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Mutation in the Class II trans-Activator Leading to a Mild Immunodeficiency This information is current as of June 18, 2017. Wojciech Wiszniewski, Marie-Claude Fondaneche, Françoise Le Deist, Maria Kanariou, Françoise Selz, Nicole Brousse, Viktor Steimle, Giovanna Barbieri, Catherine Alcaide-Loridan, Dominique Charron, Alain Fischer and Barbara Lisowska-Grospierre References Subscription Permissions Email Alerts This article cites 33 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/167/3/1787.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 J Immunol 2001; 167:1787-1794; ; doi: 10.4049/jimmunol.167.3.1787 http://www.jimmunol.org/content/167/3/1787 Mutation in the Class II trans-Activator Leading to a Mild Immunodeficiency1 Wojciech Wiszniewski,*‡ Marie-Claude Fondaneche,* Françoise Le Deist,* Maria Kanariou,§ Françoise Selz,* Nicole Brousse,† Viktor Steimle,储 Giovanna Barbieri,¶ Catherine Alcaide-Loridan,¶ Dominique Charron,储 Alain Fischer,* and Barbara Lisowska-Grospierre2* T he expression of HLA class II molecules is essential for Ag-specific immune responses and is very tightly regulated at the transcriptional level. The impairment of both constitutive and IFN-␥-induced HLA class II gene expression is characteristic of MHC class II immunodeficiency, an autosomal recessive disorder. It results from the defective transcription of all MHC class II genes (1, 2). Since this disease was first described, ⬃70 patients, in 50 families, have been reported. Four major complementation groups (A, B, C, and D) have been described by analyzing B cell lines from patients and experimental mutant HLA-deficient cell lines (3–5). The genes responsible for this deficiency encode the proteins that coordinately control MHC class II locus expression; these are class II *Unité 429 and †Department d’Anatomie Pathologique, Hôpital Necker, Paris, France; ‡Department of Genetics, Mother and Child Institute, Warsaw, Poland; §Department of Immunology-Histocompatibility, Aghia Sophia Children’s Hospital, Athens, Greece; ¶Institut National de la Santé et de la Recherche Médicale Unité 396, Institut Biomedical des Cordeliers, Paris, France; and 储Hans-Spemann Laboratory, Max Planck Institut für Immunbiologie, Freiburg, Germany Received for publication January 22, 2001. Accepted for publication May 23, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Institut National de la Santé et de la Recherche Médicale. trans-activator (CIITA),3 regulatory factor binding to the X box (RFX) protein containing ankyrin repeats, RFX5, and RFXassociated protein (6 –9). In a clinical survey of 30 patients, MHC class II deficiency resulted in combined T and B cell immunodeficiency, with an early onset and an average life expectancy of 4 years (10). Bone marrow transplantation was proposed as the only curative treatment (11) due to the very poor prognosis of most patients (despite appropriate medical care). We report herein an unusual MHC class II deficiency phenotype in three affected siblings. Two siblings, now 21 and 22 years old, are mildly affected, and the third, who is 24 years old, is asymptomatic. However, apart from residual HLA-D staining in PBMC and rare HLA-DR-positive dermal macrophages, HLA class II expression was not detected in these siblings. Consistent with the biological manifestations, but not the clinical status of the patients, a mutation in CIITA gene was detected, which is responsible for the defect in bare lymphocyte syndrome (BLS) complementation group A. This homozygous L469P substitution in the coding region of the CIITA cDNA was shown to be responsible for defective expression of MHC-II. Materials and Methods Cell culture: proliferation of mitogen- and Ag-induced blasts Isolation of PBMC, mitogen-, Ag-, and allogenic cell-induced lymphocyte proliferation and MLR were conducted as previously described (12). EBV 2 Address correspondence and reprint requests to Dr. Barbara Lisowska-Grospierre, Institut National de la Santé et de la Recherche Médicale Unité 429, Hôpital Necker Enfants-Malades, 149 rue de Sèvres, 75743 Paris Cedex 15, France. E-mail address: [email protected] Copyright © 2001 by The American Association of Immunologists 3 Abbreviations used in this paper: CIITA, class II trans-activator; RFX, regulatory factor binding to the X box; LCD, leucine-charged domain; LRR, leucine-rich repeat; GFP, green-fluorescent protein; BLS, bare lymphocyte syndrome; WT, wild type. 0022-1767/01/$02.00 Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 The expression of MHC class II molecules is essential for all Ag-dependent immune functions and is regulated at the transcriptional level. Four trans-acting proteins control the coordinate expression of MHC class II molecules: class II trans-activator (CIITA), regulatory factor binding to the X box (RFX)-associated protein; RFX protein containing ankyrin repeats, and RFX5. In humans, defects in these genes result in MHC class II expression deficiency and cause combined immunodeficiency. Most patients with this deficiency suffer from severe recurrent infections that frequently lead to death during early childhood. We investigated three sisters, now ages 21, 22, and 24 years, in whom MHC-II deficiency was detected. Even though the eldest sibling was asymptomatic