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Role of B cell in autoimmunity. Application
to lupus and Sjögren’s syndrome
Xavier Mariette, Rhumatologie, Hôpital Bicêtre, AP-HP, INSERM U802,
Université Paris-Sud
Autoimmune diseases : T or B ?
Until recently: T cell The cell which recognizes the peptide In number of animal models, transfer of the
disease by T cells Auto-Ab: hallmark of the disease but no
pathogenic role
Autoantibodies play a role Pathogenic in some AID :
Autoimmune cytopenia, Graves disease, Myasthenia gravis
Possibly pathogenic in some AID : Anti-DNA in lupus Anti-Pr3 in Wegener Anti-2GP1 in antiphospholipid syndrome
KRN / NOD mice : only spontaneous model of RA
Transferable by anti-GPI Ab
Autoimmune diseases : T or B ?
B-cells = efficient Ag presenting cells Mainly RF positive B cells
MRL lpr/lpr mice Model of lupus, Sjögren, RA Infiltration by T cells JH -/- MRL lpr/lpr (no membrane Ig, no secretion
of Ab) No T-cell infiltrate mIgM JH -/- (express membrane Ig but do not
secrete Ig) T-cell infiltrate
Chan et al, J immunol 1998 et J Exp Med 1999
Autoimmune disease : T or B ?
3 fonctions du lymphocyte B dans les MAI
IL-6
Cartilage lossCartilage loss
IL-6
T cell
Macrophage
Dendritic cell
TNF-IL-10
RF
Fix complementFix complement
Inflamed Inflamed synoviasynovia
Inflammatory Inflammatory damagedamage
TNF-
Plasma cell
RF RF
RF
RF
IL-10TNF-
IL-1
B cell B cellB cell
Présentation de l’ antigène au LT et Activation du LT
Productiond’Ac
Secrétion decytokines proinflammatoires
CD40L
T Lymphocyte
CD4
CD3
TCRCMH
BAFF-R
CD40
B Lymphocyte
CD 28 CD80
CD86
CTLA4
B-cell differentiation
Memory B IgD+ IgM +
Pro-B
T
T
T
T
T
TT
T
Centroblasts
Memory BIgG+, IgA+
Plasma-cell
Naive B
T indépendent
Marginal zone
Transitional
Immature B
Pre-B
Tdépendent
Bone marrow
Secondary lymphoid organs (lymph nodes …)
Germinal centre
centrocytes
Ab
Blood
Bonemarrow
Blood
Expression of CD20 in the different Steps in the Maturation of B Cells
c souche Pro B Pre B Naîf Activé Mémoire Plasmo
CD20
Importance of subsets of B-cells
CD27+ memory B cells : 1/3 of PBMC B cells Increased in lupus Dorner et al ART 2002
Decreased in Sjögren’s Hansen et al Arthritis Rheum 2002
Normal in RA (only 1 study: Bonhorst et al JI 2001)
2 kinds of CD27 memory B cells Classical post-GC IgM- (mainly IgG) CD27 memory B cells Marginal zone IgM+ CD27 memory B cells
B cells are polarized like T cells Harris et al, Nat Immunol 2000
Be1: produce IFN Be2 : produce IL-4
Bm5
Bm4
Bm3
Bm2’
Bm2
Bm1
Bm1-Bm5 classification
Sjögren’s syndrome: an example of a disease with
B-cell hyperactivity
Subsets of B-cells in blood
CD27+ memory B cells in blood Bonhorst et al J Immunol 2001, Hansen et al Arthritis Rheum 2002
Bm2 (IgD+, CD38+) and Bm2’ B cells (pre-germinal center cells)
Bonhorst et al J Immunol 2001
Correlated with increase of serum BAFF CD19 and retention of BCR in lipid rafts
D’Arbonneau et al Arthritis Rheum 2006
Bm2 and Bm2’ and Bm5 and e Bm5 Ratio > 5 = diagnostic marker ?
