Regulatory B cells in autoimmunity

Liwei Lu University of Hong Kong, China

Multiple functions of B cells in immunity

LeBien and Tedder, Blood (2008)

B cell functions in autoimmune pathogenesis

The Immunopathogensis of Rheumatoid Arthritis

Nature 423 (2003)

Role of BAFF in B cell maturation and function

B cell activating factor

(BAFF, BLyS) * A member of TNF family cytokines

* A survival factor for B cell maturation

Serum BAFF levels: increased in SLE & RA

Increased BAFF

in DC from CIA mice

BAFF enhances the survival of plasma cells

0

20

40

60

80

100

120

0 1 2 3 4 5

Days in culture

Via

ble

pla

sm

a c

ells (

%)

Control

BAFF

BCMA:Fc

* *

* *

0

0.5

1

1.5

2

2.5

3

3.5

OD

40

5

Control Acute

CIA

Chronic

CIA

0

2

4

6

8

10

12

14

16

18

20

BA

FF

(n

g/m

l)

Control Acute

CIA

Chronic

CIA

BAFF Auto-Ab

BAFF overproduction correlates with elevated autoAb in CIA

Effect of BAFF on B cell maturation and function

BAFF-silenced DCs are defective in B cell maturation and Ab production

BAFF gene silencing by lentivirus expressing shRNA in joint tissue LV-shActin LV-shBAFF

BAFF silencing suppresses CIA development

Reduced CIA incidence Delayed CIA onset

Intra-articular injection of lentivirus expressing shRNA-BAFF

Long term, localized effect Bone erosion Joint damage

BAFF silencing inhibits Th17 cells in the joint and LN

Detection of IL-17 producing cells by ELISPOT assays

*

*

A pathogenic role of BAFF in autoimmune arthritis

Lam Q, et al, PNAS (2010)

Validation of BAFF as a therapeutic target in experimental arthritis

BAFF induces IL-10 production in B cells

BAFF induces IL-10 production in MZ B cells

Lymphoid follicle

I: B:T

II: Induced B (IL-10-/-):T

III: Induced B:T

IV: Induced B+a-IL-10:T Yang M, et al, J Immunol (2010)

IL-10-producing B cells suppress T cell function

Existence of IL-10-producing B cells in vivo

Spleen CIA Joint tissue

IL-10-producing B cells

Kinetic changes of Breg and Th17 cells during CIA induction

Breg cells

Th17

DBA/1 CIA

Localization of transferred Breg cells In vivo

In vitro expansion of Breg cells

B10 cells

Transfer of Breg cells suppresses CIA development

Transfer of Breg cells inhibits Th17 response in vivo

Breg cells inhibit Th17 expansion in vitro

Breg cells inhibit Th17-mediated CIA in IL-17-/- mice

Yang M et al. A J Pathol ( 2012)

Role of Breg cells in Sjögren Syndrome

Henrik Sjögren (1899-1986) Dry eyes / dry mouth

CD20 CD4

Adamson TC et al. J Immunol (1983)

Available mouse models for Sjögren Syndrome

• Spontaneous SS models:

NOD, Fas(lpr), (NZB+NZW)F1

• Gene-modified animal models:

IL-12-Tg, BAFF-Tg，Id3-KO, IL-14a-Tg

SG protein Immunization C57BL/6

Induction of Experimental Sjögren Syndrome in normal mice

Enhanced Th17 responses during ESS development

CD4+ CD8+ CD19+0

2

4

6

8

10

5 wk

30 wk

******

**

Cell N

um

ber (

x 1

04)

