B cells: Beyond Antibodies

November 1st, 2006

Amit Bar-Or, MD, FRCPC

Neuroimmunology Unit Montreal Neurological Institute Montreal, QC, Canada

Antibody-independent roles of B cells

• antigen presentation (APC)

• innate immunity (environment/immune interface)

• immune regulation

• lymphoneogeneis

Outline

B Cell Development

B Cell

B cell roles in Neuroimmunological diseases

Antibody-independent roles of B cells

• antigen presentation (APC)

• innate immunity (environment/immune interface)

• immune regulation

• lymphoneogeneis

Outline

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.

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

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 (MT) mice have given rise to contradictory results.

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.

Observations (i) T cell clonal expansion is significantly reduced

(ii) 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.

Reduced T cell proliferation in B cell deficient (MT) mice

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

MHC class II on B cells in chimeric versus Wild type

Preserved architecture in LN of chimerics and wild type(T cell: Green; B cell: red)

Decreased ova-II T cell expansion in absence of MHC class II on B cells

Deficient T cell expansion could be restored with addition of wild type B cells

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

Primary response Recall response

Impaired T cell cytokine production in chimeric mice

T cell expansion in the chimerics is not dependent on CD40 signaling by B cells

Similarly, T cell expansion in the chimerics is not dependent on LT signaling by B cells

Conclusions

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

Model of Human Autoimmune Disease (eg: MS)

2. Adhesion

3. Attraction

Periphery

BBB

CNS

5. Reactivation

1. Activation

Th

4. Invasion

6. Neural/glialresponses

B Cell

B Cell

CD19

CD27

IgD CD80

Bar-Or et al, J Immunol, 2001

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

1. Lower threshold of activation

2. 3-10 more immunoglobulin

3. Significantly stronger T cell responses

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

18%

13%

CD

80

CD27

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.

CD27+CD80+ Memory B Cell Subset

Increased percent of CD27+CD80+ B cells in the CSF of patients with Progressive forms of MS

BLOOD

RRMS SPMS PPMS OND0

10

20

30

40

% B

cel

ls

RRMS SPMS PPMS OND0

25

50

75

% B

cel

ls

One way ANOVA (p<0.0001)

p<0.001

p<0.05

p<0.01

CSF

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

Antibody-independent roles of B cells

• antigen presentation (APC)

• innate immunity (environment/immune interface)

• immune regulation

• lymphoneogeneis

Outline

TLR9 stimulation (CpG) provides an essential signal for

induction of human naive B cell proliferation

Sorting human naïve 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

Sorting human naïve B cells

Naïve B cell proliferation (various stimuli)

with or without CpG addition

Naive (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

Various TLR agonists provide ‘signal three’ either

directly on naïve B cells, or indirectly through DC activation

Anti-Ig + T Cell help + different TLR …

TLR agonists provide ‘signal three’ either

directly on naïve B cells, or indirectly through DC activation

Anti-Ig + T Cell help + different TLR …

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

1

2

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

1

2

3

4

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 and Toll-like receptors (TLRs):

Model of Human Autoimmune Disease (eg: MS)

2. Adhesion

3. Attraction

Periphery

BBB

CNS

5. Reactivation

1. Activation

Th

4. Invasion

6. Neural/glialresponses

B Cell

B Cell

CD3

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

Fluorescence

Co

un

ts

Alter et al, J Immunol 2003

Blocking VLA-4 inhibits B cell migration

+ IgG1+IL-8+MCP-1+ICAM-1+VLA-40

10000

20000

30000

num

ber m

igra

ted

ControlIgG

MCP-1

ICAM-1

VLA-4IL

-820,000

30,000

10,000

0Blocking conditions

Molecular Mechanisms of B cell Migration across Brain Endothelial Barriers

Alter et al, J Immunol, 2003

MMP 11 B MMP 11 T0.0

2.5

5.0

7.57.5

5.0

2.5

0B cells T cells

MMP 11

p = 0.03

MMP 17 B MMP 17 T0.0

2.5

5.0

7.57.5

5.0

2.5

0B cells T cells

p = 0.02

MMP 17

MMP 28 T MMP 28 T0

1000

20002000

1000

0B cells T cells

p = 0.03

MMP 28

TIMP 1 B TIMP 1 T0

10

20

3030

20

10

0B cells T cells

TIMP-1 p =0 .02

Distinct MMPs expressed by B cells and T cells

Bar-Or et al, Brain 2003

B Cell

B cell roles in Neuroimmunological diseases

Antibody-independent roles of B cells

• antigen presentation (APC)

• innate immunity (environment/immune interface)

• immune regulation

• lymphoneogeneis

Outline

Reciprocal regulation of human B cell effector cytokines is context dependant

LT (pg/ml)

IL-

10 (

pg/m

l)