Binard et al, Ann Rheum Dis on line , 9 sept 2008
B-cells in salivary glands
Represent 10 to 30% of the infiltrate CD27+ memory B cells
Hansen et al Arthritis Rheum 2002
Germinal centre like in 20% of patients Salomonsson et al Arthritis Rheum 2003
Correlated with CXCL13 and CCL21 expression Barone et al Arthritis Rheum 2005
Contain transitional (TII) and marginal zone B cells
Daridon et al Arthritis Rheum 2006
B-cells biomarkers and Sjögren’s
Immunoglobulin level serum light chain level
Moutsopoulos et al J Immunol 1983 Youinou et al Clin Exp Rheumatol 1988 Gottenberg et al Ann Rheum Dis 2006 (Free light chains)
beta2 microglobulin level Michaski et al N Engl J Med 1975
Correlated with extra glandular signs Gottenberg et al Ann Rheum Dis 2005
Correlation between auto-antibodies and extraglandular complications
Extraglandular involvement
p ValuePresent (n = 73) Absent (n = 104)
Age (years) 56.2 (14.6) 57.2 (12.5) 0.6
Disease duration (years) 12.1 (7.0) 12.4 (7.1) 0.75
Anti-SSA only 28.8% 30.8% 0.29
Anti-SSA and anti-SSB 43.8% 25% 0.004
BLyS (ng/ml) 6.1 (9.4) 5.0 (5.0) 0.39
ß2 Microglobulin (mg/l) 2.2 (0.9) 1.7 (0.8) <10–4
Gamma globulin (g/l) 13.5 (7.3) 13.2 (5.9) 0.8
IgG (g/l) 15. 8 (6.7) 14.5 (6.8) 0.28
IgA (g/l) 3.0 (1.4) 3.0 (1.5) 0.26
IgM (g/l) 1.6 (1.2) 1.5 (1.0) 0.44
RF (IU/ml) 159.2 (293.1) 135.8 (330.4) 0.66
Decreased C3 level 13.8% 12.3% 0.99
Decreased C4 level 29.3% 17.1% 0.12
ESR (mm/h) 30.6 (26.2) 27.2 (25.9) 0.42
C reactive protein (mg/l) 10.5 (10.9) 6.1 (2.8) 0.003
From polyclonal B-cell activation to B-cell lymphoma
Lymphoma and Sjögren’s
The autoimmune disease where the risk is the most important Kassan et al Ann Intern Med 1978: RR = 44 Méta-analysis Zintzaras et al, Arch Intern Med 2005: RR = 19 Theander Ann Rheum Dis 2006: RR = 16
Frequency of mucosal localization Royer 1997: 12/16 Voulgarelis 1999: 27/33 Smedby 2005: 6/12 Theander 2006: 4/11
Histology: frequency of MALT marginal zone B-cell lymphomas
Lymphoma and Sjögren’s
B-cell lymphoma No association with viruses or specific genetic
abnormalities Predictive factors (Tzioufas et al 1996, Skopouli et al 2000)
Mixed cryoglobulinemia Low C4 Purpura
Frequency of rheumatoid factor activity of membrane Ig of lymphomatous B cellsMartin et al, Arthritis Rheum 2000
Rheumatoid factor activity of lymphomas complicating Sjögren’s
Localization of MALT
n t(11, 14) n Homology CDR3-RF
Salivary glands 114 2 (2%) 32 13 (41%)
Stomach 209 50 (24%) 45 8 (18%)
Lung 113 47 (42%) 19 0
Others ND ND 4 0
Bende et al J Exp Med 2005
Analogies between hepatitis C lymphomas and Sjögren’s lymphomas
HCV
Virus ?Auto-Ag ?
