We are currently investigating these possibilities to understand better the likely epigenetic control over CIITA and MHC class II gene expression in Melanoma 202 and uveal melanocytes. 
 
Figure 3.
 
MHC class I and II polypeptide expression on IFN-γ–treated melanoma cells. Western blot analyses were performed using antibodies to HLA-DR (A), HLA-DP (B), HLA-DQ (C), and MHC class I (D). Cells were incubated with IFN-γ for 0, 16, 24, 36, and 48 hours. Specific antibodies to the MHC molecules were used to monitor expression of these molecules at the indicated time points. Priess cells, which are MHC class I and II–positive B LCL cells, were used as a positive control. Lane 1: 1 μg of MHC class II (A, B, C) or HLA-class I (D) molecules immunopurified from Priess cells. Lane 2: 2.5 × 105 protein extract from Priess cells. Lanes 3 to 7: 5 × 105 Melanoma 285 cells harvested at 0, 16, 24, 36, and 48 hours, respectively, after treatment with IFN-γ. Lanes 8 to 12: 5 × 105 Melanoma 202 cells harvested at 0, 16, 24, 36, and 48 hours, respectively, after treatment with IFN-γ.
Figure 3.
 
MHC class I and II polypeptide expression on IFN-γ–treated melanoma cells. Western blot analyses were performed using antibodies to HLA-DR (A), HLA-DP (B), HLA-DQ (C), and MHC class I (D). Cells were incubated with IFN-γ for 0, 16, 24, 36, and 48 hours. Specific antibodies to the MHC molecules were used to monitor expression of these molecules at the indicated time points. Priess cells, which are MHC class I and II–positive B LCL cells, were used as a positive control. Lane 1: 1 μg of MHC class II (A, B, C) or HLA-class I (D) molecules immunopurified from Priess cells. Lane 2: 2.5 × 105 protein extract from Priess cells. Lanes 3 to 7: 5 × 105 Melanoma 285 cells harvested at 0, 16, 24, 36, and 48 hours, respectively, after treatment with IFN-γ. Lanes 8 to 12: 5 × 105 Melanoma 202 cells harvested at 0, 16, 24, 36, and 48 hours, respectively, after treatment with IFN-γ.
Figure 1.
 
Expression of HLA-DR molecules on IFN-γ–stimulated and unstimulated primary skin and uveal melanocytes assessed by flow cytometry. Primary skin melanocytes expressed class II MHC molecules on the cell surface after incubation with recombinant IFN-γ (top). In contrast, primary uveal melanocytes were resistant to IFN-γ–mediated induction of class II MHC expression on the cell surface (bottom).
Figure 1.
 
Expression of HLA-DR molecules on IFN-γ–stimulated and unstimulated primary skin and uveal melanocytes assessed by flow cytometry. Primary skin melanocytes expressed class II MHC molecules on the cell surface after incubation with recombinant IFN-γ (top). In contrast, primary uveal melanocytes were resistant to IFN-γ–mediated induction of class II MHC expression on the cell surface (bottom).
Figure 2.
 
Expression of HLA-DR on the surface of HeLa and Melanoma 202 cells after stimulation with IFN-γ. HeLa (A) and Melanoma 202 (B) cells were treated with IFN-γ for 24 hours. Untreated cells were used as the control. Solid line: untreated cells; dashed line: treated cells.
Figure 2.
 
Expression of HLA-DR on the surface of HeLa and Melanoma 202 cells after stimulation with IFN-γ. HeLa (A) and Melanoma 202 (B) cells were treated with IFN-γ for 24 hours. Untreated cells were used as the control. Solid line: untreated cells; dashed line: treated cells.
Figure 4.
 
