MHC class II-deficient tumor cell lines with a defective expression of the class II transactivator
Rodrigo Naves1,
Ana Maria Lennon2,
Giovanna Barbieri3,4,
Lilian Reyes1,
Gisella Puga1,
Laura Salas2,
Virginie Deffrennes3,
Mario Rosemblatt1,
Marc Fellous2,
Dominique Charron3,
Catherine Alcaïde-Loridan3 and
Maria Rosa Bono1
1 Departamento de Biologia, Facultad de Ciencias, Universidad de Chile, and Millennium Institute for Fundamental and Applied Biology, Casilla 653, Santiago, Chile 2 Unité dImmunogénétique Humaine, INSERM U276, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France 3 INSERM U396, Centre de Recherches Biomédicales des Cordeliers, 15 rue de lEcole de Médecine, 75006 Paris, France 4 Permanent address: Istituto di Biologia dello Sviluppo, CNR 152 via Ugo La Malfa, 90146 Palermo, Italy
The first two authors contributed equally to this work
Correspondence to: M. R. Bono; E-mail: mrbono{at}uchile.cl
Transmitting editor: D. R. Littman
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Abstract
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MHC class II expression defects have been evidenced in several human tumor cell lines originating from lung cancers or retinoblastoma. Accordingly, the mouse adenocarcinoma and fibrosarcoma cell lines, RAG and L(tk), do not express I-A and I-E molecules even when treated with IFN-
. Here we show that fusion of both cell lines restores the inducible expression of MHC class II, thereby demonstrating that they present different and recessive alterations outside the MHC class II locus. CIITA, the MHC class II transactivator, controls the tissue-specific expression of MHC class II genes and creates the architecture of the transcriptional complex that binds to the MHC class II gene promoters. In L(tk) cells, C2ta transcripts, expressed from the gene encoding CIITA, were indeed detected in severely limited amounts, with a defect in C2ta transcription initiation. In agreement we show here that the L(tk) cell line does not express the CIITA protein. In contrast, in the RAG cell line, C2ta transcripts were expressed at normal levels, from the proper initiation site. The nucleotide sequencing of the CIITA cDNA from RAG did not reveal any mutation. However, the CIITA protein was not detected. These data evidence a new type of defect in a MHC class II-defective tumor cell line, as we show here that the alteration in the RAG cells occurs downstream of C2ta transcription. The RAG mutation might therefore reside in the C2ta transcript nuclear export or translation, or in the stability of the CIITA protein.
Keywords: CIITA, MHC class II, tumor cell line, transcription
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Introduction
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MHC class II molecules play a central role in the presentation of antigens to CD4+ T lymphocytes during the initiation of the immune response (1). Constitutive expression of MHC class II molecules is restricted to professional antigen-presenting cells (APC) including B lymphocytes, dendritic cells and activated macrophages. Non-professional APC like endothelial cells, astrocytes, epithelial cells or fibroblasts can be induced to express MHC class II by IFN-
(2). MHC class II gene expression is mainly regulated at the transcriptional level by four conserved cis-acting promoter elements termed W, X1, X2 and Y boxes (2,3).
Genetic complementation analysis carried out with cell lines derived from patients affected by MHC class II deficiency (4) defined four complementation groups (groups AD) (5). Cell lines from complementation groups B, C and D display mutations in RFXANK (6), RFX5 (7) and RFXAP (8) respectively. These proteins correspond to the three subunits of the RFX transcription factor that binds to the X1 motif of the MHC class II gene promoters. Group A patients present mutations in the class II transactivator (CIITA) (9). This non-DNA-binding factor interacts with different transcription factors [regulatory factor X (RFX), nuclear factor Y (NFY) and cAMP-responsive element binding protein (CREB)] (10), the CREB-binding protein (CBP) transactivator (11) and the transcription machinery (12,13). Through these multiple interactions CIITA creates the transcriptional scaffold that permits the initiation of MHC class II gene transcription (14). In addition, CIITA is considered to be the master regulator of MHC class II expression and it has been demonstrated through mutant cell lines that it mediates the IFN-
induction of MHC class II molecules (15). Indeed the tissue-specific pattern of expression of the MHC class II genes is controlled by CIITA, the expression of which precedes that of MHC class II (16,17). Cell- and tissue-specific patterns of MHC2TA or C2ta (the human and mouse genes respectively) expression are controlled by the alternative usage of three distinct promoters. Promoters I and III regulate the constitutive expression of CIITA in dendritic cells and B lymphocytes respectively, whereas promoter IV drives the IFN-
-induced transcription of CIITA (18,19).
Modulation of MHC class II expression has been evidenced during the development of different types of tumors (20). The role of MHC class II deregulation in tumor cells is not clearly understood. It has been shown that the re-expression of MHC class II molecules in mouse sarcoma cells favors tumor regression (21). In contrast, in patients with melanoma tumors the abnormal constitutive expression of HLA-DR in melanoma cells has been associated with a bad prognosis (22). One main common point to the MHC class II deregulation is that CIITA expression is usually affected. Indeed, the analysis of the molecular events leading to MHC class II deficiency in human breast cancer and in non-small lung cancer cell lines has shown evidence of defects in MHC2TA transcription (23). In addition we have demonstrated the constitutive transcription of MHC2TA abnormally initiating from promoter III in melanoma cell lines constitutively expressing the MHC class II molecules (24).
Accordingly, here we described two additional tumor cell lines defective for expression of CIITA and explore the molecular mechanisms responsible of their defects. We show that in one cell line, RAG, transcription of C2ta is normal, while CIITA protein cannot be detected in these cells, suggesting that they display a defect in C2ta mRNA processing or protein stability. In contrast, we find that the second cell line, L(tk), expresses an aberrant form of the C2ta transcript.
