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Amit Bar-Or, MD, FRCPC Neuroimmunology Unit Montreal Neurological Institute Montreal, QC, Canada . B cells: Beyond Antibodies November 1 st , 2006. Outline. Antibody-independent roles of B cells antigen presentation (APC) innate immunity (environment/immune interface)

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B cells beyond antibodies november 1 st 2006

Amit Bar-Or, MD, FRCPC

Neuroimmunology Unit

Montreal Neurological Institute

Montreal, QC, Canada

B cells: Beyond AntibodiesNovember 1st, 2006


B cells beyond antibodies november 1st 2006

Outline

  • Antibody-independent roles of B cells

    • antigen presentation (APC)

    • innate immunity (environment/immune interface)

    • immune regulation

    • lymphoneogeneis



B cells beyond antibodies november 1st 2006

B Cell

B cell roles in Neuroimmunological diseases


B cells beyond antibodies november 1st 2006

Outline

  • Antibody-independent roles of B cells

    • antigen presentation (APC)

    • innate immunity (environment/immune interface)

    • immune regulation

    • lymphoneogeneis


B cells beyond antibodies november 1st 2006

B cells as antigen-presenting cells (APC):

T cell expansion and differentiation in vivo requires antigen presentation by B cells. Crawford et al. J Immunol 2006

Antigen-presenting B cells: role in induction of peripheral T cell tolerance Raimondi et al. J Immunol 2006.


B cells beyond antibodies november 1st 2006

The Journal of Immunology, 2006, 176: 3498–3506.


B cells beyond antibodies november 1st 2006

Background

Though B cells are well documented as APC, their role in supporting and programming the T cell response in vivo is still unclear

Studies using B cell-deficient (mMT) mice have given rise to contradictory results.


B cells beyond antibodies november 1st 2006

Methods

Mixed BM chimeric mice to define the contribution that B cells make as APC.

Can generate TCR-Tg mice where B cell compartment is deficient in MHC class II, while other APC are largely normal.

Other mice: CD40 deficient, LT deficient.


B cells beyond antibodies november 1st 2006

  • Observations

  • T cell clonal expansion is significantly reduced

  • Differentiation of T cells into cytokine-secreting effector cells is impaired (in particular, Th2 cells)

  • (iii) Development of the memory T cell populations is also decreased.

  • Furthermore: Although MHC class II-mediated presentation by B cells was crucial for an optimal T cell response, neither a B cell-specific lack of CD40 (influencing costimulation) nor lymphotoxin (influencing lymphoid tissue architecture) had any effect on the T cell response.



B cells beyond antibodies november 1st 2006

Good reconstitution in mixed BM chimera (non-B cells mostly wild-type, all B cells MHC class II deficient)


B cells beyond antibodies november 1st 2006

MHC class II on B cells in chimeric versus Wild type wild-type, all B cells MHC class II deficient)


B cells beyond antibodies november 1st 2006

Preserved architecture in LN of chimerics and wild type wild-type, all B cells MHC class II deficient) (T cell: Green; B cell: red)


B cells beyond antibodies november 1st 2006

Decreased ova-II T cell expansion in absence wild-type, all B cells MHC class II deficient) of MHC class II on B cells



B cells beyond antibodies november 1st 2006

Impaired memory response to viral antigen in MHC-deficient B cell chimerics – assessed by T cell tetramer

Primary response

Recall response


B cells beyond antibodies november 1st 2006

Impaired T cell cytokine cell chimerics – assessed by T cell tetramer

production in chimeric mice


B cells beyond antibodies november 1st 2006

T cell expansion in the chimerics cell chimerics – assessed by T cell tetramer

is not dependent on CD40 signaling by B cells


B cells beyond antibodies november 1st 2006

Similarly, T cell expansion in the chimerics cell chimerics – assessed by T cell tetramer

is not dependent on LT signaling by B cells


B cells beyond antibodies november 1st 2006

Conclusions cell chimerics – assessed by T cell tetramer

In vivo, B cells provide extra and essential Ag presentation capacity over and above that provided by dendritic cells

In this context, B cells optimize expansion and allow the generation of memory and effector T cells


B cells beyond antibodies november 1st 2006

1. cell chimerics – assessed by T cell tetramerActivation

4. Invasion

3. Attraction

Th

B Cell

B Cell

2. Adhesion

5. Reactivation

6. Neural/glial

responses

Periphery

BBB

CNS

Model of Human Autoimmune Disease (eg: MS)


