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The Journal of Immunology, 2001, 166: 5681-5687.
Copyright © 2001 by The American Association of Immunologists

Uncoordinated HLA-D Gene Expression in a RFXANK-Defective Patient with MHC Class II Deficiency1

Ana-Maria Lennon-Duménil2,3,*, Mohamed-Ridha Barbouche2,{dagger}, Jocelyn Vedrenne2,{ddagger}, Thomas Prod’Homme{ddagger}, Mohamed Béjaoui§, Salma Ghariani{dagger}, Dominique Charron{ddagger}, Marc Fellous*, Koussay Dellagi{dagger} and Catherine Alcaïde-Loridan4,{ddagger}

* Institut National de la Santé et de la Recherche Médicale Unité 276, Institut Pasteur, Paris, France; {dagger} Laboratoire d’Immunologie, World Health Organization Collaborative Center for Research and Training in Immunology, Institut Pasteur de Tunis, Tunis, Tunisie, France; {ddagger} Institut National de la Santé et de la Recherche Médicale U396, Centre de Recherches Biomédicales des Cordeliers, Paris, France; and § Centre de Greffe de Moelle Osseuse, Tunis, Tunisie, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We describe the analysis of a patient, JER, presenting classical immunological features of MHC class II deficiency. Unexpectedly, some HLA transcripts (HLA-DRA, HLA-DQA, and HLA-DMA) were found to be expressed in the JER cell line at nearly wild-type levels, while HLA-DPA and the HLA-D {beta}-chain transcripts were not detected. Gene reporter experiments confirmed the differential transcriptional activities driven by the HLA-D promoters in the JER cells. A defect in RFXANK was first suggested by genetic complementation analyses, then assessed with the demonstration of a homozygous mutation affecting a splice donor site downstream exon 4 of RFXANK. Because the severe deletion of the resulting protein cannot account for the expression of certain HLA-D genes, minor alternative transcripts of the RFXANK gene were analyzed. We thereby showed the existence of a transcript lacking exon 4, encoding a 28-aa-deleted protein that retains a transcriptional activity. Altogether, we characterize a new type of mutation in the RFXANK gene in a MHC class II-defective patient leading to an uncoordinated expression of the HLA-D genes, and propose that this phenotype is ensured by severely limited amounts of an active, although truncated RFXANK protein.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The MHC class II molecules (HLA-DR, HLA-DP, and HLA-DQ) present antigenic peptides to CD4+ T lymphocytes, and represent key elements in the onset of the cellular and humoral immune response (1). The crucial role of these molecules is clearly illustrated by a severe immunodeficiency that manifests upon their defective expression (2). MHC class II-deficient patients suffer from recurrent bacterial and viral infections appearing at an early age, and generally present reduced levels of serum Igs and CD4+ T lymphocytes, in addition to the lack of in vitro T lymphocyte proliferation in response to Ags (3). This pathology is caused by autosomal recessive genetic defects, unlinked to the HLA genes themselves, but rather affecting proteins regulating their transcription. Fusion experiments with cells from patients suffering from this immunodeficiency have evidenced four complementation groups (A, B, C, and D), demonstrating the genetic heterogeneity of the disease (4, 5).

The HLA-DR, HLA-DQ, and HLA-DP genes are coordinately expressed in B lymphocytes, and this coordinated regulation is ensured at the transcriptional level by conserved promoter motifs, the W, X1, X2, and Y boxes. Similar motives were found as well in the promoters of Ii (invariant chain) and HLA-DM, whose products are also involved in Ag presentation (6). The Y box of MHC class II promoters binds NFY5 (nuclear factor binding to Y box), a ubiquitous transcription factor that includes three subunits, NFY-A, NFY-B, and NFY-C (7, 8, 9). The factor binding the X2 box, X2BP, was recently characterized as the homodimeric cAMP response element-binding protein (CREB) (10). MHC class II deficiency group B, C, and D cells are defective in the binding of the RFX (regulatory factor binding to X box) complex to the X1 box (11, 12). The RFX complex is a heterotrimer, composed of RFXANK (13, 14), RFX5 (15), and RFXAP (RFX-associated protein) (16), respectively mutated in groups B, C, and D (13, 14, 17, 18, 19). Several publications demonstrate that the RFX, NFY, and X2BP complexes bind cooperatively to MHC class II promoters, and that their interaction increases the stability of the complexes to the promoters (20, 21). More recently, RFX5 was shown to create a bridge in between NFY, RFXAP, and RFXANK (22). Functional complementation of group A mutants has led to the identification of the CIITA gene (class II transactivator) (23). It encodes a non-DNA-binding protein, in agreement with data demonstrating that MHC class II promoters are normally occupied in group A mutants (11, 24). The transactivating capacity of CIITA was established through experiments showing its interaction with RFX5, RFXANK, CREB, and subunits of the NFY complex (25). Most importantly, CIITA is the key molecule controlling the tissue-specific expression pattern of the MHC class II genes (26).