and the other two had only mild immunodeficiency, none of the three class II isotypes was expressed on T cell blasts, fibroblasts, EBV B cell lines, or epidermal dendritic cells. Residual HLA-II expression was detected in fresh PBMC. Somatic complementation identified the disease as CIITA deficiency. A homozygous T1524C (L469P) substitution was found in the coding region of the CIITA cDNA and was shown to be responsible for the defect in MHC-II expression. This missense mutation prevents the normal functioning of MHC-II but does not lead to the nuclear exclusion of the L469P CIITA. Transfection experiments demonstrated that the CIITA L469P mutant had residual MHC class II trans activation activity, which might explain the unusual clinical course of the patients studied. This study shows that an attenuated clinical phenotype or an asymptomatic clinical course can be observed in patients despite a profound defect in the expression of MHC class II genes. The frequency of the inherited MHC class II deficiency might thus be underestimated. The Journal of Immunology, 2001, 167: 1787–1794. 1788 MUTATION IN THE CIITA LEADING TO A MILD IMMUNODEFICIENCY B cell lines and SV40-transformed skin fibroblasts were obtained and cultured as described previously (4). Fibroblasts, or their heterokaryons, were treated by IFN-␥ (Genex, 250 and 500 IU/ml) for 48 h before analyzing MHC class II expression. DLD1 is a gut epithelial cell line, which was donated by Dr. N. Cerf-Bensussan. RJ 2.5.5 is a CIITA-deficient variant of the Raji cell line. ABL, SJO, and ZM are EBV-transformed cell lines from MHC-II-deficient patients from the B, C, and D complementation groups, respectively (5). The RC SV40-transformed fibroblast cell line was established from another CIITA-deficient patient (4). Immunofluorescence Somatic complementation analysis B and fibroblasts cell lines from the patients and the RJ 2.5.5, ABL, SJO, and ZM B cell lines, previously classified into complementation groups A, B, C, and D, respectively, and fibroblasts RC (group A) and ZM (group D) were used to obtain transient heterokaryons, as previously described (4). KER B cell lines (13, 14) from the patients were also used. Phenotypic complementation was tested by immunofluorescence 48 –72 h after cell fusion. Fibroblasts were treated with IFN-␥ for 48 h before immunofluorescence analysis. Nucleic acid analysis RNA extraction and RT-PCR analysis were conducted as previously described (4, 5). The CIITA was sequenced according to standard methods. PCR products were purified with the Aquick Kit (Qiagen, Chatsworth, CA). The DNA sequence of both strands was determined by Taq polymerase cycle sequencing with fluorochrome-labeled dideoxy terminators and resolved by a laser detection system (310 ABI sequencer; Applied Biosystems, Foster City, CA). Mutagenesis of pIRES- and pEGFP-WT-CIITA vectors was conducted by use of the Transformer Site-Directed Mutagenesis kit (Clontech Laboratories, Palo Alto, CA), according to the manufacturer’s instructions. The primers used were: F, 5⬘-CAG GAT CTG CCC TTC TCC CTG-3⬘; and R, 5⬘-CAG GGA GAA GGG CAG ATC CTG-3⬘. The pIRES- and pEGFPCIITA-L469P clones were sequenced before the transfection experiments. Transfections Transfection experiments with wild-type (WT) and mutated CIITA vectors were conducted as previously described (5). The plasmids used were pIRES-WT-CIITA, pIRES-L469P-CIITA, pEGFP-WT-CIITA, pEGFPL469P-CIITA, pEGFP-CIITA-MT1 (15) and the corresponding empty vectors, pIRES-neo and pEGFP (both from Clontech). EGFP-CIITA is an N-terminal fusion of EGFP to the second in-frame ATG of the gene encoding CIITA. Stable pIRES-WT-CIITA and -L469P-CIITA transfectants were analyzed 2– 6 wk after transfection. CIITA protein sequence homologies Sequences homologous to CIITA were identified through BLASTp and tBLASTN (16) searches with aa 400 – 600 of human CIITA in the nonredundant databases of the National Center for Biotechnology Information. Multiple sequence alignments were performed with CLUSTALW 1.8 (BCM Search launcher) and rendered with BOXSHADE (Swiss EMBnet). Accession numbers: HSCIITA, emb兩X74301.1兩 (6); Mus musculus CIITA mRNA, gb兩U60653.1兩 (17); Rattus norvegicus MHC class II trans-activator, gb兩AF251307.1兩AF251307 (18); Homo sapiens chromosome 19 clone CTD-3022G6, gb兩AC008753.8兩; H. sapiens NOD2 protein (NOD2), ref兩NM_022162.1兩 (19); H. sapiens caspase recruitment domain 4 (NOD1/ CARD4), gb兩AF298548.1兩 (20, 21); H. sapiens caspase recruitment domain protein 7 mRNA, AF298548 (22). Results Clinical and immunological investigations MHC class II deficiency was detected in siblings SaE, SaM, and SaA at 15, 12, and 11 years of age, respectively. The patients are of Greek origin and were born to nonconsanguinous parents. Immunodeficiency was diagnosed in SaM and SaA (Tables I and II) and clinical and biological findings were consistent throughout the 7-year follow-up period. The immune status of SaE was tested because of her sisters’ disease, but she never underwent treatment. In addition to the HLA-D expression defect, the siblings had hypoglobulinemia and an absence of Ag-induced in vivo and in vitro Table I. Clinical history SaE (Born 1/9/76) Clinical history Several episodes of gastroenteritis in infancy Two episodes pneumonia during childhood SaM (Born 3/11/80) Septicemia at the age of 3 mo Pneumonia at the age of 5 yr Recurrent upper respiratory infections since the age of 9 yr SaA (Born 10/5/79) Recurrent respiratory infections from early childhood Chronic pulmonary infections Bronchiectasis Relapses of HSVa infections Present status Healthy Apparently asymptomatic for the last 3 yr Scars from chicken pox and HSV infections Short stature Hepatosplenomegaly Swelling lymph nodes Atrial-septal defect (corrected by surgery in 1994) No recent follow-up Treatment No treatment at present Antibiotics (occasional) in childhood Antibiotics (occasional IVIG (1990–1995) Antibiotics Chemoprophylaxis with Septrim (1990–1994) IVIG (1990–1995) Left lower lung lobectomy a HSV, herpes simplex virus; IVIG, intravenous Ig injections every 4 wk. