in the Class II trans-Activator Leading to a Mild Wojciech Wiszniewski, Marie-Claude Fondaneche, Françoise Le Deist, Maria Kanariou, Françoise Selz, Nicole This information is current as Brousse, Viktor Steimle, Giovanna Barbieri, Catherine of September 26, 2021. Alcaide-Loridan, Dominique Charron, Alain Fischer and Barbara Lisowska-Grospierre J Immunol 2001; 167:1787-1794; ;

doi: 10.4049/jimmunol.167.3.1787 Downloaded from http://www.jimmunol.org/content/167/3/1787

References This article cites 33 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/167/3/1787.full#ref-list-1 http://www.jimmunol.org/

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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. Mutation in the Class II trans-Activator Leading to a Mild Immunodeficiency1

Wojciech Wiszniewski,*‡ Marie-Claude Fondaneche,* Franc¸oise Le Deist,* Maria Kanariou,§ Franc¸oise Selz,* Nicole Brousse,† Viktor Steimle,ʈ Giovanna Barbieri,¶ Catherine Alcaide-Loridan,¶ Dominique Charron,ʈ Alain Fischer,* and Barbara Lisowska-Grospierre2*

The expression of MHC class II molecules is essential for all Ag-dependent immune functions and is regulated at the transcrip- tional level. Four trans-acting control the coordinate expression of MHC class II molecules: class II trans-activator (CIITA), regulatory factor binding to the X box (RFX)-associated ; RFX protein containing ankyrin repeats, and RFX5.

In humans, defects in these result in MHC class II expression deficiency and cause combined immunodeficiency. Most Downloaded from patients with this deficiency suffer from severe recurrent 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 blasts, fibroblasts, EBV 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. http://www.jimmunol.org/ 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.

he expression of HLA class II molecules is essential for trans-activator (CIITA),3 regulatory factor binding to the X box Ag-specific immune responses and is very tightly regu- (RFX) protein containing ankyrin repeats, RFX5, and RFX- by guest on September 26, 2021 T lated at the transcriptional level. The impairment of both associated protein (6–9). constitutive and IFN-␥-induced HLA class II expression is In a clinical survey of 30 patients, MHC class II deficiency characteristic of MHC class II immunodeficiency, an autosomal resulted in combined T and B cell immunodeficiency, with an early recessive disorder. It results from the defective of onset and an average life expectancy of 4 years (10). Bone marrow all MHC class II genes (1, 2). Since this disease was first transplantation was proposed as the only curative treatment (11) described, ϳ70 patients, in 50 families, have been reported. due to the very poor prognosis of most patients (despite appropri- Four major complementation groups (A, B, C, and D) have been ate medical care). We report herein an unusual MHC class II deficiency phenotype described by analyzing B cell lines from patients and experi- in three affected siblings. Two siblings, now 21 and 22 years old, mental mutant HLA-deficient cell lines (3–5). The genes re- are mildly affected, and the third, who is 24 years old, is asymp- sponsible for this deficiency encode the proteins that coordi- tomatic. However, apart from residual HLA-D staining in PBMC nately control MHC class II locus expression; these are class II and rare HLA-DR-positive dermal macrophages, HLA class II ex- pression 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 *Unite´ 429 and †Department d’Anatomie Pathologique, Hoˆpital Necker, Paris, France; ‡Department of Genetics, Mother and Child Institute, Warsaw, Poland; §De- the defect in bare syndrome (BLS) complementation partment of Immunology-Histocompatibility, Aghia Sophia Children’s Hospital, Ath- group A. This homozygous L469P substitution in the coding re- ¶ ens, Greece; Institut National de la Sante´et de la Recherche Me´dicale Unite´396, gion of the CIITA cDNA was shown to be responsible for defec- Institut Biomedical des Cordeliers, Paris, France; and ʈHans-Spemann Laboratory, Max Planck Institut fu¨r Immunbiologie, Freiburg, Germany tive expression of MHC-II. 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 Materials and Methods charges. This article must therefore be hereby marked advertisement in accordance Cell culture: proliferation of mitogen- and Ag-induced blasts with 18 U.S.C. Section 1734 solely to indicate this fact. Isolation of PBMC, mitogen-, Ag-, and allogenic cell-induced lymphocyte 1 This work was supported by the Institut National de la Sante´et de la Recherche proliferation and MLR were conducted as previously described (12). EBV Me´dicale. 2 Address correspondence and reprint requests to Dr. Barbara Lisowska-Grospierre, Institut National de la Sante´et de la Recherche Me´dicale Unite´429, Hoˆpital Necker 3 Abbreviations used in this paper: CIITA, class II trans-activator; RFX, regulatory Enfants-Malades, 149 rue de Se`vres, 75743 Paris Cedex 15, France. E-mail address: factor binding to the X box; LCD, leucine-charged domain; LRR, leucine-rich repeat; [email protected] GFP, green-fluorescent protein; BLS, ; WT, wild type.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 1788 MUTATION IN THE CIITA LEADING TO A MILD IMMUNODEFICIENCY