Immunofluorescent staining of MHC class II expressed on the surface of Melanoma 202 cells transfected with a CIITA expression vector. Melanoma 202 cells were passed onto chambered slides and then transfected with 1.5 μg of either the empty vector PcDNA/INOnk2 (A) or the CIITA expression vector PcDNA I/CIITA (B). The transfected cells were incubated for 25 hours and then stained with anti-HLA-DR and analyzed by immunofluorescence.
Figure 4.
 
Immunofluorescent staining of MHC class II expressed on the surface of Melanoma 202 cells transfected with a CIITA expression vector. Melanoma 202 cells were passed onto chambered slides and then transfected with 1.5 μg of either the empty vector PcDNA/INOnk2 (A) or the CIITA expression vector PcDNA I/CIITA (B). The transfected cells were incubated for 25 hours and then stained with anti-HLA-DR and analyzed by immunofluorescence.
Figure 5.
 
Transactivation of a HLA-DRA reporter construct in HeLa and Melanoma 202 cells after stimulation with IFN-γ. (A) Schematic of the promoter–reporter construct that contains the HLA-DRA promoter. The 271-bp region contains the essential cis-elements necessary for the transcription of the HLA-DRA and other class II MHC genes. The S, X, and Y elements are depicted (boxes) followed by the transcriptional start site (arrow). (B) HeLa and Melanoma 202 cells were transfected with either the control PGL2-Basic vector or the HLA-DRA reporter construct. After transfection, the cells were either stimulated or not with recombinant IFN-γ for 60 hours. Cell extracts were prepared and analyzed on a luminometer. The luciferase activity was measured as relative light units per microgram of protein. Induction (x-fold) was determined by dividing the relative light units of stimulated cells by the relative light units of the unstimulated cells. The data shown are averages of results in three independent experiments; error bars, SEM.
Figure 5.
 
Transactivation of a HLA-DRA reporter construct in HeLa and Melanoma 202 cells after stimulation with IFN-γ. (A) Schematic of the promoter–reporter construct that contains the HLA-DRA promoter. The 271-bp region contains the essential cis-elements necessary for the transcription of the HLA-DRA and other class II MHC genes. The S, X, and Y elements are depicted (boxes) followed by the transcriptional start site (arrow). (B) HeLa and Melanoma 202 cells were transfected with either the control PGL2-Basic vector or the HLA-DRA reporter construct. After transfection, the cells were either stimulated or not with recombinant IFN-γ for 60 hours. Cell extracts were prepared and analyzed on a luminometer. The luciferase activity was measured as relative light units per microgram of protein. Induction (x-fold) was determined by dividing the relative light units of stimulated cells by the relative light units of the unstimulated cells. The data shown are averages of results in three independent experiments; error bars, SEM.
Figure 6.
 
RT-PCR analysis of MHC class II (DRA, DRB, DQA, and DQB) gene expression in ocular Melanoma 285 (A) and 202 (B) cells treated with IFN-γ. Total RNA was obtained from ocular melanoma cells after treatment with IFN-γ for 0, 16, or 24 hours. The RNA was subjected to RT-PCR analysis using gene-specific primers. GAPDH mRNA levels were used as the control for RNA loading and stability.
Figure 6.
 
RT-PCR analysis of MHC class II (DRA, DRB, DQA, and DQB) gene expression in ocular Melanoma 285 (A) and 202 (B) cells treated with IFN-γ. Total RNA was obtained from ocular melanoma cells after treatment with IFN-γ for 0, 16, or 24 hours. The RNA was subjected to RT-PCR analysis using gene-specific primers. GAPDH mRNA levels were used as the control for RNA loading and stability.
Figure 7.
 
Dilution of cDNA in INF-γ–induced Melanoma 202 and 285 cells as detected by RT-PCR using DRA and GAPDH primers. After reverse transcription, the cDNAs generated from IFN-γ–treated Melanoma 202 (top) and 285 (bottom) cells (identical concentrations of IFN-γ and times of incubation) were diluted 1:5, 1:25, 1:75, and 1:125, of which 2 μL each was used as a template in the PCR reactions.
Figure 7.
 