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 The anti-HLA Abs used were anti-class HLA II (-DR, -DQ, -DP) clone IQU9 (BioDesign, Carmel, NY), and the anti-class I Ab was W6/32 (SeraLab, Crowley, U.K.). The HLA-II isotype-specific mAbs were: anti-DR L243 IgG2a (BD Biosciences, San Jose, CA) or L112, anti-DP L227; antiDQ Genox or L2. The anti-CIITA Abs were IgG1 clone 7-1H (R&D Systems, Minneapolis, MN). Ab binding was revealed by incubation with an anti-mouse Ig coupled to FITC (Immunotech, Luminy, France). Anti-DR L243 mAb, directly coupled to FITC (BD Biosciences) was also used. Anti-CD4, -CD8, -CD14, -CD19, and -CD25 have been described elsewhere (12). PBMC, B cells, untreated and IFN-␥-induced fibroblasts, and different stable transfectants were stained in suspension and analyzed with a BD Biosciences cytofluorograph. Fibroblasts transfected with pEGFP vectors were fixed after transfection with 0.1% glutaraldehyde for 48 h and 2% formaldehyde in PBS for 5 min, permeabilized with cold (⫺20°C) 100% methanol for 5 min, stained with 4⬘,6⬘-diamidino-2-phenylindole and analyzed with a Leitz Ortoplan microscope. Mutagenesis The Journal of Immunology 1789 Table II. Immunologic investigations SaE SaM SaA Control Lymphocytes/l 3784 4770 6030 1500–4800 CD3 CD3 CD8 CD19 CD20 NK (CD16) 2195 908 1968 567 303 946 2767 715 2385 620 382 668 IgG (mg/dl) IgG1 IgG2 IgG3 IgG4 IgM IgA 973 ND ND ND ND 79 276 1160 1080 50a 100 0 47 5b 875 640 70 100 7 72 4b 970–2000 500–1450 80–630 30–200 20–330 70–370 60–330 Absent Absent ND 1/160–1/3200 IgG: 140 Absent ND 100–800 IgM: ⬍50 22% Absent ND 10–30% Proliferation THY f cpm ⫻ 103 PHA Tetanus Classification of HLA class II deficiency in complementation group A in the Sa family 51 3 48 1 59 ND 25–80 10–30 a Low level. Very low level. c Indirect hemagglutination. d ELISA test (EIU). e Competition assay. f Thymidine incorporation test. b B cell somatic fusions induced the expression of HLA-class II in heterokaryons between ABL, SJO, and ZM cells, belonging to the complementation groups B, C, and D, respectively, in SaE. Heterokaryons between SaE and RC fibroblasts from group A were HLA class II negative. Somatic complementation was also obtained between B cells from SaE and patient KER (14), who did not belong to the A–D complementation groups. Correction of the HLA-II expression was obtained by transfection of SaE and RC fibroblasts with the CIITA cDNA (see below). immune responses after immunization. Serum Abs to common germs (Streptococcus pneumoniae and Haemophilus influenzae) were detected in SaE. A minor CD4 lymphopenia was detected in SaA ,whereas her sisters had normal CD4 T cell counts. Patients SaM and SaA were treated symptomatically, and a prophylactic treatment with intravenous Ig was then started and has been continued since for sibling SaM. SaM became asymptomatic and has remained so for the last 3 years, as is the eldest sibling, SaE, without treatment. Currently, both SaM and SaE refuse to be followed by the immunology department. No information is available on the follow-up of SaA. CIITA mutation Membrane expression of HLA-DR, -DQ, and -DP molecules in family Sa and in other MHC-II-deficient patients The CIITA-negative epithelial cell lines DLD1 and HeLa, the CIITA-deficient Burkitt lymphoma cell line RJ2.2.5, and the fibroblast cell line RC were stably transfected with the expression vector pIRES containing WT-CIITA or L469P-CIITA and tested for HLA-DR expression. Sixty percent of the DLD1 and HeLa cells transfected with WT-CIITA expressed HLA-DR (Fig. 4 b and e), whereas only 0.5% of cells transfected with the empty vector were DR⫹ (Fig. 4, a and d). From 1 to 4% of the DLD1 and HeLa L469P-CIITA transfectants were DR⫹ cells (Fig. 4, c and f). Of the RJ2.2.5 cells transfected with the same vectors, 80% of the WT transfectants became HLA-DR⫹ (Fig. 4h), whereas 14% of the L468P transfectants expressed HLA-DR (Fig. 4i). Ninety-six percent of the RC cells transfected with WT-CIITA expressed HLA-DR at a mean fluorescence intensity of 1 ⫻ 10⫺4 (Fig. 4k). Thirty-one percent of cells transfected with L469P-CIITA displayed HLA-DR staining, although at a mean fluorescence intensity of between 10- and 1000fold lower (Fig. 4l). Western blots of total cell lysates from transfected fibroblasts were used to assess the level of transgene expression and Defective expression of HLA-DR, -DQ, and -DP molecules was observed by immunofluorescence analysis in all three patients (Fig. 1 and data not shown). In resting PBMC (Fig. 1a), B cells (CD19 panels) and