B cell lines and SV40-transformed skin fibroblasts were obtained and cul- Mutagenesis tured as described previously (4). Fibroblasts, or their heterokaryons, were treated by IFN-␥ (Genex, 250 and 500 IU/ml) for 48 h before analyzing Mutagenesis of pIRES- and pEGFP-WT-CIITA vectors was conducted by MHC class II expression. DLD1 is a gut epithelial cell line, which was use of the Transformer Site-Directed Mutagenesis kit (Clontech Labora- donated by Dr. N. Cerf-Bensussan. RJ 2.5.5 is a CIITA-deficient variant of tories, Palo Alto, CA), according to the manufacturer’s instructions. The Ј Ј the Raji cell line. ABL, SJO, and ZM are EBV-transformed cell lines from primers used were: F, 5 -CAG GAT CTG CCC TTC TCC CTG-3 ; and R, Ј Ј MHC-II-deficient patients from the B, C, and D complementation groups, 5 -CAG GGA GAA GGG CAG ATC CTG-3 . The pIRES- and pEGFP- respectively (5). The RC SV40-transformed fibroblast cell line was estab- CIITA-L469P clones were sequenced before the transfection experiments. lished from another CIITA-deficient patient (4). Transfections Immunofluorescence Transfection experiments with wild-type (WT) and mutated CIITA vectors The anti-HLA Abs used were anti-class HLA II (-DR, -DQ, -DP) clone were conducted as previously described (5). The plasmids used were IQU9 (BioDesign, Carmel, NY), and the anti-class I Ab was W6/32 (Sera- pIRES-WT-CIITA, pIRES-L469P-CIITA, pEGFP-WT-CIITA, pEGFP- Lab, Crowley, U.K.). The HLA-II isotype-specific mAbs were: anti-DR L469P-CIITA, pEGFP-CIITA-MT1 (15) and the corresponding empty vec- L243 IgG2a (BD Biosciences, San Jose, CA) or L112, anti-DP L227; anti- tors, pIRES-neo and pEGFP (both from Clontech). EGFP-CIITA is an DQ Genox or L2. The anti-CIITA Abs were IgG1 clone 7-1H (R&D Sys- N-terminal fusion of EGFP to the second in-frame ATG of the gene en- tems, Minneapolis, MN). Ab binding was revealed by incubation with an coding CIITA. Stable pIRES-WT-CIITA and -L469P-CIITA transfectants anti-mouse Ig coupled to FITC (Immunotech, Luminy, France). Anti-DR were analyzed 2–6 wk after transfection. L243 mAb, directly coupled to FITC (BD Biosciences) was also used. CIITA protein sequence homologies Anti-CD4, -CD8, -CD14, -CD19, and -CD25 have been described else- where (12). PBMC, B cells, untreated and IFN-␥-induced fibroblasts, and Sequences homologous to CIITA were identified through BLASTp and different stable transfectants were stained in suspension and analyzed with tBLASTN (16) searches with aa 400–600 of human CIITA in the nonre- Downloaded from a BD Biosciences cytofluorograph. Fibroblasts transfected with pEGFP dundant databases of the National Center for Biotechnology Information. vectors were fixed after transfection with 0.1% glutaraldehyde for 48 h and Multiple sequence alignments were performed with CLUSTALW 1.8 2% formaldehyde in PBS for 5 min, permeabilized with cold (Ϫ20°C) (BCM Search launcher) and rendered with BOXSHADE (Swiss EMBnet). 100% methanol for 5 min, stained with 4Ј,6Ј-diamidino-2-phenylindole Accession numbers: HSCIITA, emb͉X74301.1͉ (6); Mus musculus CIITA and analyzed with a Leitz Ortoplan microscope. mRNA, gb͉U60653.1͉ (17); Rattus norvegicus MHC class II trans-activa- tor, gb͉AF251307.1͉AF251307 (18); Homo sapiens 19 clone Somatic complementation analysis CTD-3022G6, gb͉AC008753.8͉; H. sapiens NOD2 protein (NOD2), ref͉NM_022162.1͉ (19); H. sapiens caspase recruitment domain 4 (NOD1/ http://www.jimmunol.org/ 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, CARD4), gb AF298548.1 (20, 21); H. sapiens caspase recruitment domain B, C, and D, respectively, and fibroblasts RC (group A) and ZM (group D) protein 7 mRNA, AF298548 (22). were used to obtain transient heterokaryons, as previously described (4). KER B cell lines (13, 14) from the patients were also used. Phenotypic Results complementation was tested by immunofluorescence 48–72 h after cell Clinical and immunological investigations fusion. Fibroblasts were treated with IFN-␥ for 48 h before immunofluo- rescence analysis. MHC class II deficiency was detected in siblings SaE, SaM, and SaA at 15, 12, and 11 years of age, respectively. The patients are Nucleic acid analysis of Greek origin and were born to nonconsanguinous parents. Im- by guest on September 26, 2021 RNA extraction and RT-PCR analysis were conducted as previously de- munodeficiency was diagnosed in SaM and SaA (Tables I and II) scribed (4, 5). The CIITA was sequenced according to standard methods. and clinical and biological findings were consistent throughout the PCR products were purified with the Aquick Kit (Qiagen, Chatsworth, 7-year follow-up period. The immune status of SaE was tested CA). The DNA sequence of both strands was determined by Taq polymer- ase cycle sequencing with fluorochrome-labeled dideoxy terminators and because of her sisters’ disease, but she never underwent treatment. resolved by a laser detection system (310 ABI sequencer; Applied Biosys- In addition to the HLA-D expression defect, the siblings had hy- tems, Foster City, CA). poglobulinemia and an absence of Ag-induced in vivo and in vitro

