Transcript Human ICOS Deficiency – model to study Common variable

Molecular Defects leading to
defective humoral immunity
Bodo Grimbacher, M.D.
Dept. Rheumatology and Clinical Immunology
Freiburg, Germany
[email protected]
Possible causes of CVID
Block in B cell
Development
Circulating
B cells
autoantibodies
Disturbed
TH-cell regulation
Defective
glycosylation
or rapid turnover
Burmester, Pezzutto:
Taschenatlas der Immunologie
Plasma cell
Molecular Defects in CVID
The CD19 deficiency
The BAFF receptor deficiency
The TACI deficiency
The ICOS deficiency
Dual antigen recognition
Antigen
C3d
CD19
CD81
CD21
Leu-13
y
y
y
y
B-cell receptor
y
y
y y
y
CD19-complex
Van Noesel CJ et al. (1993) Immunol Today 14:8-11
CVID Family R006OI
Clinical and Laboratory Presentation of Disease: Patient III:13
39 yo, male
Referral: pneumologist due to recurrent pneumonias
Dx of CVID at age 35
Age
Clinical manifestations
7
Onset of infections: URTI (otitis, sinusitis, tonsillitis)
18
Pneumonia
34
Surgery for chronic sinusitis
35
2 pneumonias (1 with pleural effusion)
35
Bronchopneumonia and chronic bronchitis
35
Conjunctivitis
37
Chronic gastritis with intestinal metaplasia (H. pylori).
• IgG :
204 mg/dl (804-2212)
• IgM:
46.6 mg/dl (58-324)
• IgA:
17.6 mg/dl (99- 489)
Patient III:15 and Patient III:16
37 yo, female
Dx of CVID at age 33
52 yo, female
Dx CVID at age 49
Age
Diagnosis
Age
6
otitis, tonsillitis, pharyngitis, laryngitis
5
12
Tonsillectomy
15
Chickenpox
20
Chronic sinusitis
8
Pneumonia
20
Pneumonia
12
Chronic diarrhea
20
Chronic bronchitis
20
Chronic bronchitis
25
Surgery chronic sinusitis
41
Surgery for chronic otitis and sinusitis
30
Herpes zoster infection
…
Recurrent bacterial conjunctivitis
30
Rec. bacterial conjunctivitis
…
Chronic gastritis
34
Frequent diarrheas
• IgG: 198 mg/dl (814-2047)
• IgM: 29.6 mg/dl (68-379)
• IgA: 6.7 mg/dl (99-489)
7-9
Diagnosis
otitis, tonsillitis, pharyngitis, laryngitis
erysipelas
Multiple skin abscesses (neck)
• IgG: 256 mg/dl (814-2047)
• IgM: 63 mg/dl (42-600)
• IgA: 18.9 mg/dl (81-538)
CD19 Expression at Diagnosis
DNA Sequencing of CD19 in Patients and Family Members
CD19 Deficiency in Family R006OI
B Cell Markers in CD19 deficient Patients and Carriers
Peripheral Blood B cells in Patients and Carriers
Morphology and Cellularity of Tonsils in CD19 deficient Patient
Morphology and Cellularity of Tonsils in CD19 deficient Patient
CD3
CD79a
CD20
BCl-2
CD5
Ki67
CD19 Deficiency: Phenotype
• Recurrent Infections: mainly RT (U/L), other less frequent.
• Low IgM, IgG and IgA
• Low numbers of isotype switched IgD-CD27+ B cells
• Normal architecture and cellularity of secondary lymphoid organs,
high proliferation in GCs.
