Adaptive immunity 2 - Immune regulatory mechanisms through B cell axis

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Immune regulatory mechanisms

through B cell axis

Tomohiro Kurosaki

Osaka University

Adaptive Immunity 2

Number 1

Number 2

Number 3

Antigen presenting cell

Helper T cell

inflammatory cytokine(IL-6,M1P1α,M1P1)

inflammatory cytokine

T cell activation

B cell

1


Inflammatory cytokine(IL-6,M1P1α,M1P1)

Anti-inflammatory cytokine(IL-10)

Breg cells in mice

2


DTH: Delayed-type hypersensitivityEAE: Experimental autoimmune encephalomyelitisIBD: Inflammatory bowel diseases

MS: Multiple sclerosis SLE: Systemic lupus erythematosus

Year Mouse Human

1974 B cells suppress DTH (Katz SI et al., Nature)

1996 B cell-deficient mice develop EAE (Wolf SD et al., J. Exp. Med.)

2002 IL-10+/+ B cells suppress EAE (Fillatreau S et al., Nature Immunol.)B cells control IBD (Mizoguchi A et al., Immunity)

2007 B cells have an impaired IL-10 production in MS (Duddy M et al., J. Immunol.)

2010 B cells have an impaired IL-10 production in SLE (Blair PA et al., Immunity)

Highlights in regulatory B cell research



Year Mouse Human







3


Wolf SD et al. J. Exp. Med. 184: 2271-2278,1996

Data from individual mice were plotted from three consecutive B10.PL (squares) (B) and four consecutive B10.PLμMT (circles) (C) mice. The data was collected from one fifth of the experiments set up.

Dis

ease

Sev

erit

y

ＤＡＹ



Year Mouse Human







4


Fillatreau S. et al., Nature Immunol. 10, 944-950, 2002

IL-10-producing B cells suppress EAE development

Clin

ical

sco

re

Time (d)

IL-10-/- B cells

B6 B cells

T2-MZPBREG cell B10

cell

IgMhigh IgMhigh

CD21high

CD21high CD24high

CD24highCD1dhigh

CD1dhigh

CD19CD19

5




Year Mouse Human







Duddy M et al. J Immunol. 178: 6092-6099, 2007

IL-10

Cyt

oki

ne

secr

etio

n (

pg

/ml)

CD40 alone Dual Stimulation

p=0.008

p=0.037

Normal

Multiple Sclerosis

6


CD19+CD24hiCD38hi B cells from SLE patients express lower amounts of IL-10

CD24hi

CD38hi

CD24hi

CD38hiCD24hi

CD38intCD24hi

CD38-

CD24hi

CD38-CD24hi

CD38int

Blair P.A. et al, Immunity 32, 129-140, 2010

Healthy SLE

CD19+CD24hiCD38hi B cells from healthy human individuals suppress T helper cell differentiation through IL-10

:CD24hiCD38hi B Cells

Blair P.A. et al, Immunity 32, 129-140, 2010

7


Th17 cells

Th1 cells

Antigen TLR ligand

TLRBCR

CD40

MHCII

TCR

B10 cells

ActivatedB10 cells

CD4+

T cells

CD40

IL-10

IL-17

IFN-γ

CD40L

MHCII

The model of regulatory B cell development

Mauri, C. et al., Annu. Rev. Immunol., 30:221-241, 2012.Yoshizaki, A., et al., Nature, 491:264-268, 2012.

Our working model

8


What lineage of B cell is producing IL-10 in vivo ?

Lymph node B cells

FAS+GL7+B cells (GC B)

CD138+CD44+ cells are main IL-10-producing B cells

IL-10-Venus

B220+ cells CD138+CD44+ cells

IL-10-Venus

Naïve mice (day 0)

EAE-induced mice (days 14)

B220+ cells

WT mice

IL-10-Venus mice

Matsumoto M. et al, Immunity 41, 1040-1051, 2014

9


Draining LN cells

0

2

4

6

8

10

12

days0

days7

days14

days21

days28

Cel

ls (

x 10

4)

Gated on CD138+CD44+ cells

Blimp1-GFP

EAE-induced Blimp1-GFP mice

CD

138

Plasmablast (Blimp1int)

CD138+CD44+ cells in dLN are plasmablasts


Oracki, S. A. et al., Immunol. Review, 237:140-159, 2010

Blimp1 is a master gene to differentiate into CD138+ plasmablasts and plasma cells