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Methods
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Cell lines and IFN
RAG (a mouse adenocarcinoma cell line), L(tk) (a mouse fibrosarcoma cell line), WISH (a human amniotic cell line), RAW264.7 (a mouse monocyte-macrophage cell line), J777.4 (a mouse pre-monocyte cell line) and A20 (a mouse B cell lymphoma) were obtained from ATCC (Rockville, MD). The LM and GES human B cell lines were immortalized with EpsteinBarr virus. The RAW264.7 and WISH cell lines express MHC class II molecules after IFN-
induction. The LM and GES cell lines constitutively express MHC class II molecules. The R.GES.2.1 cell hybrid originates from the fusion of the RAG and GES cell lines, and contains different human chromosomes including chromosome 16 (25). The RR2.16 cell line is a radiation somatic cell hybrid prepared from the R.2417.2 somatic cell hybrid (25). The resulting RR2.16 hybrid presents the genetic background of the RAG cell line and contains fragments of human chromosome 16 (our unpublished data). The RAG, J777.4, R.GES.2.1, RR2.16 and L(tk) cell lines were grown in DMEM supplemented with 10% FCS, antibiotics and 2 mM glutamine. The A20, RAW264.7, WISH, GES and LM cells were maintained in RPMI 1640 supplemented with 10% FCS, antibiotics and 2 mM glutamine under standard conditions. The specific activity of the mouse IFN-
(kindly provided by Roussel-Uclaf, Romainville, France) was estimated by antiviral assay as previously described (26) and it was used at 150 or 250 U/ml as indicated in the figure legends.
Flow cytometric analyses
Subconfluent cell cultures were treated for 72 h with IFN-
. Indirect immunofluorescence assays were performed using the following primary mAb: 20.8.4 (anti-H-2d) (27), MKD6 (anti-I-Ad) (28), 11.4.1 (anti-H-2k; ATCC TIB 95) and H39.64.5 (anti-I-Ak) (28). Cells were next labeled with FITC-conjugated sheep anti-mouse Ig from Biosys (Compiègne, France). Flow cytometric analysis was performed on a FACScan (Becton Dickinson, Mountain View, CA) using the Lysys II program (Becton Dickinson).
Cell fusions
Somatic cell hybrids were prepared by the fusion of 107 cells of each fusion partner using PEG 4000 (Merck, Darmstadt, Germany) following previously described protocols (29). Adherent hybrids generated by the fusion of the L(tk) cell line (thymidine kinase-deficient) with GES (L.GES) or LM (L.LM) cells were selected in DMEM plus HAT while non-adherent GES and LM cells were eliminated by washing. Hybrids obtained by the fusion of L(tk) and WISH cell lines (L.W) were selected on DMEM containing HAT plus 1.0 mg/ml G418 (Geneticin; GIBCO, Rockville, MD) since the adherent WISH cells were made G418-resistant by transfection with a pSV2neo plasmid (30) by calcium phosphate precipitation (31) prior to the fusion
PCR
All PCR reactions were performed on a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT) with the following cycles: one cycle of 4 min at 95°C, 30 cycles of 1 min at 95°C, 1 min at 5565°C, 2 min at 72°C and finally one cycle of 10 min at 72°C. For each PCR reaction, the annealing temperature used was near to the melting temperature of each primers. Human genes were amplified with the following human-specific sense (S) and antisense (AS) primers sets: CIITA: H.C-S (5'-CGCCCTATTTGAGCTGTC-3') and H.C-AS (5'-GGTC AATGCTAGGTACTG-3'); RFX-5: HRFX5-S (5'-CCAATCCC AGTCTCTCCAC-3') and HRFX5-AS (5'-CACCACCTATGC CTACCTCT-3'); RFXAP: HRFXAP-S (5'-GTTCTCATCAAGG GTCAATT-3') and HRFXAP-AS (5'-TAGTTAATCCGTCAG GTTTT-3'); protamine I: PRM1-S (5'-GCTTGGCACACTAG GAGCTATTAG-3') and PRM1-AS (5'-CCCTTGCCTTGCC TGTAAAGCATG-3'); adenine phosphoribosyl transferase: APRT-S (5'-GGTGCATGTCACTGGCCTTTCAGCT-3') and APRT-AS (5'-TGTCT CAACCTCTCTGAGCTCCCAA-3').
RT-PCR
Total RNA was isolated from subconfluent cultures using the guanidinium thiocyanate method (32). One microgram of total RNA was incubated for 1 h at 42°C in the presence of 200 ng anti-sense oligonucleotides, 2 U AMV reverse transcriptase (Promega, Madison, WI), 15 U RNAsin, 1 mM dNTP and 2.5 mM MgCl2, in the Taq buffer from the AmpliTaq kit (Perkin-Elmer Cetus), in 20 µl final volume. PCR reactions were performed on the resulting cDNAs in a 100 µl final volume (200 µM dNTP; 2.5 mM MgCl2) after addition of the sense oligonucleotides and 5 U Taq DNA polymerase. PCR were performed on a DNA thermal cycler (Perkin-Elmer Cetus) using the amplification conditions: one cycle of 5 min at 95°C, 30 cycles of 1 min at 95°C, 1 min at 5565°C, 2 min at 72°C and one cycle of 10 min at 72°C. For each PCR reaction, annealing temperature was close to the melting temperature of each primer set. Mouse C2ta mRNA was amplified using the sense and antisense primers described in Table 1. Amplification efficiency was assessed with non species-specific ß-actin primers (15 ng) (ß-Act-S: 5'-CACCCTGTGCTGCTCAC CGAGGCC-3'; ß-Act-AS: 5'-CCACACAGAGTACTTGCGC TCAG G-3') (17) added to each RT-PCR reaction.
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Table 1. Sequence and position of the different primers used for the amplification and sequencing of the CIITA cDNA
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Semiquantitative RT-PCR
Reverse transcription and PCR were performed as described above using the set of primers M3'-S (5'-CTGGATCGTCT CGTGCAG-3') and M3'-AS (5'-CCATGTCCGGAAGTACTT-3') except that amplification was for 20 cycles and that 1 nCi/ml [32P]dCTP was added per tube. The 424-bp amplification product (nucleotides 27283152 of the mouse CIITA cDNA) (33) was resolved on a 6% PAGE and autoradiographed.
RACE-PCR
RACE-PCR assays were performed with the 5'-RACE-PCR kit (Gibco/BRL) according to the manufacturers instructions. CIITA cDNAs were obtained by RT-PCR using CM12-AS primer (5'-CTGAGGCTGCTTGAAGGGAG-3', nucleotides 758738). TdT-tailing of cDNA was carried out for 1 h at 4°C by incubating 10 µl of the resulting cDNA, 200 µM dCTP, 5 µl 5 x tailing buffer and 1 µl of TdT. A specificity control reaction was performed in the absence of TdT. For PCR amplification, CM20-AS primer 5'-TGCTGAACTGGTCGCAGTTGATGG-3' (nucleotides 331308) and Abridged Anchor Primer (AAP; Gibco) were used following the procedures described above. Nested-PCR was performed using 1/10 dilution of the PCR amplification product and CM21AS primer 5'-AGGTA GCTGCCCTCTGGAGATCC-3' (nucleotides 197175) and Abridged Universal Amplification Primer (AUAP; Gibco).