RFXANK, also named RFX-B, is coded by a 10-kbp gene composed of 10 exons (13, 14). This 33-kDa protein is characterized by three ankyrin domains located in its carboxyl terminus. The ankyrin motifs have been described as protein/protein interaction domains. In MHC class II-defective group B patients, two defects (del26 and del58) were first described, resulting from deletions overlapping exon 6 affecting either the intron upstream (del26) or downstream exon 6 (del58) (13, 14). More recently, a founder effect has been described for the del26 mutation in 17 patients of North African origin (27). Two additional patients with mutations in exon 5 or 8 have been evidenced (19).

We describe in this work the analysis of the JER cell line that was established from a patient affected by a MHC class II deficiency with classical immunological characteristics. Hereby, we characterize a new type of genetic defect in RFXANK, with an unexpected uncoordinated expression of MHC class II genes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Flow cytometric analyses

Immunofluorescence assays were done following classical procedures with a FACScan (Becton Dickinson, Franklin Lakes, NJ) using a CellQuest program. The phenotype of the JER PBLs, purified through Ficoll-Hypaque, was performed with CD3 (clone UCHT1), CD4 (clone 13B8.2), CD8 (clone B9.11), PanB (clone DBB42), and HLA class II (clone 9-49) mAbs from Immunotech (Luminy, France). Before labeling of B lymphoblastoid cell lines (B-LCLs), FcRs were saturated by preincubation with heat-inactivated FCS. Indirect immunofluorescence was performed on the cell lines with the following primary mAbs: L112 (anti-DR) (28), D112 (anti-DR) (29), B7/21 (anti-DP) (30), L2 (anti-DQ) (31), and MHC class I (W6/32; Serotec, Oxford, U.K.). Direct immunofluorescence was done with the anti-DR L243 mAb FITC from Becton Dickinson.

Immunological analysis of the patient JER

Serum Ig concentration was measured by radial immunodiffusion. The expression of HLA class II Ags was studied on patient and control PHA-stimulated blasts with the 9-49 mAb. The immune response was evaluated by proliferation assays, with PBLs cultured in 96-well plates (106 cells/ml) in RPMI 1640 supplemented with 10% human AB serum. PHA (10 µg/ml) or specific Ags (tuberculin (purified protein derivative, PPD, 10 U/ml) or tetanus toxoid (TT, 4 µg/ml) were added, and the proliferation was assessed 3 days (PHA) or 5 days (PPD and TT) later by the incorporation of [3H]thymidine. HLA-DR typing was performed on genomic DNA by PCR-sequence-specific oligonucleotide procedure (JER, DR13/DR7; JER’s father (TAO), DR3/DR7; JER’s mother (HAS), DR13/-).

Cell culture

The analyzed cell lines are B-LCLs grown in RPMI 1640 supplemented with antibiotics, 2 mM glutamine, and 10% of heat-inactivated FCS (for MHC class II-positive B-LCLs) or Myoclone (Life Technologies, Cergy-Pontoise, France) (for B-LCLs from MHC class II-defective patients). The JER, HAS, and TAO EBV-transformed B-LCLs were established using standard protocols. The BLS-2 and BCH cell lines belong to complementation group A; the THF B-LCL to group C; and the ZAR cell line, a kind gift from B. Grospierre-Lisowska, belongs to group D. The HAD B-LCL belongs to group B (32), as well as the KHE cell line (unpublished data), and the BLS-1 cell line (33). The HAD and BLS-1 are respectively del26 and del58 RFXANK mutants. The unrelated COM and GES B-LCLs were used as positive controls for MHC class II expression.

Northern blot analysis

Total RNA was isolated by the guanidinium thiocyanate procedure, as described previously (34). RNA samples (15 µg/lane) were loaded on agarose gels containing 2.2 M formaldehyde. The gels were transferred on Nylon N membranes (Amersham, Arlington Heights, IL), and the filters were hybridized overnight with HLA-DRA, HLA-DQA, HLA-DPA, HLA-DRB, HLA-DQB, or HLA-DPB isotype chain-specific cDNA probes (35) labeled by random priming using the Multiprime Labeling Kit from Amersham. The mouse Ii, which cross-hybridizes with its human homologue, and {beta}-actin probes were previously described (34).

Western blot analysis

Cells were lysed in a solution containing 0.5% Triton X-100, 300 mM NaCl, 50 mM Tris-HCl (pH 7.5), and a cocktail of protease inhibitors. Proteins (10 µg) were loaded per lane on a 12% polyacrylamide gel, then transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). Western blotting was performed with 1/5000 dilution of the anti-HLA-DR DA6.147 mAb (36) and 1/4000 dilution of peroxidase-labeled anti-mouse Ig Ab from Pierce (Rockford, IL), then revealed with the ECL detection kit (Amersham).