Dilution of cDNA in INF-γ–induced Melanoma 202 and 285 cells as detected by RT-PCR using DRA and GAPDH primers. After reverse transcription, the cDNAs generated from IFN-γ–treated Melanoma 202 (top) and 285 (bottom) cells (identical concentrations of IFN-γ and times of incubation) were diluted 1:5, 1:25, 1:75, and 1:125, of which 2 μL each was used as a template in the PCR reactions.
Figure 8.
 
RT-PCR analysis of MHC Class II accessory molecule (DMA, DMB, and Ii) gene expression in ocular Melanoma 285 (A) and 202 (B) cells treated with IFN-γ. Total RNA was obtained from the cells after treatment with IFN-γ for 0, 16, or 24 hours. The RNA was subjected to RT-PCR analysis with gene-specific primers. GAPDH mRNA levels were used as the control for RNA loading and stability.
Figure 8.
 
RT-PCR analysis of MHC Class II accessory molecule (DMA, DMB, and Ii) gene expression in ocular Melanoma 285 (A) and 202 (B) cells treated with IFN-γ. Total RNA was obtained from the cells after treatment with IFN-γ for 0, 16, or 24 hours. The RNA was subjected to RT-PCR analysis with gene-specific primers. GAPDH mRNA levels were used as the control for RNA loading and stability.
Figure 9.
 
RT-PCR analysis of CIITA, HMG I/Y, and NFX.1 gene expression in ocular Melanoma 285 (A) and 202 (B) cells treated with IFN-γ. Total RNA was obtained from ocular melanoma cells after treatment with IFN-γ for 0, 16, or 24 hours. The RNA was subjected to RT-PCR analysis with gene-specific primers. GAPDH mRNA levels were used as the control for RNA loading and stability.
Figure 9.
 
RT-PCR analysis of CIITA, HMG I/Y, and NFX.1 gene expression in ocular Melanoma 285 (A) and 202 (B) cells treated with IFN-γ. Total RNA was obtained from ocular melanoma cells after treatment with IFN-γ for 0, 16, or 24 hours. The RNA was subjected to RT-PCR analysis with gene-specific primers. GAPDH mRNA levels were used as the control for RNA loading and stability.
Figure 10.
 
Stat1 translocation and DNA-binding in Melanoma 202 cells. Jurkat (a positive control) and Melanoma 202 cells were stimulated with IFN-γ for 16 hours and then nuclear extracts were obtained from the cells. For the determination of Stat1 activation, the nuclear extracts (5 μg) were incubated with a Stat1-specific 32P-labbled oligonucleotide GAS probe. The DNA–protein complexes formed were separated from the free probe by electrophoresis on 5% polyacrylamide gel. The Stat1 complex is indicated by the labeled bar and unbound probe is at the bottom of the gel.
Figure 10.
 
Stat1 translocation and DNA-binding in Melanoma 202 cells. Jurkat (a positive control) and Melanoma 202 cells were stimulated with IFN-γ for 16 hours and then nuclear extracts were obtained from the cells. For the determination of Stat1 activation, the nuclear extracts (5 μg) were incubated with a Stat1-specific 32P-labbled oligonucleotide GAS probe. The DNA–protein complexes formed were separated from the free probe by electrophoresis on 5% polyacrylamide gel. The Stat1 complex is indicated by the labeled bar and unbound probe is at the bottom of the gel.
Figure 11.
 
IRF-1 nuclear translocation and DNA-binding in Melanoma 202 cells. Jurkat, Melanoma 285 (positive controls) and Melanoma 202 cells were stimulated with IFN-γ for 16 hours and then nuclear extracts were obtained from the cells. The nuclear extracts (5 μg) were incubated with a 32P-labeled oligonucleotide IRF-1–binding element. The DNA-protein complexes formed were separated from the free probe by electrophoresis on a 5% polyacrylamide gel. The IRF-1–binding complex is indicated by a labeled bar and unbound probe is at the bottom of the gel.
Figure 11.
 