Table I. Clinical history

SaE SaM SaA (Born 1/9/76) (Born 3/11/80) (Born 10/5/79)

Clinical history Several episodes of gastroenteritis in Septicemia at the age of 3 mo Recurrent respiratory infections from infancy early childhood Two episodes pneumonia during Pneumonia at the age of 5 yr Chronic pulmonary infections childhood Recurrent upper respiratory infections since the age of 9 yr 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 Antibiotics Antibiotics (occasional) in childhood IVIG (1990–1995) Chemoprophylaxis with Septrim (1990–1994) IVIG (1990–1995) Left lower lung lobectomy

a HSV, herpes simplex virus; IVIG, intravenous Ig injections every 4 wk. The Journal of Immunology 1789

ϩ Table II. Immunologic investigations observed for Sa siblings was not detected on CD19 B cells with either anti-HLA-DR Abs (Fig. 1e, DR1 and DR2). In contrast, SaE SaM SaA Control monocytes of one 14-year-old RFX protein containing ankyrin re- peats-deficient patient, KhM, displayed a similar residual expression /␮l 3784 4770 6030 1500–4800 of HLA-DQ and -DP on CD14ϩ cells (Fig. 2f). Interestingly, patient CD3 2195 2767 4100 1500–4000 KhM also suffered from a milder form of immunodeficiency. CD3 908 715 1447 900–2900 Skin biopsies from SaM and SaE (Fig. 2, D and E) were studied CD8 1968 2385 3136 600–1900 for HLA-DR expression (left) of dendritic cells identified by anti- CD19 567 620 1145 100–1200 CD1a staining (middle) and macrophages, identified by anti-CD68 CD20 303 382 663 150–920 NK (CD16) 946 668 724 90–950 staining (right), and compared with control skin (top). HLA-DR expression was not detected on patients’ CD1a-postive epidermal IgG (mg/dl) 973 1160 875 970–2000 dendritic cells (Fig. 2, D and G) in contrast to control dendritic IgG1 ND 1080 640 500–1450 cells (Fig. 2A). However, several HLA-DR-positive dermal mac- IgG2 ND 50a 70 80–630 IgG3 ND 100 100 30–200 rophages were detected in patients (Fig. 2, D and G), albeit in IgG4 ND 0 7 20–330 lower numbers than in control biopsies. (Fig. 2A). Similar findings, IgM 79 47 72 70–370 i.e., HLA-DR expression of dermal macrophages, were observed b b IgA 276 5 4 60–330 in skin of two other patients ages 21 and 14 years, both with a mild clinical presentation and belonging to complementation group B Abs to Candidac Absent Absent ND 1/160–1/3200 (C. Picard, W. Wiszniewski, M. C. Fondaneche, V. Pinet, F. Le Downloaded from Streptococcus IgG: 140 Absent ND 100–800 Deist, S. Blanche, J. F. Eliaou, J. L. Casanova, A. Fischer, and B. pneumoniaed IgM: Ͻ50 Lisowska-Grospierre, unpublished observations). Haemophilus 22% Absent ND 10–30% influenzaee Classification of HLA class II deficiency in complementation Proliferation group A in the Sa family f ϫ 3 THY cpm 10 B cell somatic fusions induced the expression of HLA-class II in http://www.jimmunol.org/ PHA 51 48 59 25–80 Tetanus 3 1 ND 10–30 heterokaryons between ABL, SJO, and ZM cells, belonging to the complementation groups B, C, and D, respectively, in SaE. Het- a Low level. b Very low level. erokaryons between SaE and RC fibroblasts from group A were c Indirect hemagglutination. HLA class II negative. Somatic complementation was also ob- d ELISA test (EIU). tained between B cells from SaE and patient KER (14), who did e Competition assay. f Thymidine incorporation test. 