Direct Genetic Correction As a New Method for Diagnosis and Molecular Characterization of MHC Class II Deﬁciency

METHOD doi:10.1006/mthe.2002.0804

Direct Genetic Correction as a New Method for Diagnosis and Molecular Characterization of MHC Class II Deﬁciency

Franck Matheux,1 Aydan Ikinciogullari,2 David A. Zapata,3 Emmanue`le Barras,1 Madeleine Zufferey,1 Figen Dogu,2 Jose´ R. Regueiro,3 Walter Reith,1,* and Jean Villard1,4,*,†

1Department of Genetics and Microbiology, University of Geneva Medical School, Geneva, Switzerland 2Department of Pediatric Immunology–Allergy, Ankara University School of Medicine, Ankara, Turkey 3Department of Immunology, School of Medicine, Complutense University, Madrid, Spain 4Immunology and Transplant Unit, University Hospital of Geneva, 1211 Geneva 4, Switzerland

*Both authors contribute equally to this work.

†To whom correspondence and reprint requests should be addressed at the Immunology and Transplant Unit, University Hospital of Geneva, 24 Rue Micheli-du-Crest, 1211 Geneva 4, Switzerland. Fax: ϩ41 22 3729390. E-mail: [email protected].

Major histocompatibility complex class II (MHCII) deficiency is a primary immunodeficiency resulting from defects in one of four different MHCII-specific transcription factors—CIITA, RFX5, RFXAP, and RFXANK. Despite this genetic heterogeneity, the phenotypical manifestations are homogeneous. It is frequently difficult to establish a definitive diagnosis of the disease on the basis of clinical and immunological criteria. Moreover, the phenotypical homogeneity precludes unambiguous identification of the regulatory gene that is affected. Identification of the four genes mutated in the disease has now allowed us to develop a rapid and straightforward diagnostic test for new MHCII-deficiency patients. This test is based on direct correction of the genetic defect by transduction of cells from patients with lentiviral vectors encoding CIITA, RFXANK, RFX5, or RFXAP. We have validated this approach by defining the molecular defects in two new patients. The RFXANK vector restored MHCII expression in a T cell line from one patient. The RFXAP vector corrected primary cells (PBL) from a second patient. Molecular analysis confirmed the presence of homozygous mutations in the RFXANK and RFXAP genes, respectively. Direct genetic correction represents a valuable tool for the diagnosis and classification of new MHCII-deficiency patients.

Key Words: MHC class II deﬁciency; gene therapy; diagnostic test; lentiviral vector; human disease; bare lymphocyte syndrome.

INTRODUCTION ments performed with cell lines derived from MHCII- deficiency patients have defined four genetic complemen- Major histocompatibility complex class II (MHCII) defi- tation groups (A, B, C, and D) reflecting the existence of ciency—also referred to as the bare lymphocyte syndrome four distinct regulatory genes [6–10]. The genes that are (BLS)—is an autosomal recessive disorder resulting from mutated in these four complementation groups have the lack of MHCII expression [1–4]. The major clinical been identified (Fig. 1A). The gene (MHC2TA) affected in manifestations of MHCII deficiency are the same as those complementation group A encodes the class II transacti- that are typically associated with other severe immuno- vator (CIITA) [11]. CIITA is a differentially expressed co- deficiency syndromes [3–5]. Although the disease is a activator protein that functions as the master control direct consequence of the lack of MHCII expression, the factor determining the cell-type specificity and induction primary genetic defects do not disrupt the MHCII genes of MHCII expression [6,11–14]. The genes affected in themselves. Instead, they reside in genes encoding trans- complementation groups B, C, and D encode RFXANK, acting regulatory factors required for the transcription of RFX5, and RFXAP, respectively [6,15–18]. These three pro- MHCII genes [6,7] (Fig. 1A). Somatic cell fusion experi- teins constitute the three subunits of RFX, a ubiquitously

on classification of the patients into one of the four genetic complementation groups by means of tedious and time-consuming cell fusion experiments performed with stable cell lines established from the patients [8–10]. Con- sequently, the availability of a simple and rapid method for confirming the diagnosis obtained by standard approaches, and for determining which of the four regulatory genes is affected in newly identified patients, would represent major progress. We have developed a straightforward complementation approach relying on direct correction of the genetic defect. We have set up a system in which cells derived from suspected MHCII-deficiency patients are transduced with lentiviral vectors expressing the four regulatory genes—MHC2TA, RFXANK, RFX5, and RFXAP. Correction of the genetic defect in the transduced cells can be scored within a few days by FACS analysis of cell surface MHCII expression. The approach can be used both for cell lines and for readily accessible primary cells such as peripheral blood lymphocytes (PBL). We have validated this approach by characterizing the genetic defects in two new MHCII-deficiency patients. The first patient belongs to complementation group B and carries a previously unknown mutation in the RFXANK gene. The second patient belongs to complementation group D and has a novel mutation in the RFXAP gene.