monocytes (CD14 panels) were faintly stained with anti-HLA-DR mAbs L243 and L112. HLA-DQ was weakly expressed on B cells and monocytes, and HLA-DP was faintly detected on monocytes (Fig. 1a). PHA-induced T cell blasts were HLA-DR, -DQ, and -DP negative (Fig. 1b), as were CD4 and CD8 MLR-induced blasts (Fig. 1c). In contrast, PHA and MLR blasts expressed CD25 normally (shown for MLR blasts (Fig. 1c)). EBV B cell lines from SaE and SaM did not express HLA-DR, -DP, or -DQ (Fig. 1d), although FACS staining revealed low, but detectable, levels of HLA-DQ and -DP expression on fresh B CD19 cells. In another BLS patient, HeJ (BLS complementation group A) with a severe clinical phenotype, the residual HLA-DR expression The CIITA mRNA from EBV cell lines from Sa patients was amplified by RT-PCR and analyzed by sequencing. A single homozygous T1524C mutation, causing a leucine to proline substitution at position 469 (L469P), was found in all three patients. This mutation was confirmed in the genomic level (Fig. 3). A heterozygous mutation was found in the mother. No cells were available from the father. No other mutations were found in the entire 4.5-kb CIITA cDNA. Functionional analysis of the L469P CIITA Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 Abs to Candidac Streptococcus pneumoniaed Haemophilus influenzaee 4100 1500–4000 1447 900–2900 3136 600–1900 1145 100–1200 663 150–920 724 90–950 observed for Sa siblings was not detected on CD19⫹ B cells with either anti-HLA-DR Abs (Fig. 1e, DR1 and DR2). In contrast, monocytes of one 14-year-old RFX protein containing ankyrin repeats-deficient patient, KhM, displayed a similar residual expression of HLA-DQ and -DP on CD14⫹ cells (Fig. 2f). Interestingly, patient KhM also suffered from a milder form of immunodeficiency. Skin biopsies from SaM and SaE (Fig. 2, D and E) were studied for HLA-DR expression (left) of dendritic cells identified by antiCD1a staining (middle) and macrophages, identified by anti-CD68 staining (right), and compared with control skin (top). HLA-DR expression was not detected on patients’ CD1a-postive epidermal dendritic cells (Fig. 2, D and G) in contrast to control dendritic cells (Fig. 2A). However, several HLA-DR-positive dermal macrophages were detected in patients (Fig. 2, D and G), albeit in lower numbers than in control biopsies. (Fig. 2A). Similar findings, i.e., HLA-DR expression of dermal macrophages, were observed in skin of two other patients ages 21 and 14 years, both with a mild clinical presentation and belonging to complementation group B (C. Picard, W. Wiszniewski, M. C. Fondaneche, V. Pinet, F. Le Deist, S. Blanche, J. F. Eliaou, J. L. Casanova, A. Fischer, and B. Lisowska-Grospierre, unpublished observations). 1790 MUTATION IN THE CIITA LEADING TO A MILD IMMUNODEFICIENCY showed that it was even higher for the L469P CIITA protein than for the WT-CIITA (not shown). We assessed intracellular expression of L469P CIITA by transfecting the RC CIITA-deficient fibroblasts with pEGFP-WTCIITA and pEGFP-L469P-CIITA and comparing the expression pattern with that of the experimental cytoplasmic retention mutant, MT1-CIITA (15). A preferentially nuclear localization of CIITA was observed in both the WT- and L469P-CIITA transfectants (Fig. 5, top and middle). An exclusively cytoplasmic CIITA staining was detected in the pEGFP-MT1-CIITA transfectants, as previously reported (Fig. 5, bottom, and Ref. 15). L469 is conserved in homologous CIITA proteins L469 is part of a so-called leucine-charged domain (LCD); 465LQDLL469) conforming to the consensus LxxLL. Replacement of all theree leucines by alanine severely impaired CIITA function (23). Recently, several sequences were discovered that contained homologies to both the nucleotide-binding domain and the C-terminal leucine-rich repeats (LRRs) of CIITA (19 –22). To test whether the region containing L469 is conserved among these proteins, we identified CIITA-homologous sequences through Blast searches and performed multiple sequence alignments of the corresponding region with the three known CIITA sequences (human, mouse, rat) and four homologous sequences (NOD1/CARD4, NOD2, CARD7, and sequence AC008753). Fig. 6 shows the part of CIITA containing both the P loop region (positions 420 – 427), the Mg2⫹ coordination region (461DAYG464), and the LCD motif (23, 24). Whereas the P loop region is very highly conserved among all sequences, the DxxG motif in human CIITA is only partly conserved in mice and rats and not at all in the other CIITAhomologous sequences. The LxxLL motif is also highly conserved among the different sequences with an acidic amino acid at position 3 (D/E). The most highly conserved amino acid is L468, which is invariant among all sequences. Residue L469 is also highly conserved with only two conservative exchanges of the leucine to isoleucine (Fig. 6). Discussion The MHC class II deficiency disease has been described as being lethal in childhood, with a mean survival age of 5 years, despite Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 1. HLA class II expression by PBMC, in vitro activated T cell blasts, and EBV-transformed B cells from the Sa patients (a– d) and from two other MHC II-deficient patients, HeJ and KhM (e and f). PBMC and MLR-activated blasts in panels a, e and f were double-stained