IRF-1 nuclear translocation and DNA-binding in Melanoma 202 cells. Jurkat, Melanoma 285 (positive controls) and Melanoma 202 cells were stimulated with IFN-γ for 16 hours and then nuclear extracts were obtained from the cells. The nuclear extracts (5 μg) were incubated with a 32P-labeled oligonucleotide IRF-1–binding element. The DNA-protein complexes formed were separated from the free probe by electrophoresis on a 5% polyacrylamide gel. The IRF-1–binding complex is indicated by a labeled bar and unbound probe is at the bottom of the gel.
Figure 12.
 
All necessary transcription factors for CIITA promoter IV expression were present and functional in Melanoma 202 cells. (A) A schematic diagram of PIVCIITA.Luc. Stat1 binds to the GAS cis-element and IRF-1 to the IRF-1 cis–element. This 396-bp promoter was inserted into the PGL2-Basic vector. Arrow: transcriptional start site. (B) HeLa and Melanoma 202 cells were transfected with either the control PGL2-Basic vector or the HLA-DRA reporter construct vector. After transfection, the cells were either stimulated or not with IFN-γ for 24 hours. Cell extracts were prepared and analyzed on a luminometer. The luciferase activity was measured as relative light units per microgram of protein. Induction (x-fold) was determined by dividing the relative light units of stimulated cells by the relative light units of the unstimulated cells. The data shown are averages of results in six independent experiments; error bars, SEM.
Figure 12.
 
All necessary transcription factors for CIITA promoter IV expression were present and functional in Melanoma 202 cells. (A) A schematic diagram of PIVCIITA.Luc. Stat1 binds to the GAS cis-element and IRF-1 to the IRF-1 cis–element. This 396-bp promoter was inserted into the PGL2-Basic vector. Arrow: transcriptional start site. (B) HeLa and Melanoma 202 cells were transfected with either the control PGL2-Basic vector or the HLA-DRA reporter construct vector. After transfection, the cells were either stimulated or not with IFN-γ for 24 hours. Cell extracts were prepared and analyzed on a luminometer. The luciferase activity was measured as relative light units per microgram of protein. Induction (x-fold) was determined by dividing the relative light units of stimulated cells by the relative light units of the unstimulated cells. The data shown are averages of results in six independent experiments; error bars, SEM.
Figure 13.
 
All necessary transcription factors for CIITA promoter III expression were present and functional in Melanoma 202 cells. (A) A schematic diagram of PIIICIITA.Luc. This more than 6-kb region of PIII has been inserted into the PGL-2Basic vector. Arrow: transcriptional start site. (B) HeLa and Melanoma 202 cells were transfected with either the control PGL2-Basic vector or the HLA-DRA reporter construct vector. After transfection, the cells were either stimulated or not with IFN-γ for 24 hours. Cell extracts were prepared and analyzed on a luminometer. The luciferase activity was measured as relative light units per microgram of protein. Induction (x-fold) was determined by dividing the relative light units of stimulated cells by the relative light units of the unstimulated cells. The average induction in six independent experiments is shown; error bars, SEM.
Figure 13.
 
All necessary transcription factors for CIITA promoter III expression were present and functional in Melanoma 202 cells. (A) A schematic diagram of PIIICIITA.Luc. This more than 6-kb region of PIII has been inserted into the PGL-2Basic vector. Arrow: transcriptional start site. (B) HeLa and Melanoma 202 cells were transfected with either the control PGL2-Basic vector or the HLA-DRA reporter construct vector. After transfection, the cells were either stimulated or not with IFN-γ for 24 hours. Cell extracts were prepared and analyzed on a luminometer. The luciferase activity was measured as relative light units per microgram of protein. Induction (x-fold) was determined by dividing the relative light units of stimulated cells by the relative light units of the unstimulated cells. The average induction in six independent experiments is shown; error bars, SEM.
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Figure 3.
 
