Autoimmunity: Lessons from Experimental Therapies and Special Cohorts

November 3rd, 2006

Amit Bar-Or, MD, FRCPC

Neuroimmunology Unit Montreal Neurological Institute Montreal, QC, Canada

Why study Special populations?

(i) Pediatric MS

• Environmental contributors - Virology (EBV)• Immune dysregulation: T cell responses (self and

environmental antigens)• Autoreactive antibodies

(ii) Experimental Therapies (ETP)

• Antigen Specific • Global versus selective immune depletion• Immune modulation

Model of Multiple Sclerosis

2. Adhesion

3. Attraction

Periphery

BBB

CNS

5. Reactivation

1. Activation

Th

4. Invasion

6. Neural/glialresponses

B Cell

B Cell

Immune cell infiltration and Dual MS Pathology

Demyelination and Axonal compromiseTCRT cell receptor) antibody

Trapp et al. NEJM, 1998

T cells reactive to the CNS become activated in the periphery

Inflammation and injury

IFNTNFLT

TTHH

11TTHH

11TTHH

11 Th 1Th 1

MBP-reactiveIL-12

B7.1

APC

(Monocytes)

T Cell

MBP-reactive

Foreign/self Ag? Molecular mimicry

Peripheral activation of Myelin-reactive T cells

Experimental Autoimmune Encephalomyelitis (EAE)

‘Altered Peptide Ligand’ (APL) therapy of MS

ENPVVHFFKNIVTPRTP MBP (85-101)

XXPVVHXFXNIVTPRTP APL CGP 77116

• APL induced ‘anti-inflammatory’ T cells andwas beneficial in EAE (Karin et al, J Exp Med, 1994).

APL induced Antigen-specific T cells responses

Week 4:

Week 5: Relapse

MBP APL

1 / (4.9x106) 0

1/840 1/620

Frequency

CrossReactivity

85% 78%

(background < 1 %) Bielekova, et al Nat. Med, 2000Kim et al, Clin Immunol, 2003

APL induced Antigen-specific T cells responses

Week 4:

Week 5: Relapse

MBP APL

1 / (4.9x106) 0

1/840 1/620

Frequency

Bielekova, et al Nat. Med, 2000Kim et al, Clin Immunol, 2003

CrossReactivity

85% 78%

(background < 1 %)

Lessons

MBP may be a real target in MS

Induction of MBP-reactivity in periphery can result in human CNS inflammatory demyelination

However, studies aimed at evaluating myelin-specific (T cell, antibody) responses in MS patients have been challenging

GeneticPredisposition Immune system Target organ (CNS)

Birth

Injury of the CNSNew targets exposed (Epitope Spread)Issues of repair

Compromise of the BBB

Proliferation of T cells, B cells, Antibody formation

Altered hostImmune Response

EnvironmentalExposure/sViruses, etc

?? 1st attack ofdemyelination(CIS)

Diagnosis of MS

Wadsworth Pediatric MS Study Group

Wadsworth Foundation

Epstein-Barr Virus Serological Results from Pediatric Multiple Sclerosis Patients and Controls

Epstein-Barr virus (EBV) serologies multiple sclerosis (MS), Neurological controls, Autoimmune (AI) controls, and healthy controls. Children with MS were more likely to be positive for remote EBV infection and less likely to be EBV-negative than control patients. ** p < 0.001; * p < 0.05

EBV

0%

20%

40%

60%

80%

100%

Acute Remote Negative Indeterminant

MS (n=83)Neurol Control (n=29)AI Control (n=47)Healthy Control (n=26)

** ** ****

*

Tellier Lab

T cell reactivities to self and environmental antigens in pediatric MS and Controls

1. Children with MS harbor a circulating pool of peripherally primed myelin-reactive T cells

2. Children with other autoimmune diseases harbor circulating T cells reactive against disease-specific target tissues

3. Nutritional exposures early in life are associated with heightened reactivity of T cells against target organs

Methods

Primary stimulation assay using fresh PBMC

Adding no antigen or specific test antigens

Assessing T cell proliferation at day 7

Blinded to source of PBMC (HSC Dosch lab)

T cell proliferative responses to tissue antigens in pediatric demyelinating disease and control cohorts