Fig. 1. MHCII deficiency is a heterogeneous genetic disease. (A) Molecular RESULTS defects in MHCII deficiency. Elucidation of the molecular defects in MHCII Lentiviral Vectors for the Correction of deficiency has led to the identification of four essential and specific transacti- MHCII-Deficient Cells vators of MHCII genes. A prototypical MHCII promoter is depicted with conserved S, X, X2, and Y motifs. The four transactivators affected in MHCII To develop a diagnostic test for MHCII deficiency we deficiency are highlighted. CIITA is a highly regulated non-DNA-binding co- generated a series of lentiviral vectors driving the expres- activator that is responsible for cell-type specificity and inducibility of MHCII sion of CIITA, RFXANK, RFXAP, and RFX5. Lentiviral expression. RFX5, RFXAP, and RFXANK are the three subunits of the X box- expression vectors were chosen because they are very binding RFX complex. (B) Complementation of mutant MHC class II-deficient cell lines with lentiviral vectors. EBV-transformed B cell lines derived from efficient for the stable transduction of both established MHCII-deficiency patients (SJO and BLS-1) and in vitro-generated mutants cell lines [21,22] and a variety of primary cell types [23– (RJ2.2.5 and 6.1.6) belonging to the four complementation groups were 26]. The four lentiviral vectors efficiently restore MHCII transduced with the lentiviral vectors encoding CIITA, RFXANK, RFX5, or expression when used to transduce MHCII-negative cell RFXAP. lines lacking the corresponding transcription factor (Fig. 1B). The CIITA vector specifically corrects CIITA-deficient cell lines belonging to complementation group A, such as expressed protein complex that binds to the X-box cis- the in vitro-generated mutant RJ2.2.5 [11,27]. The RFX- acting sequence present in MHCII promoters [6,7]. De- ANK vector specifically corrects RFXANK-deficient cell fects in RFX account for the majority (over 70%) of all lines classified in complementation group B, such as the known patients. patient cell line BLS-1 [17,28]. The RFX5 vector specifi- The hallmark of MHCII deficiency is the absence of cally corrects RFX5-deficient cell lines from complemen- MHCII molecules at the surface of all cells that should tation group C, such as the patient cell line SJO [16,29]. normally express them. Together with the typical clinical Finally, the RFXAP vector specifically corrects RFXAP-de- and immunological features, the demonstration of this ficient cell lines classified in complementation group D, lack of MHCII expression currently remains the mainstay such the in vitro-generated mutant 6.1.6 [18,30]. of diagnosis. However, patients presenting a milder clinical phenotype have recently been described [19,20], Correction of a T Cell Line Derived from an which renders diagnosis uncertain. Moreover, the classi- MHCII-Deficient Patient cal diagnostic approaches do not permit identification of As a first step toward the development of a diagnostic test the precise genetic defect that is responsible for the ab- for MHCII deficiency we used the four lentiviral vectors to sence of MHCII expression. Until now this relied largely characterize the genetic defect in a herpesvirus saimiri-

sequence [17] (Fig. 3A). This leads to a frameshift followed by a premature out-of-frame stop codon at nucleotide 263. The 5-nucleotide insertion in the RFXANK cDNA introduces a new SacI restriction site. We took advantage of this SacI site to type the family of patient 1 (Fig. 3B). The patient is homozygous for the 5-nucleotide insertion. The two parents and a healthy brother are heterozygous (Fig. 3B).