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). by guest on September 26, 2021 immune responses after immunization. Serum Abs to common CIITA mutation germs (Streptococcus pneumoniae and Haemophilus influenzae) were detected in SaE. A minor CD4 lymphopenia was detected in The CIITA mRNA from EBV cell lines from Sa patients was am- SaA ,whereas her sisters had normal CD4 T cell counts. Patients plified by RT-PCR and analyzed by sequencing. A single homozy- SaM and SaA were treated symptomatically, and a prophylactic gous T1524C mutation, causing a leucine to proline substitution at treatment with intravenous Ig was then started and has been con- position 469 (L469P), was found in all three patients. This muta- tinued since for sibling SaM. SaM became asymptomatic and has tion was confirmed in the genomic level (Fig. 3). A heterozygous remained so for the last 3 years, as is the eldest sibling, SaE, mutation was found in the mother. No cells were available from without treatment. Currently, both SaM and SaE refuse to be fol- the father. No other were found in the entire 4.5-kb lowed by the immunology department. No information is available CIITA cDNA. on the follow-up of SaA. Functionional analysis of the L469P CIITA Membrane expression of HLA-DR, -DQ, and -DP molecules in The CIITA-negative epithelial cell lines DLD1 and HeLa, the family Sa and in other MHC-II-deficient patients CIITA-deficient Burkitt lymphoma cell line RJ2.2.5, and the fi- Defective expression of HLA-DR, -DQ, and -DP molecules was broblast cell line RC were stably transfected with the expression observed by immunofluorescence analysis in all three patients vector pIRES containing WT-CIITA or L469P-CIITA and tested (Fig. 1 and data not shown). In resting PBMC (Fig. 1a), B cells for HLA-DR expression. Sixty percent of the DLD1 and HeLa (CD19 panels) and monocytes (CD14 panels) were faintly stained cells transfected with WT-CIITA expressed HLA-DR (Fig. 4 b and with anti-HLA-DR mAbs L243 and L112. HLA-DQ was weakly e), whereas only 0.5% of cells transfected with the empty vector expressed on B cells and monocytes, and HLA-DP was faintly were DRϩ (Fig. 4, a and d). From 1 to 4% of the DLD1 and HeLa detected on monocytes (Fig. 1a). PHA-induced T cell blasts were L469P-CIITA transfectants were DRϩ cells (Fig. 4, c and f). Of the HLA-DR, -DQ, and -DP negative (Fig. 1b), as were CD4 and CD8 RJ2.2.5 cells transfected with the same vectors, 80% of the WT MLR-induced blasts (Fig. 1c). In contrast, PHA and MLR blasts transfectants became HLA-DRϩ (Fig. 4h), whereas 14% of the L468P expressed CD25 normally (shown for MLR blasts (Fig. 1c)). EBV transfectants expressed HLA-DR (Fig. 4i). Ninety-six percent of the B cell lines from SaE and SaM did not express HLA-DR, -DP, or RC cells transfected with WT-CIITA expressed HLA-DR at a mean -DQ (Fig. 1d), although FACS staining revealed low, but detect- fluorescence intensity of 1 ϫ 10Ϫ4 (Fig. 4k). Thirty-one percent of able, levels of HLA-DQ and -DP expression on fresh B CD19 cells transfected with L469P-CIITA displayed HLA-DR staining, cells. although at a mean fluorescence intensity of between 10- and 1000- In another BLS patient, HeJ (BLS complementation group A) fold lower (Fig. 4l). Western blots of total cell lysates from transfected with a severe clinical phenotype, the residual HLA-DR expression fibroblasts were used to assess the level of transgene expression and 1790 MUTATION IN THE CIITA LEADING TO A MILD IMMUNODEFICIENCY Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