MHC class I and II polypeptide expression on IFN-γ–treated melanoma cells. Western blot analyses were performed using antibodies to HLA-DR (A), HLA-DP (B), HLA-DQ (C), and MHC class I (D). Cells were incubated with IFN-γ for 0, 16, 24, 36, and 48 hours. Specific antibodies to the MHC molecules were used to monitor expression of these molecules at the indicated time points. Priess cells, which are MHC class I and II–positive B LCL cells, were used as a positive control. Lane 1: 1 μg of MHC class II (A, B, C) or HLA-class I (D) molecules immunopurified from Priess cells. Lane 2: 2.5 × 105 protein extract from Priess cells. Lanes 3 to 7: 5 × 105 Melanoma 285 cells harvested at 0, 16, 24, 36, and 48 hours, respectively, after treatment with IFN-γ. Lanes 8 to 12: 5 × 105 Melanoma 202 cells harvested at 0, 16, 24, 36, and 48 hours, respectively, after treatment with IFN-γ.
Figure 3.
 
MHC class I and II polypeptide expression on IFN-γ–treated melanoma cells. Western blot analyses were performed using antibodies to HLA-DR (A), HLA-DP (B), HLA-DQ (C), and MHC class I (D). Cells were incubated with IFN-γ for 0, 16, 24, 36, and 48 hours. Specific antibodies to the MHC molecules were used to monitor expression of these molecules at the indicated time points. Priess cells, which are MHC class I and II–positive B LCL cells, were used as a positive control. Lane 1: 1 μg of MHC class II (A, B, C) or HLA-class I (D) molecules immunopurified from Priess cells. Lane 2: 2.5 × 105 protein extract from Priess cells. Lanes 3 to 7: 5 × 105 Melanoma 285 cells harvested at 0, 16, 24, 36, and 48 hours, respectively, after treatment with IFN-γ. Lanes 8 to 12: 5 × 105 Melanoma 202 cells harvested at 0, 16, 24, 36, and 48 hours, respectively, after treatment with IFN-γ.
Figure 1.
 
Expression of HLA-DR molecules on IFN-γ–stimulated and unstimulated primary skin and uveal melanocytes assessed by flow cytometry. Primary skin melanocytes expressed class II MHC molecules on the cell surface after incubation with recombinant IFN-γ (top). In contrast, primary uveal melanocytes were resistant to IFN-γ–mediated induction of class II MHC expression on the cell surface (bottom).
Figure 1.
 
Expression of HLA-DR molecules on IFN-γ–stimulated and unstimulated primary skin and uveal melanocytes assessed by flow cytometry. Primary skin melanocytes expressed class II MHC molecules on the cell surface after incubation with recombinant IFN-γ (top). In contrast, primary uveal melanocytes were resistant to IFN-γ–mediated induction of class II MHC expression on the cell surface (bottom).
Figure 2.
 
Expression of HLA-DR on the surface of HeLa and Melanoma 202 cells after stimulation with IFN-γ. HeLa (A) and Melanoma 202 (B) cells were treated with IFN-γ for 24 hours. Untreated cells were used as the control. Solid line: untreated cells; dashed line: treated cells.
Figure 2.
 
Expression of HLA-DR on the surface of HeLa and Melanoma 202 cells after stimulation with IFN-γ. HeLa (A) and Melanoma 202 (B) cells were treated with IFN-γ for 24 hours. Untreated cells were used as the control. Solid line: untreated cells; dashed line: treated cells.
Figure 4.
 
Immunofluorescent staining of MHC class II expressed on the surface of Melanoma 202 cells transfected with a CIITA expression vector. Melanoma 202 cells were passed onto chambered slides and then transfected with 1.5 μg of either the empty vector PcDNA/INOnk2 (A) or the CIITA expression vector PcDNA I/CIITA (B). The transfected cells were incubated for 25 hours and then stained with anti-HLA-DR and analyzed by immunofluorescence.
Figure 4.
 