• Normal T lymphocyte responses to mitogens, variable to antigens
• Normal B cell proliferation in response to SAC and IgM
• Normal Ca++ influx
• Vaccination responses to neo-antigen (rabies) pending
• 2nd family from Turkey (consanguineous):
• Earlier onset (7-12 y/o), persistent IgA
• Abnormal B cell proliferation to SAC and IgM
• Abnormal Ca++ flux studies
• Different mutation in the intracellular region
University of Freiburg:
Cristina Woellner
Dörte Thiel
Ulrich Salzer
Michael Schlesier
University of Antioquia:
Jose-Luis Franco
Diana Castaño
Pablo Javier Patiño G
Julio César Orrego
Carlos Julio Montoya
Claudia Rugeles
Beatriz Vieco
University of Rotterdam:
Jacques J.M. van Dongen
Menno C. van Zelm
Mirjam van der Burg
Carel J. van Noesel
Selçuk University:
Ismail Reisli
Molecular Defects in CVID
The CD19 deficiency
The BAFF receptor deficiency
The TACI deficiency
The ICOS deficiency
TNF receptor : ligand systems
Nature Reviews Immunology
2, 465 -475 (2002);
Fabienne Mackay &
Jeffrey L. Browning
BAFF: A
FUNDAMENTAL
SURVIVAL
FACTOR FOR
B CELLS
Role of BAFF(R) in the life of a B cell
B cell
B cell
Nature Reviews Immunology 2, 465 -475 (2002);
Fabienne Mackay & Jeffrey L. Browning
BAFF: A FUNDAMENTAL SURVIVAL FACTOR FOR B CELLS
Human BAFFR Deficiency
Clinical Data:
BAFF-R deficient family
*1929
*1934
*1937
*1940
*1942
(g/l)
*1928
Gender: male
Occupation: farmer
Age of onset: 37(?)
Age of diagnosis CVID: 57
Infections: recurr. pneumonias,
Rhizopus infection of oral cavity
Splenomegaly: no
Lymphadenopathy: no
Autoimmunity: no
*1967
*1969
*1972
8
6
4
2
Normal
range
0
IgM
IgG
Serum Immunoglobulins
IgA
In Frame deletion of 24bp causes BAFF-R Deficiency
Genomic Sequence
Exon 3
RT-PCR
Exon2
HD
Patient
(CVID 5)
Site of deletion
(exon 2:128-152)
Exon1
Deletion affects transmembrane region
BAFFR PROTEIN
HD
HLR
1MRRGPRSLRG
RDAPAPTPCV PAECFDLLVR HCVACGLLRT
1MRRGPRSLRG RDAPAPTPCV PAECFDLLVR HCVACGLLRT
HD
HLR
41
PRPKPAGASS PAPRTALQPQ ESVGAGAGEA ALPLPGLLFG
41PRPKPAGASS PAPRTALQPQ ESVVAGAGEA ALPLPGLLFG
HD
HLR
81
APALLGLALV LALVLVGLVS WRRRQRRLRG ASSAEAPDGD
81APALLGLA.. ......GLVS WRRRQRRLRG ASSAEAPDGD
HD
HLR
121KDAPEPLDKV
IILSPGISDA TAPAWPPPGE DPGTTPPGHS
121KDAPEPLDKV IILSPGISDA TAPAWPPPGE DPGTTPPGHS
HD
HLR
161VPVPATELGS
TELVTTKTAG PEQQ184
161VPVPATELGS TELVTTKTAG PEQQ184
Transmembrane domain
Deletion affects protein expression
Sister
Cord Blood
R2
R3
R4
R4
R2
R3
R4
CD21
CVID5
Western Blot of BAFF-R
CVID4
R4
R2
R3
CVID3
R3
CVID2
R2
CVID1
Anti BAFF R
Healthy control
BAFF-R-/(CVID 5)
B cell phenotype
71.2
IgM
IgM
Healthy control
12.0
R7
R4
2.7
R2
R3
8.6
R5
15.3
CD19
CD38
IgM
BAFF R-/-
IgM
CD27
R4
1.9
R2
52.2
93.5
R5
1.2
R3
CD19
Transitional
B cells
R7
2.8
CD27
CD38
BAFFling Surprise
Clinical Data:
(g/l)
*1928
*1929
*1934
*1937
*1940
*1942
8
6
4
2
*1967
0
IgG
IgA
*1969
*1972
IgM
IgM
Serum Immunoglobulins
IgM
BAFF-R
SSC
Age: 70
Gender: female
Occupation: nurse