0 wk 5 wk 30 wk0

100

200

300 Th1 cells

Th17 cells

*

*

SF

C / 5

x 1

03 c

ells

A

D

IL-17 CD4 Hoechst

CD19

CD

4

66

20 10

Gated on CD45+

5 wk 30 wk

33

29 37

F

E

0

1

2

3

4

Ctrl ESS

His

tolo

gic

al S

co

re

CB

ESS (5 w k ) ESS (30 w k )Ctrl

50 mm

20 mm0d

3d

10d

17d

24d

35d

30w

k

0.00

0.05

0.10

0.150.2

0.3

0.4 ESS - Th1 cells

ESS - Th17 cells

*

***

*****

Ctrl - Th1 cells

Ctrl - Th17 cells

*

Ce

ll N

um

be

r (

x 1

06)

IL-1

7

0.3 0.5 0.6 1.2 0.7 0.3

0.3 0.4 0.5 0.3 1.4 0.8

IFN

-γ

CD4

0d

A

B

3d 10d 17d 24d 30wk

IL-1

7

30wk25wk15wk10wk5wk12.910.33.42.10.3

CD4

5wk

10wk

15wk

25wk

30wk

0

100

200

300

400

500

** ** **

***

Cell N

um

ber

0d

3d

10d

17d

24d

35d

30wk

0.00

0.05

0.10

0.150.2

0.3

0.4 ESS - Th1 cells

ESS - Th17 cells

*

***

*****

Ctrl - Th1 cells

Ctrl - Th17 cells

*

Ce

ll N

um

be

r (

x 1

06)

IL-1

7

0.3 0.5 0.6 1.2 0.7 0.3

0.3 0.4 0.5 0.3 1.4 0.8

IFN

-γ

CD4

0d

A

B

3d 10d 17d 24d 30wk

IL-1

7

30wk25wk15wk10wk5wk12.910.33.42.10.3

CD4

5wk

10wk

15wk

25wk

30wk

0

100

200

300

400

500

** ** **

***

Cell N

um

ber

Ctrl ESS0.0

0.5

1.0

1.5 Ctrl-peptide

M3R-peptide

Anti-ANA

Se

ru

m A

uto

-an

tib

od

ies

(O

D V

alu

e a

t 4

50

nm

)

******SSA-peptide

***

IL-17KO mice are resistant to ESS induction

0.75 0.02

IFN-γ

0.9 0.6

WT IL-17 KO

IL-1

7A

1 7 14 21 280

100

200

300

400

WT

IL-17 KO

**

**

***

Days post-immunization

Sa

liv

a F

low

Ra

te

(µl/1

5m

in)

WT IL-17 KO

0

1

2

3

4

WT IL-17A KO

His

tolo

gic

al S

co

re

T+

CD4

T+

CD8

Gam

ma-delta T

NKT

Neu

trophil

0

20

40

60

80

% o

f IL

-17 P

ro

du

cin

g C

ells in

CL

N

WT IL-17 KO

A B

C D E

F CD4 Th1 Th170.000.02

0.04

0.2

0.4

5

10 IL-17 KO

WT

***

***

**

Ce

ll N

um

be

r (

x 1

06)

20 µm

0.75 0.02

IFN-γ

0.9 0.6

WT IL-17 KO

IL-1

7A

1 7 14 21 280

100

200

300

400

WT

IL-17 KO

**

**

***

Days post-immunization

Sa

liv

a F

low

Ra

te

(µl/1

5m

in)

WT IL-17 KO

0

1

2

3

4

WT IL-17A KO

His

tolo

gic

al S

co

re

T+

CD4

T+

CD8

Gam

ma-delta T

NKT

Neu

trophil

0

20

40

60

80

% o

f IL

-17 P

ro

du

cin

g C

ells in

CL

N

WT IL-17 KO

A B

C D E

F CD4 Th1 Th170.000.02

0.04

0.2

0.4

5

10 IL-17 KO

WT

***

***

**

Ce

ll N

um

be

r (

x 1

06)