ActivatedB cell

Plasmablast Short-livedPlasmablast

Long-livedPlasmablast

Surface lg+

B220+

Synd-1-

Flt3+

MHC ll++

CXCR4-

Pax5+++

Irf4+

Surface lg+

B220 lo

Synd-1+

Flt3-

MHC ll+

CXCR4+

Pax5-

Irf4+++

Blimp-1+

Surface lg-

B220-

Synd-1+

Flt3-

MHC ll-

CXCR4+++

Pax5-

Irf4+++

Blimp-1+++

Surface lg-

B220-

Synd-1+

Flt3-

MHC ll+

CXCR4++

Pax5-

Irf4+++

Blimp-1++

(CD138+)

10


EA

E s

core

Days

Blimp1 BKO mice exhibit increased severity of EAE

0

1

2

3

4

5

0 10 20 30

MBC

mbprdm1

P<0.05

Blimp1 BKO

Control


Where can B cells suppress EAE ?

11


dLNs

zz

CD138+

Medullary sinus

Is the spleen a suppression site?

Splenectomy

Spleen

CD138+

VeinArtery VeinArtery

24

Splenectomy mice show normal EAE development

0

1

2

3

4

5

0 10 20 30

Sham Splenectomy

EA

E s

core

Days


12


zz

Medullary sinus

CD138+

WT

VeinArtery

CD138+

WT

HEV

B cell-deficient mice

Are LNs a suppression site?

WT B cells

Artery Vein

Spleen dLNs

zz

Medullary sinus

KO

CD138+

KO

HEV

B cell-deficient mice

Are LNs a suppression site?

L-selectin KO B cells

VeinArtery VeinArtery

Spleen dLNs

13


0

1

2

3

4

5

0 10 20 30

WT B cells

L-sel KO B cellsE

AE

Sco

re

Days

L-sel KO B cells do not suppressEAE development


How does B cell IL10 suppress EAE ?

14


Th17 cells

B10 cellsCD4+

T cells

IL-10

TLRBCR

CD40

MHCIITCR

CD40L

Dendriticcell

Plasmablast MHCII

Naive T cells Th1

cells

IL-10R

Antigen

TCR

The model of regulatory function of plasmablasts

CD4+ T cells DCCD8+ T cells B cells

Isotype control

Anti-IL-10R

DC in the draining LN express IL-10 receptor

EAE-induced WT mice (days 14)

IL-10R

Th1 cells Th17 cells

IL-10RMatsumoto M. et al, Immunity 41, 1040-1051, 2014

15


0

200

400

600

800

1000

1200

1400

LPS LPS+IL-10

IL-10 can inhibit the production of IL-6 and IL-12 by DC

IL-6

(p

g/m

l)

LPS LPS + IL-10

LPS LPS + IL-10

IL-6 IL-6DendriticCell (DC)

DendriticCell (DC)


TGF- + MOG35-55

IL-10 can inhibit the differentiation of naïve T cells into Th17 cells

IFN

-

IL-17

IFN

-

IL-17

LPS LPS + IL-10

IL-6 IL-6DC

Naïve T cells

MOG TCR


16


Th17 cells

B10 cellsCD4+

T cells

IL-10

TLRBCR

CD40

MHCIITCR

CD40L

Dendriticcell

Plasmablast MHCII

Naive T cells Th1

cells

IL-10R

Antigen

TCR


ArteryVein

Afferent lymphatic vessels

Medullary sinus

Girard, J.P. et al., Nature Rev. Immunol., 12:762-773, 2012.Wehrlii, N., et al., Eur. J. Immunol., 31:609-616, 2001.

Dendritic Cell

Efferent lymphatic vesselsHEV

B

T

PB

T

B

17


Plasmablasts are mainly localized in the T-B border area

B

BT

Plasmablast (CD138) DC (CD11c)

Merge

B

BT

B

BT

Efferent lymphatic

Medullary sinus

Afferent lymphatic

B cell follicle

T cell area

Matsumoto et. al. Immunity 41, 1040-1051, 2014

Central nervous system (CNS)

CNS autoantigen

Neuron

Myelin sheath

CNS autoantigen (MOG35-55)

