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The Journal of Immunology, 1999, 163: 794-801.
Copyright © 1999 by The American Association of Immunologists

Discoordinate Expression of Invariant Chain and MHC Class II Genes in Class II Transactivator-Transfected Fibroblasts Defective for RFX51

Ad Peijnenburg*, Marja J. C. A. Van Eggermond*, Sam J. P. Gobin*, Rian Van den Berg*, Barbara C. Godthelp*, Jaak M. J. J. Vossen{dagger} and Peter J. Van den Elsen2,*

Departments of * Immunohematology and Blood Bank and {dagger} Pediatrics, Leiden University Medical Center, Leiden, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
MHC class II deficiency or bare lymphocyte syndrome is a severe combined immunodeficiency caused by defects in MHC-specific transcription factors. In the present study, we show that fibroblasts derived from a recently identified bare lymphocyte syndrome patient, SSI, were mutated for RFX5, one of the DNA-binding components of the RFX complex. Despite the lack of functional RFX5 and resulting MHC class II-deficient phenotype, transfection of exogenous class II transactivator (CIITA) in these fibroblasts can overcome this defect, resulting in the expression of HLA-DR, but not of DP, DQ, and invariant chain. The lack of invariant chain expression correlated with lack of CIITA-mediated transactivation of the invariant chain promoter in transient transfection assays in SSI fibroblast cells. Consequently, these CIITA transfectants lacked Ag-presenting functions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Major histocompatibility complex class II genes encode cell surface glycoproteins that fulfill a crucial function in the immune response (1). The importance of these molecules is illustrated in patients suffering from MHC class II deficiency or bare lymphocyte syndrome (BLS),3 a severe combined immunodeficiency. This disease is characterized by an absence of MHC class II expression and frequently also by a reduced level of MHC class I expression. As a consequence, these patients have severely impaired humoral and cellular immune responses and are extremely susceptible to bacterial and viral infections. MHC class II deficiency is inherited in an autosomal recessive fashion and is the result of defects in trans-acting factors that regulate the expression of MHC class II genes by binding directly or indirectly to conserved regulatory elements in the proximal promoter of these genes (2, 3).

Cell fusion experiments among BLS-derived cell lines have led to the definition of at least four complementation groups, A, B, C, and D (4). The genes affected in these complementation groups have been identified. The gene mutated in BLS complementation group A encodes the class II transactivator (CIITA; 5). CIITA is a coactivator that lacks DNA-binding activity but has strong transactivation properties (6, 7). The genes affected in groups B, C, and D encode the 33-kDa subunit (RFXANK/RFX-B; Refs. 8 and 9), the 75-kDa subunit (RFX5; Ref. 10), and the 41-kDa subunit (RFXAP; Refs. 9 and 11) of the multimeric phosphoprotein complex, RFX (12).

A number of conserved DNA sequences, termed W/S, X (comprising X1 and X2 halves), and Y box, have been identified in the proximal promoter of MHC class II genes, and each has been shown to play a key role in the regulation of these genes (reviewed in Refs. 3 and 13, 14, 15). Together these boxes form a regulatory module, which is also found in the promoters of other functionally related genes, such as the invariant chain gene (Ii) and the HLA-DM genes (reviewed in Refs. 3 and 16). These conserved DNA sequence motifs have been demonstrated to bind protein complexes that include RFX, which binds to the X1 box (17); X2BP, which interacts with the X2 box (18, 19); and NF-Y, which binds to the Y box (20). These protein complexes bind cooperatively to DNA, and together they are engaged in the formation of a highly stable quaternary multiprotein/X-Y DNA complex (19, 21, 22, 23). Although individual binding of RFX or X2BP to some of the class II promoters has been shown to be absent or poor (18, 23), the RFX/X2BP/NF-Y complex is formed on all class II isotype X-Y DNAs, thus allowing the class II isotypes to be regulated in a coordinate fashion (23). Inversely, the lack of expression of all MHC class II genes in BLS-derived B cell lines defective for one of the RFX subunits might be explained by the lack of stable RFX/X2BP/NF-Y complex formation. In line with this latter hypothesis are in vivo DNA footprint data that show that the promoters of the various class II isotypes and functionally related genes in RFX-deficient cells exhibit a "bare" phenotype with unoccupied X1, X2, and Y box elements (24, 25, 26, 27).