20 µm

Th17 cell transfer restores disease pathology in IL-17KO mice

CD4+ T cells Transfer Th17 cells Transfer

98.6

IL-1

7

CD3CD4

36.70.4

WTIL-17 KO

IL-1

7

A

CD4CD4CD4

from IL-17 KO micefrom WT mice

B

C

CD4 Hoechst

20 µm

20 µm

Tunnel Hoechst

20 µm

4 | ADVANCE ONLINE PUBLICATION www.nature.com/ nrrheum

infiltrates, but interestingly without any detectable CMV

in salivary glands at the onset of auto immunity.27 Another

potential explanation for the elusiveness of the trigger for

pSS could be that overexpression of some endogenous

retroviral sequences has been overlooked, and should

be considered instead of searching for an external virus

responsible for disease-associated activation of the IFN

system. For example, epigenetic abnormalities related to

expression of endogenous retroviral elements could be

responsible for activation of the type I IFN pathway.

NFκB pathway dysfunction in pSS pathogenesis

One of the consequences of the stimulation of innate

immunity is the activation of nuclear factor κB (NFκB),

which can occur in a number of different cell types. In

salivary gland epithelial cells from patients with pSS,

hyperactivation of NFκB has been associated with

decreased expression of one of the regulators of NFκB

activation, A20 (also known as TNF-α-induced protein 3

[TNFAIP3]).28 Interestingly, defective function ing of

another gene involved in feedback regulation of NFκB,

NFκB inhibitor α (also known as IκBα), leads to develop-

ment of a pSS phenotype in mice.29 Furthermore, the

first GWAS in pSS14 identified an association between

this disease and a gene (TNIP1) encoding TNFAIP3-

interacting protein 1, a protein that interacts with A20

and is involved in the regulation of NFκB activation.

Together, these observations suggest that dysregu-

lated activation of NFκB signalling might influence

su sceptibility to development of pSS.

B cells in pSS pathogenesisCytokines and B-cell activation

BAFF—linking innate and adaptive immunity

BAFF promotes B-cell maturation, proliferation and

survival. Transgenic mice that overexpress this cytokine

develop features of SLE, and go on to develop clinical

characteristics of pSS, such as sialadenitis.30 Further-

more, BAFF-transgenic mice have an increased risk

of lymphoma as they age, compared with wi ld-type

mice, although lymphomas are rare in these transgenic

mice (2.9% in homozygotes) unless they also lack TNF

expression, which increases the frequency of lymphoma

to 38%.31

Over the past decade, several studies have focused

on elucidating the role of BAFF in pSS. First, serum

BAFF levels were demonstrated to be increased and to

corre late with levels of anti-Ro/SSA antibodies, anti-La/

SSB antibodies and rheumatoid factor (RF) in patients

with pSS.32 Second, elevated levels of BAFF have been

detected in the salivary glands in patients with pSS.33,34

Interestingly, BAFF was found to be secreted not only

by typically BAFF-producing cell types, such as mono-

cytes, macrophages and dendritic cells (DCs), but also by

T cells and B cells, as well as salivary epithelial cells that

are the target of autoimmunity in pSS.33,34 This finding

strongly supports the hypothesis that epithelial cells are

not only the passive victim of the immune response in

pSS, but also an active contributor that might promote

overactivation of immune system.

Virus? Other activatorof innate immunity?

Epithelial cell

pDC DC

Autoantigen

ICs

IFN-α

BAFF

NKcell

IL-12

TH1

cell

IFN-γ

TFH

cell

B cell

GC-likestructure

Plasmacell

B-cellactivation

T-cellactivation

IL-12R

NK-cellactivation

Figure 1 | Schematic representation of the pathophysiology processes

hypothesized to underlie pSS, based on our current understanding. Environmental

factors such a virus or other activators of innate immunity cause epithelial cell and