Correction of Primary PBL from an MHCII-Deficient Patient Successful complementation of the TRG T cell line suggested that the direct genetic complementation approach was also likely to be feasible with primary PBL isolated from MHCII-deficiency patients. In preliminary experiments we confirmed that primary PBL can be readily transduced by our lentiviral vectors, as shown in Fig. 4B for a control vector encoding GFP. PBL isolated from a recently identified MHCII-deficiency patient (patient 2) were cultured in the presence of IL-2 and PHA (Fig. 4A). Under these culture conditions, wild-type PBLs differentiate mainly into T lymphoblasts expressing MHCII. As expected in the case of MHCII deficiency, the cells from patient 2 remain MHCII negative Fig. 2. Complementation of cells derived from patient 1 with the RFXANK (Fig. 4C, bottom). The lymphoblasts from patient 2 were lentiviral vector. TRG is a herpesvirus saimiri-transformed T cell line derived transduced during the expansion phase with the lentiviral from patient 1. ANZ is a herpesvirus saimiri-transformed T cell line derived from a normal individual. TRG cells were cultured in the presence of IL-2 and transduced with the four lentiviral vectors. The RFXANK lentiviral vector restores cell surface MHCII expression on 93% of the cells. Open profiles, untransduced cells from patient 1 (TRG). Black profiles, transduced cells. Gray profile, wild-type (WT) cell control (ANZ). immortalized T cell line (TRG) derived from an MHCII- deficiency patient in which the molecular defect was not known (patient 1). In contrast to wild-type herpesvirus saimiri-immortalized T cells, which exhibit an activated phenotype and express MHCII genes, TRG T cells are MHCII negative (Fig. 2, bottom). The TRG cell line was transduced with the lentiviral vectors encoding CIITA, RFXANK, RFXAP, and RFX5. Ten days after transduction, the expression of MHCII molecules at the cell surface was examined by FACS. The RFXANK vector clearly restored MHCII expression in a major fraction (93%) of the TRG cells (Fig. 2). This correction is specific because MHCII expression was not restored by the CIITA, RFX5, or RFXAP Fig. 3. Molecular analysis of the mutation in patient 1. (A) The RFXANK gene expression vectors (Fig. 2). is mutated in patient 1. RFXANK cDNA clones from TRG were amplified by The finding that the TRG cell line is specifically com- RT-PCR, subcloned, and sequenced. All clones contain an insertion of 5 nu- plemented by the RFXANK vector suggested that patient 1 cleotides at position 203. This results in a frameshift that leads to a premature belongs to complementation group B, which is character- stop codon at position 263. (B) Analysis of the family of patient 1. The insertion of 5 nucleotides in RFXANK introduces a new SacI restriction site. A 370-bp ized by defects in the RFXANK gene. We therefore isolated fragment spanning this region of RFXANK mRNA was amplified from all family and sequenced full-length RFXANK cDNA clones from members and was then digested with SacI. The SacI digestion cleaves the TRG. Several independent RFXANK clones were se- mutant fragments into two fragments of 146 and 225 bp. The brother and quenced. All clones contained a 5-nucleotide insertion at both parents of patient 1 are heterozygous for the mutation. The patient is nucleotide 203 (amino acid 68) of the published RFXANK homozygous.

that this patient belongs to complementation group D, which is characterized by mutations in the RFXAP gene. We therefore isolated and sequenced full-length RFXAP cDNA clones from the PBL of patient 2. Several independent RFXAP clones were sequenced. All clones contained a point mutation converting a CAG glutamine codon at nucleotide 761 (amino acid 251) into a premature TAG stop codon (Fig. 5A). To determine whether patient 2 is homozygous for the point mutation in RFXAP we performed an oligotyping experiment (Fig. 5B). Genomic DNA fragments encompassing the mutation were ampli- ﬁed from patient 2 and his parents, spotted on a mem- brane, and hybridized with oligonucleotide probes corresponding to the wild-type and mutated RFXAP sequence. The results demonstrate that patient 2 is homozygous for the mutation. The two parents are heterozygous.

DISCUSSION In this report we describe a new method for the diagnosis and molecular characterization of MHCII deficiency based on direct genetic correction of cells from the patients. We have generated a series of lentiviral vectors encoding CIITA, RFXANK, RFX5, and RFXAP. These vectors can efficiently restore MHCII expression in cells derived from patients in each of the four known MHCII- deficiency complementation groups. Moreover, this genetic correction can be achieved using PBL, a readily available source of primary cells. We have validated the approach by identifying the genetic defects in two MHCII-deficiency patients.