Luciferase constructs and assays

The pGL3 control, containing an SV40 promoter upstream the luciferase gene, and the promoterless pGL3-basic plasmid constructs were purchased from Promega (Madison, WI). The pGL3-DRA, pGL3-DQA, pGL3-DQB, and pGL3-DPA plasmids contain 250-bp promoter fragments including the conserved W, X1, X2, and Y boxes (37). Transient transfections were performed by electroporation with a mixture of the luciferase constructs and pSV{beta}gal vector (Promega) (10:1 molar ratio) using a Gene Pulser apparatus (Bio-Rad, Richmond, CA) with a 960 µF/300 V/200 {Omega} pulse, under conditions previously described (38). Luciferase activities were measured with a Lumat luminometer (Berthold Instruments, Nashua, NH), and were further divided by the {beta}-galactosidase activities (expressed as A420) to correct for transfection efficiency. All the values correspond to an average of three to four independent experiments.

Cell fusions

Two days before the fusion, all cell lines were depleted of L112-positive cells (caused either by reversion of the phenotype or nonspecific binding of the Ab) through cell sorting using anti-mouse Ig-coupled magnetic beads (Dynal, Great Neck, NY), as described previously (34). Cell fusions were performed with 107 cells of each fusion partner using PEG 4000 (Merck, West Point, PA). As a control, homocaryons were prepared with each fusion partner to assess the complete lack of MHC class II-positive cells. After fusion, cells were cultured in RPMI 1640 supplemented with 10% Myoclone serum for 48–72 h. Indirect immunofluorescence analysis was then performed on cells incubated with either anti-HLA-DR L112 or D112 or with the anti-HLA-DQ L2 mAbs. Cells were analyzed under a fluorescence microscope without any step of permeabilization of the cells.

RT-PCR analysis

cDNA preparation and PCR were performed with the reverse transcriptase and Taq polymerase from Qiagen (Chatsworth, CA) following the instructions of the manufacturer. Amplification was conducted as follows: 95°C, 5 min, one cycle; 95°C, 1 min; 55°C, 1 min; 72°C, 2 min, 30 cycles; 72°C, 10 min, one cycle. Amplification of a 811-bp cDNA fragment of RFXANK, corresponding to nt 416-1227 (exons 3–10) of the cDNA, was performed with primers S-5'p33 (5'-CCATGGAGCTTACCCAGCCTGCAGA-3') and AS-3'p33 (5'-AGTGTCTGAGTCCCCGGCA-3'), kindly providedby K. Masternak (University of Geneva Medical School, Geneva,Switzerland). cDNA amounts were verified through 24-cycle PCR with GAPDH-specific primers that amplify a 255-bp fragment (GAPDH sense, 5'-GTCGTATTGGGCGCCTGGTCAC-3'; GAPDH antisense, 5'-CACGACGTACTCAGCGCCAGCA-3'), used in a 1/15 concentration compared with the RFXANK primers. The analysis of the different RFXANK products in the JER cell line was performed with sets of primers (whose sequences are available upon request) hybridizing on exons 3, 4, 5, 6, 7, and 8, or on intron 4. HLA-DMA mRNA was amplified with primers DMA sense (5'-CTAAAGCTGGGTTGGTAGC-3') and DMA antisense (5'-GCTGGCATCAAACTCTGGT-3'). DMB was amplified with primers DMB sense (5'-ATCTTTACAGAGCAGAGCAT-3') and DMB antisense (5'-CCTTCTCACTTGGAGTGGA-3'). To control the amplification efficiency, 15 ng of {beta}-actin primers ({beta}-Act sense, 5'-CACCCTGTGCTGCTCACCGAGGCC-3'; {beta}-Act-antisense, 5'-CCACACAGAGTACTTGCGCTCAGG-3') was introduced in each of the HLA-DM amplifications.

DNA sequencing of the genomic RFXANK products

A DNA fragment encompassing exons 4–6 of the RFXANK gene was amplified from the patient and parent genomic DNA using the sense ANK-Si3.4 primer that hybridizes to intron 3 (5'-GAACCCAGGAGGCAGAGGTTG-3') and the antisense primer ANK-ASe6 that recognizes exon 6 (5'-AGCAGGAAGCGAACGGTC-3'). The PCR products (831 bp) were subcloned into the pCR2.1-TOPO plasmid (Invitrogen, San Diego, CA), and nucleotide sequencing was performed with M13 and M13 reverse primers by the Big Dye Terminator cycle sequencing (PE Applied Biosystems, Foster City, CA) using an ABI 377 automatic sequencer (Perkin-Elmer, Norwalk, CT).