B cells beyond antibodies november 1st 2006

CD19 cell chimerics – assessed by T cell tetramer

CD80

IgD

CD27

Bar-Or et al, J Immunol, 2001

A human memory B cell subset expresses the CD80 (B7.1) costimulatory molecule


B cells beyond antibodies november 1st 2006

18% cell chimerics – assessed by T cell tetramer

CD80

13%

CD27

CD27+CD80+ Memory B Cell Subset

Lower threshold of activation

3-10  more immunoglobulin

Significantly stronger T cell responses

Additional molecules (CD11b, CD72) important to B cell:T cell interaction

Bar-Or A, et al, J Immunol. 2001;167:5669-5677.

Alter A, et al. J Immunol. 2003;170:4497-4505.

Duddy ME, et al. J Immunol. 2004;172:3422-3427.

Bar-Or A. Adv Neurol. 2006;98:91-109.


B cells beyond antibodies november 1st 2006

BLOOD cell chimerics – assessed by T cell tetramer

CSF

p<0.01

p<0.05

p<0.001

40

75

30

50

% B cells

% B cells

20

25

10

0

0

RRMS

SPMS

PPMS

OND

RRMS

SPMS

PPMS

OND

One way ANOVA (p<0.0001)

RRMS; n=11, SPMS; n=8, PPMS; n=11, OND; n=8

Increased percent of CD27+CD80+ B cells in the CSF

of patients with Progressive forms of MS


B cells beyond antibodies november 1st 2006

Outline cell chimerics – assessed by T cell tetramer

  • Antibody-independent roles of B cells

    • antigen presentation (APC)

    • innate immunity (environment/immune interface)

    • immune regulation

    • lymphoneogeneis


B cells beyond antibodies november 1st 2006

Sorting human naïve cell chimerics – assessed by T cell tetramer

B cells

Naïve B cell proliferation

(various stimuli)

with or without CpG addition

TLR9 stimulation (CpG) provides an essential signal for

induction of human naive B cell proliferation


B cells beyond antibodies november 1st 2006

TLR9 stimulation (CpG) provides an essential signal for cell chimerics – assessed by T cell tetramer

induction of human naive B cell proliferation

Sorting human naïve

B cells

Naïve B cell proliferation

(various stimuli)

with or without CpG addition


B cells beyond antibodies november 1st 2006

Naive cell chimerics – assessed by T cell tetramer (TN) CD4+CD45RO–CCR7+

Central memory (TCM) CD4+CD45RO+CCR7+CXCR5–

Follicular help (TFH) CD4+CD45RO+CCR7+CXCR5+

Effector memory (TEM) CD4+CD45RO+CCR7–

Regulatory T cells (TREG) CD4+CD45RO+CD25+

Requirement for CpG is seen regardless

of the source of T cell help


B cells beyond antibodies november 1st 2006

Various TLR agonists provide ‘signal three’ either cell chimerics – assessed by T cell tetramer

directly on naïve B cells, or indirectly through DC activation

Anti-Ig

+ T Cell help

+ different TLR …


B cells beyond antibodies november 1st 2006

TLR agonists provide ‘signal three’ either cell chimerics – assessed by T cell tetramer

directly on naïve B cells, or indirectly through DC activation

Anti-Ig

+ T Cell help

+ different TLR …


B cells beyond antibodies november 1st 2006

1 cell chimerics – assessed by T cell tetramer

Anti-Ig, T help

and CpG, induce strong

RNA expression of:

1. AID (Ig Class switch)

2. xbp-1 (Plasma cell)

2

IgA G M

TLR stimulation is required for induction of Ig isotype switch and differentiation to Ig-secreting (plasma) cells.


B cells beyond antibodies november 1st 2006

1 cell chimerics – assessed by T cell tetramer

3

2

4

TLR stimulation is required for induction of Ig isotype switch and differentiation to Ig-secreting (plasma) cells.

Anti-Ig, T help

and CpG, induce strong

RNA expression of:

1. AID (Ig Class switch)

2. xbp-1 (Plasma cell)

IgA G M


B cells beyond antibodies november 1st 2006

B cells and Toll-like receptors (TLRs): cell chimerics – assessed by T cell tetramer

Toll-like receptors (TLRs) make it possible for B cells to ‘sense’

the environment (viruses, bacteria) and respond to external stimuli

TLR signaling is important for normal B cell activation

Ruprecht, Lanzavecchia, 2006; Medzhitov. Nature. Pasare, 2006.