Immunofluorescent staining of MHC class II expressed on the surface of Melanoma 202 cells transfected with a CIITA expression vector. Melanoma 202 cells were passed onto chambered slides and then transfected with 1.5 μg of either the empty vector PcDNA/INOnk2 (A) or the CIITA expression vector PcDNA I/CIITA (B). The transfected cells were incubated for 25 hours and then stained with anti-HLA-DR and analyzed by immunofluorescence.
Figure 5.
 
Transactivation of a HLA-DRA reporter construct in HeLa and Melanoma 202 cells after stimulation with IFN-γ. (A) Schematic of the promoter–reporter construct that contains the HLA-DRA promoter. The 271-bp region contains the essential cis-elements necessary for the transcription of the HLA-DRA and other class II MHC genes. The S, X, and Y elements are depicted (boxes) followed by the transcriptional start site (arrow). (B) HeLa and Melanoma 202 cells were transfected with either the control PGL2-Basic vector or the HLA-DRA reporter construct. After transfection, the cells were either stimulated or not with recombinant IFN-γ for 60 hours. Cell extracts were prepared and analyzed on a luminometer. The luciferase activity was measured as relative light units per microgram of protein. Induction (x-fold) was determined by dividing the relative light units of stimulated cells by the relative light units of the unstimulated cells. The data shown are averages of results in three independent experiments; error bars, SEM.
Figure 5.
 
Transactivation of a HLA-DRA reporter construct in HeLa and Melanoma 202 cells after stimulation with IFN-γ. (A) Schematic of the promoter–reporter construct that contains the HLA-DRA promoter. The 271-bp region contains the essential cis-elements necessary for the transcription of the HLA-DRA and other class II MHC genes. The S, X, and Y elements are depicted (boxes) followed by the transcriptional start site (arrow). (B) HeLa and Melanoma 202 cells were transfected with either the control PGL2-Basic vector or the HLA-DRA reporter construct. After transfection, the cells were either stimulated or not with recombinant IFN-γ for 60 hours. Cell extracts were prepared and analyzed on a luminometer. The luciferase activity was measured as relative light units per microgram of protein. Induction (x-fold) was determined by dividing the relative light units of stimulated cells by the relative light units of the unstimulated cells. The data shown are averages of results in three independent experiments; error bars, SEM.
Figure 6.
 
RT-PCR analysis of MHC class II (DRA, DRB, DQA, and DQB) gene expression in ocular Melanoma 285 (A) and 202 (B) cells treated with IFN-γ. Total RNA was obtained from ocular melanoma cells after treatment with IFN-γ for 0, 16, or 24 hours. The RNA was subjected to RT-PCR analysis using gene-specific primers. GAPDH mRNA levels were used as the control for RNA loading and stability.
Figure 6.
 
RT-PCR analysis of MHC class II (DRA, DRB, DQA, and DQB) gene expression in ocular Melanoma 285 (A) and 202 (B) cells treated with IFN-γ. Total RNA was obtained from ocular melanoma cells after treatment with IFN-γ for 0, 16, or 24 hours. The RNA was subjected to RT-PCR analysis using gene-specific primers. GAPDH mRNA levels were used as the control for RNA loading and stability.
Figure 7.
 
Dilution of cDNA in INF-γ–induced Melanoma 202 and 285 cells as detected by RT-PCR using DRA and GAPDH primers. After reverse transcription, the cDNAs generated from IFN-γ–treated Melanoma 202 (top) and 285 (bottom) cells (identical concentrations of IFN-γ and times of incubation) were diluted 1:5, 1:25, 1:75, and 1:125, of which 2 μL each was used as a template in the PCR reactions.
Figure 7.
 