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. showed that it was even higher for the L469P CIITA protein than for proteins, we identified CIITA-homologous sequences through the WT-CIITA (not shown). Blast searches and performed multiple sequence alignments of the We assessed intracellular expression of L469P CIITA by trans- corresponding region with the three known CIITA sequences (hu- fecting the RC CIITA-deficient fibroblasts with pEGFP-WT- man, mouse, rat) and four homologous sequences (NOD1/CARD4, CIITA and pEGFP-L469P-CIITA and comparing the expression NOD2, CARD7, and sequence AC008753). Fig. 6 shows the part pattern with that of the experimental cytoplasmic retention mutant, of CIITA containing both the P loop region (positions 420–427), MT1-CIITA (15). A preferentially nuclear localization of CIITA the Mg2ϩ coordination region (461DAYG464), and the LCD motif was observed in both the WT- and L469P-CIITA transfectants (23, 24). Whereas the P loop region is very highly conserved (Fig. 5, top and middle). An exclusively cytoplasmic CIITA stain- among all sequences, the DxxG motif in human CIITA is only ing was detected in the pEGFP-MT1-CIITA transfectants, as pre- partly conserved in mice and rats and not at all in the other CIITA- viously reported (Fig. 5, bottom, and Ref. 15). homologous sequences. The LxxLL motif is also highly conserved among the different sequences with an acidic amino acid at posi- L469 is conserved in homologous CIITA proteins tion 3 (D/E). The most highly conserved amino acid is L468, L469 is part of a so-called leucine-charged domain (LCD); which is invariant among all sequences. Residue L469 is also 465LQDLL469) conforming to the consensus LxxLL. Replace- highly conserved with only two conservative exchanges of the ment of all theree leucines by alanine severely impaired CIITA leucine to isoleucine (Fig. 6). function (23). Recently, several sequences were discovered that contained homologies to both the nucleotide-binding domain and Discussion the C-terminal leucine-rich repeats (LRRs) of CIITA (19–22). To The MHC class II deficiency disease has been described as being test whether the region containing L469 is conserved among these lethal in childhood, with a mean survival age of 5 years, despite 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). Downloaded from http://www.jimmunol.org/