RFXANK constructs and transfections

The RFXANK wild-type and ANK{Delta}4 cDNAs were obtained through RT-PCR amplification of mRNA preparation from the control COM and JER B-LCLs, using the S-5'p33 and AS-3'p33 sets of primers described above. These PCR products were cloned in the pCR2.1-TOPO plasmid, and further subcloned into the EcoRI site of the pIRES-Neo expression vector (Clontech Laboratories, Palo Alto, CA). Sequencing was performed on both inserts to assess the lack of mutations created during the process. A total of 20 µg of these constructs was next cotransfected with 5 µg of the red fluorescent DsRed1 construct (Clontech) through electroporation with the ECM 630 electroporator from BTX (975 µF, 200 {Omega}, 270 V). Cell surface HLA-DR expression was determined 2 days later by flow cytometry on Red-positive transfected cells stained with fluorescein-conjugated L243 mAb from Becton Dickinson.

Southern blot

Oligonucleotides were 3'-end labeled with the digoxigenin oligonucleotide 3'-end labeling kit from Roche following the instructions of the manufacturer. The gels containing the RT-PCR products were transferred on Nylon N membranes (Amersham), and the filters were hybridized overnight with the labeled probes. The membranes were further revealed with ready-to-use CSPD from Roche (Meyan, France). The sequences of the exons 4- and 5-specific oligonucleotides were respectively: 5'-CCGGTTGGTGAGAGTGGTGGAGT-3' and 5'-CCATCCACCAGCTCGCAGCAC-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Clinical features of the MHC class II-deficient patient JER

The clinical features of the patient JER included a severe chronic diarrhea, recurrent pulmonary infections, and mucocutaneous candidiasis detected at the age of 5 mo. He died at the age of 7.5 mo following an overwhelming infection. Consanguinity was observed in the family with a second degree marriage. The familial survey indicates the death at an early age of a first degree maternal female cousin. Consistent with a diagnosis of MHC class II deficiency (3), phenotyping of the JER patient revealed decreased numbers of CD4+ T lymphocytes (Table IGo) and normal counts of B lymphocytes. In addition, PBLs and PHA blasts from patient JER did not stain with anti-pan MHC class II mAbs (data not shown). The PBLs from the JER did not proliferate in vitro in response to specific Ags (PPD and TT), although they retained normal proliferative capacities to PHA. Finally, evaluation of serum immunoglobulins also revealed reduced levels compared with an age-matched normal control (Table IGo).


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  		Table I. Immunological features of the patient JER

 
Lack of cell surface HLA-D expression in the JER B-LCL

EBV-transformed cell lines were established from JER as well as from his healthy parents, HAS (the mother) and TAO (the father), to characterize the molecular defect of the JER patient. Cell surface expression of the HLA-DR, HLA-DQ, and HLA-DP molecules in the three established cell lines was first analyzed by flow cytometry (Fig. 1Go for the JER and HAS B-LCLs, not shown for the TAO cell line). As initially observed on fresh PBLs, the JER cell line is completely negative for the cell surface expression of the three MHC class II isotypes, in contrast to HAS and TAO B-LCLs, which express high levels of these molecules. As often evidenced in MHC class II-deficient patients (3), MHC class I expression in the JER cell line was slightly decreased and appeared heterogeneous.



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  		FIGURE 1. Analysis by indirect immunofluorescence of HLA-DR, HLA-DP, HLA-DQ, and HLA-ABC cell surface expression, using the L112, B721, L2, and W6/32 mAbs, respectively, on JER (dark histogram) and HAS (light histogram) B-LCLs.

 
The JER cell line displays normal expression of DRA and DQA mRNAs

We next investigated by Northern blot analysis the expression of the transcripts encoding the {alpha}- and {beta}-chains of MHC class II isotypes in the JER B-LCL, and we compared it with that of HAS, TAO, and a MHC class II-expressing unrelated B-LCL. The results were quite striking showing a HLA-DRA and HLA-DQA transcript expression in the JER cell line quite similar to that of the wild-type controls (Fig. 2GoA). However, transcripts corresponding to the HLA-DRB, HLA-DQB, HLA-DPA, and HLA-DPB mRNAs were not detected in the JER B-LCL. Through semiquantitative RT-PCR techniques (20-cycle amplifications), we observed that the HLA-DRA mRNA of the JER cells is about one-half to one-third of the levels observed in a B-LCL control cell line (data not shown). The HLA-DRA transcript is functional, as the corresponding chain was evidenced by Western blot analysis (Fig. 3Go). We then analyzed the expression of HLA class II-related molecules that are implicated in Ag presentation. Similar to many MHC class II-deficient cell lines, the expression of Ii mRNA in the JER cell line was comparable with positive control cell lines (data not shown). In contrast, the analysis of HLA-DM was puzzling because the expression of these mRNAs appeared to be unstable: we initially observed that HLA-DMA mRNA was expressed in the JER B-LCL at levels quite similar to that of control cell line, whereas HLA-DMB mRNA expression was residual. After several weeks in culture, the expression of these mRNAs was progressively lost (Fig. 2GoB), whereas that of HLA-DRA and HLA-DQA remained stable under these conditions (data not shown).