Patient SSI was found to lack MHC class II expression in combination with a reduced expression level of MHC class I (16, 28). The defect in SSI was found to reside in RFX5. Since we had previously shown that constitutive expression of exogenous CIITA was able to drive expression of HLA-DR, but not of HLA-DP and HLA-DQ in RFXAP-defective fibroblasts, which resulted in HLA-DR-mediated Ag-presenting functions (29), we have investigated whether a defect in RFX5 could also be compensated for by exogenous CIITA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

BLS patient SSI is of mixed Dutch and Indonesian origin, and was characterized by oligotyping as DR7/DR10. Primary fibroblasts from a skin biopsy of the patient were first transformed with an SV40 ori- plasmid and subsequently stably cotransfected with pCMVEBNA and pRSVneo (29). WSI (DR7/DR10) and FSI (DR7/DR11) are SV40-transformed fibroblasts derived, respectively, from the mother and father of BLS patient SSI. SV40-transformed fibroblasts derived from group D BLS patient ABI (DR16/DR7) were described previously (29). SV40-transformed JVH (DR17, DR11) and ABL fibroblasts (DR1, DR15) were derived from a group A and group B BLS patient, respectively (30). CCRF-SB (ATCC CCL 120) is a B lymphoblastoid cell line derived from an individual with acute lymphoblastic leukemia. Fibroblasts and B lymphoblastoid cell lines were grown in Iscove’s modified DMEM (Life Technologies, Paisley, Scotland), supplemented with 10% (v/v) FCS (Life Technologies), penicillin (100 IU/ml), streptomycin (100 µg/ml), and, if cells contained the neomycin-resistance marker neo, G418 (200 µg/ml; Life Technologies). C2116 is an allospecific Th cell clone specific for HLA-DR4/DR7/DR9 (a generous gift of Drs. S. De Koster and A. Termijtelen, Leiden University Medical Center, Leiden, The Netherlands). RKPVB2, also referred to as D(UPN53), is a tetanus toxoid (TT)-specific DR2/DR7-restricted T cell line (29, 31). T cells were stimulated and cultured as described before (29). WEHI-164 clone 13 (W13) is a mouse fibrosarcoma cell line used in the TNF-{alpha} release assay (32). For induction with IFN-{gamma}, cells were grown in the presence of 500 U/ml human rIFN-{gamma} (Boehringer Mannheim, Ingelheim, Germany) for 48 h.

Plasmids and DNA transfections

Plasmid pREP4 (Invitrogen, San Diego, CA) is a cDNA cloning vector in which the expression of the cDNA is driven by promoter sequences from the Rous sarcoma virus long terminal repeat. Plasmids pREP4-CIITA and pGL3-DRA have been described previously (33). Plasmid pREP4-RFX5 was constructed by insertion of a 3.4-kb HindIII/SalI fragment of EBO-pLPP/RFX5 (10) into pREP4. The Ii -300/+1 Ii promoter fragment of the Ii gene was isolated by PCR, and its nucleotide sequence was confirmed (34). In pGL3-Ii, the -300/+1 promoter fragment was fused in front of the luciferase gene of pGL3. The -300/+1 Ii promoter fragment is sufficient to drive expression of a reporter gene (35). Plasmids were introduced into the fibroblasts by the calcium phosphate method (36).

Promoter activity assay

In each of four wells of a six-well plate, 0.15 x 106 fibroblast cells were transfected by the calcium phosphate method, with a DNA mix containing 1 µg firefly luciferase pGL3 reporter plasmid (pGL3-DRA or pGL3-Ii), 1 µg Renilla luciferase pRL-TK control plasmid (Promega, Madison, WI), and 1 µg of pREP4, pREP4-RFX5, and/or 0.5 µg of pREP4-CIITA. Cells were harvested 3 days after transfection. Luciferase activity was determined using the dual-luciferase reporter assay system (Promega) and a luminometer (Tropix, Bedford, MA).

Flow cytometric analysis

To measure MHC class II surface expression, cells were stained with mAbs against the HLA-DR backbone (B8.11.2; Ref. 37), HLA-DQ backbone (SPV-L3; Ref. 38), and HLA-DP backbone (B7/21; Ref. 39), and FITC-conjugated goat anti-mouse IgG (Becton Dickinson, Mountain View, CA). For each sample, 5000 cells were analyzed on a FACScan flow cytometer (Becton Dickinson).