Sequencing
Sequencing of each RT-PCR product was performed with a DNA sequencer 370A (Perkin-Elmer Cetus). Fluorescent deoxynucleotide labeling of DNA was performed by PCR with the ABI prism kit, following the manufacturers instruction (Perkin-Elmer Cetus). The primers used to sequence each RT-PCR product were the same than those used for the RT-PCR experiments.
Immunoprecipitation and Western blot of CIITA
Immunoprecipitation was made with a polyclonal rabbit anti-human CIITA directed against a peptide (726GEIKDKELPQYLALTR741) described in (34) and immunoaffinity purified through a CNBr-activated Sepharose 4B column (Amersham Pharmacia Biotech, Piscataway, NJ) coupled to the peptide. Western blots were performed with a mAb anti-human CIITA clone 7-1H from R & D Systems (Minneapolis, MN). Cells were incubated with mouse IFN-
(250 U/ml) and lysed either in RIPA buffer or in high salt buffer (10 mM HEPES, pH 7.9, 0.4 M NaCl, 1.5 mM MgCl2, 0.1 mM EGTA and 5% glycerol) containing 0.5% NP-40 (Sigma). Lysates were cleared for 2 h with Protein GSepharose (Amersham Pharmacia Biotech) and CIITA proteins were next immunoprecipitated overnight with 2.5 µg of affinity-purified polyclonal anti-CIITA antibodies per sample. Immune complexes were recovered by binding for 30 min to Protein GSepharose, resolved on a 6% SDSPAGE and absorbed to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech). Blocking was performed overnight in PBS/Tween buffer containing 2.5% low-fat milk. Incubation with 1.5 µg/ml of mAb anti-CIITA was performed for 2 h at room temperature in PBS/Tween buffer containing 0.25% low-fat milk. Immunoreactive bands were visualized with the ECL Plus Western blotting system (Amersham Pharmacia Biotech). To test for proteolytic activity in the RAG cell line, 2 mg of total cell extracts of RAW264.7 and RAG cells was mixed and incubated for 1 h at 37°C with or without proteases inhibitors from Sigma (0.4 mM PMSF, 3 µg/ml aprotinin, 1 µg/ml leupeptin and 0.5 µg/ml pepstatin A) followed by immunoprecipitation as described above. Moreover, the same type of experiments were performed using 10 µM of the proteasome inhibitor NLVS, kindly donated by Dr Hidde Ploegh (Boston, MA)
Northern blot analysis
Total RNA was isolated by the guanidinium thiocyanate procedure. Northern blot analysis was performed as previously described (35). The IFN regulatory factor (IRF)-1 and IRF-2 full-length cDNA probes were kindly provided by Hitoshi Ueda (Hyogo, Japan), and the guanylate binding protein (GBP)-1 cDNA probe was kindly provided by Mark Richter (Paris, France).
Luciferase constructs and assays
Plasmid constructs were made in the pGL3-basic vector from Promega (Madison, WI). The 445 bp pIV promoter fragment was obtained by PCR on mouse genomic DNA from L(tk) and RAW264.7 cells using the primer set P1-S (5'-GGTTGG GCTGAGATAGAGTG-3') and P1-AS (5'-CTGTGCCCCGT GCTTCTGAG-3') designed after the published sequence (18). Both PCR fragments were subcloned in the SmaI site of pGL3-basic and sequenced. The resulting constructs were called PIV-L(tk).1, PIV-L(tk).8 and PIV-RAW5.1. Assays were performed with the luciferase assay system from Promega as described previously (19). The pGL3-control vector contains the SV40 promoter driving the luciferase expression. Transfection efficiency was monitored using the pSVß-galactosidase vector from Promega.
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Results
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MHC class II-defective RAG and L(tk) cell lines display different genetic defects
The RAG and L(tk) cell lines do not express MHC class II genes after IFN-
treatment even though IFN-
induces MHC class I cell surface expression in both cell lines (Fig. 1). In order to determine if RAG and L(tk) display defects in the same gene, RAG x L(tk) hybrid cells were generated. These hybrids were analyzed by indirect immunofluorescence for surface MHC class II expression using two allele-specific mAb that allowed us to distinguish between MHC class II molecules from each cell lines [haplotypes d and k for RAG and L(tk) respectively]. RAG x L(tk) hybrids display cell surface expression of both I-Ad and I-Ak MHC class II molecules when treated with IFN-
demonstrating that I-A genes are functional in both cell lines (Fig. 1). Additionally, we conclude from these results that the RAG and L(tk) cell lines display mutations in different genes, and that both defects are recessive.

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Fig. 1. Flow cytometric analysis of the RAG, L(tk) and RAG x L(tk) cell lines for the cell surface expression of MHC molecules. Cells were either untreated or treated with 250 U/ml of IFN- for 72 h. H-2k and H-2d molecule expression was detected with mAb 11.4.1 and 20.8.4 respectively. The MKD6 and H39.64.5 mAb were used to identify the I-Ad and I-Ak molecules respectively.
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The RAG cell line
Expression of C2ta transcripts in the RAG cell line
We have previously described that MHC class II expression can be restored in RAG cells by transfection of the human CIITA cDNA. However, analysis of hybrid cells between RAG and CIITA-deficient cell lines indicated that C2ta was not mutated in the RAG cell line (26). These data therefore suggest that a factor required for CIITA expression might be missing or defective in these cells.
Defects in MHC2TA transcription associated with a general alteration on the IFN-
transduction pathway have been described for different tumor cells. Indeed, defects on the transcripts of GBP-1 and MHC2TA were found after IFN-
induction of the non-small cell lung cancer and small cell lung cancer tumor cell lines (23). We therefore investigated if the IFN-
transduction pathway was fully functional in the RAG cell line through the analysis of different IFN-responsive gene expression. The hybrid cell lines R.GES.2.1 and RR2.16, which express MHC class II molecules after an IFN-
incubation, were used as positive controls. These somatic cell hybrids were previously described and they were generated by the fusion of RAG cells with two different human cell lines. They contain human chromosome 16 or fragments of this chromosome (R.GES2.1 and RR2.16 respectively) and we have demonstrated that the recovery of mouse MHC class II expression was correlated with the presence of this chromosome (25). The expression of the IRF-1, IRF-2 and GBP-1 IFN-
-responsive genes was assayed through Northern blot analysis. Figure 2 demonstrates that all three genes are inducible in RAG cells with similar intensities when compared to the somatic cell hybrids. Therefore, the defect in the RAG cell line does not affect the IFN-
transduction pathway and is limited to the expression of MHC class II molecules.