EX-2

MBP-1

MOG

GFAP

S-100

Tep69

69-3

49GAD

Gad55

5 PI0

1

2

3

4

5

6

PancreaticMyelin Glial

EX-2

MBP-1

MOG

GFAP

S-100

Tep69

69-3

49GAD

Gad55

5 PI

0

1

2

3

4

5

6

PancreaticMyelin Glial

Diabetes Cohort

EX-2

MBP-1

MOG

GFAP

S-100

Tep69

69-3

49GAD

Gad55

5 PI0

1

2

3

4

5

6

PancreaticMyelin Glial

EX-2

MBP-1

MOG

GFAP

S-100

Tep69

69-3

49 GAD

Gad55

5 PI0

1

2

3

4

5

6

PancreaticMyelin Glial

MS CohortCIS Cohort

Healthy Cohort

T c

ell p

rolif

erat

ion

(SI)

CIS Cohort MS Cohort Diabetes Cohort

Number 37 26 24 29

Average age (range) 14.3(5.8-19.4)

9.7 (1-20)

Healthy Cohort

9.8 (3.0-16.0)

10.6 (1.1-18.1)

Dosch Lab (Banwell, Dosch, Bar-Or, Submitted; Wadsworth Foundation)

CIS cohort

BLGCas

ein

BSABSA-1

93

BSA-394

Abbos

OVA

0

1

2

3

4

5

6MS cohort

BLGCas

ein

BSABSA-1

93

BSA-394

Abbos

OVA

0

1

2

3

4

5

6

T1D cohort

BLGCas

ein

BSABSA-1

93

BSA-394

Abbos

OVA

0

1

2

3

4

5

6 Healthy Control cohort

BLGCas

ein

BSABSA-1

93

BSA-394

Abbos

OVA

0

1

2

3

4

5

6

T cell responses to dietary antigens in inflammatory demyelination and controls

Dosch Lab (Banwell, Dosch, Bar-Or, Submitted; Wadsworth Foundation)

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

5.50

0 5 10 15 20Age

Activity

CIS Patients

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

5.50

6.00

0 5 10 15 20

Age

Acti

vit

y

MS Patients

1.00

2.00

3.00

4.00

5.00

6.00

0 2 4 6 8 10 12 14 16 18

Age

Act

ivit

y

BLG Casein BSA BSA-193 BSA-394 Abbos

Tep69 69-349 GFAP S-100 IS4 GAD

Gad555 PI b-IA2 EX-2 MBP-1 MOG

Healthy Controls

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

5.50

6.00

0 5 10 15 20

A ge

Ac

tiv

ity

Neurological Controls

T-cell reactivities to multiple antigens as a function of Age

Dosch Lab (Banwell, Dosch, Bar-Or, Submitted; Wadsworth Foundation)

B Cell

B cell roles in Neuroimmunological diseases

Oligoclonal bands (OCB) in cerebrospinal fluid (CSF)

B cells + Plasma Cells: ‘intra-clonal’ expansion Qin, 1998; Colombo, 2000; Baranzini, 2000; Smith-Jensen, 2000; Owens, 2000

MS lesion Pathology:

Ig/complement Lucchinetti, Bruck, Lassmann, 1996, 2000

Anti-myelin Antibodies in phagocytes within lesions of MS and EAE Genain, 1999; Linnington; others

Anti-myelin antibody extracted from MS brain pathology O’Connor 2005

Auto-antibodies in Multiple Sclerosis: History

Auto-antibodies in Neurological disease

Role in peripheral diseases

MG, neuropathies

Treatment responsiveness

Role in CNS inflammatory disease

NMO-Ig and Neuromyelitis optica

Multiple sclerosis?

Antel and Bar-Or, NEJM, 2003

Anti-MBP (rabbit)

Anti-GAD T1D

Scatchard analysis of serum autoantibodies

boun

d/fr

ee

0.04

0.09

0.14

0.19

0.24

1800 3800 5800

bound [cpm]

MS serumNHD serum

Anti-MBP ADEM

O’Connor et al, JNI, 2003

MS Cohort

Healthy Controls

Neurological Controls

Autoimmune Controls

Number 137 80 59 48

Average age at enrollment (range)

14.1

(2.2-19.6)

12.4

(2.0-20.1)

12.1

(2.2-18.5)

11.4

(5.8-17.6)

F:M 1.5 1.4 0.8 2.2

Western blot for MBP immune reactivity

Lopez- Amaya et al

Cohort

MS

Autoimmune

Neurological

Healthy

% Positive

22.3

28.6

23.1

19.2

Frequency of Seropositivity for IgG/M/A anti-MBP antibodies

Lopez- Amaya et al

Suggests that development of humoral (antibody) responses to myelin antigen can be normal

Anti-MBP antibody: no major role in MS initiation

In the MS children with anti-MBP ‘+’: significant higher proportion of ‘ADEM-like presentation’

Acute Disseminated EncephaloMyelitisTypically MONOPHASIC, seen post-immunization

Multi-focal, often quite dramatic inflammatory demyelinating attack, but is not a chronic disease

Suggests that: IF you have first attack of MS, AND IF you have circulating anti-myelin antibodies, you are more likely to get ‘ADEM-like’ episode.