Sequence analysis

Sequence analysis was performed on 2 µg of plasmid DNA using the dideoxynucleotide chain-termination method and T7 DNA polymerase sequencing kit (Pharmacia LKB, Uppsala, Sweden).

Northern blot analysis

Total cellular RNA was prepared using RNAzolB (Cinna/Biotecx Laboratories, Houston, TX), following the manufacturer’s instructions. Twenty micrograms of total RNA was separated on a 1.2% agarose gel containing 2.2 M formaldehyde, transferred to a Hybond N membrane (Amersham, Little Chalfont, U.K.), and hybridized using probes that were labeled with 32P by random priming (DuPont-NEN, Brussels, Belgium). Transfer and hybridization were performed according to the instructions of the membrane manufacturer. The human cDNA probes for MHC class I, MHC class II (HLA-DRA), Ii, ß2m, ß-actin, and the rat GAPDH probe were described before (29, 40, 41).

Complementation analysis

The generation and analysis of transient fibroblast homo- and heterokaryons were essentially as described before (40). Upon treatment of fibroblasts with polyethylene glycol 4000, cells were cultured in the absence or presence of 500 U/ml IFN-{gamma} for 48 h. Subsequently, the cells were harvested, and RNA was isolated and subjected to RT-PCR using HLA-DRB haplotype-specific oligonucleotides as 5' primer and a generic HLA-DRB oligonucleotide as 3' primer. PCR products were size fractionated on a 1% agarose gel, transferred to Hybond N+ membranes (Amersham), and hybridized with biotin-labeled HLA-DRB haplotype-specific probes. The hybridization probes and the 3' generic primer were localized within exon 3 of the HLA-DRB gene, whereas the 5' primers were localized within exon 2. The generic 3' primer as well as the 5' primer and biotin-labeled hybridization oligonucleotide specific for DR7 were described before (40). The other PCR primers used were DR1, 5'-TTGTGGCAGCTTAAGTTTGAAT-3'; DR11, 5'-CTGGGGCGGCCTGATGAGGA-3'. The other biotin-labeled probes were DR1, '-TGTGGCAGCTTAAGTTTGAA-3'; DR11, 5'-GCCTGATGAGGAGTACT-3'. To assure that the quality and the amount of the various cDNAs were similar, GAPDH-specific PCR and hybridization were performed as described before (29).

RT-PCR of RFX5

Upon annealing with oligo(dT) (Pharmacia, Uppsala, Sweden), first strand cDNA was synthesized from 5 µg of total RNA in a final volume of 25 µl, using SuperScript II RNase H- reverse transcriptase (Life Technologies), according to the instructions of the manufacturer. Subsequently, the cDNA was subjected to PCR using oligonucleotide primers specific for RFX5. The following primer pair was used: 5' primer, 5'-TACAAGCTTTGGGCATATATGGGCCTGGCGAAG-3' (HindIII; nt 76–102 of the published RFX5 cDNA sequence), and 3' primer, 5'-TGAGCGGCCGCCTCTACTAGGCAAAGTTAACG-3' (NotI; nt 2060–2082 of the published RFX5 cDNA sequence). For the PCR, 1 µl of cDNA was incubated in a final reaction volume of 100 µl XL buffer (Perkin-Elmer, Roche Molecular Systems, Branchburg, NJ) supplemented with 1.1 mM Mg(OAc)2, 0.2 mM of each dNTP, 30 pmol 5' primer, and 30 pmol 3' primer, according to the manufacturer’s instructions. Before PCR, samples were heated to 94°C for 2 min and cooled to 85°C, at which point 4 U Tth DNA polymerase (Perkin-Elmer, Roche Molecular Systems) was added. PCR was performed for 10 cycles, at 94°C for 1 min, at 50°C for 1 min, and at 68°C for 2 min, followed by 20 cycles, at 94°C for 1 min and at 50°C for 1 min, and at 68°C for 2 min with a 20-s elongation per cycle. The full-length RFX5 PCR product was, upon digestion with HindIII and NotI, inserted in pBluescript (Stratagene, San Diego, CA) and completely sequenced. To determine the functionality of the cloned PCR product in transfection experiments, the HindIII/NotI fragment was transferred from pBluescript to pREP4.

