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Antibodies: Role in MS and Relationship to Triggers

Antibodies: Role in MS and Relationship to Triggers

Jeffrey L. Bennett, MD, PhD

ProfessorDepartments of Neurology and Ophthalmology

University of Colorado Denver

Jeffrey L. Bennett, MD, PhD

ProfessorDepartments of Neurology and Ophthalmology

University of Colorado Denver

B Cells and Antibody in MS LesionsB Cells and Antibody in MS Lesions

Type II: Mixed T and B cell infiltrate

• ~ 50% Lesions of RRMS lesions

Ig-Complement deposition• Hallmark of active pathology

Type II: Mixed T and B cell infiltrate

• ~ 50% Lesions of RRMS lesions

Ig-Complement deposition• Hallmark of active pathology

Acute ON Nerve: IgG DepositionOptic Nerve Biopsy

Acute ON Nerve: IgG DepositionOptic Nerve BiopsyAcute MS Plaque: IgG Deposition

Brain BiopsyAcute MS Plaque: IgG Deposition

Brain Biopsy

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Oligoclonal IgG in MS CSFOligoclonal IgG in MS CSF

Kabat et al. (1942)• Elevated amount of antibodies in MS CSF

Yahr et al. (1954)• Intrathecal synthesis

Link (1967)• Oligoclonal bands (OCB)

Kabat et al. (1942)• Elevated amount of antibodies in MS CSF

Yahr et al. (1954)• Intrathecal synthesis

Link (1967)• Oligoclonal bands (OCB)

pH

9.3

8.6

8.0

6.0

4.5

Cry

ptoc

occa

lm

enin

gitis

MS

Ser

um

MS

CS

F

Nor

mal

Oligoclonal IgGSensitive marker of disease

95% of MS patientsOCBs do not disappear

Present in all stages of disease

Oligoclonal IgGDisease Directed Against Reference

Subacute sclerosing panencephalitis Measles virus Vandvik et al., 1976

Cryptococcal meningitis Cryptococcus Porter et al., 1977

Mumps meningitis Mumps virus Vandvik et al., 1978

Chronic rubella panencephalitis Rubella virus Coyle et al., 1981

(HSV) encephalitis HSV glycoprotein B Grimaldi et al., 1988

Progressive multifocal JC virus Sindic et al., 1997leukoencephalopathy

Neurosyphilis Treponema pallidum Vartdal et al., 1981

VZV vasculopathy VZV Burgoon et al., 2003

Theiler’s virus-induced demyelination Theiler’s virus Roos et al., 1987

Theiler’s virus Pachner et al., 2007

Coronavirus-induced demyelination JHM coronavirus Dorries et al., 1987

Oligoclonal IgGDisease Directed Against Reference

Subacute sclerosing panencephalitis Measles virus Vandvik et al., 1976

Cryptococcal meningitis Cryptococcus Porter et al., 1977

Mumps meningitis Mumps virus Vandvik et al., 1978

Chronic rubella panencephalitis Rubella virus Coyle et al., 1981

(HSV) encephalitis HSV glycoprotein B Grimaldi et al., 1988

Progressive multifocal JC virus Sindic et al., 1997leukoencephalopathy

Neurosyphilis Treponema pallidum Vartdal et al., 1981

VZV vasculopathy VZV Burgoon et al., 2003

Theiler’s virus-induced demyelination Theiler’s virus Roos et al., 1987

Theiler’s virus Pachner et al., 2007

Coronavirus-induced demyelination JHM coronavirus Dorries et al., 1987

Oligoclonal Bands in Neurologic DiseaseOligoclonal Bands in Neurologic Disease

Multiple Sclerosis Negative for major myelin proteins and common virusesNegative for major myelin proteins and common viruses

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What is the Nosology of CNS Demyelinating Disorders?

What is the Nosology of CNS Demyelinating Disorders?

CNS

Demyelinating Disease

CNS

Demyelinating Disease

Multiple SclerosisMultiple

Sclerosis

Neuromyelitis Optica

Neuromyelitis Optica

Schilder

Disease

Schilder

DiseaseMarburg DiseaseMarburg Disease

BaloConcentric Sclerosis

BaloConcentric Sclerosis

Multiple Sclerosis

Schilder’sDisease

Marburg Disease

BaloConcentric Sclerosis

Neuromyelitis Optica

A Candidate Approach?Myelin Proteins

MOGMBPPLPαB-crystallin

Cell Adhesion Moleculesneurofascincontactin

Glycolipidsgalactocerebrosidessulfatides

Foreign Antigens“God, Collings, I hate to start a Monday with a case like this.”

EAE

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Reindl et al. J Neuroimmunol 180:50 (2006).

Reindl et al. J Neuroimmunol 180:50 (2006).