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  		FIGURE 2. A, Northern blot analysis of MHC class II mRNA expression in GES (a control MHC class II-positive B-LCL), JER, HAS, and TAO cell lines. B, RT-PCR analysis of the expression of HLA-DMA and HLA-DMB genes in a control MHC class II-expressing (Cont) and in JER B-LCLs. The JER cell line was cultured for 8 wk, and RNA preparations were made on aliquots taken each 2 wk from the cell culture.

 


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  		FIGURE 3. Western blot analysis of the HLA-DR{alpha}-chain expression detected with the DA6.147 mAb in cell extracts from the TAO, HAS, and JER B-LCLs, in addition to an unrelated MHC class II-expressing control cell line (GES) and a group B cell line presenting a del26 mutation in the RFXANK gene (HAD).

 
To establish the involvement of trans-acting factors in the unusual HLA-D expression in the JER B-LCL, the transcriptional activity of different HLA-D promoters was next analyzed through luciferase reporter gene experiments. In agreement with the data presented above, the HLA-DRA and HLA-DQA promoters drive, respectively, a luciferase expression in the JER cell line ~two-thirds or one-half of the HAS-positive cell line, while the HLA-DQB and HLA-DPA promoters lead to weak transcriptional activities close to that of the BLS-1 B-LCL (Fig. 4Go). These data indicate a transcriptional defect of HLA-D genes in the JER cells like classical MHC class II-deficient mutants, even though HLA-DRA and HLA-DQA genes remain transcribed at nearly wild-type level.



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  		FIGURE 4. Luciferase assay performed with pGL3-DRA, pGL3-DQA, pGL3-DQB, and pGL3-DPA promoter constructs in the JER, HAS, and BLS-1 B-LCLs, in addition to a MHC class II-expressing cell line (GES). Background luciferase expression determined with the promoterless pGL3-basic vector was deduced from these values. Luciferase relative counts were standardized through the measurement of the cotransfected pSV{beta}gal plasmid.

 
Genetic complementation of the JER cell line

To further understand the particular defect in the JER B-LCL, genetic complementation analysis was undertaken. These experiments were performed by cell fusion between the JER cells and cell lines belonging to the four MHC class II deficiency complementation group, followed by an analysis of cell surface expression of HLA-DR or HLA-DQ on the heterokaryons by immunofluorescence.

The heterokaryons obtained after fusion of JER with either BCH or BLS-2 cell lines from group A, with THF cells from group C, or with ZAR cells from group D, displayed HLA-DR Ag expression at their surface, with a slightly weaker expression of HLA-DQ (Table IIGo). These results indicate that the JER cells are not defective for CIITA, RFX5, or RFXAP.


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  		Table II. Results obtained from the fusion experiments of JER cells and MHC class II deficient cell lines from complementation groups A–Da

 
The situation was more complex for heterokaryons between JER and group B mutants. Three unrelated group B cell lines, BLS-1, HAD, and KHE, were assayed to assess the complementation analysis. Strikingly, when using either the anti-DR L112 or D112 mAbs, a faint cell surface staining was detected with all types of heterokaryons. This labeling was much weaker than that observed in fusions involving JER and the other groups, but was perfectly reproductive with all three group B cell lines. However, when using the L2 anti-DQ Ab, staining was not detected for any of the JER x group B heterokaryons. As controls, detection of HLA-DR expression was neither obtained with any homokaryon, nor with heterokaryons obtained with the BLS-1, HAD, and KHE fusion partners. Therefore, these data suggested a defect in the RFXANK gene in the JER cell line.

RFXANK is defective in the JER cell line

To test this possibility, we amplified by RT-PCR the whole coding sequence of RFXANK mRNA using specific oligonucleotides hybridizing to exons 3 and 10 of the RFXANK gene. A major band was detected corresponding to the RFXANK mRNA form containing exons 3–10 (wild type) in control MHC class II-positive B-LCL, and in HAS and TAO cell lines. A minor band ({Delta}5), described by others as the outcome of an alternative splicing of exon 5 (14), was also observed in these cell lines. In BLS-1 group B cell line, the major and minor bands detected correspond respectively to mRNAs lacking both exons 5 and 6 ({Delta}5/6) or lacking exon 6 only ({Delta}6), as previously described (13).

