Supplementary Table 1. Immunological Characteristics of Patients
Patie nts
P1.1
P1.3
P1.4
Age (years)
Leukocyte
a
counts
Polynuclear
a
counts
Monocyte
a
counts
Lymphocyte
a
counts
T
a
lymphocytes
(CD3)
CD4
a
lymphocytes
CD45RO/CD4
(%)
CD8
a
lymphocytes
CD45RO/CD8
(%)
B
a
lymphocytes
(CD19)
CD27/CD19
(%)
a
NK cells
(CD16/CD56)
PHA-T cell
b
Proliferation
c
IgG
8
7500
(4400-9500)
3900
(1900-8000)
225
(200-600)
3300
(1900-3700)
2740
(1200-2600)
10
5900
(4400-9500)
3700
(1900-8000)
400
(200-600)
1800
(1900-3700)
1556
(1200-2600)
40
n.d
1122
(650-1500)
n.d
306
(650-1500)
n.d
n.d
1650
(370-1100)
n.d
1044
(370-1100)
n.d
n.d
264
(270-860)
216
(270-860)
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
P2.1
P2.2
P3.3
mother
mother
P2.1
P2.2
23
11
36
47
35
5100
4800
4900
4900
11500
(4400-9500) (4400-9500) (4400-9500) (4400-9500) (4400-9500)
2500
2000
2700
3500
8200
(1900-8000) (1900-8000) (1900-8000) (1900-8000) (1900-8000)
500
400
600
200
500
(200-600)
(200-600)
(200-600)
(200-600)
(200-600)
2100
2400
1600
1200
2800
(1000-2800) (1900-3700) (1000-2800) (1000-2800) (1000-2800)
1174
1962
1066
633
2322
(700-2100) (1200-2600) (700-2100)
(700-2100) (700-2100)
661
(460-1230)
26
(16-34)
687
(190-850)
17.6
(2.6-22.9)
517
(110-570)
646
(650-1500)
12.2
(13-30)
1396
(370-1100)
26.5
(4-21)
125
(270-860)
677
(460-1230)
n.d
599
(190-850)
n.d
67
(110-570)
40.3
n.d
15.6
13.7
62.1
(10-50)
(10-50)
(10-50)
(10-50)
297
28
n.d
436
51
215
(100-480)
(100-480)
(92-420)
(100-480)
(92-420)
88
119
n.d
n.d
n.d
n.d
(>40)
(>40)
12.8
4.17
2.5
11.7
9.71
6.04
(5.7-13.2)
(5.7-13.2)
(9.2-14.8)
(9.2-14.8)
(5.7-13.2)
(9.2-14.8)
c
IgA
0.58
0.08
0.49
2.99
3.42
1.29
(0.65-2.4)
(0.65-2.4)
(1.42-2.62) (1.42-2.62)
(0.65-2.4)
(1.42-2.62)
c
IgM
0.75
2.05
1.38
0.43
1.2
0.27
(0.6-1.75)
(0.6-1.75)
(0.88-1.84) (0.88-1.84
(0.6-1.75)
(0.88-1.84)
Parameters have been checked at least two times and found to be stable over the time.
n.dК:not determined
Age matched control values are indicated in italics in brackets
a
3
Numbers of cells/mm
b
-3
Phytohemagglutinin (PHA)-induced proliferations are expressed as couts per minute x10
c
-1
in mg.ml
464
(460-1230)
42.2
(16-34)
325
(190-850)
11.2
(2.6-22.9)
196
(110-570)
1465
(460-1230)
26.6
(16-34)
890
(190-850)
7
(2.6-22.9)
302
(110-570)
57.9
(10-50)
235
(92-420)
n.d
42.2
(10-50)
87
(92-420)
n.d
n.d
n.d
n.d
n.d
n.d
n.d
Supplementary Figure 1. Expression of SAP in cells from affected individuals of
XLP families without SAP mutations. Cell lysates from PHA blasts of healthy control
individuals (Ctr.), patients (P1.3, P2.1 and P3.1) and a SAP-deficient patient (SAP) were
analyzed by Western blotting with anti-SAP antibodies (upper panel). Immunoblot with anti-Fyn
antibodies (lower panel) is shown as control of loading.