Fig. 4. Complementation of PBL from patient 2 with the RFXAP lentiviral vector. (A) Culture and transduction of primary PBL. PBL were purified over a Ficoll gradient and cultured in the presence of IL-2 and PHA. Lentiviral supernatants were added after 2.5 days of culture. Cells were analyzed by FACS after 7.5 and 12.5 days of culture. (B) Efficient transduction of PBL with lentiviral vectors. PBL from a normal donor or from patient 2 were transduced with a control lentiviral vector encoding GFP. Expression of GFP was analyzed by FACS. Open profiles, untransduced cells. Black profiles, transduced cells. (C) PBL from patient 2 are complemented by the RFXAP vector. PBL from the patient were transduced with the four lentiviral vectors. The RFXAP vector restores cell surface MHCII expression on 48% of the cells. Open profiles, untransduced cells form patient 2. Black profiles, transduced cells. Gray profile, PBL from a wild-type (WT) donor.

vectors encoding GFP (Fig. 4B) or CIITA, RFXANK, RFX5, and RFXAP (Fig. 4C). Five and ten days after transduction, Fig. 5. Molecular analysis of the mutation in patient 2. (A) The RFXAP gene is the expression of MHCII molecules at the cell surface was mutated in patient 2. RFXAP cDNA clones from patient 2 were amplified by analyzed by FACS. MHCII expression was restored in a RT-PCR, subcloned, and sequenced. All clones contain a point mutation at significant fraction (48%) of the cells transduced with the position 761. This replaces a glutamine codon with a premature stop codon. (B) Oligotyping of the family of patient 2. Genomic DNA fragments spanning RFXAP vector (Fig. 4C). This correction is specific because the mutation were amplified by PCR from the father, the mother, and patient MHCII expression was not restored by the CIITA, RFX5, or 2. The PCR products were then blotted onto membranes and hybridized with RFXANK expression vectors (Fig. 4C). oligonucleotides containing the mutated (C 3 T) and wild-type sequence The finding that the primary cells from patient 2 were (WT) of RFXAP. The patient is homozygous for the mutation. Both parents are complemented specifically by the RFXAP vector suggested heterozygous.

