cytokines and autoimmunity
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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
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