We next examined the transcription of C2ta in RAG through a semiquantitative RT-PCR assay (20 cycles). As controls we used RNA from mouse CIITA+ (RAW264.7) and CIITA (J777.4) cell lines. As shown in Fig. 3, RAG cells display a significant amount of C2ta transcripts after induction with IFN-
. These levels of C2ta mRNA are only slightly weaker than those detected in RAW264.7, as expected from the monocytic origin of the latter. In addition, the lack of C2ta transcript in the J777.4 pre-monocytic cells demonstrates that the CIITA fragment amplified in RAG and RAW264.7 cells is specific.

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Fig. 3. Semiquantitative RT-PCR analysis of C2ta expression in the RAG cell line. Total RNA was isolated from cells either untreated or treated with 250 U/ml of IFN- for 6 h. The monocyte/macrophage RAW264.7 cell line was used as a positive control. The J777.4 pre-monocyte cell line was used as a negative control. [32P]dCTP was introduced in the reaction mix and a 20-cycle amplification was applied. Amplification products were run on a 6% acrylamide gel and autoradiographed. Mouse ß-actin primers were introduced in the reaction to control the amount of cDNA per lane.
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In order to assess if in RAG cells the C2ta messenger results from an appropriate transcription initiation site, 5'-RACE-PCR was performed. The RAW264.7 and the B lymphocyte A20 cell lines were used as positive controls for transcription initiations on promoters IV and III respectively (18,19). Nucleotide sequencing identified the amplification products obtained from three independent experiments. The amplification products obtained from the A20 cell line were found to correspond to transcripts initiated from promoter III at position 1. In IFN-
-treated RAW264.7 cells, transcription was initiated from promoter IV at positions 13, 14 or 15. In the RAG cell line, transcription was initiated from promoter IV at positions +16 or +32. Since initiation of C2ta transcription from promoter IV can take place within positions 54/+69 with two major initiation sites at positions +1 and +17 (18), we conclude that transcription of C2ta in RAG is initiated from promoter IV using adequate start sites for its expression.
Abnormal splicing events within C2ta transcripts in RAG might account for its inability to induce the expression of MHC class II molecules. We thus amplified through RT-PCR the CIITA cDNA from RAG cells using 13 sets of primers allowing the recovery of short and overlapping amplification products. For each set of primers we found that the size of the products was similar in RAG and in RAW264.7 cells, thereby suggesting the absence of major abnormalities in the splicing of C2ta (data not shown). In order to assess the absence of minor deletions that could take place during the splicing in the C2ta mRNA, all PCR amplification products from both cell lines were directly sequenced without further subcloning. This approach has the additional advantage to permit the detection of putative heterozygous mutation. Differences found in C2ta sequences of both the RAW264.7 and RAG cDNAs compared to the published sequences are summarized in Table 2.
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Table 2. Differences in nucleotide sequences obtained for the RAW264.7, RAG and L(tk) cell lines as compared with the published sequence (33)
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All together, our data converge towards the demonstration that the transcription of C2ta in the RAG cell line is comparable to that observed in a monocytic cell line in which the expression of MHC class II is normally induced by IFN-
. This suggested that the defect in MHC class II expression in RAG cells is not the result of a defect in C2ta transcript, leading us towards the hypothesis of a defect in the biosynthesis or stability of CIITA protein in this cell line.
Absence of the CIITA protein in the RAG cell line
The expression of CIITA was analyzed through immuno precipitation and detection by Western blot with a mAb directed against the protein. As shown in Fig. 4(A), multiple CIITA forms were detected in the control RAW264.7 cell line treated with IFN-
for 1221 h, as previously observed in human cell lines (Barbieri et al., submitted and our unpublished data). However, CIITA was not detected in cell extracts from RAG cells. Following the hypothesis that CIITA might be improperly folded or located in a specific compartment in the RAG cells that would hamper its solubilization in mild detergents, the immunoprecipitation was also performed using RIPA lysis buffer that includes SDS (Fig. 4B). Here again CIITA could not be detected in RAG cell extracts, even when increasing the IFN-
induction period up to 72 h. These data thereby demonstrate that even though C2ta transcripts are present, the CIITA protein is not detectable in the RAG cell line.

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Fig. 4. Expression of CIITA protein is not observed in RAG cells. Expression of the CIITA protein was studied by immunoprecipitation with a purified polyclonal rabbit anti-CIITA antibody and revealed by Western blot using a mAb directed against CIITA. RAG or RAW264.7 cells were untreated or treated with 250 U/ml of IFN- for different times. (A) Cells were lysed in a high salt buffer. (B) Cells were lysed in RIPA buffer containing SDS. Immunoprecipitations were performed using 660 µg of extracts. Data are representative of three independent experiments.
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A high abnormal proteolytic activity might account for the lack of CIITA protein in RAG cells. To test this point, we analyzed the stability of the CIITA protein expressed by the RAW264.7 cells in the presence of different amounts of cytosolic extracts from RAG cells prepared using mild detergent conditions known to preserve proteolytic activity (36). These experiments did not reveal any evidence of an enhanced degradation of the CIITA protein by RAG cell extracts (data not shown).
The L(tk) cell line
Complementation of MHC class II expression
We have already shown that the mouse fibrosarcoma cell line L(tk) does not express MHC class II molecules after IFN-
treatment (37). Moreover, transfection of MHC2TA genomic DNA in the L(tk) cells was shown to restore cell surface I-A expression (17), thereby suggesting that L(tk) cells are defective in MHC2TA. This led us to study further the defect in MHC class II expression in the L(tk) cell line.
We prepared several somatic cell hybrids between L(tk) cells and human B lymphoblastoid cell lines (GES and LM) or with the human amniotic cell line WISH. Out of the 13 hybrid clones and subclones analyzed here, eight were induced by IFN-
to express mouse MHC class II molecules (Fig. 5 and data not shown). Consequently, human genetic material is able to complement the expression of MHC class II in L(tk) cells.

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Fig. 5. Flow cytometric analysis of I-Ak cell surface expression in somatic cell hybrids (L.GES, L.W or L.LM) obtained by the fusion of the L(tk) cells with different human cell lines (GES, WISH or LM). Cells treated with 250 U/ml of IFN- for 72 h are represented as light histograms. Untreated cells appear as dark histograms.