appropriate medical care. We describe herein three affected sib- ertheless, the main immunological manifestations of MHC class II lings who are still alive at 21, 22, and 24 years of age. The eldest expression defect were, with one exception, similar to those re- patient suffered very few infections in early childhood and since ported in other cases of MHC II deficiency, namely, IgG2 and IgA then has been asymptomatic without any treatment. Her sisters and the absence of Ag-induced re- suffered from mild immunodeficiency. None of them was prone to sponses in vivo and in vitro. Unexpectedly, in contrast to most by guest on September 26, 2021 opportunistic infections. These patients do not suffer from the pro- other MHC II-deficient patients who are T CD4 lymphopenic, CD4 tracted that affects most MHC II-deficient patients. Nev- cell counts were normal in SaE and SaA and only slightly lower in

FIGURE 3. CIITA mutation. Top, CIITA coding re- gion. Positions of the LCD 1 and 461DAYG465 (GTP- binding) motifs and the L469P substitution are indicated. Bottom, Control and SaE CIITA DNA se- quences. The CIITA sequences for the other Sa siblings were identical. TF, Transcription activation factors; NLS, nuclear localization sequence. 1792 MUTATION IN THE CIITA LEADING TO A MILD IMMUNODEFICIENCY

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 CIITA- deficient 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 immunode- ficiency, with severe clinical consequences (10). In one case, pa- tient 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 3Ј 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 Downloaded from 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 FIGURE 4. HLA-DRA expression in different cell types stably trans- fected with the pIRES WT- and L469P-CIITA vectors. HeLa cells (a–c), with alanines, was unable to drive transcription from the DR-X1- DLD1 epithelial cells (d–f), CIITA-deficient RJ 2.5.5 (g–i), and RC fibro- X2-Y or -W-X-Y promoters (23). Recently, several sequences blasts (j–l) were transfected with an empty pIRES vector, WT-CIITA showing homology to the nucleotide binding and LRR regions of http://www.jimmunol.org/ pIRES, or L469P-CIITA pIRES, as indicated in each panel. FITC-coupled CIITA have been published (19–22). A search for protein se- mAb 243 anti-HLA-DR Ab was used. Isotypic controls are not shown. quence homologies revealed that the LCD motif containing L469 FSC, Forward scatter. 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 SaM. This suggests that in the three Sa patients the MHC-II ex- proteins (Fig. 6). It also shows that other human proteins share this pression defect has led to less severe consequences on T cell dif- motif. In the CIITA gene of the Sa family, only leucine 469 was ferentiation than observed in other patients. replaced (by proline), indicating the importance of this motif The HLA-II molecule expression defect in the Sa siblings was in vivo. by guest on September 26, 2021 found in B lymphocytes, monocytes, IFN-␥-induced fibroblasts, Functional analysis revealed that the L469P allele of CIITA is dendritic cells, and T and B cell blasts. However, faint but detect- not completely inactive. Stable transfection of DLD1 or HeLa cells able HLA-DR, -DQ and -DP staining was observed on B cells and with the L469P-CIITA cDNA did not lead to the trans activation monocytes. Similarly, there was faint but detectable HLA-D stain- of