Claudia Mauri

University College London

United Kingdom

The regulatory outcome of cross-talk between regulatory B cells and invariant

natural killer T cells

PlasmaCell

B cell

Pathogenicantib

odi es

“Normal”antib

odi es

PlasmaCell

B cell

T cell

MHCIIpeptide

iNKTcell

CD1diTCR lipid

B cellB cell

iNKTcell

T cell

TCR

Antigen presentation Antibody production

B cells play a pleiotropic role in the immune system

B cell subsets

Bregs

immunosuppression

Francis Lund et al. Nature Immunol 2000

Defense against parasites

Contribute to allergies and asthma

Defense against intracellular pathogens

Contribution in the pathogenesis of autoimmune diseases

Naïve B cells

B effector 1

IFNg Th1

B effector 2

IL-4

Th2

Naïve B cells

B cells can suppress inflammation

Mice lacking IL-10 producing B cells develop an exacerbated arthritis

IL-10-/- B cell mice: lower Treg numbers but higher Th17 and Th1 cells than WT mice

A

100 101 102 103 104100

101

102

103

104

100 101 102 103 104100

101

102

103

104

CD

4

FoxP3

WT B cells IL-10-/- B cells 8.75% 3.94%

Fo

xP

3

WT B cells

100 101 102 103 104100

101

102

103

104

100 101 102 103 104100

101

102

103

104

0.42% 0.16%

CD4

CD4+CD25-

IL-10-/- B cells

Synovia draining LN

Janus face at the Vatican (Rome) Habemus papam!

CD19

CD24hi

CD21hi

CD23hi

AA4+/int

CD1dhi

IgMhi

CD21hi

CD5

CD1dhi

CD24hi

IgMhi

Yan

ab

a K

. 20

08

(B1

0) Ev

ans

J. 2

007

T2-M

ZP

Markers for Bregs

immature memory

mature

;

Sims, G. P. et al. Blood; 2005

Anolik J. et al. Arthritis and Rheum.2007 Carsetti R. Immunol. Rev. 2004 Blair P. et al Immunity. 2010

B cell subsets in periphereal blood mononuclear cells

CHO-CD154

Blair et al. Immunity 2010

Memory

mature

Immature

IL-10 producing B cells are enriched within the CD24hiCD38hi gate

Blair et al Immunity 2010;32(1):129-40. 2010

Flores-Borja et al. Science Trans Med Feb 20;5(173):2013

CD19+CD24hiCD38hiB cells from healthy individuals suppress T helper cell differentiation

T cells are converted into FOXP3+T regs

FoxP

3

Bregs induce regulatory T cells

Flores Borja et al. (Science Trans Med. 2013)

CD4+ +

-

+ +

-

+

++

cp

m (

10

3)

CD25+

CD127-

0

25

50

75

100

125 *

Healthy

8.78.4

100

101

102

103

104

1.4 11.4H

4

no B cells

CD4+ + anti-CD3

CD4+ CD24intCD38int CD24hiCD38hi

4

6

8

10

0

2

CD24int

CD38int

CD24hi

CD38hi

CD

4+F

oxP

3+ (

%)

**

-

+

- +

- -

CD4

Treg

Beff

Th17

Th1Breg

Treg

Breg

Autoantibodies IL-17, IFNg, TNFa

BeffTh17

Th1Breg

Treg

Healthy Autoimmunity

Systemic lupus erythematosus (SLE)

• Systemic autoimmune disease affects approximately

0.1% of overall population

• Extremely diverse with manifestations in major organ

systems

• Treatments range from steroids to cell-targeted

treatments, such as Rituximab, a chimeric monoclonal

antibody targeting and depleting CD20 expressing B cells

HEALTHY

SLE

Blair et al Immunity 2010;32(1):129-40.

CD19+CD24hiCD38hi B cells from SLE patients fail to

suppress IFNg+ /TNFa+CD4+ T cell differentiation

• Immature B cells isolated from SLE are not

numerically different from healthy individuals

• Effector T cells from SLE are not refractory to

suppression

• Intrinsic defect in Bregs in SLE patients

IL-10 production in SLE patients is reduced on immature B cells, but not on other B cell subsets

Paul Blair et al Immunity 2010

B cell IL-10 production

Uns

timulat

ed

anti CD3

0

1

2

3

4

5Healthy controls

SLEIL

-10

%

healthy

SLE

Paul Blair Madhvi Menon

%im

mat

ure

B c

ell

IL-1

0+

CHO-CD40L medium

CD1d expression in SLE patients is reduced on immature B cells, but not on other B cell subsets