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It has already been shown that in human x mouse hybrids, human chromosomes are segregated over the mouse genetic background (38). This phenomenon can be used to identify the human chromosomes which allow the restoration of a normal phenotype in mutated mouse cell lines (25,38). However, this methodology could not be applied here as the hybrids with the L(tk) background contained multiple fragmented human chromosomes, thereby hampering any significant analysis through karyotyping. Therefore, we analyzed by PCR the segregation of human MHC2TA in the whole panel of somatic cells hybrids described above. Two other genes, encoding protamine I (PRMI) (16p13.13) and adenine phosphoribosyl transferase (APRT) (16q24.2), located on human chromosome 16 were used as markers for this chromosome. Results are shown in Table 3. A positive correlation was evidenced between the presence of human MHC2TA and PRMI and expression of mouse MHC class II molecules. No association was found with other transcription factor encoding genes (RFX5 and RFXAP) described to be mutated in MHC class II-defective cells nor with the APRT gene. These results are in agreement with a mutation in the C2ta gene in the L(tk) cell line.
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Table 3. Correlation between the inducible expression of MHC class II molecules in a panel of L(tk) somatic cell hybrids and the presence of human genes implicated in MHC class II regulation
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Analysis of CIITA expression in the L(tk) cell line
The analysis of C2ta transcription in the L(tk) cell line revealed a high degree of complexity. The level of C2ta transcripts was much lower in the L(tk) cell line than that observed in the RAW264.7 or in the RAG cells (Fig. 6). Indeed, when examining the activity of C2ta promoter IV through luciferase reporter gene experiments, the L(tk) cells were 4 times less active than the RAW264.7 cells when IFN-
was used to induce transcription from this promoter (Fig. 7). In contrast, the activity of the SV40 promoter did not present any major difference between both cell lines (Fig. 7, pGL3-control vector).

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Fig. 6. RT-PCR analysis of C2ta mRNA expression in the L(tk) cell line. RAW264.7 and A20 cells were used as positive controls. Total RNA was isolated from cells either untreated or treated with 250 U/ml of IFN- for 6 h. Primers CM2S-2AS and CM5S-5AS allow the amplification of products 741761 and 14331417 nucleotides respectively.
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In addition, depending on the primer set used, the relative level of expression of C2ta transcripts in L(tk) compared to the RAW264.7 cells was highly variable (Fig. 6). This phenomenon was particularly visible in the 5' region of the CIITA cDNA in L(tk) where amplification of the cDNA was not observed with primer sets hybridizing on the first 950 bp of the C2ta transcript. RACE-PCR did not allow the precise determination of an intragenic transcription initiation site. A highly G-rich region and the weak amount of transcripts hampered this methodology. We additionally did not evidence alternative splicing events with transcripts initiated from either promoter III and IV. The sequencing of both alleles of promoter IV of C2ta did not reveal any mutation. Nucleotide sequencing of the CIITA cDNA from L(tk) cells (nucleotides 9524989) revealed different substitutions in the coding region compared to the published sequence which are summarized in Table 2. Moreover, a deletion of two out of 14 repetitive motifs (ccaaaccaaa) present in the non-coding cDNA sequence was found. Immunoprecipitation of the CIITA protein was next attempted using L(tk) cell extracts. As seen in Fig. 8, and in agreement with the above observations, the protein could not be evidenced in the L(tk) cell extracts whatever induction period with IFN-
(1648 h) was applied.

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Fig. 8. Expression of CIITA protein is not observed in the L(tk) cells. Expression of the CIITA protein was studied by immunoprecipitation with a purified polyclonal rabbit anti-CIITA antibody and revealed by Western blot using a mAb directed against CIITA. Cells were untreated or treated with 250 U/ml of IFN- for different times. Immunoprecipitations were performed using 660 µg of extracts. Data are representative of three independent experiments.
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Taken as a whole our data indicate that the L(tk) cell line presents altered C2ta transcripts and is devoid of the CIITA protein.
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Discussion
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Loss of MHC class II expression has been observed in numerous types of tumor cell lines: small cell lung cancers (39), non-small cell lung cancers, neuroblastoma (40), cervical carcinoma and retinoblastoma cell lines (23), pancreatic tumors (41), certain melanoma cell lines (24), choriocarcinoma (42) or multiple myeloma (43). In the latter two cases MHC class II expression has been shown to be repressed in association with the differentiation state of the cell lines (42,43). In contrast, the other tumor cells were expected to express MHC class II following IFN-
stimulation. Several of them (neuroblastoma, retinoblastoma or plasmacytoma cell lines, small cell lung cancers and pancreatic tumors) were shown to lack MHC2TA transcription (23,39,40,41). However the defects causing the lack of MHC2TA transcription have not been established, except for the pancreatic tumor cells and a corresponding cell line, where a mutation in the IRF-2 gene has been evidenced (41). Therefore, we have studied two mouse cell lines presenting a defect in MHC class II expression in order to define more precisely the types of mutations that can affect tumor cells.
Different from the above cell lines, the L(tk) fibrosarcoma cell line displays a weak amount of C2ta transcripts. Interestingly, defects hampering CIITA expression have been observed at different levels in this cell line. We had previously shown that the L(tk) cells exhibit a poor induction by IFN-
of certain IFN-
-responsive genes [genes 18 and (2'5')oligoadenylate synthetase] (37), although it is normally responsive for the stimulation of MHC class I molecules (17,37). However, the IFN-
response is unaltered (37), suggesting that this cell line displays a selective and partial defect in the IFN-
transduction pathway. In agreement with this phenotype, the expression of C2ta promoter IV driving a luciferase reporter gene was reduced 4-fold in the L(tk) cells when compared with the RAW264.7 cell line (Fig. 7). Defects in the IFN-
transduction pathway is not a rare phenomenon in tumor cell lines, although to our knowledge these defects usually totally inhibit the IFN-
response, as shown in cervical carcinoma cell lines (23), pancreatic tumors (41), melanoma (44) or renal cell carcinoma cell lines (45).
In addition to the poor C2ta transcript expression in the L(tk) cell line, we have shown that C2ta transcripts are truncated of
950 nucleotides in their 5' region. RACE-PCR techniques have not allowed the identification of the actual transcription initiation site of these transcripts. They might either originate from illegitimate transcription start site(s) or from an intragenic promoter as suggested in a previous report (19). Based on these data we have attempted to identify a truncated form of the CIITA protein which was not detected (data not shown).