Blood-brain barrier

IFN-

IL-17

Subcutaneous tissue

Lymph node

CD4+

T cells

B cell follicle

DC

DC

EAE, a mouse model of multiple sclerosis, is caused by autoreactive T cells

Bloodcirculation

Tfh

Th17

Th1

Bloodcirculation

18


CD4+

T cells

Tfh

Th17

Th1

B cell follicle

PB

Dendritic cell

B cell follicle

Lymph node

Tfh

Tfh

CD4+

T cells

Dendritic cell

BCR

Antigen

Naïve B cells

ActivatedB cells

PB Tfh


Central nervous system (CNS)

CNS autoantigen

Neuron

Bloodcirculation

CNS autoantigen (MOG35-55)

Blood-brain barrier

Subcutaneous tissue

Th17

B cell follicleIFN-

IL-17

IL-10

TregTreg

Th1

Th17

Th1

DC

DC

EAE, a mouse model of multiple sclerosis, is caused by autoreactive T cells

Myelin sheath

19


What are essential factors for IL-10 production inside B cells?

IRF4 is essential for NFAT-dependent IL-10 production

B cells Plasmablasts

BCR BCRTLR

Differentiation

TLR

Irf4NF-B IRF4 NFAT Il-10

20


IRF4 expression is induced by TLR stimulation

Day 0 Days 2 Days 4

WT

IRF4

-actin

LPS

48 hr WB (IRF4 and -actin)Naive B cells


IRF4 is essential for IL-10 production

IL-1

0 (p

g/m

l per

105

cells

)

0

2000

4000

6000

8000

10000

No anti-IgM PI

WT

IRF4 KO

LPS+

BCR stimulation

LPS

ELISALPS

48 hr

BCR stimulation

24 hrPBPBB


21


IRF4 BKO mice show increased severity of EAE

EA

E S

core

Days

0

1

2

3

4

5

6

0 7 14 21 28 35

Control

IRF4 BKO


1000

IL-1

0 (p

g/m

l pe

r 10

5 cel

ls)

0

500

2000

1500

STIM1&2 BKOControl

STIM KO plasmablasts impair IL-10 production

LPS+

BCR stimulation

LPS

ELISALPS

48 hr

BCR stimulation

24 hrPBPBB


22


ControlSTIM1 BKO

ControlSTIM2 BKO

ControlSTIM1&2 BKO

2mM Ca2+

Time (sec)

anti-IgM

EGTA

Flu

ores

cenc

e ra

tioSpleen: B220+ gate

Control: mb1-Cre mice

anti-IgM

anti-IgM

Ca2+

Ca2+

Ca2+

Calcium influx in STIM-deficient B cells


0

1

2

3

4

5

0 10 20 30

Clin

ical

sco

re

P < 0.05

Time (d)

STIM1&2 BKO

Control

STIM-deficiency exacerbates the development of EAE


23


IRF4 is essential for NFAT-dependent IL-10 production

B cells Plasmablasts

47

BCR BCRTLR

Differentiation

TLR

Irf4NF-B IRF4 NFAT Il-10

How about humans?

24


None IL-2/6/IFN CpG+IL-2/6/IFNCpG

CD38

CD

27

CD27int plasmablast

Naïve BMemory B

CD27hi plasmablast

Concomitant treatment with CpG and cytokines induces the generation of CD27intCD38+ cells


Naïve B Memory B

CD27int

plasmablastCD27hi

plasmablast

CD27intCD38+ cells consist of plasmablasts


25


0

200

400

600

800

0

2000

4000

6000

8000

0

2000

4000

6000

8000

10000

IL-1

0 (p

g/m

l)

None Anti-IgM PMA+Ionomycine

CD27int plasmablasts selectively secrete IL-10


0

500

1000

1500

CD38

CD

27

Naïve B Memory B

IL-1

0 (p

g/m

l)

IL-10-producing CD27int plasmablastsare derived from naïve B cells

Culture with CpG+IL-2/6/IFN

CD

27


26


IL-10-producing CD27int plasmablastsare derived from naïve B cells

IL-10

Cytokinereceptors

BCR BCRTLR

Differentiation

TLR

Naive B cells

TLR

Memory B cells Differentiation

BCRTLR

CD27hi

plasmablasts

CD27int

plasmablasts

Conclusions

1) Plasmablasts in LNs produce IL-10 to suppress EAE

2) Plasmablasts move across interfollicular and T cellzones, probably interacting with DCs

3) IL-10 inhibits DC functions to generate Th17 cells

4) The above mechanism is probably operating inhumans

→ Plasmablasts control T-cell autoimmunity

through their IL-10 production54

27

Adaptive immunity 2 - Immune regulatory mechanisms through B cell axis

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