Dilution of cDNA in INF-γ–induced Melanoma 202 and 285 cells as detected by RT-PCR using DRA and GAPDH primers. After reverse transcription, the cDNAs generated from IFN-γ–treated Melanoma 202 (top) and 285 (bottom) cells (identical concentrations of IFN-γ and times of incubation) were diluted 1:5, 1:25, 1:75, and 1:125, of which 2 μL each was used as a template in the PCR reactions.
Figure 8.
 
RT-PCR analysis of MHC Class II accessory molecule (DMA, DMB, and Ii) gene expression in ocular Melanoma 285 (A) and 202 (B) cells treated with IFN-γ. Total RNA was obtained from the cells after treatment with IFN-γ for 0, 16, or 24 hours. The RNA was subjected to RT-PCR analysis with gene-specific primers. GAPDH mRNA levels were used as the control for RNA loading and stability.
Figure 8.
 
RT-PCR analysis of MHC Class II accessory molecule (DMA, DMB, and Ii) gene expression in ocular Melanoma 285 (A) and 202 (B) cells treated with IFN-γ. Total RNA was obtained from the cells after treatment with IFN-γ for 0, 16, or 24 hours. The RNA was subjected to RT-PCR analysis with gene-specific primers. GAPDH mRNA levels were used as the control for RNA loading and stability.
Figure 9.
 
RT-PCR analysis of CIITA, HMG I/Y, and NFX.1 gene expression in ocular Melanoma 285 (A) and 202 (B) cells treated with IFN-γ. Total RNA was obtained from ocular melanoma cells after treatment with IFN-γ for 0, 16, or 24 hours. The RNA was subjected to RT-PCR analysis with gene-specific primers. GAPDH mRNA levels were used as the control for RNA loading and stability.
Figure 9.
 
RT-PCR analysis of CIITA, HMG I/Y, and NFX.1 gene expression in ocular Melanoma 285 (A) and 202 (B) cells treated with IFN-γ. Total RNA was obtained from ocular melanoma cells after treatment with IFN-γ for 0, 16, or 24 hours. The RNA was subjected to RT-PCR analysis with gene-specific primers. GAPDH mRNA levels were used as the control for RNA loading and stability.
Figure 10.
 
Stat1 translocation and DNA-binding in Melanoma 202 cells. Jurkat (a positive control) and Melanoma 202 cells were stimulated with IFN-γ for 16 hours and then nuclear extracts were obtained from the cells. For the determination of Stat1 activation, the nuclear extracts (5 μg) were incubated with a Stat1-specific 32P-labbled oligonucleotide GAS probe. The DNA–protein complexes formed were separated from the free probe by electrophoresis on 5% polyacrylamide gel. The Stat1 complex is indicated by the labeled bar and unbound probe is at the bottom of the gel.
Figure 10.
 
Stat1 translocation and DNA-binding in Melanoma 202 cells. Jurkat (a positive control) and Melanoma 202 cells were stimulated with IFN-γ for 16 hours and then nuclear extracts were obtained from the cells. For the determination of Stat1 activation, the nuclear extracts (5 μg) were incubated with a Stat1-specific 32P-labbled oligonucleotide GAS probe. The DNA–protein complexes formed were separated from the free probe by electrophoresis on 5% polyacrylamide gel. The Stat1 complex is indicated by the labeled bar and unbound probe is at the bottom of the gel.
Figure 11.
 
IRF-1 nuclear translocation and DNA-binding in Melanoma 202 cells. Jurkat, Melanoma 285 (positive controls) and Melanoma 202 cells were stimulated with IFN-γ for 16 hours and then nuclear extracts were obtained from the cells. The nuclear extracts (5 μg) were incubated with a 32P-labeled oligonucleotide IRF-1–binding element. The DNA-protein complexes formed were separated from the free probe by electrophoresis on a 5% polyacrylamide gel. The IRF-1–binding complex is indicated by a labeled bar and unbound probe is at the bottom of the gel.
Figure 11.
 