Our data show that transcription from promoter IV of C2ta does not occur in a detectable fashion in the L(tk) cell line. Data from this report and (17) suggest that the defect of the L(tk) cell line resides in the C2ta gene, thus we examine the existence of mutations in the L(tk) C2ta promoter IV. We show here that a 445-bp promoter fragment from L(tk) drives normal expression of the luciferase reporter gene in the RAW264.7 cell line (Fig. 7) and displays a normal nucleotide sequence (data not shown). The lack of expression from promoter IV might then be explained by mutations outside the minimal promoter. However, it is possible that the C2ta endogenous promoter is not functional due to an epigenetic phenomena. Indeed, in a human choriocarcinoma cell line, methylation of MHC2TA promoter IV has been shown to inhibit MHC2TA transcription and thereby the expression of HLA-D genes (42). Alternatively, binding of histone deacetylases to MHC2TA promoter IV might have similar effects on CIITA expression, as exemplified in a plasmacytoma cell line (40). Blockade of C2ta promoter IV might then favor a residual transcript initiation from an intragenic position or illegitimate transcription initiation(s). In summary, in addition to different mutations (or polymorphisms) in the CIITA cDNA sequence, alterations have been observed in the L(tk) cell line with a lack of transcription from the promoter IV and partial modifications in the response to IFN-
.
In contrast, RAG cells have retained the transcription of the C2ta gene after treatment with IFN-
, even though they lack MHC class II expression. Interestingly, this unusual phenotype has been described in another mouse adenocarcinoma cell line (40). Our data assess that the RAG cell line is not defective for the C2ta gene and that the IFN-
transduction pathway is functional. In addition, the cDNA CIITA nucleotide sequencing has confirmed the absence of mutation in the coding region and the lack of splicing defects. RACE-PCR experiments have demonstrated that transcription initiation occurs within sites that have been described previously in normal cell lines. All together our data show that the C2ta transcript of the RAG cell line is expressed and that it is devoid of defects. However, we have not shown that the factors required, downstream of CIITA, for MHC class II gene expression were functional in this cell line (35).
We have shown in this report that the CIITA protein is not detected in the RAG tumor cell line, in agreement with the lack of MHC class II transcription and cell surface expression. Different hypotheses are compatible with our data. One possibility is that the C2ta transcript is not translated either through a blockade of its nuclear export or through the lack of a factor required for the translation of CIITA. It is also possible that the CIITA protein is expressed; however, in such a labile manner that we did not evidence this protein. Data obtained on cells infected with Chlamydia showed that this organism expresses a protease degrading RFX5 and up-stream stimulatory factor (USF)-1 (36,46), and thereby inhibits MHC class II expression in the infected cells. As the RAG cell line could be infected by an oncogenic virus presenting similar capacities, a highly active viral protease might degrade the CIITA protein. This phenomenon was not evidenced in this report, in agreement with the recessivity of the RAG mutation that does not fit with this hypothesis. We also hypothesized that CIITA might be rapidly degraded by an over-active immune proteasome in the RAG cell line. However, treatment of these cells with a proteasome inhibitor did not alter MHC class II expression, although it reduced MHC class I expression (data not shown). This suggests that C2ta and MHC class II gene-specific transcription factors are not submitted to proteasome degradation. One additional possibility is that a specific chaperone, allowing the proper intracellular trafficking or editing of the CIITA protein, is inactive or missing in the RAG cells. Interestingly, we have shown that the transfection of a human CIITA cDNA allows for the immunoprecipitation of the human CIITA protein (data not shown) and restores MHC class II expression. This might indicate that the over-expression of a recombinant form of CIITA bypasses these putative defects or that the missing or mutated factor is not required for the human form of the CIITA protein or transcript. Even though we have shown that both the C2ta gene and transcript are normal in the RAG cell line, numerous hypotheses remain concerning the defect on these cells, as this defect can affect various stages of CIITA protein expression as proposed above. Further study of this cell line will therefore help in the understanding of CIITA synthesis and MHC class II expression.
The importance of MHC class II molecule expression in tumor cells is quite controversial (21,47,48). The loss of MHC class II molecules is not essential with regard to immune escape. Recent data suggest that the lack of CIITA itself might be the crucial step. Indeed CIITA, through the sequestration of NFAT (49,50), has been shown to modulate the expression of genes like Fas ligand (49) or IL-4 (50). In addition, CIITA can interact with cyclins (51) or the transactivator CBP (11). One can therefore hypothesize that CIITA, through an indirect effect, might alter tumor growth, tumor progression or immune escape. The loss of CIITA expression might therefore confer a selective advantage to tumors from certain cell types. This thereby reinforces the necessity of the identification of the defects altering CIITA expression in tumor cell lines and the precise targets of CIITA effects.
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Acknowledgements
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This research was supported by the Association pour la Recherche sur le Cancer, INSERM, Fondecyt (8000011) and Ecos/Conicyt (C99S02). R. N. was supported by a grant from Fondecyt (4980004 and 2950018). G. B. was supported by a grant from INSERM and Foundation of France. A. M. L. was supported by a fellowship from the Ligue Nationale contre le Cancer. V. D. was supported by a grant from the Association pour la Recherche sur le Cancer.