MHC-II genes, but we observed a residual trans activation po- ing on PBMC from KhM, another MHC-II-deficient patient from tential of L469P-CIITA in RC and in RJ2.2.5 cells (Fig. 4, i and l). complementation group B suffering from a milder form of immu- In the patient-derived, CIITA-deficient RC fibroblasts transfected nodeficiency (Fig. 1f). In contrast, B cells from HeJ, a CIITA- with L469P-CIITA, DR expression was restored in 30% of cells, deficient patient with a severe immunodeficiency, were HLA-DRϪ albeit at a much lower level than that observed in WT-CIITA (Fig. 1e). Therefore, it appears that a residual HLA-D staining of transfectants. In the RJ2.5.5 B cell line, which has genomic dele- PBMC correlates with less severe clinical symptoms. We have no tions of the CIITA gene (6), transfection with L469P CIITA led to explanation why there was no residual HLA-D expression on any an abnormally low but clearly detectable HLA class II expression blast types. Although mitogen and MLR stimulation led to blas- in 14% of the cells. The mutated CIITA alleles from patients togenesis and the expression of CD25 on both CD4 and CD8 T cell BLS-2 (⌬940–963) and BCH (BCH-1 ⌬1079–1106; BCH-2 blasts (shown for SaA, Fig. 1c), in both patients Sa and HeJ T and E381Stop) had been tested functionally in RJ2.2.5 earlier. None of B cell blasts were HLA-II negative (Fig. 1, b, c, and e). However, these alleles, which were derived from patients with severe immu- in patients Sa, in addition to PBMC, some dermal macrophages nodeficiency, led to residual HLA class II expression in RJ2.2.5 (6, were HLA-DRϩ, although dermal dendritic cells were not. 32). Thus, partial HLA-DR expression in the RJ2.5.5 and RC MHC II-deficient patients with mild clinical presentation have transfectants shows that the expression of the L469P-CIITA cDNA previously been reported, e.g., the 7-year-old KER twins. Their T allows partial trans activation of MHC II genes. This probably cells were able to respond in vivo to antigenic challenge (13, 14). corresponds to the residual MHC class II expression detected on Although the molecular basis of the MHC II defect was not elu- fresh PBMC from the patients. cidated in these twins, the Sa patients do not share the same de- The immunofluorescence data suggest that the recombinant fects, because their cells complemented the KER cell line for L469P-CIITA protein can translocate into the nucleus (Fig. 5). MHC-II expression. This result is confirmed by Western blotting, which revealed the Complementation experiments by somatic cell fusion assigned presence of full length L469P-CIITA protein in nuclear extracts the Sa family defect to complementation group A, indicating that (G. Barbieri, T. Prod’homme, J. Vedrenne, B. Lisowska-Grospi- the CIITA gene was affected in cis. CIITA encodes a 1130-aa erre, D. Charron, and C. Alcaide-Loridan, manuscript in prepara- protein, the N-terminal region of which acts as a transcriptional tion). Therefore, the L469P mutant is the first loss-of-function mu- activator and the C-terminal region of which provides MHC-II tant that retains the ability to translocate into the nucleus. promoter specificity (6, 25, 26). CIITA controls both constitutive 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 -MT1- CIITA, stained with 4Ј,6Ј-diamidino-2-phenylindole 48 h later, and analyzed. Left, GFP fluorescence; right, nuclear staining. Downloaded from http://www.jimmunol.org/