Hépatitis C
Sjögren
pc RF B
B
B
B
B
Chromosomicabnormalities
mc RF B
Lymphoma
B B
Mariette, Ann Rheum Dis 2001
Unusual roles of B cells in AID
B-cells and bone destruction in RA
Synovial and bone tissue in 8 RA patients Lymphoid infiltrates in bone marrow of subchondral bone with more B
cells (CD20) than T cells (CD3) Presence of B-cell attracting chemokines (CCL21 et CXCL13)
CD20 CD3
Bugatti et al, Arthritis Rheum 2005, 52: 3448-59
B-cells and bone destruction in RA
Correlation between osteoclast count and bone marrow inflammatory score
ACR 2005 – D’après Bugatti (1311)Bugatti et al, Arthritis Rheum 2005, 52: 3448-59
B cells and periodontal bone erosion
Model of infectious periodontitis (actinomycete) Ag-Specific B cells in periodontal bone
Expressing RANK Ligand Increasing osteoclast formation and bone resorption Inhibited by OPG
Han et al, JI 2005; 176: 625-31
A new axis in pathogeny of lupus and Sjögren
Interferon type 1 or type 2
BAFF (B-cell Activator Factor of the TNF family) or BLyS
Activation of B cells
Interferon signature in Sjogren’s like in lupus
SO
CS
3
CC
L1
8
EP
HA
4
PR
KC
D
CO
L1
6A
1
CC
L2
0
MA
TK
MA
P3
K1
B2
M
TL
R8
PT
K9
LD
IAB
LO
TA
P2
ET
V5
SY
KIF
ITM
2
IFIT
M3
IL4
R
MIC
B
NA
LP
2P
ML
CD
6IF
ITM
1
0,010
0,100
1,000
10,000
Ra
tio
of
Me
an
s (
log
sc
ale
)
TLR8, TLR9, IFITM1, MICB, TAP2, B2M, CCL20
CCL-18
SOCS-3
Hjermelvik at al Arthritis Rheum. 2005 May;52(5):1534-44. Gottenberg et al, PNAS 2006 Feb 21;103(8):2770-5
424 differentially expressed genes
Presence of type 1 IFN-secreting cells in salivary glands of patients
Bave at al Arthritis Rheum. 2005 Apr;52(4):1185-95.
Interferon signature in Sjogren’s like in lupus
Presence of pDC in salivary glands
CD123
BDCA2
Sjogren Controls
A new axis in pathogeny
Interferon type 1 or type 2
BAFF (B-cell Activator Factor of the TNF family) or BLyS
Activation of B cells
The BAFF system
IFNIFNIL-10 Mono
M
B lymphocyte
BR3 BCMA TACI
CD40
APRILBAFF
BCR
sBAFF
SurvivalAutoantibody
secretion
BAFF Transgenic mice
Biological features Increase in peripheral B cells Increase in serum Ig Increase in serum auto-antibodies (RF, Anti-
DNA)
Clinical features Autoimmune glomerulonephritis Polyarthritis Auto-immune Sialadenitis B-cell lymphoma x2 (x30 in TNF ko mice)
Mackay, J Exp Med 1999; Khare, PNAS 2000; Groom, JCI 2002; Batten JI 2004
Serum BAFF is increased and correlates with the presence of autoantibodies
0
10
20
30
40
50
60
normal Sjogren
BL
ys, n
g/m
l
0
10
20
30
40
50
60
- + - +
SSA RF
BL
ys, n
g/m
l
Mariette et al. Ann Rheum Dis, 2003
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Lo
g B
lys
(ng
/ml)
1.25 1.5 1.75 2 2.25 2.5 2.75 3 3.25 3.5 3.75
Log Serum RF (iu/l)
R=0.70, p<0.0001
C
Serum BAFF is increased and correlates with the level of autoantibodies
Increase in BAFF in Sjögren: other reports
Increase in serum level of BAFF Groom JCI 2002 Correlated with hypergammaglobulinemia Szodoray et al, J
Clin Immunol. 2004; 24:600-11
Correlated with anti-SSA secretion Pers JO at al Ann N Y Acad Sci 2005;1050:34-9
Increase in saliva level of BAFF Correlated with complications of periodonditis Pers JO at al,
Arthritis Rheum 2005;52:2411-4
Increase in serum level of APRIL Correlated with BAFF level Jonsson et al, J Clin Immunol. 2005
IFNIFNTNF
Monocyte
Epithelial
T
BAFF
SurvivalAuto-Ab
Auto-immunity
B
NFB
Fibroblastic