Clinic: no increased infections
Splenomegaly: no
Lymphadenopathy: no
Autoimmunity: no
R6
R4
R2
R3
31.3
1.3
5.8
R3
R7
R2
R4
R5
8.2
CD19
CD21
CD38
CD27
Acknowledgements BAFF-R
Dpt. of Clinical Immunology and
Rheumatology, Freiburg
Klaus Warnatz
Ulrich Salzer
Sylvia Gutenberger
Lukas Bossaller
Michael Schlesier
Hermann Eibel
Hans Hartmut Peter
Referring physicians:
Christine Zeidler
Friedrich von Flotow
Molecular Defects in CVID
The CD19 deficiency
The BAFF receptor deficiency
The TACI deficiency
The ICOS deficiency
TNF receptor : ligand systems
Nature Reviews Immunology
2, 465 -475 (2002);
Fabienne Mackay &
Jeffrey L. Browning
BAFF: A
FUNDAMENTAL
SURVIVAL
FACTOR FOR
B CELLS
Phenotype of TACI -/- mice
TACI -/- Mouse
Autoimmunity
• high titers of ANAs
• SLE
Lymphoproliferation
• splenomegaly
• lymphadenopathy
Immunodeficiency
• low IgA and IgM titers
• impaired T independent
antibody response
Seshasayee et al., Immunity. 2003 Feb;18(2):279-88.
Phenotype of TACI -/- mice
TACI -/- Mouse
Autoimmunity
• high titers of ANAs
• SLE
Lymphoproliferation
• splenomegaly
• lymphadenopathy
Immunodeficiency
• low IgA and IgM titers
• impaired T independent
antibody response
Seshasayee et al., Immunity. 2003 Feb;18(2):279-88.
Phenotype of TACI -/- mice
TACI -/- Mouse
Autoimmunity
• high titers of ANAs
• SLE
Lymphoproliferation
• splenomegaly
• lymphadenopathy
Immunodeficiency
• low IgA and IgM titers
• impaired T independent
antibody response
Seshasayee et al., Immunity. 2003 Feb;18(2):279-88.
AR CVID family 10
Hashimoto Thyreoiditis
I.1
I.2
IgA , IgM
Hypogammaglobulinemia
IgG
positive ANAs
, IgM
CVID:
II.1
II.2
age 15 months diarrhea, emesis, splenomegaly,
severe hypogammaglobulinemia, EBV +
Splenomegaly
IVIG up to age 4, with distinct improvement
Gastrointestinal infections,
Malabsorbtion,
Re-occurence of gastrointestinal complaints at
age 16, splenomegaly
Salmonella infection age 16
Ig levels age 17 (before IVIG restart)
– IgG 3,41 g/l
– IgA 0,37 g/l
– IgM absent
July 2003 / IVIG therapy re-started
Nodular lymphatic
Hyperplasia
Hypogammaglobulinemia
IgG , IgM
IVIG
AR-CVID family 10
I.1
I.1
I.2
I.2
II.1
II.1
II.2
II.2
HD
TACI exon 3 genomic DNA sequence
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
C104R
I.1
II.1
I.2
II.2
II.3
Vorechovski, Webster, Hammarstroem et al.
AR-CVID family cv79
I.1
I.2
CVID
recurrent respiratory
infections
splenomegaly,
splenectomy at age 19
lymphoid infiltration of
the liver, cirrhosis
nodular lymphoid
hyperplasia
Hypogammaglobulinemia
II.1
II.2
II.3
AR-CVID family cv79
II.1
I.1
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
I.2
II.2
II.3
II.1
II.2
II.3
HD
HD
TACI exon 3 genomic DNA sequence
S144STOP
AR-CVID family cv79
0.06%
Patient II.1
I.1
I.2
SSC
Control
II.2
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
R2
6.78%
II.1
S-S
II.3
R2
CD19
S144STOP
I.1
II.1
I.2
II.2
Vorechovski, Webster, Hammarstroem et al.