Interestingly, the JER cell line presented an unusual pattern with six main bands, in which none of these bands has the size of the wild-type mRNA (Fig. 5Go). The more abundant form (band A) detected in the JER cells was observed as well, in some experiments, however very faintly, in both parents’ cell lines, but not in control B-LCLs. The size of band A was compatible with the presence of the intron (Int.4–5) located between exons 4 and 5. The presence of this intron was further confirmed through RT-PCRs and Southern blotting of the RT-PCR products with Int.4–5-specific oligonucleotides (data not shown). Sequencing of the cDNA obtained through RT-PCR amplification with primers hybridizing with exons 3 and 6 showed the presence of Int.4–5 in the mRNA and the existence of a G to C point mutation in the canonical donor splice site downstream exon 4 (not shown).



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  		FIGURE 5. RT-PCR analysis of RFXANK expression on RNA extracted from the patient JER, its parent HAS and TAO B-LCL, BLS-1 (group B), and MHC class II-expressing B-LCLs. GAPDH amplification was also performed to assess that equal amounts of cDNA were used for these samples.

 
Accordingly, genomic PCR of the intron 3 to exon 6 region from RFXANK was amplified in both parents’ and patient JER DNA, and further subcloned. Sequencing of these DNA fragments confirmed the presence of the same G to C mutation in all six sequenced clones from the patient JER (Fig. 6Go). Concerning the parents’ DNA, the mutation was found in two of four clones for the mother and in three of four clones in the father, indicating the heterozygocity of both parents. Therefore, these data show that the JER cell line presents a defect in a splice donor site downstream exon 4 of the RFXANK gene. The lack of splicing of Int.4–5 generates a premature stop codon in the RFXANK cDNA, thereby producing an RFXANK mutant protein lacking all three ankyrin domains.



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  		FIGURE 6. Sequence of the exon 4/intron 4–5 junction of the RFXANK gene from the patient JER and HAS cell lines.

 
We next performed a functional complementation analysis through transient transfection of the wild-type cDNA encoding RFXANK. As seen in Fig. 7Go, the construct was able to restore the cell surface expression of HLA-DR in both the JER and BLS-1 cell lines, further assessing the belonging of this cell line to group B.



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  		FIGURE 7. Immunofluorescence analysis of cell surface expression of HLA-DR in the JER and BLS-1 B-LCLs transiently cotransfected with the red fluorescent DsRed1 vector and the indicated RFXANK cDNA construct or empty vector. HLA-DR expression was detected with the L243 mAb stained with fluorescein (FL1-H). Values in the upper right of the quadrants represent the number of events. The mean fluorescence intensity appears in between parentheses.

 
The JER cell line expresses a mutant form of RFXANK

We next investigated the uncoordinated pattern of expression of the HLA-D genes in the JER B-LCL and the peculiar genetic complementation data obtained when the JER cells were fused with group B B-LCLs. Bandshift experiments (data not shown) suggested that this mutant protein would be in low amounts or that it would render the RFX complex unstable: these experiments did not show the binding of RFX-containing complexes with nuclear extracts from the JER cells using the HLA-DRA promoter, thereby resembling classical group B cell lines. Given the detection of six RFXANK RT-PCR products in the JER B-LCL, we hypothesized that a mutant form of RFXANK might retain a residual activity and a selective affinity for certain HLA-D promoters. This was investigated through size analysis of the PCR products and Southern blotting with oligonucleotides specific for different introns and exons of the RFXANK gene. We thereby identified different alternative splicing forms of the RFXANK mRNA. Four of these bands, including band A, were either containing Int.4–5, or were splice variants in which the exon junctions were not in a proper reading frame. The fifth band presented an alternative splicing of both exons 4 and 5, with the junction of exons 3 and 6 remaining in a proper open reading frame (Fig. 8Go). This RT-PCR product was dismissed as a possible candidate for the HLA-DRA transcription, as it has been previously demonstrated that a cDNA lacking exon 5 ({Delta}5) restored HLA-DRA expression 15 times less efficiently than the wild-type construct (19).



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  		FIGURE 8. Southern blot of the RT-PCR products (nt. 416-1227, exons 3–10) obtained by the amplification of RFXANK transcripts from JER (ethidium bromide staining, C) and control (COM) B-LCLs using the S-5'p33 and AS-3'p33 primers. Blots were next hybridized with oligonucleotides corresponding to exon 4 (A) and exon 5 (B).