Supplementary Figure 2. Expression of genes surrounding XIAP, THOC2, STAG2 and SAP is
normal in XIAP-deficient patients. The relative expression of THOC2, XIAP, STAG2, SAP and G3PDH was
examined by RT-PCR in lymphocytes from two XIAP-deficient patients (P1.1 and P2.1) and two healthy control
individuals (Ctr.). Human G3PDH RT-PCRs are shown as normalization controls of the samples. Three fold-serial
dilutions of cDNAs (indicated as 1, 0.3 and 0.1) were used for amplification of each gene. Products were resolved
by 1% agarose gel electrophoresis and were detected by ethidium bromide staining.
a
b
c
Supplementary Figure 3. Expression of XIAP in the different hemopoetic lineages (a) and lack of expression of
truncated or altered XIAP proteins in cells from XIAP-deficient patients (b,c). a, Cells were purified as stated in the
“Methods” section. Cell lysates from PBMCs, B cells, CD4 T cells (CD4+), CD8 T cells (CD8+), monocytes (monocytes), dendritic
cells (DC), MAB11 NK T cells (NKT) and NK cells (NK). b and c, Cell lysates of PHA blasts from healthy control (Ctr.) and XIAPmutated (P3.1, P2.1 and P1.3) individuals were electrophoresed in 15% (a) or in 8% (b) SDS-polyacrylamide gels and immunoblotted
with anti-XIAP antibodies raised against the N-terminus of XIAP (H202). Immunoblot with anti-Ku80 antibodies is shown as control
of loading.
a
b
80
% specific lysis
% specific lysis
cont. Ab
anti-2B4
60
anti-CD16
40
20
0
C1
C2
Ctr.
SAP
XIAP
SIM
(n=11)
(n=8)
(n=8)
(n=7)
P1.7
P2.2
SAP1
SAP2
60
40
20
0
c
% of NK cells (PBMC)
15
10
5
0
Supplementary Figure 4. Analysis of NK cells from XIAP-deficient patients. a, The 2B4-mediated cytolytic activity is impaired
in SAP-deficient NK cells (SAP) but not in XIAP-deficient NK cells. Lysis of L1210 target cells by NK cells from healthy control (C1 and
C2), SAP-deficient (SAP1 and SAP2) or XIAP-deficient (P1.7 and P2.2) individuals that have been stimulated by anti-2B4, anti-CD16 or
control antibodies (cont. Ab). b, Lysis of K562 target cells by NK cells from normal (C1, C2 and C3), SAP-deficient (SAP1 and SAP2) or
XIAP-deficient (P1.1 and P2.2) individuals. c, Percentages of NK cells (CD56+/CD3-) in PBMCs of blood samples from Ctr., SAP, XIAP
and SIM individuals. Bars correspond to mean values of each group of NK cell percentages. Values of means  s.d.: Ctr, 6.24.8 ; SAP,
2.62.5 ; XIAP, 3.472.9 ; SIM, 3.23.8. SIM =Sporadic Severe Infectious Mononucleosis.
a
6
b
c
Supplementary Figure 5. Analysis of NKT cells of female XIAP-mutated carriers. a, Comparison of the numbers of NKT cells (V24
TCR+ V11 TCR+) in CD3+ lymphocytes from two female XIAP-mutated carriers (Mothers) and their XIAP-deficient sons (Patients). NKT cells
were analyzed after gating on CD3+ cells by the staining with anti-V24 and anti-V11 TCR antibodies. The XIAP patients tested are P2.1, P2.2.
Relatives are indicated by a trait. Values of means  s.d.: Mothers, 24317.7 ; Patients, 8121. b, c. Non-random X chromosome inactivation in NKT
cells from a female XIAP-mutated carrier. X chromosome inactivation was examined by analyzing the methylation pattern of the human androgenreceptor gene (AR) b, X chromosome inactivation analysis in total blood cells from two female XIAP-mutated carriers and one control healthy female
(Ctr.). Extracted DNA of blood from control (Ctr.) and XIAP-mutated carriers (mothers of P2.1 and P2.2) were digested (+) or not (-) with HpaII, a
methylation-sensitive enzyme. Maternal and paternal polymorphic alleles of AR were then amplified by PCR from digested or non-digested DNAs.