Paracrine or autocrine BAFF secretion Autoimmune B cell
Autoimmunity
Quantification of BAFF by Q-PCR in salivary glands
RNA extracted from labial salivary glands
0
2
4
6
8
10
12
pSS Patients Controls
BA
FF
mR
NA
/act
ine
3.8 fold, P = 0.003
Gottenberg et al, PNAS 2006
Lavie et al, J Pathol 2004
Detection of BAFF in salivary glands
Sjögren
Normal
Sarcoidosis
Sjögren BAFF Sjögren CD3 Sjögren BAFF + CD3
Detection of BAFF in salivary glands
Lavie et al, J Pathol 2004
BAFF can be expressed by salivary ducts
[ BAFF ] = 0 [ BAFF ] = [ AC ] [ BAFF ] = 2 [ AC ]
BAFF
Presence of BAFF in salivary B cells
Daridon et al Arthritis Rheum. 2007
BAFF in salivary glands : T cells, epithelial cells and B cells
BAFF BAFF
Daridon et al Arthritis Rheum. 2007
Inhibition with an anti-IFNAR1 Ab (n=3x2)
0
5
10
15
20
25
IFNa IFNa+anti-IFNAR1
FO
LD
(m
RN
A B
AF
F)
Expression of BAFF mRNA by SGECs after stimulation with different doses of IFN
0
1
2
3
4
5
6
7
8
9
10
50 UI 100 UI 500 UI 1000 UI 2500 UI
Ra
tio
: B
AF
F/a
cti
ne
Effect of IFN on BAFF mRNA in salivary epithelial cells
Ittah et al Arthritis
Research Therapy, 2006
BAFF mRNA
BAFF Protein
0
20
40
60
80
100
120
140
CpG R837 Poly IC PGN HSV1 Reovirus
Rat
io m
RN
A:B
AFF
/Act
in
0
20
40
60
80
100
CpG R837 Poly IC PGN HSV1 Reovirus
BA
FF (p
g/m
L)
TLR2 TLR3 -actinTLR7 TLR9
BAFF expression by SGECs after TLR stimulation
Ittah et al Eur J immunol, 2008Apr;38(4):1058-64
BAFF is highly induced by viral infection or poly (I:C) stimulation of epithelial cells Salivary epithelial cells* Airway epithelial cells** Oral epithelial cells*** Epidermal and intestinal epithelial cells***
This induction of BAFF may be TLR dependant or independent Type 1 IFN dependent or independent
* Ittah et al Eur J immunol 2008
** Kato et al, JI 2006
*** Xu et al, Nature Immunol 2007
BAFF is induced by viral infection
Cross-regulation between type 1 IFN and TNF
Banchereau, Pascual Immunity 2006
TRIPSS: Design of the study
Visits
Pre-inclusion W0 W2 W6 W10 W14 W18 W22D-14
Infliximab 5mg/kg or placebo
Evaluation
Mariette et al, Arthritis Rheum 2004
Primary end point:Decrease of at least 30% in 2 of the 3 VAS
0
5
10
15
20
25
30
30% Response at W10
INFPlacebo
P=0.89
Mariette et al, Arthritis Rheum 2004
Anti-TNF may increase IFN and BAFF
Controlled trial in Sjogren’s with etanercept (n=17)
Etanercept Placebo
IFN activity baseline (units)
3.81 ± 1.86 4.61 ± 3.47
IFN activity
3 months (units)
7.87 ± 5.95
p=0.04
3.92 ± 3.81
p=0.67
BAFF baseline
(ng/ml)
0.86 ± 0.28 0.80 ± 0.23
BAFF 3 months
(ng/ml)
1.12 ± 0.25
p=0.03
0.95 v 0.32
p=0.24
Mavragani et al, Arthritis Rheum 2007
Change in gammaglobulin level between W10 and baseline in TRIPSS
Placebo n=38 Infliximab n=39
Gammaglobulin (g/l) 0.121.16 1.031.94 0.02
IgG (g/l) 0.061.19 0.872.47 0.10
IgA (g/l) 0.030.56 0.130.42 0.40
IgM (g/l) -0.010.40 0.480.87 0.005
Mariette et al, Arthritis Rheum 2004
Induction of anti-nuclear antibodies by infliximab
ATTRACT: RA (102 weeks): ANA Placebo: 27% Infliximab: 62%
anti-dsDNA Placebo: 0% Infliximab: 15%
ACCENT I: Crohn (54 weeks):ANA All patients: 49%
anti-dsDNA All patients: 26%
Lupus induced by anti-TNF
The CRI study Retrospective study in France 22 cases :
10 cutaneous reactions with ANA and anti-DNA Ab
6 infliximab, 4 etanercept
12 true SLE with 4 ACR criteria 9 infliximab, 3 etanercept Recovery in all patients after withdrawal of anti-TNF but