with the conversion of CIITA to the GDP-bound state leads to its immunodeficiency than that affecting other patients. Residual HLA exclusion from the nucleus (24). The mutation (proline 469) is class II expression was not observed on fresh B cells from two very close to the 461DAYG465 motif and it may therefore be other CIITA-deficient patients, patient HeJ (Fig. 1e) and patient by guest on September 26, 2021 informative to test the GTP binding of mutated CIITA, even if BCH (31). However, a milder immunodeficiency associated with a nuclear exclusion is not observed in transfectants. The LCD1 motif residual HLA class II expression has been described for the Ker/ mutated in Sa CIITA conforms to the short LxxLL consensus of a Ken twins (14) and is shown here for a patient KhM from BLS putative nuclear export sequence, but the fact that the L469P mu- group B (Fig. 1f). An unrelated patient with MHC II deficiency tant protein is found in the nucleus argues against such a function caused by a CIITA defect presenting similarities with the Sa pa- for this sequence. The role of neither the LCD nor the GTP-bind- tients has been described (33). This patient was not diagnosed until ing motif (24) or of a recently shown (35) interaction between the the age of 27 years, well beyond the life expectancy of most BLS GTP-binding region (residues 336–702) and the C-terminal patients. Clinical and immunological data for the patient have not leucine-rich region, LRR (15), are well understood. Thus, further been reported. Interestingly, a single amino acid substitution, studies on the L469P CIITA displaying these unusual features will F962S, was found in the coding region of CIITA (33). The patients contribute to our understanding of the mechanisms that govern we describe are the first in which a mild phenotype of the disease nuclear translocation and transcriptional activation of CIITA. can be correlated with a residual trans activation potential of the Although it cannot be formally proved, it is tempting to specu- mutated regulatory factor (Fig. 4). It can be assumed that the en- late that the residual HLA class II expression in cells from the Sa suing residual HLA class II expression in the patients is respon- siblings and indeed the residual trans activation potential of the sible for a substantial T cell differentiation and the capacity to L469P allele of CIITA are responsible for the lesser severity of the 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), Mg2ϩ 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. 1794 MUTATION IN THE CIITA LEADING TO A MILD IMMUNODEFICIENCY the patients did not develop the protracted diarrhea that affects marrow transplantation in major histocompatibility complex class II deficiency: most patients with MHC II deficiency may be a consequence of a single-center study of 19 patients. Blood 85:580. 12. Le Deist, F., G. Thoenes, J. Corado, B. Lisowska-Grospierre, and A. Fischer. residual MHC II expression in intestinal epithelium, like that 1991. Immunodeficiency with low expression of the T cell receptor/CD3 com- in PBMC. plex: effect on T cell activation. Eur. J. Immunol. 21:1641. 13. Wolf, H. M., I. Hauber, H. Gulle, V. Thon, H. Eggenbauer, M. B. Fischer, These observations on MHC II deficiency in the Sa family have S. Fiala, and M. M. Eibl. 1995. Twin boys with major histocompatibility complex important medical implications. They show that an asymptomatic class II deficiency but inducible immune responses. N. Engl. J. Med. 332:86. clinical course or an attenuated clinical phenotype can be observed 14. Hauber, I., H. Gulle, H. M. Wolf, M. Maris, H. Eggenbauer, and M. M. Eibl. 1995. Molecular characterization of major histocompatibility complex class II in patients with a profound defect in the expression of HLA class and demonstration of antigen-specific T cell response indicate a II genes. Therefore, in patients with mild symptoms of immuno- new phenotype in class II-deficient patients. J. Exp. Med. 181:1411. deficiency, an inherited MHC II expression defect should be con- 15. Hake, S. B., K. Masternak, C. Kammerbaouer, C. Janzen, W. Reith, and V. Steimle. 2000. CIITA leucine-rich repeats control nuclear localisation, in vivo sidered. In CIITA-deficient patients, residual HLA-DR expression recruitment to the MHC class II enhanceosome, and MHC class II gene trans- in peripheral blood leukocytes might be of prognostic value. When activation. Mol. Cell. Biol. 20:7716. 16. Altschul, S. F., T. L. Madden, A. A. Schoffer, J. Zhang, Z. Zhang, W. Miller, and such residual HLA-DR expression is detected and coincides with D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of pro- an absence of severe infections, bone marrow transplantation tein database search programs. Nucleic Acids Res. 25:3389. should not be recommended. However, even those MHC II-defi- 17. 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 cient patients whose clinical status is good should be kept under with the human class II transactivator. Immunogenetics 45:220. close medical surveillance because late onset immunodeficiency 18. Mori-Aoki, A., M. Pietrarelli, M. Nakazato, P. Caturegli, L. D. Kohn, and can be fatal. K. Suzuki. 2000. Class II transactivator (CIITA) suppresses transcription of thy- roid-specific genes. Biochem. Biophys. Res. Commun. 11:278:58. 19. Ogura, Y., N. Inohara, A. Benito, F. F. Chen, S. Yamaoka, and G. Nunez. 2001. Downloaded from Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates Acknowledgments NF-␬ B. J. Biol. Chem. 276:4812. We thank the staff of the First Department of Pediatrics of the University 20. Bertin, J., W. J. Nir, C. M. Fischer, O. V. Tayber, P. R. Errada, J. R. Grant, of Athens, especially X. Nikolaidou. We thank E. Jouanguy for expert J. J. Keilty, M. L. Gosselin, K. E. Robison, G. H. Wong, M. A. Glucksmann, and advice; V. Pinet for conducting the in vitro induction assays; J.-F. Eliaou 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. and P. Louis-Plence for helpful discussions; C. Harre´, C. Jacques, and O. 21. Inohara, N., T. Koseki, L. del Peso, Y. Hu, C. Yee, S. Chen, R. Carrio, J. Merino,

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