A certain degree of variability in the efficiency of cor- Haploidentical peripheral CD34ϩ stem cell transplantation was performed rection (percentage of cells that are complemented) has but the patient died due to interstitial pneumonia. been observed. This depends on the cells (primary or Patient 2 was the second son of consanguineous parents of Turkish origin. The medical history was marked by several episodes of bronchioli- established cell lines), the vector, and the virus prepara- tis. Immunological examinations revealed panhypogammaglobulinemia, tion. However, it is consistently between 20 and 100%, normal T and B cell counts, but a reversed CD4ϩ/CD8ϩ T cell ratio. No HLA-DR expression was detected on B cells, monocytes, or activated T which exceeds by many fold the residual level of MHCII ϩ expression occasionally observed in cells from certain lymphocytes. Haploidentical peripheral CD34 stem cell transplantation was performed but the boy died due to veno-occlusive disease despite MHCII-deficiency patients. The latter rarely exceeds a few heparin prophylactic. The patient’s sister also suffers from the disease and percent. has been affected from pneumonia. Her cells are also MHCII negative. Due to the low incidence of the disease, it has as yet Lentiviral vectors. Wild-type CIITA, RFX5, and RFXAP cDNAs were in- not been possible to extend our procedure to a larger serted into the pHRЈCMV lentiviral vector [23,24]. The vector pHRЈCMV- panel of patients. In the meantime, to ensure future suc- GFP was used as a control [23,24]. The RFXANK cDNA clone was inserted cess, we are in the process of improving the existing into the pHRЈCMV-IRES-mCD8 lentiviral vector [22]. Details of the clon- vector system. These improvements include the replace- ing strategies are available upon request. The production of virus from the ␣ lentiviral plasmids was done as follows. The packaging plasmid (pCMV ment of the current CMV promoter by a promoter (EF1 ) ⌬R8.91) [23,24] was used to provide all of the viral proteins except for the that is more strongly expressed in hematopoietic cells [26] envelope protein. A second plasmid (pMD-G) was used to provide the and the reintroduction of an HIV-1 sequence (cPPT) that heterologous envelope G glycoprotein of vesicular stomatitis virus [23,35]. increases the transduction efficacy [31]. Virus was generated by cotransfection of 293T cells by calcium phosphate ␮ Ј The two mutations characterized in this report have DNA precipitation in 10-cm plates with 20 g of the pHR CMV vector encoding CIITA, RFXANK, RFX5, or RFXAP; 13 ␮g of pCMV ⌬R8.91; and 7 not been described previously in other patients. Both ␮g of pMD-G [23]. Supernatants were collected 36 h after transfection, mutations are very severe and probably represent com- filtered (0.45 ␮m), and concentrated by ultracentrifugation at 20,000 rpm plete loss-of-function mutations. Patient 1 is homozygous for 90 min in an SW 28 rotor. Virus pellets were then resuspended in 1/100 for a 5-bp insertion in the RFXANK gene. This insertion volume of RPMI without serum. leads to a C-terminal truncation removing the entire Cell culture and transduction. The B cell line BLS-1 [28] derived from an ankyrin-repeat domain of RFXANK. This ankyrin-repeat RFXANK-deficient patient [15,17], the B cell line SJO [29] derived from an domain is absolutely essential for the function of RFX- RFX5-deficient patient [16], and the in vitro-generated cell lines RJ 2.2.5 [27] and 6.1.6 [30]—mutated respectively in CIITA and RFXAP ANK [32]. Patient 2 is homozygous for a point mutation [11,18,36]—were grown in RPMI 1640. 293T cells used for the production generating a premature stop codon in RFXAP. Although of virus were grown in DMEM. RPMI and DMEM were supplemented with this nonsense mutation removes only the last 20 amino 2 mM glutamine, 10% heat-inactivated fetal calf serum, and antibiotics. acids of RFXAP, it lies within a critical C-terminal region TRG is a T cell line derived from patient 1 [34]. ANZ is a control T cell line derived from a normal individual. TRG and ANZ were immortalized with rich in glutamine residues. The integrity of this glu- herpesvirus saimiri and cultured as described [34]. Primary cells from tamine-rich region is crucial for the function of RFXAP patient 2 were prepared as follows. PBL were isolated from peripheral [22]. blood by standard Ficoll gradient centrifugation and were grown in RPMI Bone marrow transplantation (BMT) is currently the 1640 supplemented with 2 mM glutamine, 10% human AB serum, anti- only curative treatment for MHCII deficiency. The overall biotics, and either 1% PHA plus 40 IU/ml rIL-2 or only 40 IU/ml rIL-2. All cells were grown at 37°Cin5%CO2. success rate of BMT in this disease is, however, relatively For the transduction experiments, 5 ϫ 105 cells were infected by poor compared to other immunodeficiency syndromes incubation in 100 ␮l of the concentrated viral supernatants for 2 h at 37°C. [33]. Moreover, compatible bone marrow donors are not Then, 1 ml of RPMI supplemented as described above was added to the available for all patients. Alternative therapeutic ap- culture. At 5 and 10 days after infection, transduced cells were washed twice with PBS, stained with the HLA-DR mAb 2.06 [37] followed by proaches, particularly somatic gene therapy, should fluorescein-conjugated rabbit anti-mouse IgG (Serotec), and analyzed by therefore be envisaged. Introduction of the wild-type FACS. MHC2TA, RFXANK, RFX5, or RFXAP genes into hemato- Mutation analysis. Full-length RFXANK cDNA was amplified by RT-PCR poietic stem cells of MHCII-deficiency patients in comple- from patient 1 (TRG cell line). Full-length RFXAP cDNA was amplified by mentation group A, B, C, or D, respectively, would repre- RT-PCR from primary PBL from patient 2. Several independent RFXANK sent a logical strategy. The lentiviral delivery vectors we and RFXAP cDNA clones were cloned in pBluescript and sequenced on are generating and optimizing for CIITA, RFXANK, RFX5, both strands. For the analysis of the family of patient 1, a 133-bp fragment of RFXANK cDNA was amplified with primers 5Ј-CCTGGGGGACAGACG- and RFXAP therefore pave the way for the development of GAGAC-3Ј and 5Ј-GCGAGCTGGTGGATGGACAG-3Ј and digested with somatic gene therapy for MHCII deficiency. SacI. For the oligotyping experiment, genomic PCR products were amplified with primers 5Ј-TACTAAGAAGTCC-3Ј and 5Ј-CATGTAGATGTTCTT- GGTAAG-3Ј from the family of patient 2, blotted onto nylon membranes, MATERIALS AND METHODS and hybridized with oligonucleotide probes as described [38]. RFXAP probes were 5Ј-CAGAAACAGCAACA-3Ј (wild type) and 5Ј-CAGAAATAG- Patients. Patient 1 (also called TGR) [34] was a Spanish girl born to healthy CAACA-3Ј (mutated nucleotide underlined). unrelated parents. Severe pneumonitis, recurrent fever, and hepatospleno- megaly prompted immunological examinations that revealed the absence of MHCII molecules at the surface of peripheral blood mononuclear cells, ACKNOWLEDGMENTS which were as a consequence unable to elicit a normal allogeneic response. We thank Solange Vischer (Immunology and Transplant Unit, University Hos- She had normal numbers of T and B cells, but CD4ϩ T cell counts were low. pital of Geneva) for expert technical assistance. We thank Didier Trono (Depart-

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