need of steroids in 6 cases
M De Bandt, Arthritis Research Therapy 2005
B-cell targeted therapy in lupus and Sjögren’s
Rituximab in lupus
18 open studies including 2 to 32 patients Efficacy on most symptoms including
nephritis Decrease of anti-DNA Ab No decrease of anti-ENA Ab Decrease of memory B cells and
plasmablast Re-establishment of tolerance ? Randomized control studies are going on
Rituximab in lupus
Press release - 29 avril 2008 -- « Genentech, Inc. (NYSE: DNA) and Biogen Idec, Inc. (Nasdaq: BIIB) announced today that a Phase II/III study of Rituxan® (rituximab) for systemic lupus erythematosus (SLE, commonly called lupus) did not meet its primary endpoint defined as the proportion of Rituxan treated patients who achieved a major clinical response (MCR) or partial clinical response (PCR) measured by BILAG, a lupus activity response index, compared to placebo at 52 weeks. The study also did not meet any of the six secondary endpoints. »
This study excluded patients with lupus nephritis (LN). A total of 257 patients from approximately 55 sites in the U.S. and Canada were randomized 2:1 to receive Rituxan plus prednisone or placebo plus prednisone in two infusions 15 days apart.
Other positive open studies with rituximab
Lupus Sjögren’s Syndrome ANCA-associated vasculitis Cryoglobulinemia Inflammatory myopathies Aquired anti-F VIII antibodies Pemphigus Multiple sclerosis ++ (positive RC) Myasthenia gravis Anti-MAG-associated demyelinating neuropathy
AIR registry: non RA autoimmune AIR registry: non RA autoimmune diseasesdiseases
179 patients 46 SLE 42 primary Sjögren’s syndrome 21 vasculitis 17 myositis 53 other diseases
116 patients already had at least 1 follow-up visit Mean follow-up : 35.2 weeks 79 patient years
Lupus and Sjögren
SLE (data on 36 patients) Glomerulonephritis : 10 patients Mean corticosteroid dosage : 22.4 mg/day No concomitant IS : 8 patients Concomitant IS : 28 patients
HQ : 13, MTX : 5, MMF : 7, AZA : 2, CPH : 1 Efficacy : 21/36 (58%) Retreatments : 11 patients
pSS (data on 36 patients) 33/36 (92%): systemic involvement Mean corticosteroid dosage : 13.5 mg/day No concomitant IS : 30.5 % Efficacy on systemic complications : 23/36 (64%) Retreatments : 7
Overall tolerance in non RA AIDs
2 deaths pSS patient with metastatic Paget’s disease SLE patient with Endocarditis due to Staph Aureus after the
second course (γglob 4.3 g/l before the 2nd course)
• 5 severe infections : 6.3/100 patient years Staphylococcus Aureus endocarditis Staphylococcus pneumonia Corynebacteria pneumonia undocumented pneumonia Staphylococcus Aureus arthritis
4 moderate serum-sickness like reactions SLE : 2 Vasculitis : 1 pSS: 1
Rituximab in Sjögren’s: 3 open studies
Study Dutch1 French3
Patients 15 patients
6 lymphoma
9 early disease (<4yrs)
16 patients
5 lymphomas
11 systemic features
Efficacy Lymphoma: 3/7
Dryness: only in patients with residual glandular function
Lymphoma: 4/5
Systemic signs: 9/11
Subj dryness: 5/16
Obj dryness: 2/16
French2 (Brittany)
16 patients
Disabling subjective symptoms
Dryness: subjective improvement in patients with early pSS
1. Pijpe et al. Arthritis Rheum, 2005 2. Devauchelle-Pensec et al.. Arthritis Rheum, 20073. Seror et al. Ann Rheum Dis, 2006
Sjögren’s syndrome: the dutch Study
Open study of 15 patients 9 with early pSS 6 with pSS and MALT
