Download Leading to a Mild Immunodeficiency Activator trans

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