Frequency of Anti-Myelin Antibodies Are Assay Dependent

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Myelin Antigen Arrays: High Throughput Antigen Screening

Robinson et al. Nat Biotechnol 21:1033 (2003).

Enhanced Dynamic Range

Epitope Spreading in EAEMyelin Proteome Microarray

Myelin Antigen Arrays: Application in Multiple Sclerosis

Quintana et al. Neurology 78:532 (2012).

• 334 myelin and inflammation related CNS Ags• CSF: MS vs. OND (non-inflammatory controls)• 115 Ags > 3SD over OND mean; 29 with ↑ freq

• Antibody Index: Ag (CSF/Ser) / Albumin (CSF/Ser)• Suggestive of intrathecal synthesis

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Immunoprecipitation• Serum Ab; 2D PAGE• Proteomics

Kir 4.1• inwardly rectifying K(+) channel• Astrocytes and oligodendrocytes-Kir4.1 Ab

– 47% of MS patients– 0.9% of OND

Immunoprecipitation• Serum Ab; 2D PAGE• Proteomics

Kir 4.1• inwardly rectifying K(+) channel• Astrocytes and oligodendrocytes-Kir4.1 Ab

– 47% of MS patients– 0.9% of OND

Antibody Index (AI)• AI > 2 considered intrathecal synthesis• 3/30 positive

Antibody Index (AI)• AI > 2 considered intrathecal synthesis• 3/30 positive

FACS Sort of Normal CSF

CD19+

CD3 +

T Cells (CD3)

B C

ells

(C

D1

9)

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FACS: CSF B and Plasma Cells in ON, MS & NMOFACS: CSF B and Plasma Cells in ON, MS & NMO

Light Scattering

CD19+ B Cell Population104

CD19

3.7% 0.4%

88.7%7.2%

CD3

100

101

102

103

100 101 102 103 104

FITC Comp

PE

Co

mp

CD138+ Plasma Cell Population

CD138

CD3

3.5%

6.7%

0.1%

89.7%

100

101

102

103

104

100 101 102 103 104

FITC Comp

PE

Co

mp

0 64 128 192 2560

64

128

192

256

FSC

SS

C

• Recover Ab-secreting B cells (plasma cells) from CSF • Clone and produce rAbs that duplicate their Ag specificity

CSF as a Surrogate to Study Humoral Immunity in CNS Demyelinating Disease

B cells cultured from MS CSF produce oligoclonal bands

(CSF Cells) Culture in vitro Oligoclonal banding

CS

F

(

)IEF

Strategy

Nat Med, vol. 14, pp. 688–693, (2008).

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Isolation of Single Plasma or B Cells by FACS

Patient CSF

Pellet CSF cellsand label with conjugate antibodies

CD19-APCCD138-PECD3-FITCCD14-APC cy7

FACS

Deposit single CD138+ CD3- plasma cell

Reverse Transcriptase PCR and PCR Amplification

RT-PCR

LEADER V-REGION D J CH1

1 2 3FR 1 FR 3

Primary PCR

Nested PCR

CH3

VH Leader 1 CH1

cDNA Synthesis

Fr1 CH2

VH PCR Products

Vk PCR Products

Electrophoresis of PCR product

DNA Sequence Positive VH and Vk products

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MS and NMO B Cells Are Antigen-TargetedMS and NMO B Cells Are Antigen-Targeted

Restricted and dynamic population of clonally expanded antibody secreting cells with a phenotype of plasma blasts are consistent with ongoing stimulation

IgG V regions display somatic hypermutation indicating progression through germinal center reactions

Restricted and dynamic population of clonally expanded antibody secreting cells with a phenotype of plasma blasts are consistent with ongoing stimulation

IgG V regions display somatic hypermutation indicating progression through germinal center reactions

MS Tissue• Owens et al., 1998• Baranzini et al., 1999

MS Tissue• Owens et al., 1998• Baranzini et al., 1999

MS CSF• Qin et al., 1998• Colombo et al., 2000• Owens et al., 2003• Ritchie et al., 2004• Monson et al., 2005

CIS• Qin et al., 2003• Haubold et al., 2004• Monson et al., 2005

C

Transfect 293 Cells

YYY

YYYY

YY Y

Y

Recombinant IgG1B

CH3

CH 2CH 2

CH3

CH1

Ck

Vk

VH

Ck

Vk

VH

CH1

FLAG Epitope

250 kD

150 kD100 kD

75 kD50 kD37 kD

25 kD

1 2 3 4 5 6

A

VHDJHPCMV IgG1 C RegionVL

VL – VHDJH - C

Flag

pIgG1 Flag

VJPCMV CkVL

VL – VJ - Ck

pCEP4

Xho1Hind III

Pin A1Kpn 1

Generation of Monoclonal Human IgG1 Recombinant Antibodies

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Staining of HEK 293 Cells Expressing MOG with MS CSF rAbs