 
Therefore, we focused on band B (Fig. 8Go), which is lacking exon 4 and whose alternative splicing maintains a proper open reading frame. We hypothesized that the ANK{Delta}ex4 mutant protein, which presents a 28-aa deletion, was responsible for the selective expression of HLA-DRA and HLA-DQA mRNAs in the JER cell line. The corresponding cDNA, the ANK{Delta}ex4 form, was subcloned, and the lack of exon 4 was confirmed by nucleotide sequencing. Considering the complementation analysis, where the fusion of the JER and BLS-1 cell lines led to a residual expression of HLA-DR at the cell surface of the heterokaryons, we thus investigated the possibility of the rescue of HLA-DR expression in the BLS-1 transfectants by the ANK{Delta}ex4 cDNA. The ANK{Delta}ex4 construct was transiently transfected in the JER and BLS-1 B-LCLs, and cell surface HLA-DR expression was analyzed by cytofluorometry. In agreement with the above hypothesis concerning the BLS-1 transfectants, HLA-DR expression was detected in these cells. Therefore, these data show that the ANK{Delta}ex4 cDNA can rescue both HLA-DRA and HLA-DRB expression. In the JER-transfected cells, the ANK{Delta}ex4 cDNA restored HLA-DR expression with values quite similar to those of the BLS-1 cell line. Therefore, these data establish that the ANK{Delta}ex4 protein, although deleted of 28 aa, retains, when overexpressed in transfected cells, a transcriptional activity quite similar to that of the wild-type protein.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In most group B cell lines from patients of North African origin with MHC class II deficiency, a similar 26-bp deletion upstream exon 6 of the RFXANK gene was described (13, 14, 27). It was recently shown that this phenomenon is not due to a hot-spot site of mutagenesis, but is linked to the genetic transmission of the mutation in these patients (27). In the JER patient, who is of North African origin, we have characterized a new type of defect in RFXANK. A homozygous single nucleotide substitution abolishing the splice site downstream of exon 4 was assessed by gene sequencing and by functional complementation with the wild-type RFXANK cDNA. Interestingly, mutations affecting exon 5 or 8 of the RFXANK gene have been recently described in two other patients from group B (19). In contrast to CIITA mutations altering the carboxyl-terminal part of the protein (26), or RFXAP defects, in which all the mutations have been found in exon 2 (17), it appears that in the RFXANK-defective patients different regions of the gene can be affected.

The presence of intron 4 in the most abundant RFXANK transcript form from the JER cells creates a premature stop codon that leads to a protein lacking all three ankyrin domains. The resulting mutant protein, if expressed, cannot be active, as it has been shown that the lack of the three domains suppresses the function of this protein in ~20 group B BLS cell lines (13, 14, 19, 27). However, two characteristics of the JER cell line therefore remained unexplained: the nearly wild-type expression of HLA-DRA and HLA-DQA mRNAs, in addition to HLA-DMA and Ii, and the residual expression of HLA-DR in the heterokaryons generated by the fusion of the JER B-LCL with three group B cell lines.

Differential expression of the HLA-D genes has been described mainly in tumor cell lines (reviewed in Refs. 39 and 40). CIITA-independent selective expression of HLA-DQ in clone 13 (41) and the expression of HLA-DP in a neuroblastoma cell line (42), for instance, have been reported. In these cells, compensation mechanisms mediated by the dysregulation of expression or activity of certain oncogenes (ras), antioncogenes (Rb), or cellular transcriptional regulators (CREB) (40) may account for the unusual phenotype. In cell lines originating from MHC class II-deficient patients, uncoordinated HLA-D expression has been occasionally described. However, this expression is residual in these cells and is most likely due to the mutant CIITA or RFX proteins they contain. As an example, HLA-DP transcripts are detected at low levels in certain RFXAP-defective cells (17). The only case of a clearly uncoordinated pattern of expression of HLA-D are B-LCLs established from two brothers, KEN and KER, which express HLA-DRA, HLA-DQA, HLA-DPB, and Ii (43), but not HLA-DM (33) mRNAs. This phenotype does not entirely match the JER phenotype, in which HLA-DPB is not expressed, although HLA-DMA is. The observation that both KEN and KER brothers displayed very mild immunodeficiency symptoms compared with patient JER (44) further differentiates these patients. In addition, genetic complementation analysis showed that KER cells do not belong to groups A, B, or C, and are therefore not defective for CIITA, RFXANK, or RFX5 (33). Indeed, to our knowledge, the genetic defect in the KEN and KER cell lines has not been identified.

With the absence of a clear indication concerning noncoordinate HLA-D expression, we hypothesized that a truncated RFXANK protein might be responsible for the peculiar phenotype displayed by the JER cells. One of the transcripts of RFXANK could be encoding such a protein, ANK{Delta}ex4, as the skipping of exon 4 maintains a proper open reading frame. We show in this work that this mutant protein, although lacking 28 aa, is retaining the capacity to rescue cell surface expression of the HLA-DR molecules (Fig. 7Go). This 28-aa region is a Thr- and Ser-rich region (four Thr and four Ser residues) that is highly conserved in the mouse protein (27 of 28 aa) (13). Its localization between the acidic amino-terminal region and the ankyrin domains of the RFXANK protein may indicate that this region is a hinge between these two domains. However, our data exclude an essential role for this region in MHC class II gene transcription.