Genomic PCR of RFX5

Genomic DNA of patient SSI and her relatives was amplified using Tth DNA polymerase and the 5' primer, 5'-TCTCGCACGTGGAGAGCGGAA-3' (nt 1049–1069 of the published RFX5 cDNA sequence), and 3' primer, 5'-ATTGGCGGAATTAGTGAGCGA-3' (nt 1163–1183 of the published RFX5 cDNA sequence). PCR was performed as described above for the RT-PCR, except that 1 µg of genomic DNA was used and exposed to 40 cycles at 94°C for 1 min, at 55°C for 1 min, and at 68°C for 1.5 min. The PCR products were either cloned into pMOSBlue T-vector (Amersham) and subjected to sequence analysis or used for oligotyping experiments.

Genomic oligotyping

Genomic PCR products were transferred to Hybond N+ nylon membranes (Amersham) by using a Bio-Dot microfiltration apparatus (Bio-Rad, Hercules, CA) and hybridized to biotin-labeled oligonucleotides, as described (42). For hybridization of the genomic PCR products of SSI and relatives, the wild-type and mutated oligonucleotides were 5'-ATAACCTGCAGGTTAAT-3' and 5'-ATAACCTGTAGGTTAAT-3', respectively, with a hybridization temperature of 30°C and a washing temperature of 35°C.

TNF-{alpha} assay

The degree of stimulation of the TT-specific T cell line RKPVB2 and the allospecific T cell clone C2116 by mock and CIITA/RFX5-transfected fibroblasts was determined using the TNF-{alpha} release assay, as described previously (29).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression profile of MHC and accessory genes in a recently identified BLS patient

Primary fibroblasts from BLS patient SSI were transformed with SV40 and subjected to Northern blot analysis to examine the expression characteristics of MHC class II (HLA-DRA), MHC class I, Ii, and ß2m genes. No HLA-DRA mRNA could be detected upon induction of SSI fibroblasts with IFN-{gamma}, whereas fibroblasts derived from the maternal control WSI produced a large amount of HLA-DRA transcripts (Fig. 1Go). Furthermore, Ii expression in SSI was negligible upon treatment with IFN-{gamma}. In line with our previous observations, in RFX-deficient cells the amount of MHC class I was greatly reduced (16, 28). Although in patient SSI constitutively generated ß2m mRNA was not detectable, the IFN-{gamma}-induced amount of ß2m transcripts was similar to that in the control WSI.



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  		FIGURE 1. Northern blot analysis. Before RNA isolation, fibroblasts derived from BLS patient SSI and fibroblasts of the maternal control WSI were treated (+) or not treated (-) with IFN-{gamma}.

 
Characterization of the genetic defect in SSI

Transient heterokaryon analysis. In a first attempt to determine the gene affected in SSI, cell fusion experiments were performed. SSI fibroblasts were fused with fibroblasts derived from BLS patients JVH, ABL, and ABI, which were representative for complementation groups A, B, and D, respectively. No fibroblasts belonging to group C were available at the time of analysis. Transient heterokaryons were analyzed for restoration of HLA-DRB expression by RT-PCR and Southern blotting. As shown in Fig. 2Go, SSI could be complemented in a reciprocal fashion by JVH, ABL, and ABI. These results demonstrate that SSI belongs to a complementation group that is different from groups A, B, and D.



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  		FIGURE 2. Characterization of transient heterokaryons. SSI fibroblasts were cocultured with fibroblasts belonging to BLS complementation groups A, B, and D. The monolayers were exposed (P) or not exposed to polyethylene glycol 4000 and, subsequently, grown in the presence (+) or in the absence (-) of IFN-{gamma}. The transient heterokaryons were analyzed for reexpression of HLA-DRB by RT-PCR and Southern blotting using oligonucleotide primers and probes specific for a particular HLA-DRB haplotype (underlined). Monocultures of each of the fibroblasts were treated and analyzed as above and served as control. The amount of cDNA that was used as template for the various reactions did not differ significantly, as could be deduced from the signals obtained after GAPDH-specific PCR and hybridization (results not shown).

 
Transfection of exogenous RFX5 into SSI fibroblasts. To determine whether patient SSI would belong to group C, fibroblasts were stably transfected with pREP4-RFX5. RFX5-transfected fibroblasts of SSI showed expression of HLA-DR and HLA-DP, but not HLA-DQ at the cell surface upon treatment with IFN-{gamma} (Fig. 3GoA). This expression profile is similar to the one found for IFN-{gamma}-induced fibroblasts of the maternal control WSI (Fig. 3GoB). These results indicate that patient SSI is affected in the RFX5 gene.