DC activation (see Box 1 for a description of how viruses might instigate

development of pSS). pDCs can also be activated by ICs formed as a consequence

of the adaptive immune response. Such stimulation of DCs promotes activation of

the type I and type II IFN pathways. Furthermore, IL-12 secreted by conventional

DCs leads to activation of NK cells and TH1 cells that increase IFN-γ production and

mediate tissue damage. IFN-α and IFN-γ enhance BAFF secretion—leading to

overproduction of BAFF—and activation of B cells and T cells. In individuals

susceptible to pSS, activation of B cells is thought to drive autoantibody production

within GC-like structures. Epithelial cells release autoantigens that participate in

formation of ICs and in the perpetuation of the vicious circle of immune system

activation. Abbreviations: BAFF, B-cell-activating factor; DC, dendritic cell; GC,

germinal centre; ICs, immune complexes; IL-12R, IL-12 receptor; NK, natural killer

(cell); pDC, plasmacytoid DC; pSS, primary Sjögren’s syndrome; TFH

, follicular

helper T (cell); TH1, type 1 T-helper (cell).

Box 1 | Viral infection as a trigger for autoimmunity in pSS

The development of primary Sjögren’s syndrome (pSS) results from the successive

activation of the innate and adaptive immune system. On the basis of the

environment within the mouth and of the discovery of the IFN signature in the

disease, viral infection of salivary epithelial cells, the primary cell type affected

in pSS, has long been suspected as the initial trigger of inflammation and thus

development of the disease. Epithelial activation and injury resulting from the viral

infection could promote release of pSS-associated ribonucleoprotein autoantigens,

such as Ro/ SSA and La/ SSB, and chemokines that attract both conventional

dendritic cells (DCs) and plasmacytoid DCs (pDCs) within salivary glands (Figure 1).

Such epithelial cell activation in response to viral infection is likely to be promoted

in individuals who carry susceptibility alleles of genes encoding proteins of the

interferon pathways. Subsequent secretion of IFN-α by pDCs and of proinflammatory

cytokines such as IL-6 and IL-12 by DCs could lead to the migration of lymphocytes

to salivary glands, and to the secretion of B-cell-activating factor by epithelial

cells and DCs. These conditions promote B-cell activation and the secretion of

autoantibodies, especially in predisposed individuals. These autoantibodies can

form immune complexes with the aforementioned autoantigens, which participate in

the maintenance of IFN-α production. Together, these interacting processes promote

persistent immune system activation leading to tissue damage.

REVIEWS

© 2013 Macmillan Publishers Limited. All rights reserved

How about macrophages?

Th17

A critical role of Th17 cells in ESS development

(Lin X, at al Ann Rheum Dis. 2015)

Functional defects of Breg cells in ESS development

Transfer of Breg cells suppresses ESS progression

Breg transfer suppresses Th1/Th17 cells in ESS mice

Breg transfer suppresses inflammatory cytokine production

IL-17

Ctrl PBS Breg Bve0

2

4

6

8

10

ESS

**

***

Seru

m level (n

g/m

l)

IL-12

Ctrl PBS Breg Bve

40

80

120

160

ESS

Seru

m lev

el (n

g/m

l)IL-21

Ctrl PBS Breg Bve0.0

0.5

1.0

1.5

ESS

****

Seru

m level (n

g/m

l)

IL-6

Ctrl PBS Breg Bve0.0

0.5

1.0

1.5

2.0

ESS

* *S

eru

m level (n

g/m

l)

Cell therapy?

(Manuscript in preparation)

Mechanisms of action for Bregs in immune responses

Yang M et al, Cell Mol Immunol (2014)

Acknowledgements

Queenie Lam

Min Yang

Jun Deng

Xiang Lin

Xiaohui Wang

Qian Chen

Kongyang Ma

Otis Ko

This project was funded by Hong Kong Research Grants Council,

National Science Foundation of China, 973 Program of MOST,

and Hong Kong Croucher Foundation.

BAFF in regulating B cell maturation and function

TACI

BAFF-R

TBK1

NIK

IKKa

NEMO IKKb

p52

RelB

p50

RelA

Proliferation Survival

Ig Class

switching Differentiation

BAFF

BAFF