with the indicated Abs. Anti-CD14, CD19, CD4, CD8, and CD25 were used with following anti-HLA-D Abs: FITC-coupled anti-DR mAb 243 as a direct stain (DR1); mAb 1.12 (DR2); anti-DQ and -DP in combination with an FITC-coupled anti-mouse Ig. The anti-DQ mAb was Genox. Anti-Ig isotype-matched control staining. PHA-activated cells (b and e) and B EBV cell lines (d) were labeled with anti-DR, -DQ, and -DP. a, Double-stained PBMC from SaE. FACS profiles for SaA and SaM were identical. b, PHA-activated blasts from SaE and SaM. c, MLR-induced blasts from SaA and a control. d, B EBV cell lines from SaA and SaM. e, HLA-D expression by CD19 cells and PHA blasts from patient HeJ. f, HLA-D expression by CD19 and CD14 cells from patient KhM. The Journal of Immunology 1791 FIGURE 2. Immunohistochemical study of the skin biopsies from a control (A–C), SaM (D–F), and SaE (G–I). The anti-DR mAb used in A, D, and G was anti-HLA-DR L243 mAb. Dendritic Langerhans cells were revealed by anti-CD1a mAb IOT6 (B, E, and H). Macrophages were identified by staining with anti-CD68 mAb KiM7 (C, F, and I). ertheless, the main immunological manifestations of MHC class II expression defect were, with one exception, similar to those reported in other cases of MHC II deficiency, namely, IgG2 and IgA hypogammaglobulinemia and the absence of Ag-induced responses in vivo and in vitro. Unexpectedly, in contrast to most other MHC II-deficient patients who are T CD4 lymphopenic, CD4 cell counts were normal in SaE and SaA and only slightly lower in FIGURE 3. CIITA mutation. Top, CIITA coding region. Positions of the LCD 1 and 461DAYG465 (GTPbinding) motifs and the L469P substitution are indicated. Bottom, Control and SaE CIITA DNA sequences. The CIITA sequences for the other Sa siblings were identical. TF, Transcription activation factors; NLS, nuclear localization sequence. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 appropriate medical care. We describe herein three affected siblings who are still alive at 21, 22, and 24 years of age. The eldest patient suffered very few infections in early childhood and since then has been asymptomatic without any treatment. Her sisters suffered from mild immunodeficiency. None of them was prone to opportunistic infections. These patients do not suffer from the protracted diarrhea that affects most MHC II-deficient patients. Nev- 1792 MUTATION IN THE CIITA LEADING TO A MILD IMMUNODEFICIENCY SaM. This suggests that in the three Sa patients the MHC-II expression defect has led to less severe consequences on T cell differentiation than observed in other patients. The HLA-II molecule expression defect in the Sa siblings was found in B lymphocytes, monocytes, IFN-␥-induced fibroblasts, dendritic cells, and T and B cell blasts. However, faint but detectable HLA-DR, -DQ and -DP staining was observed on B cells and monocytes. Similarly, there was faint but detectable HLA-D staining on PBMC from KhM, another MHC-II-deficient patient from complementation group B suffering from a milder form of immunodeficiency (Fig. 1f). In contrast, B cells from HeJ, a CIITAdeficient patient with a severe immunodeficiency, were HLA-DR⫺ (Fig. 1e). Therefore, it appears that a residual HLA-D staining of PBMC correlates with less severe clinical symptoms. We have no explanation why there was no residual HLA-D expression on any blast types. Although mitogen and MLR stimulation led to blastogenesis and the expression of CD25 on both CD4 and CD8 T cell blasts (shown for SaA, Fig. 1c), in both patients Sa and HeJ T and B cell blasts were HLA-II negative (Fig. 1, b, c, and e). However, in patients Sa, in addition to PBMC, some dermal macrophages were HLA-DR⫹, although dermal dendritic cells were not. MHC II-deficient patients with mild clinical presentation have previously been reported, e.g., the 7-year-old KER twins. Their T cells were able to respond in vivo to antigenic challenge (13, 14). Although the molecular basis of the MHC II defect was not elucidated in these twins, the Sa patients do not share the same defects, because their cells complemented the KER cell line for MHC-II expression. Complementation experiments by somatic cell fusion assigned the Sa family defect to complementation group A, indicating that the CIITA gene was affected in cis. CIITA encodes a 1130-aa protein, the N-terminal region of which acts as a transcriptional activator and the C-terminal region of which provides MHC-II promoter specificity (6, 25, 26). CIITA controls both constitutive Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 4. HLA-DRA expression in different cell types stably transfected with the pIRES WT- and L469P-CIITA vectors. HeLa cells (a– c), DLD1 epithelial cells (d–f), CIITA-deficient RJ 2.5.5 (g–i), and RC fibroblasts (j–l) were transfected with an empty pIRES vector, WT-CIITA pIRES, or L469P-CIITA pIRES, as indicated in each panel. FITC-coupled mAb 243 anti-HLA-DR Ab was used. Isotypic controls are not shown. FSC, Forward scatter. and inducible MHC-class II expression (27) and is considered to be a master gene in MHC class II regulation. In all tissues tested, CIITA expression correlates with MHC class II expression (28, 29), and CIITA knockout mice reproduce the phenotype of CIITAdeficient patients with only minor differences (30). Analysis of the CIITA gene in family