TLR signaling may cause B cells to become abnormally activated,

and may lead to production of auto-antibodies Lau et al. J Exp Med. 2005;

Martin, Elkon. J Exp Med, 2005; Fields et al. J Immunol 2006


B cells beyond antibodies november 1st 2006

Model of Human Autoimmune Disease cell chimerics – assessed by T cell tetramer(eg: MS)

1.Activation

4. Invasion

3. Attraction

Th

B Cell

B Cell

2. Adhesion

5. Reactivation

6. Neural/glial

responses

Periphery

BBB

CNS


B cells beyond antibodies november 1st 2006

CD3 cell chimerics – assessed by T cell tetramer

Comparing Migration of Monocytes, B cells and T cells

across Human Brain Endothelial Cells (HBECs)

Alter et al, J Immunol, 2003; Bar-Or et al Brain 2003


B cells beyond antibodies november 1st 2006

Blocking VLA-4 inhibits B cell migration cell chimerics – assessed by T cell tetramer

Counts

Fluorescence

Alter et al, J Immunol 2003


B cells beyond antibodies november 1st 2006

Molecular Mechanisms of B cell Migration cell chimerics – assessed by T cell tetramer across Brain Endothelial Barriers

Control

IgG

30,000

20,000

aIL-8

aMCP-1

aICAM-1

aVLA-4

10,000

0

Blocking conditions

Alter et al, J Immunol, 2003


B cells beyond antibodies november 1st 2006

Distinct MMPs expressed by B cells and T cells cell chimerics – assessed by T cell tetramer

MMP 11

MMP 17

p = 0.02

7.5

7.5

p = 0.03

7.5

5.0

5.0

5.0

2.5

2.5

2.5

0

0

0.0

B cells

T cells

B cells

T cells

MMP 11 B

MMP 11 T

TIMP-1

p =0 .02

MMP 28

30

2000

2000

p = 0.03

20

1000

1000

10

0

0

0

T cells

B cells

B cells

T cells

MMP 28 T

MMP 28 T

Bar-Or et al, Brain 2003


B cells beyond antibodies november 1st 2006

B cell roles in Neuroimmunological diseases cell chimerics – assessed by T cell tetramer

B Cell


B cells beyond antibodies november 1st 2006

Outline cell chimerics – assessed by T cell tetramer

  • Antibody-independent roles of B cells

    • antigen presentation (APC)

    • innate immunity (environment/immune interface)

    • immune regulation

    • lymphoneogeneis


B cells beyond antibodies november 1st 2006

CD40 alone cell chimerics – assessed by T cell tetramer

Dual stimulation (BCR + CD40)

b

a

CD40 alone

400

p = 0.0064

p < 0.0001

Dual stimulation (BCR + CD40)

600

500

300

400

IL-10 (pg/ml)

200

pg/ml

300

200

100

100

0

LT

IL-10

0

0

100

200

300

400

500

600

LT (pg/ml)

Reciprocal regulation of human B cell effector cytokines is context dependant

Duddy et al J Immunol 2004; Duddy, Niino et al, in review


B cells beyond antibodies november 1st 2006

CD27 - Naive B cells cell chimerics – assessed by T cell tetramer

CD27 + Memory B cells

Proliferation

IL-10

p = 0.004

ns

200

20

ns

ns

100

cpm (x 10-3)

Cytokine Secretion (pg/ml)

10

0

0

CD40 alone

Dual stimulation

CD40 alone

Dual stimulation

TNFa

LT

ns

p = 0.002

ns

p = 0.002

300

200

Cytokine Secretion (pg/ml)

150

Cytokine Secretion (pg/ml)

100

0

0

CD40 alone

Dual stimulation

CD40 alone

Dual stimulation

Normal memory and naïve B cells express distinct patterns of cytokine production


B cells beyond antibodies november 1st 2006

B Cells as Active Immune Regulators via cell chimerics – assessed by T cell tetramer

Effector Cytokines - role in immune regulation

  • Memory B cells, stimulated by their Ag (BCR) and subsequent T cell help (CD40), secrete TNF and LT, thereby actively contributing to efficiency of adaptive memory immune responses

  • Naive B cells (normally harboring autoreactive repertoire), stimulated in “bystander” context (CD40 only), secrete IL-10 that could acquiesce otherwise undesired response


B cells beyond antibodies november 1st 2006

15 cell chimerics – assessed by T cell tetramer

Proliferation

IL-10

ns

ns

ns

ns

ns

ns

p = 0.008

p = 0.037

400

10

Normals

Multiple Sclerosis

5

cpm (x 10-3)