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Abbreviations
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APCantigen-presenting cell
CBPCREB binding protein
CIITAclass II transactivator
CREBcAMP-responsive element binding protein
GBPguanylate binding protein
IRFIFN regulatory factor
NFYnuclear factor Y
RFXregulatory factor X
USFup-stream stimulatory factor
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References
|
---|
- Germain, R. N. 1994. MHC-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell 76:287.[ISI][Medline]
- Glimcher, L. H. and Kara, C. J. 1992. Sequences and factors: a guide to MHC class II transcription. Annu. Rev. Immunol. 10:13.[ISI][Medline]
- Reith, W., Muhlethaler-Mottet, A., Masternak, K., Villard, J. and Mach B. 1999. The molecular basis of MHC class II deficiency and transcriptional control of MHC class II gene expression. Microb. Infect. 1:839.[ISI][Medline]
- Griscelli, C., Lisowska-Grospierre, B. and Mach B. 1989. Combined immunodeficiency with defective expression in MHC class II genes. Immunodefic. Rev. 1:135.[Medline]
- Bénichou, B. and Strominger, J. L. 1991. Class II-antigen-negative patient and mutant B-cell lines represent at least three, and probably four, distinct genetic defects defined by complementation analysis. Proc. Natl Acad. Sci. USA 88:4285.[Abstract]
- Masternak, K., Barras, E., Zufferey, M., Conrad, B., Corthals, G., Aebersold, R., Sanchez, J. C., Hochstrasser, D. F., Mach, B. and Reith, W. 1998. A gene encoding a novel RFX-associated transactivator is mutated in the majority of MHC class II deficiency patients. Nat. Genet. 20:273.[ISI][Medline]
- Steimle, V., Durand, B., Barras, E., Zufferey, M., Hadam, M. R., Mach, B. and Reith, W. 1995. A novel DNA-binding regulatory factor is mutated in primary MHC class II deficiency (bare lymphocyte syndrome). Genes Dev. 9:1021.[Abstract]
- Durand, B., Sperisen, P., Emery, P., Barras, E., Zufferey, M., Mach, B. and Reith, W. 1997. RFXAP, a novel subunit of the RFX DNA binding complex is mutated in MHC class II deficiency. EMBO J. 16:1045.[Abstract/Free Full Text]
- Steimle, V., Otten, L. A., Zufferey, M. and Mach, B. 1993. Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or bare lymphocyte syndrome). Cell 75:135.[ISI][Medline]
- Zhu, X. S., Linhoff, M. W., Li, G., Chin, K. C., Maity, S. N. and Ting, J. P. 2000. Transcriptional scaffold: CIITA interacts with NF-Y, RFX, and CREB To cause stereospecific regulation of the class II major histocompatibility complex promoter [In process citation]. Mol. Cell. Biol. 20:6051.[Abstract/Free Full Text]
- Kretsovali, A., Agalioti, T., Spilianakis, C., Tzortzakaki, E., Merika, M. and Papamatheakis, J. 1998. Involvement of CREB binding protein in expression of major histocompatibility complex class II genes via interaction with the class II transactivator. Mol. Cell. Biol. 18:6777.[Abstract/Free Full Text]
- Fontes, J. D., Jiang, B. and Peterlin, B. M. 1997. The class II trans-activator CIITA interacts with the TBP-associated factor TAFII32. Nucleic Acids Res. 25:2522.[Abstract/Free Full Text]
- Mahanta, S. K., Scholl, T., Yang, F. C. and Strominger, J. L. 1997. Transactivation by CIITA, the type II bare lymphocyte syndrome-associated factor, requires participation of multiple regions of the TATA box binding protein. Proc. Natl Acad. Sci. USA 94:6324.[Abstract/Free Full Text]
- Harton, J. A. and Ting, J. P. 2000. Class II transactivator: mastering the art of major histocompatibility complex expression [In process citation]. Mol. Cell. Biol. 20:6185.[Free Full Text]
- Chin, K. C., Mao, C., Skinner, C., Riley, J. L., Wright, K. L., Moreno, C. S., Stark, G. R., Boss, J. M. and Ting, J. P. 1994. Molecular analysis of G1B and G3A IFN gamma mutants reveals that defects in CIITA or RFX result in defective class II MHC and Ii gene induction. Immunity 1:687,[ISI][Medline]
- Steimle, V., Siegrist, C. A., Mottet, A., Lisowska-Grospierre, B. and Mach, B. 1994. Regulation of MHC class II expression by interferon-gamma mediated by the transactivator gene CIITA. Science 265:106.[ISI][Medline]
- Chang, C. H., Fontes, J. D., Peterlin, M. and Flavell, R. A. 1994. Class II transactivator (CIITA) is sufficient for the inducible expression of major histocompatibility complex class II genes. J. Exp. Med. 180:1367.[Abstract]
- Muhlethaler-Mottet, A., Otten, L. A., Steimle, V. and Mach, B. 1997. Expression of MHC class II molecules in different cellular and functional compartments is controlled by differential usage of multiple promoters of the transactivator CIITA. EMBO J. 16:2851.[Abstract/Free Full Text]
- Lennon, A. M., Ottone, C., Rigaud, G., Deaven, L. L., Longmire, J., Fellous, M., Bono, R. and Alcaide-Loridan, C. 1997. Isolation of a B-cell-specific promoter for the human class II transactivator. Immunogenetics 45:266.[ISI][Medline]
- Garrido, F., Cabrera, T., Concha, A., Glew, S., Ruiz-Cabello, F. and Stern, P. L. 1993. Natural history of HLA expression during tumour development. Immunol. Today 14:491.[ISI][Medline]
- Ostrand-Rosenberg, S., Thakur, A. and Clements, V. 1990. Rejection of mouse sarcoma cells after transfection of MHC class II genes. J. Immunol. 144:4068.[Abstract/Free Full Text]
- Brocker, E. B., Suter, L. and Sorg, C. 1984. HLA-DR antigen expression in primary melanomas of the skin. J. Invest. Dermatol. 82:244.[Abstract]
- Lu, Y., Tschickardt, M. E., Schmidt, B. J. and Blanck, G. 1997. IFN-gamma inducibility of class II transactivator is specifically lacking in human tumour lines: relevance to retinoblastoma protein rescue of IFN-gamma inducibility of the HLA class II genes. Immunol. Cell Biol. 75:325.
- Deffrennes, V., Vedrenne, J., Stolzenberg, M. C., Piskurich, J., Barbieri, G., Ting, J. P., Charron, D. and Alcaide-Loridan, C. 2001. Constitutive expression of MHC class II genes in melanoma cell lines results from the transcription of class II transactivator abnormally initiated from its B cell-specific promoter. J. Immunol. 167: 98.