Consistent with EAE animal model observations:

Anti-myelin antibody alone, cannot ‘transfer’ disease

However, with anti-myelin antibodies, less T cells are needed to transfer EAE and disease is more severe

Why study Special populations?

(i) Pediatric MS

• Environmental contributors - Virology (EBV)• Immune dysregulation: T cell responses (self and

environmental antigens)• Autoreactive antibodies

(ii) Experimental Therapies (ETP)

• Antigen Specific • Global versus selective immune depletion• Immune modulation

Immunomodulation with MBP encoding DNA plasmid (BHT-3009)

Amit Bar-Or*, Farzaneh Jalili*, Masaaki Niino*, Caroline Anita Bodner*, Jack Antel*, Fu-Dong Shi^, Mary Price^,

Susan Rhodes^, Timothy Vollmer^, Jill Gianettoni#, Frank Valone#, Lawrence Steinman#, Hideki Garren#

Promoter

Ori

pA

hMBP

Kanr

BHT-30093485 bp

IM at weeks 1, 3, 5, 9Administration of BHT-3009

0.5 mg and 1.5 mg dose cohorts complete

Current status

10Patients per dose cohort

80 mg dailyDose of atorvastatin

0.5 mg, 1.5 mg, or 3 mgDose of BHT-3009

a) Placebo

b) BHT-3009

c) BHT-3009 + atorvastatin

Arms

30Total patients

RR-MS or SP-MSPatients

I/IIPhase

ENS, 2006

Antigen-specific T cell responsesproliferation and cytokine production

using CFSE and Intracellular Cytokine Staining

No Antigen Tetanus

100 101 102 103 104100

101

102

103

104DM130904.006ÉCD4

FL1-H: CFSE

FL

2-H

: IF

Ng

PE

0.9 8.73

873.38

100 101 102 103 104100

101

102

103

104

DM130904.009ÉCD4

FL1-H: CFSEF

L2

-H: I

FN

g P

E

20.8 5.36

65.58.31

CFSE

IFN

Baseline:

IFN

Week 9 of dose:

CFSE

Patient 002 (CD4+)TT W-MBP

100 101 102 103 104100

101

102

103

104

DS041104.020ÉCD4+ cells

FL1-H: CFSE

FL

2-H

: IF

Ng

PE

11.8 4.03

75.38.81

100 101 102 103 104100

101

102

103

104DS041104.021ÉCD4+ cells

FL1-H: CFSE

FL

2-H

: IF

Ng

PE

2.27 3.73

88.55.48

100 101 102 103 104100

101

102

103

104

DSW22-030205.009ÉCD4

FL1-H: CFSE

FL

2-H

: IF

Ng

PE

16.5 3.5

68.311.6

100 101 102 103 104100

101

102

103

104

DSW22-030205.010ÉCD4

FL1-H: CFSE

FL

2-H

: IF

Ng

PE

0.79 6.31

88.93.96

100 101 102 103 104100

101

102

103

104

DM130904.011ÉCD4

FL1-H: CFSE

FL

2-H

: IF

Ng

PE

25.9 7.33

59.27.52

100 101 102 103 104100

101

102

103

104DM130904.009ÉCD4

FL1-H: CFSE

FL

2-H

: IF

Ng

PE

20.8 5.36

65.58.31

100 101 102 103 104100

101

102

103

104

DM121104.009ÉCD4+ cells

FL1-H: CFSE

FL

2-H

: IF

Ng

PE

26.5 3.24

57.812.5

100 101 102 103 104100

101

102

103

104

DM121104.011ÉCD4+ cells

FL1-H: CFSE

FL

2-H

: IF

Ng

PE

1.2 4.32

89.54.98

Patient 003 (CD4+)TT MBP 83-99

Patient 013 (CD8+)TT MBP 83-99

7.0% 13.3%

10.1% 5.4%

Immunomodulation with MBP encoding DNA plasmid (BHT-3009)

ECTRIMS, 2005

Immune ablation and AHST Reconstitution

2. Adhesion

3. Attraction

Periphery

BBBCNS

5. Reactivation

1. Activation

Th

4. Invasion

6. Neural/glialresponses

B Cell

B Cell

Periphery

BBBCNS

5. Reactivation

6. Neural/glialresponses

Immune ablation and AHST Reconstitution

B Cell

Periphery

BBBCNS

5. Reactivation

6. Neural/glialresponses

Immune ablation and AHST Reconstitution

Periphery

BBBCNS

5. Reactivation

6. Neural/glialresponses

Immune ablation and AHST Reconstitution

Th

New immune system

B Cell

Young, early diagnosis, highly active patients

Treatment had dramatic effects:

No new relapses; no new inflammatory brain lesions

However, brain continues to shrink (atrophy) …

BMT in MS

Important window into disease pathophysiology and human immune reconstitution

The continued brain atrophy over 3 years is probably beyond ‘destined’ tissue loss

May reflect under-recognized CNS toxicity of chemotherapy regimen

May emphasize ongoing process within CNS (relatively independent of periphery)

Uccelli, Corcione, Pistoia, Serafini, Aloisi

Reproduced with permission from Kutzelnigg A et al. Brain. 2005;128:2705-2712.

Subpial cortical demyelination in progressive MS

Kutzelnigg, A. et al. Brain 128:2705-2712; 2005

B cell Activating Factor (BAFF) produced by astrocytes and up-regulated in MS lesions

Krumbholz M et al; J Exp Med. 17;201(2):195-200, 2005

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

Mitoxantrone therapy reciprocally regulates B cell cytokine production

Untreated MS Mitoxantrone treated MS

Cyt

oki

ne

Se

cre

tion

(p

g/m

l)

IL-10 p = 0.007 p = 0.004

0

300

600

CD40 alone Dual stimulation

TNF

0

100

200

CD40 alone Dual stimulation

ns p = 0.006

Cyt

oki

ne

Se

cre

tion

(p

g/m

l)

LT

0

150

300

CD40 alone Dual stimulation

ns p = 0.008

Cyt

oki

ne

Se

cre

tion

(p

g/m

l)cp

m

(x 1

0-3)

Proliferation

CD40 alone Dual stimulation

0

5

10

15

ns ns

CD27 MFI

10 0 10 1 10 2 10 3 10 4

10 0

10 1

10 2

10 3

10 4

10 0 10 1 10 2 10 3 10 4

10 0

10 1

10 2

10 3

10 4

Untreated Mitoxantrone Treated

64% 36% 89% 11%

CD

19 M

FI

a

Mitoxantrone treatment results in decreased proportion of circulating CD27+ B cells

Untreated Mitoxantrone Treated

0

10

20

30

40

50

% C

D27

+ B

cel

ls

p = 0.004b

100 101 102 103 104100

101

102

103

104MT-M0-040504.011ÉLymphocytes

FL2-H: CD20

FL

3-H

: C

D1

9

0.36 3.38

0.02596.2100 101 102 103 104

100

101

102

103

104MT-D3-070504.011ÉLymphocytes

FL2-H: CD20F

L3

-H: C

D1

9

0.23 0

0.02799.7

100 101 102 103 104100

101

102

103

104

MT-M10-290305.010ÉLymphocytes

FL3-H: CD19

FL

2-H

: C

D2

0

0.083 8.99

0.1890.7

CD20

CD

19

Pre D8 M10

Rituximab (anti-CD20) depletion of circulating B cells

Stuve O, et al. Clinical stabilization and effective B-lymphocyte depletion in the cerebrospinal fluid and peripheral blood of a patient with fulminant relapsing-remitting multiple sclerosis. Arch Neurol. 2005;62(10):1620-3.

Monson NL, et al. Effect of rituximab on the peripheral blood and cerebrospinal fluid B cells in patients with primary progressive multiple sclerosis. Arch Neurol. 2005;62(2):258-64.

Cross AH, et al. Rituximab reduces B cells and T cells in cerebrospinal fluid of multiple sclerosis patients. J Neuroimmunol. 2006 Aug 10.

(Cree BA, et al. An open label study of the effects of rituximab in neuromyelitis optica. Neurology. 2005;64(7):1270-2).

Rituximab in MS

0

200

400 LT IL-10

M12BLM12BLC

yto

kin

e S

ecr

etio

n (

pg

/ml)

BL M3 M6 M9 M12Tx0

50

100

150

Ab

solu

te B

ce

ll N

um

be

r (p

er l

)

AHSCT(BMT)

Effects of selective and non-selective B cell depletion on effector cytokine network

CD27 - B cells CD27 + B cells

M12BLM12BL0

200

400 LT IL-10

Cyt

oki

ne

Se

cre

tion

(p

g/m

l)

0

50

100

150

BL M3 M6 M9 M12Tx

Ab

solu

te B

ce

ll N

um

be

r (p

er l

)

Rituximab

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

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