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  		FIGURE 3. Wild-type RFX5 is able to complement fibroblasts from SSI. A, SSI fibroblasts transfected with either pREP4 (···) or pREP4-RFX5 (——) were grown in the absence (-) or presence (+) of IFN-{gamma}. Upon staining with Abs against HLA-DR, HLA-DP, and HLA-DQ, followed by incubation with a fluorescein-labeled goat anti-mouse Ab, fibroblasts were subjected to FACS analysis. B, Healthy control fibroblasts WSI were analyzed as described under A.

 
Patient SSI has a nonsense point mutation in both RFX5 alleles. To characterize the mutation of SSI, RFX5 mRNA was amplified by RT-PCR. All cDNA clones of SSI contained a C to T point mutation at nt 1122 of the published RFX5 cDNA sequence, resulting in the conversion of a CAG glutamine codon at amino acid position 321 to a premature TAG stop codon (Fig. 4GoA). Thus, the mutated RFX5 gene of SSI encodes a 320-aa truncated protein that still contains the DNA-binding domain. Oligotyping experiments, in which genomic PCR products encompassing nt 1122 were hybridized to wild-type and mutated oligonucleotides, revealed that SSI was homozygous for the mutated allele, whereas the mother (WSI) and the father (FSI) were heterozygous (Fig. 4GoB). SB, which served as a control, was homozygous for the wild-type allele.



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  		FIGURE 4. Mutation analysis of patient SSI. A, Comparison of the sequence of RFX5 cDNA from SSI with wild-type (wt) RFX5 cDNA reveals a C to T transversion at nucleotide position 1122, replacing the Gln322 (CAG) codon by a stop (TAG) codon. B, Oligotyping on genomic PCR products spanning the point mutation indicates that SSI is homozygous for the mutated (mu) allele. Both father (FSI) and mother (WSI) of SSI are heterozygous for the mutated allele. The control (SB) hybridizes only with the wild-type (wt) probe.

 
The mutated RFX5 cDNA of SSI was cloned into the expression vector pREP4 and transfected in SSI fibroblasts. The transfectants failed to express MHC class II upon treatment with IFN-{gamma}, which confirmed that the truncated RFX5 protein encoded by the mutated cDNA was indeed inactive (results not shown).

CIITA-transfected SSI fibroblasts express only HLA-DR Ags at the cell surface

To determine whether CIITA would be able to bypass the defective RFX5 of SSI, fibroblasts of this patient were stably transfected with pREP4-CIITA. A pool of transfected cells was analyzed by flow cytometry for cell surface expression of MHC class II. Among the primary pool of CIITA-transfected SSI fibroblasts, 5–10% of the cells were MHC class II positive even without treatment with IFN-{gamma}, and these cells were enriched by one round of cell sorting (Fig. 5Go). Although CIITA-transfected SSI fibroblasts expressed HLA-DR molecules at the cell surface, no HLA-DQ and DP could be detected (Fig. 5Go). This is in contrast to CIITA-transfected maternal control (WSI) fibroblasts, which were able to express all MHC class II isotypes (see also Ref. 29). As could be deduced from the CIITA-specific RT-PCR, the CIITA transfectants of SSI and WSI generated a similar amount of CIITA mRNA (results not shown). Subsequently, we examined in a TNF-{alpha} release assay whether these CIITA-transfected SSI fibroblasts would be able to process and present TT Ag. Upon exposure to TT, the transfectants were not recognized by the TT-specific T cell line RKPVB2 (Fig. 6GoA), not even after treatment with IFN-{gamma}. In contrast, CIITA-transfected fibroblasts from the mother of SSI (WSI), BLS patient ABI, and IFN-{gamma}-treated RFX5-transfected SSI fibroblasts stimulated the TT-specific T cell line to produce TNF-{alpha} (Fig. 6GoA). Furthermore, using this functional assay, we have evaluated whether the HLA-DR molecules at the cell surface of CIITA-transfected SSI fibroblasts would be recognized by the allospecific T cell line C2116. As shown in Fig. 6GoB, C2116 was not stimulated by CIITA transfectants of SSI, but was stimulated by CIITA transfectants of WSI and ABI and IFN-{gamma}-treated SSI fibroblasts transfected with RFX5. The degree of recognition of the CIITA-transfected SSI fibroblasts by C2116 was not increased upon treatment of the transfectants with IFN-{gamma}. Also, a standard [3H]thymidine incorporation assay revealed that the CIITA-transfected SSI fibroblasts were neither recognized by RKPVB2 nor by C2116 (results not shown).