lymphoma
Tolerance: 3 of 9 patients without lymphoma developed serum sickness 1 week after the 2nd infusion
Pijpe at al, Arthritis Rheum 2005
Effect of rituximab on mouth dryness
Pijpe at al, Arthritis Rheum 2005
The French study: biological findings
A significant median level decrease of RF : 124 to 7.5 IU/mL (p=0.004, n=13 ) Gammaglobulin : 13.4 to 9.6 g/L (p=0.003, n=15) IgG : 10.8 to 7.7 g/L (p=0.003, n=11) Beta-2 microglobulin : 3.3 to 2.3 mg/L (p =0.02, n=12)
Seror et al. Ann Rheum Dis, 2006
A small randomized controlled study 8 patients with rituximab and 9 with placebo Mean decrease of fatigue VAS:
50% with rituximab 20% with placebo P=0.24 between the 2 groups but significant
decrease only in the rituximab group
1 episode of serum sickness disease in the rituximab group
Dass et al, Ann Rheum Dis 2008 online 14 Feb
Rituximab in Sjögren’s: the first RCT
TEARS
Etude multicentrique de phase II randomisée menée en double aveugle, contrôlée versus placebo
Coordonnateur : Pr Alain Saraux (Brest) Centres participants :
Pr A. Saraux / V Devauchelle (Brest)*, Pr Olivier Vittecoq (Rouen) Pr E Hachulla (Lille) Pr X Mariette (KB)* Pr J Sibilia (Strasbourg)*
Tolérance et efficacité du rituximab dans le syndrome de Sjögren
ESR ML20771Promoteur : Hôpital de Brest (PHRC)
Dr J Morel (Montpellier)
Pr A Perdriger (Rennes)
Dr X Puechal (Le Mans)
Dr J-M Berthelot (Nantes)
Dr D Alcaix (Le Havre)
Pr L Guillevin (Cochin)
Dr JJ Dubost (Clermont Ferrand)
Schéma de l’étude
S2
S16 S48
Placebo* n=60
Rituximab n=60
Patients SGSn = 120 Randomisation
S24J15J1
Sélection
Inclusion Période de suivi
Rituximab 1000mg ou Placebo (* Serum phy)
Visites de suivi
Début des inclusions : Q4 2007
Durée des inclusions : 36 semaines
J-30 J-7 S6 S36
Evaluation principale
0
10
20
30
40
50
60
70
80
0 1 2 3 4 5 6 7 8 9 10 11
Weeks after rituximab first infusion
BA
FF
to
actin
e m
RN
A r
atio
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
BA
FF
Pro
tein
lev
el
(ng
/ml) mRNA
Protein
0
10
20
30
40
50
60
70
80
t0 t1
BA
FF
to
actin
e m
RN
A r
atio
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
0
1
2
3
4
5
6
7
8
9
t0 t1
Ser
um B
AF
F le
vel (
ng/m
l)
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Serum BAFF PBMC BAFF mRNA
D
Increase in BAFF after rituximab
Lavie et al, Ann Rheum Dis 2006
• 2 SLE• 2 SS• 1 RA
The level of BAFF before RTX could predict the lenght of B-cell depletion
Pers et al, A§R 2007
Epralizumab (anti-CD22 Ab) : open studyPatient demographics and baseline disease characteristics n =15
Gender (female/male) 13/2
Age (years) 49 (33–73)
Median years post-diagnosis 2.9 (1–16)
Unstimulated salivary flow (ml/minute)
0.07 ± 0.13
Fatigue VAS (mm) 56 ± 22
Focus score ≥ 1 12 (80%)
Anti-Ro antibodies 12 (80%)
Anti-La antibodies 11 (73%)
ESR (mm/hour) 33 ± 15
IgG (mg/dl) 2,114 ± 934
Steinfeld et al ART 2006
Epralizumab (anti-CD22 Ab) : open study
Steinfeld et al ART 2006
Epralizumab (anti-CD22 Ab) : open study
Steinfeld et al ART 2006
Conclusion
B cells play a role in autoimmunity 3 RCT demonstrated efficacy of rituximab in RA But no large RCT demonstrated efficacy of
rituximab in other systemic diseases Problems with end-points The currents targets to be explored in controlled
studies in lupus and Sjögren’s The IFN pathway BAFF (or BLyS) B cells
The new treatments will come from new pathogenic concepts