- Bono, M. R., Alcaide-Loridan, C., Couillin, P., Letouze, B., Grisard, M. C., Jouin, H. and Fellous, M. 1991. Human chromosome 16 encodes a factor involved in induction of class II major histocompatibility antigens by interferon gamma. Proc. Natl Acad. Sci. USA 88:6077.[Abstract]
- Bono, M. R., Benech, P., Couillin, P., Alcaide-Loridan, C., Grisard, M. C., Jouin, H., Fischer, D. G. and Fellous, M. 1989. Characterization of human IFN-gamma response using somatic cell hybrids of hematopoietic and nonhematopoietic origin. Somat. Cell Mol. Genet. 15:513.[ISI][Medline]
- Oi, V., Jones, P. P., Goding, J. W. and Herzenberg, L. A. 1978. Properties of monoclonal antibodies to mouse Ig allotype, H2 and Ia antigens. Curr. Topics Microbiol. Immunol. 81:115.[Medline]
- Kappler, J. W., Skidmore, B., White, J. and Marrack, P. 1981. Antigen-inducible, H-2 restricted, interleukin-2-producing T cell hybridomas. Lack of independent antigen and H-2 recognition. J. Exp. Med. 153:1198.[Abstract/Free Full Text]
- Couillin, P., Mollicone, R., Grisard, M. C., Gibaud, A., Ravise, N., Feingold, J. and Oriol, R. 1991. Chromosome 11q localization of one of the three expected genes for the human alpha-3-fucosyltransferases, by somatic hybridization. Cytogenet. Cell Genet. 56:108.[ISI][Medline]
- Southern, P. J. and Berg, P. 1982. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. Appl. Genet. 1:327.[Medline]
- Chen, C. and Okayama, H. 1987. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7:2745.[ISI][Medline]
- Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. and Rutter, W. J. 1979. Isolation of biologically-active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294.[ISI][Medline]
- Sims, T. N., Goes, N. B., Ramassar, V., Urmson, J. and Halloran, P. F. 1997. In vivo class II transactivator expression in mice is induced by a non-interferon-gamma mechanism in response to local injury. Transplantation 64:1657.[ISI][Medline]
- Zhou, H., Su, H. S., Zhang, X., Douhan, J., III and Glimcher, L. H. 1997. CIITA-dependent and -independent class II MHC expression revealed by a dominant negative mutant. J. Immunol. 158:4741.[Abstract]
- Lennon, A., Ottone, C., Peijnenburg, A., Hamon-Benais, C., Colland, F., Gobin, S., van den Elsen, P., Fellous, M., Bono, R. and Alcaide-Loridan, C. 1996. The RAG cell line defines a new complementation group of MHC class II deficiency. Immunogenetics 43:352.[ISI][Medline]
- Zhong, G., Fan, T. and Liu, L. 1999. Chlamydia inhibits interferon gamma-inducible major histocompatibility complex class II expression by degradation of upstream stimulatory factor 1. J. Exp. Med. 189:1931.[Abstract/Free Full Text]
- Bono, M. R., Alcaide-Loridan, C., Letouze, B., Jouin, H., Gobin, S. J. and Fellous, M. 1991. The recombinant human interferon-gamma receptor is fully functional in a human x murine hybrid containing human chromosome 21. Res. Immunol. 142:765.[ISI][Medline]
- Gourdeau, H. and Fournier, R. E. K. 1990. Genetic analysis of mammalian cell differentiation. Annu. Rev. Cell Biol. 6:69.[ISI]
- Yazawa, T., Kamma, H., Fujiwara, M., Matsui, M., Horiguchi, H., Satoh, H., Fujimoto, M., Yokoyama, K. and Ogata, T. 1999. Lack of class II transactivator causes severe deficiency of HLA-DR expression in small cell lung cancer. J. Pathol. 187:191.[ISI][Medline]
- Magner, W. J., Kazim, A. L., Stewart, C., Romano, M. A., Catalano, G., Grande, C., Keiser, N., Santaniello, F. and Tomasi, T. B. 2000. Activation of MHC class I, II, and CD40 gene expression by histone deacetylase inhibitors. J. Immunol. 165:7017.[Abstract/Free Full Text]
- Xi, H. and Blanck, G. 2000. Interferon regulatory factor-2 point mutations in human pancreatic tumors. Int. J. Cancer 87:803.[ISI][Medline]
- Morris, A. C., Spangler, W. E. and Boss, J. M. 2000. Methylation of class II trans-activator promoter IV: a novel mechanism of MHC class II gene control. J. Immunol. 164:4143.[Abstract/Free Full Text]
- Ghosh, N., Gyory, I., Wright, G., Wood, J. and Wright, K. L. 2001. Positive regulatory domain I binding factor 1 silences class ii transactivator expression in multiple myeloma cells. J. Biol. Chem. 276:15264.[Abstract/Free Full Text]
- Wong, L. H., Krauer, K. G., Hatzinisiriou, I., Estcourt, M. J., Hersey, P., Tam, N. D., Edmondson, S., Devenish, R. J. and Ralph, S. J. 1997. Interferon-resistant human melanoma cells are deficient in ISGF3 components, STAT1, STAT2, and p48-ISGF3gamma. J. Biol. Chem. 272:28779.[Abstract/Free Full Text]
- Dovhey, S. E., Ghosh, N. S. and Wright, K. L. 2000. Loss of interferon-gamma inducibility of TAP1 and LMP2 in a renal cell carcinoma cell line. Cancer Res. 60:5789.[Abstract/Free Full Text]
- Zhong, G., Liu, L., Fan, T., Fan, P. and Ji, H. 2000. Degradation of transcription factor RFX5 during the inhibition of both constitutive and interferon-
-inducible major histocompatibility complex class I expression in Chlamydia-infected cells. J. Exp. Med. 191:1525.[Abstract/Free Full Text]
- Armstrong, T. D., Clements, V. K., Martin, B. K., Ting, J. P. Y. and Ostrand-Rosenberg, S. 1997. Major histocompatibility complex class II-transfected tumor cells present endogenous antigen and are potent inducers of tumor-specific immunity. Proc. Natl Acad. Sci. USA 94:6886.[Abstract/Free Full Text]
- Toes, R. E. M., Ossendorp, F., Offringa, R. and Melief, C. J. M. 1999. CD4 T cells and their role in antitumor immune response. J. Exp. Med. 189:753.[Free Full Text]
- Gourley, T. S. and Chang, C. H. 2001. The class II transactivator prevents activation-induced cell death by inhibiting Fas ligand gene expression. J. Immunol. 166:2917.[Abstract/Free Full Text]
- Sisk, T. J., Gourley, T., Roys, S. and Chang, C. H. 2000. MHC class II transactivator inhibits IL-4 gene transcription by competing with NF-AT to bind the coactivator CREB binding protein (CBP)/p300. J. Immunol. 165:2511.[Abstract/Free Full Text]
- Kanazawa, S. and Peterlin, B. M. 2001. Combinations of dominant-negative class II transactivator, p300 or CDK9 proteins block the expression of MHC II genes. Int. Immunol. 13:951.[Abstract/Free Full Text]
- Sims, T. N., Elliott, J. F., Ramassar, V., Denney, D. W., Jr and Halloran, P. F. 1997. Mouse class II transactivator: cDNA sequence and amino acid comparison with the human class II transactivator. Immunogenetics 45:220.[ISI][Medline]