200

Cytokine Secretion (pg/ml)

0

CD40 alone

Dual stimulation

0

CD40 alone

Dual stimulation

400

TNFa

LT

200

200

Cytokine Secretion (pg/ml)

100

Cytokine Secretion (pg/ml)

0

CD40 alone

Dual stimulation

0

CD40 alone

Dual stimulation

Abnormal B cell cytokine production in patients with Multiple Sclerosis


B cells beyond antibodies november 1st 2006

Outline cell chimerics – assessed by T cell tetramer

  • Antibody-independent roles of B cells

    • antigen presentation (APC)

    • innate immunity (environment/immune interface)

    • immune regulation

    • lymphoneogeneis


B cells beyond antibodies november 1st 2006

Many thanks to: cell chimerics – assessed by T cell tetramer

Migration/MMP

Wee Yong - Calgary

Robert Nuttall - UEA

Dylan Edwards - UEA

Fabrizio Giuliani - Edmonton

Jack Antel - MNI

Pediatric MS/CIS

Brenda Banwell

Julia Kennedy

Lauren Krupp

Doug Arnold

Dessa Sadovnick

Bar-Or Lab

Farzaneh Jalili

Christine Ghorayeb

Sarah Ekdawi

Stefan Sawoszczuk

Melissa Wright

Claudia Calder

Madeline Pool

Tarik Touil

Donald Gagne

Thierry Vincent

Isabel Rambaldi

Experimental Therapeutics

Gregory Cosentino

Boli Fan

Caroline Bodner

Sudy Alatab

Alumni

Martin Duddy - Belfast

Masaaki Niino -Sapporo

Andrea Alter - McGill

Ho Jin Kim - Seoul

anti-CNS Antibodies

Clara Lopez-Amaya - HSC

Mario Moscarello U of T

Kevin O’Connor – Harvard

Bill Robinson - Stanford

David Hafler - Harvard

Larry Steinman - Stanford

Canadian BMT Study Group

Mark Freedman - Ottawa

Harry Atkins - Ottawa

Rafick Sekaly - CHUM

Remi Cheynier - Inst. Pasteur

Clare Baecher Alan - Harvard

Nogo Project

Alyson Fournier - MNI

Tanja Kuhlmann - Gottingen

T cell Assays

Hans-Michael Dosch - HSC

Roy Chung

APC Projects

Heinz Wiendl - Wurzburg

Scott Zamvil - UCSF

Sergio Baranzini - UCSF

Jack Antel - MNI

DNA Vaccine

Hideki Garren - BHT

Virology/HERV

Raymond Tellier - HSC

Chris Power - Edmonton

CIHR, MSSC, ITN/NIH, Wadsworth, CIHR/IHRT, MSSC Scientific Research Foundation


B cells beyond antibodies november 1st 2006

Many thanks to: cell chimerics – assessed by T cell tetramer

Pediatric MS/CIS

Brenda Banwell

Doug Arnold

Dessa Sadovnick

Julia Kennedy

Maria Tantses

Lauren Krupp

Jack Antel

Pierre Duquette

Alexnadre Prat

Nathalie Arbour

Claudia Calder

Tarik Touil

Christine Ghorayeb

Donald Gagne

Farzaneh Jalili

Madelaine Pool

Alumni

Masaaki Niino – Sapporo

Martin Duddy - Belfast

Ho Jin Kim - Seoul

Canadian BMT Study Group

Mark Freedman - Ottawa

Harry Atkins - Ottawa

David Haegert - McGill

Rafick Sekaly - CHUM

Remi Cheynier - Inst. Pasteur

Clare Baecher Alan

Virology/HERV

Raymond Tellier - HSC

Chris Power - Edmonton

T cell Assays/BHT

Hans-Michael Dosch - HSC

Clare Baecher-Allan

Roy Chung

Larry Steinman

Hideki Garren

ETP Projects

Greg Cosentino

Sudy Alatab

Caroline Bodner

Boli Fan

Alyson Fournier

Tim Kennedy

Phil Barker

anti-CNS Antibodies

Clara Lopez-Amaya - HSC

Mario Moscarello U of T

Kevin O’Connor - Harvard

Bill Robinson - Stanford

Larry Steinman - Stanford

Support: CIHR/IHRT, MSSC, ITN/NIH, Wadsworth

MSSC Scientific Research Foundation