0 100 200 300 400 500 6000

100

200

300

400

500

600

a

Dual stimulation (BCR + CD40)

CD40 alone

pg/m

l

IL-10 LT0

100

200

300

400 p = 0.0064 p < 0.0001

b

CD40 alone

Dual stimulation (BCR + CD40)

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

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

TNF

0

100

200

CD40 alone Dual stimulation

ns p = 0.002

Cyt

oki

ne

Se

cre

tion

(p

g/m

l)

LT

0

150

300

CD40 alone Dual stimulation

ns p = 0.002

Cyt

oki

ne

Se

cre

tion

(p

g/m

l)

IL-10 p = 0.004 ns

0

100

200

CD40 alone Dual stimulation

Cyt

oki

ne

Se

cre

tion

(p

g/m

l)

CD27 + Memory B cells CD27 - Naive B cellscp

m

(x 1

0-3)

Proliferation

CD40 alone Dual stimulation

0

10

20

ns ns

B Cells as Active Immune Regulators via 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

Abnormal B cell cytokine production in patients with Multiple Sclerosis

IL-10

p = 0.008 p = 0.037

0

200

400

CD40 alone Dual stimulation

Cyt

oki

ne

Se

cre

tion

(p

g/m

l)

Normals

Multiple Sclerosis

cpm

(x

10-3

)

Proliferation

CD40 alone Dual stimulation

0

5

10

15 ns ns

LT

0

200

400

CD40 alone Dual stimulation

Cyt

oki

ne

Se

cre

tion

(p

g/m

l)

ns ns

TNF

0

100

200

CD40 alone Dual stimulation

Cyt

oki

ne

Se

cre

tion

(p

g/m

l) ns ns

Antibody-independent roles of B cells

• antigen presentation (APC)

• innate immunity (environment/immune interface)

• immune regulation

• lymphoneogeneis

Outline

Bar-Or LabFarzaneh JaliliChristine Ghorayeb Sarah EkdawiStefan SawoszczukMelissa WrightClaudia CalderMadeline PoolTarik TouilDonald Gagne

Thierry VincentIsabel Rambaldi

Experimental TherapeuticsGregory CosentinoBoli FanCaroline BodnerSudy Alatab

AlumniMartin Duddy - BelfastMasaaki Niino -SapporoAndrea Alter - McGillHo Jin Kim - Seoul

Many thanks to:

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

Virology/HERVRaymond Tellier - HSCChris Power - Edmonton

T cell AssaysHans-Michael Dosch - HSCRoy Chung

anti-CNS AntibodiesClara Lopez-Amaya - HSCMario Moscarello U of TKevin O’Connor – HarvardBill Robinson - StanfordDavid Hafler - HarvardLarry Steinman - Stanford

Pediatric MS/CISBrenda BanwellJulia Kennedy Lauren KruppDoug ArnoldDessa Sadovnick

DNA VaccineHideki Garren - BHT

Nogo ProjectAlyson Fournier - MNITanja Kuhlmann - Gottingen

Migration/MMPWee Yong - CalgaryRobert Nuttall - UEADylan Edwards - UEA Fabrizio Giuliani - EdmontonJack Antel - MNI

APC ProjectsHeinz Wiendl - WurzburgScott Zamvil - UCSFSergio Baranzini - UCSFJack Antel - MNI

Canadian BMT Study Group Mark Freedman - OttawaHarry Atkins - OttawaRafick Sekaly - CHUMRemi Cheynier - Inst. PasteurClare Baecher Alan - Harvard

Claudia Calder

Tarik Touil

Christine Ghorayeb

Donald Gagne

Farzaneh Jalili

Madelaine Pool

AlumniMasaaki Niino – SapporoMartin Duddy - BelfastHo Jin Kim - Seoul

Many thanks to:

Support: CIHR/IHRT, MSSC, ITN/NIH, WadsworthMSSC Scientific Research Foundation

Virology/HERVRaymond Tellier - HSCChris Power - Edmonton

T cell Assays/BHTHans-Michael Dosch - HSCClare Baecher-AllanRoy ChungLarry SteinmanHideki Garren

anti-CNS AntibodiesClara Lopez-Amaya - HSCMario Moscarello U of TKevin O’Connor - HarvardBill Robinson - StanfordLarry Steinman - Stanford

Pediatric MS/CISBrenda BanwellDoug ArnoldDessa SadovnickJulia KennedyMaria TantsesLauren Krupp

ETP ProjectsGreg CosentinoSudy AlatabCaroline BodnerBoli Fan

Alyson FournierTim KennedyPhil Barker

Canadian BMT Study Group Mark Freedman - OttawaHarry Atkins - OttawaDavid Haegert - McGillRafick Sekaly - CHUMRemi Cheynier - Inst. PasteurClare Baecher Alan

Jack Antel

Pierre DuquetteAlexnadre PratNathalie Arbour