Based on the RT-PCR analysis of RFXANK mRNA, the ANK{Delta}ex4 protein is most likely expressed in very limited amounts in the JER cell line. This is in agreement with our bandshift experiments in which RFX-containing complexes were not detected with the JER B-LCL nuclear extracts. It is also possible that the ANK{Delta}ex4-containing RFX complex, although highly likely formed in vivo, might be unstable in our experimental conditions. Our data suggest that the few ANK{Delta}ex4 molecules expressed in the JER cell line would only bind to promoters for which they present the highest affinity. However, comparison of the HLA-D promoter sequences, mainly in the 3' region of the X1 motif in which RFXANK binding sites have been identified (45), did not reveal any sequence that would correlate with the selective HLA-DRA, HLA-DQA, and HLA-DMA transcription in the JER cell line. Affinities of the RFX complex for the different HLA-D promoters were shown as DRA = DPA > DQB >> DQA = DPB > DRB (46). Therefore, our data suggest that the truncation of the RFXANK protein modifies the affinities of the mutant RFX complex for the different HLA-D promoters in the JER cell line.

Genetic complementation experiments (Table IIGo) led to highly interesting data: the JER cell line was complemented with cells from groups A, C, and D, but displayed residual expression of HLA-DR Ags when fused with three different group B cell lines. One of these latter cell lines (HAD) has the classical 26-nt deletion upstream exon 6 of RFXANK (27), while BLS-1 is displaying a 58-nt deletion downstream this exon (14). We propose that the faint staining detected on the JER/group B B-LCLs heterocaryons is due to {alpha}-complementation phenomenons between the two RFXANK mutant proteins provided by each fusion partner. In agreement with our data, Lin et al. have demonstrated the oligomerization of Tvl-1, the mouse homologue of the RFXANK protein (47). Accordingly, it was recently demonstrated that a del26 cell line, Ramia, was complemented by a fragment of RFXANK encompassing residues 69–260 (48). Therefore, our data suggest that the ANK{Delta}ex4 mutant protein from JER and the 122 amino-terminal part of the del26 or del58 RFXANK proteins from the other group B cell lines present a capacity to dimerize and to weakly activate HLA-DR expression. It was shown that a mutant Tvl-1 protein lacking the 130 carboxyl-terminal amino acids lost the capacity to oligomerize (47). Taken as a whole, these data suggest that the individual monomers interact through different domains, and that the amino-terminal domain is required as well for the dimerization of the RFXANK protein.

In summary, we report that a 28-aa truncated RFXANK protein retains a transcriptional activity. Therefore, we propose that the deleted region is not essential for the activity of the protein, but the low amount of this protein triggers the transcription of the HLA-D promoters most likely presenting the highest affinity for this protein or for the protein complex containing RFXANK. This report further emphasizes the necessity to study mutant RFXANK cDNA constructs in cell lines presenting a complete lack of the endogenous protein, to avoid misleading interpretations linked to {alpha}-complementation phenomenons occurring between the endogenous and exogenous mutant proteins.


   Acknowledgments
 
We are grateful to Drs. R. Al-Daccak. N. Setterblad, N. Mooney, and G. Barbieri for critical reading of the manuscript. We thank Dr. B. Lisowska-Grospierre for highly helpful guidance.


   Footnotes
 
1 This work was supported in part by grants from the Réseau International des Instituts Pasteurs et Instituts Associés , Progrès from the Institut National de la Santé et de la Recherche Médicale, the Tunisian State Secretariat for Research and Technology, the Association pour la Recherche contre le Cancer, and the Fondation pour la Recherche Médicale. A.-M.L.-D. was supported by a fellowship from La Ligue Nationale contre le Cancer. J.V. was supported by a fellowship from the Ligue Nationale contre le Cancer, then from the Fondation pour la Recherche Médicale. Back

2 A.-M.L.-D., M.-R.B., and J.V. contributed equally to the work. Back

3 Current address: Department of Pathology, Harvard Medical School, Boston, MA 02115. Back

4 Address correspondence and reprint requests to Dr. Cathrine Alcaïde-Loridan, Unité d’Immunogénétique Humaine, Institut National de la Santé et de la Recherche Médicale Unité 396, Centre de Recherches Biomédicales des Cordeliers, 15 rue de l’Ecole de Médecine, 75006 Paris, France. Back

5 Abbreviations used in this paper: NFY, nuclear factor binding to Y box; B-LCL, B lymphoblastoid cell line; CIITA, class II transactivator; CREB, cAMP response element-binding protein; Ii, invariant chain; PPD, purified protein derivative; RFX, regulatory factor binding to X box; RFXAP, RFX-associated protein; TT, tetanus toxoid. Back

Received for publication July 11, 2000. Accepted for publication February 22, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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