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  		FIGURE 5. CIITA-transfected SSI fibroblasts show cell surface expression of HLA-DR, but not of DP and DQ. SSI and WSI fibroblasts transfected with either pREP4 (····) or pREP4-CIITA (——) were stained with Abs directed against HLA-DR, HLA-DP, and HLA-DQ, and analyzed by FACS.

 


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  		FIGURE 6. HLA-DR Ags on CIITA-transfected SSI fibroblasts are not functional. Transfected SSI (DR7/DR10), ABI (DR2/DR7), and WSI (DR7/DR10) fibroblasts, upon cultivation in the absence or presence (+) of IFN-{gamma}, were used as targets either for the TT-specific T cell line RKPVB2 (DR7/DR2 restricted) or for the allospecific T cell clone C2116 (DR4/DR7/DR9 restricted) in a TNF-{alpha} release assay. The degree of recognition was reflected by the amount of TNF-{alpha} produced and measured by the percentage of dead WEHI W13 cells.

 
To understand the nature of the Ag presentation defect of the CIITA-transfected SSI fibroblasts, both a Northern blot analysis and a transient transfection assay were performed. It was found that Ii transcripts were absent in the CIITA transfectants of SSI, whereas CIITA transfectants of ABI were expressing Ii (Fig. 7GoA). The absence of Ii transcripts correlated with the RFX5 defect since expression of Ii in RFX5 transfectants of SSI could be induced upon treatment with IFN-{gamma} (Fig. 7GoA). Likewise, in transient transfection assays the CIITA-mediated transactivation of the Ii promoter was negligible, unless functional RFX5 was also present in SSI fibroblasts (Fig. 7GoB). It should be noted that the levels of Ii promoter-driven luciferase activity in CIITA- and RFX5-cotranfected SSI fibroblasts were similar as in CIITA-cotransfected WSI control fibroblasts (Fig. 7GoB).



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  		FIGURE 7. A, CIITA transfectants of SSI do not express Ii. Transfected SSI and ABI fibroblasts were grown in the presence (+) or absence (-) of IFN-{gamma}. RNA was isolated and subjected to Northern blot analysis. B, CIITA fails to transactivate the Ii promoter in SSI fibroblast cells. Transient cotransfection of HLA-DRA and Ii promoter-luciferase constructs (pGL3-DRA and pGL3-Ii) with pREP-4, pREP4-RFX5, and pREP4-CIITA in RFX5-defective SSI and in normal WSI fibroblasts. Luciferase activity, corrected for transfection efficiency with Renilla luciferase values, is shown as mean ± SD (n = 4).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have shown that the novel MHC class II deficiency patient SSI belongs to complementation group C. SSI has a defective RFX5 protein caused by a point mutation generating a premature stop codon. The predicted amino acid sequence of this mutated RFX5 gene reveals that the SSI-RFX5 protein still contains its DNA-binding domain.

CIITA transfectants of SSI expressed HLA-DR Ags at the cell surface, but did not express HLA-DP and HLA-DQ molecules. This is in contrast to wild-type fibroblasts that expressed all MHC class II isotypes at the cell surface upon transfection with CIITA (29). This indicates that the defect in RFX5 can partially be bypassed in the presence of exogenous CIITA.

The lack of HLA-DP and HLA-DQ cell surface expression in the CIITA transfectants might be a consequence of differential binding of transcription factors to the promoters of the various MHC class II loci. For instance, in vitro studies have shown that RFX does not bind to the X boxes of the DRB or DPB promoters, whereas binding to the other isotype promoters could be observed albeit with different efficacies (18, 43, 44). Similarly, it has been demonstrated that the X2BP has different binding affinities for the X2 box of the various MHC class II isotypes (18, 23). X2BP binds to the X2 box in the DRA and DRB promoter and to a lesser extent also the DPB promoter, but not to the X2 box of the other promoters. Despite the fact that both RFX and X2BP individually hardly bind to some of the MHC class II promoters, stable RFX/X2BP/NF-Y complexes are formed the promoters of all MHC class II isotypes and Ii by virtue of cooperative interactions (23, 27). These interactions, including those that occur on the Ii promoter, are positively influenced by CIITA, as can be deduced from in vivo DNA footprint analysis (45, 46). However, these different binding affinities of individual DNA-binding protein complexes may correlate with the allelic and isotypic differences in promoter strength in B cells (47, 48, 49, 50, 51, 52, 53, 54) or after IFN-{gamma} treatment (48, 54, 55).