Sa revealed a homozygous T1524C substitution which is responsible for a leucine to proline missense mutation at aa 469 (L469P) in the CIITA coding region. All group A patients studied thus far have T and B immunodeficiency, with severe clinical consequences (10). In one case, patient BCH, a stop codon was found at position 1256 in one CIITA allele. However, all other patients with a severe phenotype carry mutations in the 3⬘ end of the CIITA gene (6, 31–34). This is consistent with mutation analysis, which showed that MHC class II specific transcription depends on the 830 C-terminal residues of CIITA (25, 26). The L469P mutation is the first CIITA mutation to be identified in the N-terminal part of this 830-aa region which contains a LCD (LCD motif) essential for CIITA activity (23). This motif is very close to the second tripartite GTP-binding region motif, described by Harton et al. (24). A mutant, in which the conserved LCD1 motif leucine residues (positions 465, 468, and 469) were replaced with alanines, was unable to drive transcription from the DR-X1X2-Y or -W-X-Y promoters (23). Recently, several sequences showing homology to the nucleotide binding and LRR regions of CIITA have been published (19 –22). A search for protein sequence homologies revealed that the LCD motif containing L469 is very highly conserved in these sequences. Position 468 is the most conserved, but L469 is also highly conserved, indicating a functional relevance of this motif in this group of GTP-binding proteins (Fig. 6). It also shows that other human proteins share this motif. In the CIITA gene of the Sa family, only leucine 469 was replaced (by proline), indicating the importance of this motif in vivo. Functional analysis revealed that the L469P allele of CIITA is not completely inactive. Stable transfection of DLD1 or HeLa cells with the L469P-CIITA cDNA did not lead to the trans activation of MHC-II genes, but we observed a residual trans activation potential of L469P-CIITA in RC and in RJ2.2.5 cells (Fig. 4, i and l). In the patient-derived, CIITA-deficient RC fibroblasts transfected with L469P-CIITA, DR expression was restored in 30% of cells, albeit at a much lower level than that observed in WT-CIITA transfectants. In the RJ2.5.5 B cell line, which has genomic deletions of the CIITA gene (6), transfection with L469P CIITA led to an abnormally low but clearly detectable HLA class II expression in 14% of the cells. The mutated CIITA alleles from patients BLS-2 (⌬940 –963) and BCH (BCH-1 ⌬1079 –1106; BCH-2 E381Stop) had been tested functionally in RJ2.2.5 earlier. None of these alleles, which were derived from patients with severe immunodeficiency, led to residual HLA class II expression in RJ2.2.5 (6, 32). Thus, partial HLA-DR expression in the RJ2.5.5 and RC transfectants shows that the expression of the L469P-CIITA cDNA allows partial trans activation of MHC II genes. This probably corresponds to the residual MHC class II expression detected on fresh PBMC from the patients. The immunofluorescence data suggest that the recombinant L469P-CIITA protein can translocate into the nucleus (Fig. 5). This result is confirmed by Western blotting, which revealed the presence of full length L469P-CIITA protein in nuclear extracts (G. Barbieri, T. Prod’homme, J. Vedrenne, B. Lisowska-Grospierre, D. Charron, and C. Alcaide-Loridan, manuscript in preparation). Therefore, the L469P mutant is the first loss-of-function mutant that retains the ability to translocate into the nucleus. Interestingly, a mutation in the 461DAYG465 motif that correlates The Journal of Immunology 1793 FIGURE 5. Subcellular localization of the L469P CIITA. CIITA-deficient RC fibroblasts were transfected with pEGFP-WT-CIITA, -L469P-CIITA and -MT1CIITA, stained with 4⬘,6⬘-diamidino-2-phenylindole 48 h later, and analyzed. Left, GFP fluorescence; right, nuclear staining. immunodeficiency than that affecting other patients. Residual HLA class II expression was not observed on fresh B cells from two other CIITA-deficient patients, patient HeJ (Fig. 1e) and patient BCH (31). However, a milder immunodeficiency associated with a residual HLA class II expression has been described for the Ker/ Ken twins (14) and is shown here for a patient KhM from BLS group B (Fig. 1f). An unrelated patient with MHC II deficiency caused by a CIITA defect presenting similarities with the Sa patients has been described (33). This patient was not diagnosed until the age of 27 years, well beyond the life expectancy of most BLS patients. Clinical and immunological data for the patient have not been reported. Interestingly, a single amino acid substitution, F962S, was found in the coding region of CIITA (33). The patients we describe are the first in which a mild phenotype of the disease can be correlated with a residual trans activation potential of the mutated regulatory factor (Fig. 4). It can be assumed that the ensuing residual HLA class II expression in the patients is responsible for a substantial T cell differentiation and the capacity to mount CD4 T cell-dependent immune responses in vivo. The fact FIGURE 6. Protein sequence comparisons between proteins containing LCD1 motifs. An alignment of positions 419 – 474 of human CIITA with mouse and rat CIITA and four homologous sequences is presented. The P loop region (420GKAGQGKS427), Mg2⫹ coordination region (461DAYG464), LCD region (465LQDLL469), and L469 are boxed. Amino acids identical with HSCIITA are highlighted in black, and similar amino acids are highlighted in gray. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 with the conversion of CIITA to the GDP-bound state leads to its exclusion from the nucleus (24). The mutation (proline 469) is very close to the 461DAYG465 motif and it may therefore be informative to test the GTP binding of mutated CIITA, even if nuclear exclusion is not observed in transfectants. The LCD1 motif mutated in Sa CIITA conforms to the short LxxLL consensus of a putative nuclear export sequence, but the fact that the L469P mutant protein is found in the nucleus argues against such a function for this sequence. The role of neither the LCD nor the GTP-binding motif (24) or of a recently shown (35) interaction between the GTP-binding region (residues 336 –702) and the C-terminal leucine-rich region, LRR (15), are well understood. Thus, further studies on the L469P CIITA displaying these unusual features will contribute to our understanding of the mechanisms that govern nuclear translocation and transcriptional activation of CIITA. Although it cannot be formally proved, it is tempting to speculate that the residual HLA class II expression in cells from the Sa siblings and indeed the residual trans activation potential of the L469P allele of CIITA are responsible for the lesser severity of the 1794 MUTATION IN THE CIITA LEADING TO A MILD IMMUNODEFICIENCY the patients did not develop the protracted diarrhea that affects most patients with MHC II deficiency may be a consequence of residual MHC II expression in intestinal epithelium, like that in PBMC. These observations on MHC II deficiency in the Sa family have important medical implications. They show that an asymptomatic clinical course or an attenuated clinical phenotype can be observed in patients with a profound defect in the expression of HLA class II genes. Therefore, in patients with mild symptoms of immunodeficiency, an inherited MHC II expression defect should be considered. In CIITA-deficient patients, residual HLA-DR expression in peripheral blood leukocytes might be of prognostic value. When such residual HLA-DR expression is detected and coincides with an absence of severe infections, bone marrow transplantation should not be recommended. However, even those MHC II-deficient patients whose clinical status is good should be kept under close medical surveillance because late onset immunodeficiency can be fatal. 12. 13. 14. 15. 16. 17. 18. 19. We thank the staff of the First Department of Pediatrics of the University of Athens, especially X. Nikolaidou. We thank E. Jouanguy for expert advice; V. Pinet for conducting the in vitro induction assays; J.-F. Eliaou and P. Louis-Plence for helpful discussions; C. Harré, C. Jacques, and O. Boucher for expert technical assistance; and Jean-Paul Monnet for preparing the photomicrographs. References 1. Lisowska-Grospierre, B., D. J. Charron, C. de Préval, A. Durandy, C. Griscelli, and B. Mach. 1985. A defect in the regulation of major histocompatibility complex class II gene expression in human HLA-DR negative lymphocytes from patients with a combined immunodeficiency syndrome. J. Clin. Invest. 76:381. 2. Reith, W., V. Steimle, B. Lisowska-Grospierre, A. Fischer, and B. Mach. 1997. Molecular basis of major histocompatibility complex class II deficiency. In Primary Immunodeficiency Diseases, a Molecular and Genetic Approach. H. Ochs, J. Puck, and E. Smith, eds. Oxford Univ. Press, New York. 3. Hume, C. R., and J. S. Lee. 1989. Congenital immunodeficiencies associated with absence of HLA class II antigens on lymphocytes results from distinct mutations in trans-acting factors. Hum. Immunol. 26:288. 4. Lisowska-Grospierre, B., M.-C. Fondaneche, M.-P. Rols, C. Griscelli, and A. Fischer. 1994. Two complementation groups account for most cases in inherited MHC class II deficiency. Hum. Mol. Genet. 3:953. 5. Fondaneche, M.-C., J. Villard, W. Wiszniewski, A. Etzioni, F. Le Deist, A. Peijnenburg, J.-L. Casanova, W. Reith, B. Mach, A. Fischer, and B. Lisowska-Grospierre. 1998. Genetic and molecular definition of complementation group D in MHC class II deficiency. Hum. Mol. Genet. 7:879. 6. Steimle, V., L. A. Otten, M. Zufferey, and B. Mach. 1993. Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or bare lymphocyte syndrome). Cell 75:135. 7. Durand, B., M. Kobr, W. Reith, and B. Mach. 1994. Functional complementation of major histocompatibility complex class II regulatory mutants by the purified X-box-binding protein RFX. Mol. Cell. Biol. 14:6839. 8. Masternak, K., E. Barras, M. Zufferet, B. Conrad, G. Corthals, R. Aebersold, J.-C. Sanchez, D. F. Hochstrasser, B. Mach, and W. Reith. 1998. The majority of MHC class II deficient patients are affected in a novel RFX-associated transactivator containing ankyrin repeats. Nat. Genet. 20:273. 9. Villard, J., B. Lisowska-Grospierre, P. van den Elsen, A. Fischer, W. Reith, and B. Mach. 1997. Mutations of RFXAP, a regulator of MHC class II genes, in promary MHC class II deficiency. N. Engl. J. Med. 337:748. 10. Klein, C., B. Lisowska-Grospierre, F. LeDeist, A. Fischer, and C. Griscelli. 