Selective IgAD family 102
I.2
I.1
I.2
II.2
A181E
II.1
II.2
HD
A181E
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
Mutations of TACI in 10 out of 134 sporadic CVID patients
P1
P4
P2
HD
S-S
S-S S-S
S-S
S-S S-S
P5
S-S
S-S S-S
HD
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
C104R
A181E
C104R
A181E
S194STOP
P3
P6
HD
R202H
HD
S194STOP
R202H
Mutations in TACI deficient patients
A181E
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
S-S
S-S S-S
C104R
S144STOP
S194STOP
R202H
TACI expression on patients‘ B cells
TACI (mAb 1A1) EBV
TACI (mAb 1A1) PBMC
S1 (C104R heterozygote)
A.II.1 (S144X homozygote)
healthy control
S7 (A181E heterozygote)
B.II.2 (C104R homozygote)
S9 (S194X heterozygote)
B cell phenotype and
serum Ig levels
b
in TACI deficiency
100
20
Immunoglobulin [g/L]
% of lymphocytes
80
60
40
20
15
10
*
*
5
0
0
0 CD19+ IgD
2 +/ IgD+/
CD27-
CD27+
IgD
4 -/
CD27+
of CD19+ cells
6
0
IgM
1
IgG
2
IgA
3
4
Healthy control
B cells bind BAFF but not APRIL
a-BAFFR
flag-BAFF
a-TACI (1A1)
flag-APRIL
blocked with
April-Fc
C104R
S144X
SWR
DEH
APRIL induced proliferation is
impaired in TACI deficiency
WT
C104R (B.II.2)
cpm
3000
2000
1000
0
P
+A
R
2
L+I
M
Ig
M
Ig
m
iu
ed
M
Ig
M
IL
25
1:
CSR is defective in TACI deficiency
healthy donor
Ig-Cg germline
transcripts
b-actin
C104R
Tonsil of patient II.1
I.1
I.2
HE
Patient II.1
II.1
Patient II.1
II.2
IgD
Patient II.1
Control
Summary of Clinical features
Dysgammaglobulinemia (18/19):
CVID (14), sIgAD (2), sIgMD (2)
Lymphoproliferation (11/19):
splenomegaly (6), nodular lymphatic hyperplasia (2),
peribronchial lymphoid infiltrates (1), lymphoid infiltration
of the liver (1), Tonsillar hyperplasia (4)
Autoimmunity (5/19):
Vitiligo (1), Thyreoid (3), Anti-IgA antibodies (1), positive
ANA (2), pernicious anaemia (1)
Summary for TACI deficiency
• In 3 unrelated CVID families 3 distinct genetic defects in TACI could
be identified in 9 individuals
• 10 out of 134 sporadic CVID patients were heterozygous for
mutations in TACI
• TACI expression was found to be normal on peripheral B cells in
some individuals tested, but absent in patients with the S144X
STOP codon mutation
• B cells of patients proliferated normally in response to different
mitogens
• Autoimmune phenomena and lymphoproliferation occured
frequently in familial and sporadic cases
Centre for Clinical Immunology,
Oxford, UK
Royal Free Hospital School of Medicine
Centre for Clinical Immunology, London, UK
Helen Chapel
Berne Ferry
David Webster
Sarita Workman
Karolinska Institutet, Huddinge, Sweden
Institute of Biochemistry
University of Lausanne, Switzerland
Lennart Hammarström
Quiang Pan-Hammerström
Pascal Schneider
Acknowledgements TACI
Dpt. of Pathology
Freiburg
Dpt. of Clinical Immunology and
Rheumatology, Freiburg
Annette Schmitt-Graeff