MS03-1 # 5 & 15 MS04-2 #30 & 33 MS03-7 #10 & 13 ONO3-3 #69 & 77 ON04-7 #18 & #57

8-18C5 mAb 8-18C5 (Mock) 2B7 mAb Hu 2B7 rAb MS02-19 # 28 & 53

Owens et al. Ann Neurol (2009) vol. 65 (6) pp. 639-49

MS CSF rAbs Bind to Glial Cell Antigens

Courtesy of G. Owens

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MS04-2#30 binds white matter antigen in CNSMS04-2#30 binds white matter antigen in CNS

Human optic nerve

Cerebellum

Cerebrum

Hind brain

A

a b c d

B

a b c d

C

a b c d

E

a b c d

F

a b c d

D

a b c d

a b c d

G.

Spinal cord outgrowth

Courtesy of K. Blauth and G. Owens

MS05-3#38 binds neuronal targets in the cerebellum

A

B

C

a b c d

a b c d

a b c d

P

G

M

Courtesy of K. Blauth and G. Owens

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NMO Pathogenesis: Assays

Complement +Serum NMO-IgG

NMO

Saadoun et al., Brain, 2010

Ex Vivo Spinal Cord Explants

Zhang et al., Annal Neurol, 2011

+ complement

+ serum or rAb

Calcein-AMEthD-1

LiveDead

In vitro

Control Ab AQP4 Ab

Tradtrantip et al., Annal Neurol, 2011

MS rAbs Cause Complement-Mediated DemyelinationMS rAbs Cause Complement-Mediated Demyelination

Courtesy of K. Blauth and G. Owens

MS05‐3#1922B42B4 MS05‐3#192

DAPI

MBP

DAPIMBP

10 ug/ml 50 ug/ml

10% human Complement 10% human Complement

Spinal Cord Slice Explants

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rAb: Glycolipid bindingrAb: Glycolipid binding

Source % Glycolipid Reactive Specificity*

Multiple Sclerosis(10 patients 73 rAbs)

27%(5 patients, 20 rAbs)

• Sulfatide• Sulfatide complexes• Cholesterol

Inflammatory Controls(6 patients, 27 rAbs)

33%(3 patients, 9 rAbs)

• Sulfatide• Lipid complexes

Brennan et al., J. Neuroimmunol, 238: 87 (2011)

Sulfatide Lipids Inhibit Axon OutgrowthSulfatide Lipids Inhibit Axon Outgrowth

Winzeler et al., J Neurosci, 31:6481 (2011)

Sulfatide Specifically Inhibits Axonal Outgrowth

O4 Ab (Anti-Sulfatide) Antibody Restores Neurite Outgrowth

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B Cells Are Multifunctional at Neuro-immune InterfaceB Cells Are Multifunctional at Neuro-immune Interface

Produce Antibodies

Instruct T Cells• Antigen Presentation

Activate Immune Cells• Antigen Presenting Cells

Immunoregulation

Produce Antibodies

Instruct T Cells• Antigen Presentation

Activate Immune Cells• Antigen Presenting Cells

Immunoregulation

McFarland. N Engl J Med, 358: 664-5

Dependent on B cell Receptor

B Cells Are Antigen-Presenting CellsB Cells Are Antigen-Presenting Cells

Protein Antigens

Antigen Internalization BCR (Ag) Dependent Costimulatory Molecule

Expression

Protein Antigens

Antigen Internalization BCR (Ag) Dependent Costimulatory Molecule

Expression

Constant et al. J Immunol 155: 3734 (1995).

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Secretion of Toxins and CytokinesSecretion of Toxins and Cytokines

Secretory products of multiple sclerosis B cells are cytotoxic to oligodendroglia in vitro

Lisak et al., J Neuroimmunol, vol. 246: 85 (2012).

B cell depletion may ameliorate disease through ablation of IL-6–

producing B cells Barr et al., J Exp Med, vol. 209: 1001 (2012).

Acknowledgments

FundingNIHGuthy Jackson

Universitätsmedizin Göttingen

Christine Stadelmann

Stefan Nessler

Claudia Wrzos

Alan Verkman

Hua Zhang

Lukmanee Tradtrantip

Puay Phan

Julien Ratelade

Greg Owens

Corey Reiter

Alanna Ritchie

Scott Wemlinger

Kevin Blauth

John Soltys

Hannah Schumann

Markus Kowarik

Greg Owens

Corey Reiter

Alanna Ritchie

Scott Wemlinger

Kevin Blauth

John Soltys

Hannah Schumann

Markus Kowarik