1993. Major histocompatibility complex class II deficiency: clinical manifestations, immunologic features, and outcome. J. Pediatr. 123:921. 11. Klein, C., M. Cavazzana-Calvo, F. Le Deist, N. Jabado, N. Benkerrou, S. Blanche, B. Lisowska-Grospierre, C. Griscelli, and A. Fischer. 1995. Bone 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 Acknowledgments marrow transplantation in major histocompatibility complex class II deficiency: a single-center study of 19 patients. Blood 85:580. Le Deist, F., G. Thoenes, J. Corado, B. Lisowska-Grospierre, and A. Fischer. 1991. Immunodeficiency with low expression of the T cell receptor/CD3 complex: effect on T cell activation. Eur. J. Immunol. 21:1641. Wolf, H. M., I. Hauber, H. Gulle, V. Thon, H. Eggenbauer, M. B. Fischer, S. Fiala, and M. M. Eibl. 1995. Twin boys with major histocompatibility complex class II deficiency but inducible immune responses. N. Engl. J. Med. 332:86. Hauber, I., H. Gulle, H. M. Wolf, M. Maris, H. Eggenbauer, and M. M. Eibl. 1995. Molecular characterization of major histocompatibility complex class II gene expression and demonstration of antigen-specific T cell response indicate a new phenotype in class II-deficient patients. J. Exp. Med. 181:1411. Hake, S. B., K. Masternak, C. Kammerbaouer, C. Janzen, W. Reith, and V. Steimle. 2000. CIITA leucine-rich repeats control nuclear localisation, in vivo recruitment to the MHC class II enhanceosome, and MHC class II gene transactivation. Mol. Cell. Biol. 20:7716. Altschul, S. F., T. L. Madden, A. A. Schoffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389. Sims, T. N., J. F. Elliott, V. Ramassar, D. W. Denney, Jr., and P. F. Halloran. 1997. Mouse class II transactivator: cDNA sequence and amino acid comparison with the human class II transactivator. Immunogenetics 45:220. Mori-Aoki, A., M. Pietrarelli, M. Nakazato, P. Caturegli, L. D. Kohn, and K. Suzuki. 2000. Class II transactivator (CIITA) suppresses transcription of thyroid-specific genes. Biochem. Biophys. Res. Commun. 11:278:58. Ogura, Y., N. Inohara, A. Benito, F. F. Chen, S. Yamaoka, and G. Nunez. 2001. Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF- B. J. Biol. Chem. 276:4812. Bertin, J., W. J. Nir, C. M. Fischer, O. V. Tayber, P. R. Errada, J. R. Grant, J. J. Keilty, M. L. Gosselin, K. E. Robison, G. H. Wong, M. A. Glucksmann, and P. S. DiStefano. 1999. Human CARD4 protein is a novel CED-4/Apaf-1 cell death family member that activates NF-B. J. Biol. Chem. 274:12955. Inohara, N., T. Koseki, L. del Peso, Y. Hu, C. Yee, S. Chen, R. Carrio, J. Merino, D. Liu, J. Ni, and G. Nunez. 1999. Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-B. J. Biol. Chem. 274:14560. Bertin, J., and P. S. DiStefano. 2000. The PYRIN domain: a novel motif found in apoptosis and inflammation proteins. Cell Death Differ. 7:1273. Brown, J. A., E. M. Rogers, and J. M. Boss. 1998. The MHC class II transactivator (CIITA) requires conserved leucine charged domains for interactions with conserved W box promoter element. Nucleic Acids Res. 26:4128. Harton, J. A., D. E. Cressman, K.-C. Chin, C. J. Der, and J. P.-Y. Ting. 1999. GTP binding by class II transactivator: role in nuclear transport. Science 285: 1402. Zhou, H., and L. H. Glimcher. 1995. Human MHC class II gene transcription directed by the carboxyl terminus of CIITA, one of the defective genes in type II MHC combined immune deficiency. Immunity 2:545. Riley, J. L., S. D. Westerheide, J. A. Price, J. A. Brown, and J. M. Boss. 1995. Activation of class II MHC genes requires both the X box region and the class II transactivator (CIITA). Immunity 2:533. Steimle, V., C. A. Siegrist, A. Mottet, B. Lisowska-Grospierre, and B. Mach. 1994. Regulation of MHC class II expression by interferon-␥ mediated by the transactivator CIITA. Science 265:106. Rammasar, V., N. Goes, M. Hobart, and P. Halloran. 1996. Evidence for the in vivo role of class II transactivator in basal and IFN-␥ induced class II expression in mouse tissue. Transplantation 62:1901. Otten, L. A., V. Steimle, S. Bontron, and B. Mach. 1998. Quantitative control of MHC class II expression by the transactivator CIITA. Eur. J. Immunol. 28:473. Chang, C.-H., S. Guerder, S.-C. Hong, W. van Ewijk, and R. A. Flavell. 1996. Mice lacking the MHC class II transactivator (CIITA) show tissue-specific impairment of MHC class II expression. Immunity 4:167. Mannhalter, J. W., H. M. Wolf, H. Gadner, M. Potschka, and M. M. Eibl. 1991. Cell-mediated immune functions in a patient with MHC class II deficiency. Immunol. Invest. 1991;20:151. Bontron, S., V. Steimle, C. Ucla, M. M. Eibl, and B. Mach. 1997. Two novel mutations in the MHC class II transactivator CIITA in a second patient from MHC class II deficiency group A. Hum. Genet. 99:541. Quan, V., M. Towey, S. Sacks, and A. P. Kelly. 1999. Absence of MHC class II expression in a patient with a single amino acid substitution in the class II transactivator protein CIITA. Immunogenetics 49:957. Cressman, D. E., K.-C. Chin, D. J. Taxman, and J. P.-Y. Ting. 1999. A defect in the nuclear translocation of CIITA causes a form of type II bare lymphocyte syndrome. Immunity 10:163. Linhoff, M. W., J. A. Harton, D. E. Cressman, B. K. Martin, and J. P.-Y. Ting. 2001. Two distinct domains within CIITA mediate self-association: involvement of the GTP-binding and leucine reach repeat domains. Mol. Cell. Biol. 21:3001.