Rolf Knoth
Ulrich Salzer
Michael Schlesier
Ruth Dräger
Cristina Wöllner
Hermann Eibel
Klaus Warnatz
Hans Hartmut Peter
Dpt. of Internal Medicine,
Universitätsklinikum Bonn
Jürgen Rockstroh
M. Vogel
Molecular Defects in CVID
The CD19 deficiency
The BAFF receptor deficiency
The TACI deficiency
The ICOS deficiency
Hypothesis: CVID may be caused by defective T-B
collaboration in germinal centers
PC
Germinal center
B cell zone
B
B
T
B
PC
FDC
T
B
Red pulp
PC
PC
BB B
B
B BB
T cell zone
Hypothesis: CVID may be caused by defective T-B
collaboration in germinal centers
B
T
Sharpe and Freeman; Nature, 2002
ICOS-deficiency in four AR CVID families
Family A
P1
Family B
P2
P3
Family C
P4
Family D
P5
P6
P7
7 yr
P8 P9
4 yr 1.5 yr
Molecular Analysis of the ICOS-gene
Long-Range PCR
ICOSwt
3 kb
ICOSmut
1,2 kb
Grimbacher et al.,
Nat. Immunol. 2003
Screening for mutations of ICOS in CVID patients
Exon 1
Exon 2
Exon 3
sequenced
194
194
194
194
194
wildtype
194
185
185
194
194
mutation
0
0
0
49+patients
5 del Δex2+3
4 + 5 del
Exon 4 Exon 5
Three major waves of migrations in the 18th century
Clinical phenotype of ICOS-deficiency
» Recurrent upper respiratory infections (all)
» Recurrent pneumonia (4)
» Gastrointestinal tract infections (Lamblia, Salmonella) (1)
» Nodular lymphatic hyperplasia (1)
» Splenomegaly (1)
» Autoimmune Granulocytopenia (anti-neutrophil antibodies) (1)
» Childhood-onset (3) and adult-onset (6)
3/
an
tiC
D
28
S
IL-13
tiCD
tiIC
O
500
3
<62
3/
an
HD
ICOS -/-
tiCD
1500
1000
An
iu
D
S
28
tiIC
O
3
m
28
tiCD
tiC
3/
an
An
3/
an
tiCD
tiCD
An
M
ed
D
IL-10 pg/ml
2000
An
2500
2000
IL-17 pg/ml
tiC
S
0
m
28
3/
an
tiIC
O
<312
iu
D
tiCD
3
IFNg
An
tiC
S
An
3/
an
tiCD
HD
ICOS -/-
M
ed
3/
an
tiIC
O
tiCD
An
m
8000
6000
tiCD
tiCD
3/
an
3
An
iu
10000
An
An
tiCD
tiCD
m
500
0
An
iu
M
ed
IFNg pg/ml
4000
2000
An
M
ed
IL-13 pg/ml
In vitro cytokine production in ICOS -/- CD4+ T cells
IL-10
>10000
10000
8000
6000
4000
HD
ICOS -/-
0
<31
IL-17
2500
2000
1500
1000
HD
ICOS -/-
0
<31
Acknowledgements ICOS
Freiburg:
Collaborators:
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Ruth Dräger
Michael Schlesier
Klaus Warnatz
Lukas Bossaller
Hermann Eibel
Hans-Hartmut Peter
Marzenna Orlowska-Volk
Rolf Knoth
Ulrich Salzer
Charlotte Cunningham-Rundles, NY
Hans-Dieter Ochs, Seattle
Wen I. Lee, Seattle
Are Holm, Oslo
Jiri Litzman, Brno
Bernd H. Belohradsky, Munich
Simon Urschel, Munich
Jose L Franco, Medellin
Mirjam van der Burg, Rotterdam
Jacques van Dongen, Rotterdam
Andrea Skrabl-Baumgartner, Graz
Wolfgang Schwinger, Graz
Possible causes of CVID
Block in B cell
Development
autoantibodies
Circulating
B cells (CD19, TACI, BAFFR)
Disturbed ICOS
TH-cell regulation (ICOS)
Defective
glycosylation
or rapid turnover
Burmester, Pezzutto:
Taschenatlas der Immunologie
Plasma cell