CIITA-transfected fibroblasts of SSI showed differential activation not only among the MHC class II isotypes, but also between MHC class II genes and Ii. The absence of HLA-DQ, HLA-DP, and Ii, and the presence of DR in the CIITA transfectants of SSI could therefore be the consequence of differences in their promoter structure and resulting promoter strength. For instance, the DRA and Ii promoters differ in the spacing between the X and Y box, and in nucleotide composition of the conserved S-X-Y module (reviewed in Refs. 3, 16, 26). It might be envisaged that these differences further impair the assembly of a transcriptionally active Ii promoter complex in the absence of functional wild-type RFX5 and affect dramatically Ii promoter strength. The lack of Ii expression in CIITA transfectants of SSI correlates with the inability of these transfectants to process and present TT, since it is known that Ii is crucial for the reconstitution of a functional MHC class II peptide-loading compartment (56). Moreover, the lack of recognition of the CIITA-transfected SSI fibroblasts by an alloreactive T cell clone, raised against DR+Ii+ PBMCs, might be due to absence of Ii expression, since evidence has recently been presented that the absence of Ii results in an altered array of peptides displayed by HLA-DR molecules (57).

MHC class II and Ii genes are regulated in a coordinate fashion in professional APCs, such as dendritic cells and mature B cells. However, there is also evidence to suggest a general difference in transactivation between the various MHC class II isotypes and Ii. This is illustrated in normal skin fibroblasts in culture that are known to express HLA-DR and DP, but not DQ upon exposure to IFN-{gamma} (29, 58, 59). Besides the results from our studies on CIITA-transfected fibroblasts defective for RFXAP or RFX5, also the work performed by others indicates that the MHC class II isotypes can be expressed discoordinately. B cell lines derived from patients suffering from an atypical form of MHC class II deficiency have been shown to express HLA-DRA, HLA-DPB, HLA-DQA, and Ii, but not HLA-DRB, HLA-DPA, and HLA-DQB (60, 61). The B-LCL clone 13, which has a defect in CIITA, expresses HLA-DQ, but not HLA-DR and HLA-DP (62). Differential reexpression of MHC class II Ags has also been observed to spontaneously occur in cultures of the in vitro generated MHC class II-negative cell line 6.1.6 (63). The MHC class II-positive cells in these cultures were expressing HLA-DR and HLA-DP, but not HLA-DQ. Also, the (differential) expression patterns of MHC class II genes in thymic medulla, mature dendritic cells, and activated B cells from RFX5-/- mice argue for the existence of a RFX5-independent pathway of MHC class II transactivation (64). In addition, mutational analysis of the DRA and Ii promoter has revealed differences in the contribution of, for instance, the S box in the transactivation of these promoters (26). Furthermore, subtle differences in promoter occupancy in vivo between HLA-DR and Ii have been noted in uninduced IFN-{gamma}-sensitive cells (26, 65, 66).

Taken together, the present work demonstrates that exogenous CIITA in fibroblasts of an RFX5-defective BLS patient was able to rescue HLA-DR, but not DQ, DP, and Ii. These findings may correlate with differences in promoter structure and resulting promoter strength.


   Acknowledgments
 
We thank Drs. F. Ossendorp, B. Roep, J. van Bergen, and A. Thompson for critically reading the manuscript. We are grateful to Dr. B. Lisowska-Grospierre for providing ABL and JVH fibroblasts, and Drs. B. Mach and W. Reith for the kind gift of the CIITA and RFX5 cDNA.


   Footnotes
 
1 This work was supported by The Netherlands Organization for Research (NWO Grant 901-09-243) and the Macropa Foundation. Back

2 Address correspondence and reprint requests to Dr. P. J. Van den Elsen, Department of Immunohematology and Blood Bank, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. E-mail address: Back

3 Abbreviations used in this paper: BLS, bare lymphocyte syndrome; ß2m, ß2-microglobulin; CIITA, class II transactivator; Ii, invariant chain; TT, tetanus toxoid; X2BP, X2-binding protein. Back

Received for publication June 11, 1998. Accepted for publication April 29, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Saito, T., R. N. Germain. 1988. The molecular basis of MHC-restricted antigen recognition by T cells. Int. Rev. Immunol. 3:147.[Medline]
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