Bosma et al Immunity 2012

CD1d

isotype

mature

memory

immature

but not on monocytes

B cells Monocytes

Healthy

SLE

Healthy

SLE

CD1d

% o

f M

ax

Increased CD1d internalisation-rate leads to the reduction of CD1d expression on B cells from SLE

patients

He

alt

hy

S

LE

0 min 5 min

CD1d DAPI

CD1d DAPI

CD1d DAPI

CD1d DAPI

CD1d-lipid antigen presentation to iNKT cell

iNKT cells are important for B cell antibody production and memory

What is the impact of the reduction of CD1d expression in SLE B cells in iNKT cell differentiation and function

iNKT cells are reduced in number in SLE patients

****

iNK

T c

ells

(x10

6/L

ite

r)

0

1

2

3

4

51015

Hea

lthy

SLE

CD1d

isotype

mature

memory

immature

Healthy, and not SLE, iNKT cell proliferate in response to a-Gal-Cer stimulation

Bosma et al Immunity 2012

Day0 Day 7 Day0 Day 7

HEALTHY SLE

IL-2

(ng

/ml)

IFN

-g (

ng/m

l)

TN

F-a

(p

g/m

l)

IL-1

0 (

pg

/ml)

0

2

4

6

0

2

4

6

* **

*

0

50

100

150

0

10

20

30

40

50

H SLE H SLE H SLE

IFN-g (ng/ml)0 5 10 15

TNF-a (pg/ml)0 10 20 30 40

***

Healthy PBMC-B cells + Allogeneic healthy B cells

SLE PBMC-B cells + Healthy B cells

Healthy PBMC-B cells + SLE B cells

SLE PBMC-B cells + Allogeneic SLE B cells

Healthy PBMC-B cells + Allogeneic healthy B cells

SLE PBMC-B cells + Healthy B cells

Healthy PBMC-B cells + SLE B cells

SLE PBMC-B cells + Allogeneic SLE B cells

C

Healthy

PBMC -B cells

SLE

PBMC -B cells

Healthy PBMC -B cells

+ SLE B cells

SLE PBMC-B cells

+ Healthy B cells

1 2 3

Regulatory B cells induce higher iNKT cell proliferation than mature B cells

Breg iNKT

cell +

aGal-Cer+IL-2

Beff iNKT

cell +

Bosma et al Immunity 2012

Depletion of immature B cells from PBMCs

impairs iNKT cell expansion

Whole

PBMCs

iNKT cell

expansion (day 7)

a-GalCer

+ IL-2

B cell depleting (rituximab) treated patient groups

RBRnr

RBRr

BCD = B cell depleted; RBRnr = rituximab, B cell repopulated, non-responding (Global BILAG Score after repopulation);

RBRr =rituximab, B cell repopulated, responding (No change in Global BILAG Score after repopulation)

iNKT cell numbers remain low during B cell depletion but is restored upon return of B cells in responding patients

BC

DR

r B

CD

Rn

r

0 min 5 min

CD1d DAPI

CD1d DAPI

CD1d DAPI

CD1d DAPI

CD1d recycling is normalizes in repopulated B cells patients responding to B cell depletion therapy

BCD = B cell depleted BCDRnr = B cell depleted repopulated, non-responding BCDRr = B cell depleted repopulated,responding

RBRr

0.001 0.01 0.1 10.1

1

10

100

r = 0.3770

p = 0.0017

% C

D1d

hi

imm

atu

re B

cells 0

6

BILAG

% iNKT cells

Restoration of CD1d expression on immature B cells correlate with higher number of iNKT cells and

better clinical response to rituximab

Conclusions from studies in SLE patients

• Immature B cells sustain the in vitro expansion and

activation of iNKT cells in healthy individuals via CD1d

• SLE patients present a decreased expression of CD1d hi on

immature B cells

• SLE iNKT cells are numerically and functionally impaired

• iNKT cell, B cell, CD1d defect in SLE patients are

exclusively reversed in patients responding to rituximab

treatment

• Positive correlation between iNKT cell and immature B cell

and amelioration of disease.

UCL Paul Blair Fabian Flores-Borja Anneleen Bosma Azza Abdel Gadir Elizabeth Rosser Madhvi Menon Kiran Nistala Christoper Piper David Isenberg Elizabeth Jury

Washington University David Rawlings University of Oxford Vincenzo Cerundolo INSERM Agnes Lehuen