IRF-1 nuclear translocation and DNA-binding in Melanoma 202 cells. Jurkat, Melanoma 285 (positive controls) and Melanoma 202 cells were stimulated with IFN-γ for 16 hours and then nuclear extracts were obtained from the cells. The nuclear extracts (5 μg) were incubated with a 32P-labeled oligonucleotide IRF-1–binding element. The DNA-protein complexes formed were separated from the free probe by electrophoresis on a 5% polyacrylamide gel. The IRF-1–binding complex is indicated by a labeled bar and unbound probe is at the bottom of the gel.
Figure 12.
 
All necessary transcription factors for CIITA promoter IV expression were present and functional in Melanoma 202 cells. (A) A schematic diagram of PIVCIITA.Luc. Stat1 binds to the GAS cis-element and IRF-1 to the IRF-1 cis–element. This 396-bp promoter was inserted into the PGL2-Basic vector. Arrow: transcriptional start site. (B) HeLa and Melanoma 202 cells were transfected with either the control PGL2-Basic vector or the HLA-DRA reporter construct vector. After transfection, the cells were either stimulated or not with IFN-γ for 24 hours. Cell extracts were prepared and analyzed on a luminometer. The luciferase activity was measured as relative light units per microgram of protein. Induction (x-fold) was determined by dividing the relative light units of stimulated cells by the relative light units of the unstimulated cells. The data shown are averages of results in six independent experiments; error bars, SEM.
Figure 12.
 
All necessary transcription factors for CIITA promoter IV expression were present and functional in Melanoma 202 cells. (A) A schematic diagram of PIVCIITA.Luc. Stat1 binds to the GAS cis-element and IRF-1 to the IRF-1 cis–element. This 396-bp promoter was inserted into the PGL2-Basic vector. Arrow: transcriptional start site. (B) HeLa and Melanoma 202 cells were transfected with either the control PGL2-Basic vector or the HLA-DRA reporter construct vector. After transfection, the cells were either stimulated or not with IFN-γ for 24 hours. Cell extracts were prepared and analyzed on a luminometer. The luciferase activity was measured as relative light units per microgram of protein. Induction (x-fold) was determined by dividing the relative light units of stimulated cells by the relative light units of the unstimulated cells. The data shown are averages of results in six independent experiments; error bars, SEM.
Figure 13.
 
All necessary transcription factors for CIITA promoter III expression were present and functional in Melanoma 202 cells. (A) A schematic diagram of PIIICIITA.Luc. This more than 6-kb region of PIII has been inserted into the PGL-2Basic vector. Arrow: transcriptional start site. (B) HeLa and Melanoma 202 cells were transfected with either the control PGL2-Basic vector or the HLA-DRA reporter construct vector. After transfection, the cells were either stimulated or not with IFN-γ for 24 hours. Cell extracts were prepared and analyzed on a luminometer. The luciferase activity was measured as relative light units per microgram of protein. Induction (x-fold) was determined by dividing the relative light units of stimulated cells by the relative light units of the unstimulated cells. The average induction in six independent experiments is shown; error bars, SEM.
Figure 13.
 
All necessary transcription factors for CIITA promoter III expression were present and functional in Melanoma 202 cells. (A) A schematic diagram of PIIICIITA.Luc. This more than 6-kb region of PIII has been inserted into the PGL-2Basic vector. Arrow: transcriptional start site. (B) HeLa and Melanoma 202 cells were transfected with either the control PGL2-Basic vector or the HLA-DRA reporter construct vector. After transfection, the cells were either stimulated or not with IFN-γ for 24 hours. Cell extracts were prepared and analyzed on a luminometer. The luciferase activity was measured as relative light units per microgram of protein. Induction (x-fold) was determined by dividing the relative light units of stimulated cells by the relative light units of the unstimulated cells. The average induction in six independent experiments is shown; error bars, SEM.
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