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