The PCR products were resolved by 5% polyacrylamide gel electrophoresis. The two major products amplified from the non-digested DNAs of the
control and the XIAP-mutated carrier individuals correspond to the maternal and paternal polymorphic AR alleles. They are respectively indicated by
an asterisk and an arrow. In HpaII-digested DNAs from the two XIAP-mutated carriers, the two AR alleles were unequally amplified by contrast to the
non-digested DNAs and to the digested DNA from the control carrier indicating non-random X chromosome inactivation in the two XIAP-mutated
carriers. c, X chromosome inactivation in the different blood cell populations including NKT cells. T lymphocytes (T), B lymphocytes (B), NK cells
(NK), monocytes (Mono) and NKT lymphocytes (NKT) from one female XIAP-mutated carrier (mother of P2.2) were purified by FACS sorting. The
analysis of the X chromosome inactivation was then analyzed in purified populations as stated in b. In HpaII-digested DNAs from the different blood
cell populations, the two AR alleles were unequally amplified by contrast to the non-digested DNAs indicating non-random X chromosome
inactivation.
% of induced apoptosis
a
100
P1.3
C3
P2.1
C1
80
P3.3
C2
60
40
20
0
Anti-CD95 (ng ml-1) :
5
b
10
15
20
5
10
15
5
20
10
15
20
c
80
60
40
20
0
% of induced apoptosis
% of induced apoptosis
100
30
20
10
0
Supplementary Figure 6. Enhanced apoptosis of XIAP-deficient T lymphocytes to apoptotic stimuli. a,
Induced apoptosis of T cell blasts from control healthy (C1, C2 and C3) and XIAP-deficient individuals (P3.1, P2.1 and P1.3)
that were stimulated with various concentrations of anti-CD95 antibodies as indicated. b, Activation-induced cell death (AICD)
of T cells from normal (C) and XIAP-deficient individuals (P). T cell blasts were stimulated with anti-CD3 antibodies and
analyzed for apoptosis. c, Induced apoptosis of T cells stimulated with a trimeric TRAIL molecule. Data are means of
duplicates from one representative experiment.
b
a
40
30
20
10
0
P1.1
60
50
50
% of GFP positives cells
50
GFP positives cells
cells
GFP-positive
% of% of
apoptosis
% %ofofinduced
induced apoptosis
Ctr. 1
Ctr. 2
Ctr. 3
P1.1
60
Anti-CD95 (ng ml-1) :
Ctr.
60
70
40
30
20
10
10
15
20
pTRIP
pTRIP XIAP
pTRIP XIAP + anti-CD95
30
20
10
0
5
40
0
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Anti-CD95 (ng/ml)
Time (days)
Time (days)
Time (days)
Time (days)
c
Supplementary Figure 7. Expression of wild-type XIAP in XIAP-deficient EBV-transformed B cells allows their selective survival
and expansion upon anti-CD95 stimulation. a, Enhanced apoptosis of XIAP-deficient EBV-transformed B cells to anti-CD95 apoptotic stimuli.
EBV-transformed B cell lines derived from three healthy control individuals (Ctr.) and a XIAP-deficient patient, P1.1 (XIAP) were stimulated with
various concentrations of anti-CD95 antibodies as indicated, and analyzed for apoptosis. b, EBV-transformed B cell lines from healthy control (Ctr.) and
XIAP-deficient (XIAP) individuals were infected with the pTRIP-GFP-XIAP vector or the empty vector pTRIP-GFP. Infected cells with pTRIP-GFPXIAP were then repeatedly stimulated with anti-CD95 (100ng/ml) during the culture as indicated by the arrows. The percentages of GFP-positive cells
were determined by flow cytometry during the culture. c, Reconstituted expression of XIAP in XIAP-deficient EBV-transformed B cells. Cell lysates
from control (Ctr.) and XIAP-deficient (XIAP) EBV-transformed B cells at day 25 of culture, infected with pTRIP-GFP, pTRIP-GFP-XIAP and treated
or not with anti-CD95, or non-infected (/) were immunoblotted with anti-XIAP antibodies or anti-Ku80 antibodies as control of loading.