Aetiopathogenesis of

Autoimmune Hepatitis

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Aetiopathogenesis of autoimmune

hepatitis

He

pa

tite

s

Au

to-i

mm

un

es

Positive AAB and high IgG

Progressive inflammatory disorder

Exclusion of other pathology

(Wilson, HCV, HBV, NASH)

Anti-Smooth Muscle (SMA) &

Anti-Nuclear (ANA) autoantibodies

Liver Kidney Microsomal antibody

Liver Cytosol Type 1 antibody

Interface

hepatitis

M IFNγ

TNFα

IL1

CTL

IL2

IFNγ

NK

P

IL13 IL10

IL4 B

Th2

IL4 Tr

Co-

stimuli

Th0

Th1

Class I

Class II

Liver

cell

APC

IL12

Tr

Co-

stimuli

Th0

APC

Co-

stimuli

Th0

APC

Tr

Longhi MS et al - Impairment of CD4(+)CD25(+) regulatory T-cells in autoimmune liver disease. J Hepatol. 2004;41:31-7

Longhi MS, et al. Effect of CD4+CD25+ regulatory T-cells on CD8 T-cell function in autoimmune liver disease. J Autoimmunity 2005;25:63-71

Longhi MS, et al. Functional study of CD4+CD25+ regulatory T-cells in health and autoimmune hepatitis. J Immunol. 006;176:4484-91

Longhi MS, et al. Expansion and de novo generation of potentially therapeutic regulatory T cells inpatients with autoimmune hepatitis. Hepatology. 2008;47:581-91

Longhi MS, et al. Vigorous activation of monocytes in juvenile autoimmune liver disease escapes the control of regulatory T-cells. Hepatology. 2009; 50: 30-42

Longhi MS, et al. Auto-antigen-specific regulatory T-cells, a potential tool for immune-tolerance reconstitution in type-2 autoimmune hepatitis. Hepatology. 2011; 53: 536-547

Longhi MS, et al. Inhibition of interleukin-17 promotes differentiation of CD25– cells into stable T regulatory cells in patients with autoimmune hepatitis. Gastroenterology 2012;14:1526-1535

Liberal R, et al. The impaired immune regulation of autoimmune hepatitis is linked to a defective Galectin-9/Tim-3 pathway. Hepatology 2012;56:677-686

0.00%

0.50%

1.00%

1.50%

2.00%

2.50%

3.00%

3.50%

4.00%

4.50%

Diagnosis Remission

2.50%

4.20%

CD

4 C

D2

5

Elevated Immunoglobulin G

Co-

stimuli

Class I

Class II

Liver

cell

APC

Th2

NK

P

IL13 IL10

IL4 B

IL4

Th0

Tr

Class I Co-

stimuli

Class II

Liver

cell

APC

Th2

NK

P

IL13 IL10

IL4 B

IL4

Th0

Tr

Autoantigen

CYP2D6

497 aa

Immunodominant epitopes

254-271

CYP2D6

410-429 321-350

373-389

193-212

Kerkar N, et al. Cytochrome P4502D6(193-212): a new

Immunodominant... J Immunol 2003;170:1841-9

Gut 2000;46;553-561

Liver kidney microsomal antibody type 1 targets CYP2D6

on hepatocyte plasma membrane

L Muratori, M Parola, A Ripalti, G Robino, P Muratori, G Bellomo,

R Carini, M Lenzi, M P Landini, E Albano and F B Bianchi

Th2

IL4 Tr

Co-

stimuli

Th0

Th1

Class I

Class II

Liver

cell

APC

IL12

M IFNγ

TNFα

IL1

CTL

IL2

IFNγ

NK

P

IL13 IL10

IL4 B

Th2

IL4 Tr

Co-

stimuli

Th0

Th1

Class I

Class II

Liver

cell

APC

IL12

NKT

Predisposing HLA-Class II

allele in AIH-2

DR7 a

b

Prediction of DR7

binding peptides

7 predicted high binders

induced proliferation

full length CYP2D6 497 aa 1 aa

20-mer peptides, overlapping by 12 aa and spanning

the entire molecule of CYP2D6 were constructed

overlapping peptides

Sixty-one in total

DR7 positive patients

SI 3

6

9

12

1 11 21 31 41 51 61

High

Medium

Low

2 months of immunosuppressive treatment

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61

4

8

12

SI

at relapse 6 years after diagnosis

4

8

12

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61

SI

0

100

200

300

400

500

600

pg/ml

IFN

-g

1 11 21 31 41 51 61

0

100

200

300

400

500

600

IL-4

pg/ml

1 11 21 31 41 51 61

pg/ml

IL-1

0

1 11 21 31 41 51 61

0 100

200 300

400

500

600

Controls

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61

3

6

9

SI

Viral liver disease

Other autoimmune liver disease 0/46

Non autoimmune liver disease 0/17

HBV 0/3

HCV 4/7

CYP2D6313-332 TLAWGLLLMILHPDVQRRVQ

Similarity between HCV and CYP2D6

GLMALTLSP HCV825-833

WPLLLLLL HCV794-801

LLMCAVHP HCV875-882

HCV1246-1258 AQGYKVLVLNPSV

HCV402-412 LLAPGKKQNVQ

IL2

IFNγ

Tr

Co-

stimuli

Th0

Th1

Class I

Class II

Liver

cell

APC

IL12 CTL

Peptide number

Amino acid position

Binding score

1 176-185 G L L D K A V S N V 28

2 204-213 R L L D L A Q E G L 24

3 2-11 G L E A L V P L A V 22

4 245-254 K A F L T Q L D E L 22

5 393-402 T T L I T N L S S V 22

6 177-186 L L D K A V S N V I 21

7 306-314 G M V T T S T T L 21

Cytotoxicity of CYP2D6-specific CD8 T cells

% c

yto

tox

icit

y

0

10

20

30

40

50

60

70

80

p2 p3 p4 p5 p7 Hbc18-27

Diagnosis

% c

yto

tox

icit

y

0

10

20

30

40

50

60

70

80

p2 p3 p4 p5 p7 Hbc18-27

Remission

%

0

1

2

3

4

5

6

7

8

9

p2 p3 p4 p5 p7

IFNg producing CYP2D6-specific CD8 T cells

Diagnosis

%

0

1

2

3

4

5

6

7

8

9

p2 p3 p4 p5 p7

Remission

21306-31457

HLA-A*0201 nonamers

21177-1866

22393-40245

22245-25434

222-1123

24204-21312

28176-1851

Amino acid position

Binding

scoreCYP2D6 sequence

HLA-A*0201

decamers

Tetramer

number

Peptide

number

21G M V T T S T T L306-31457

HLA-A*0201 nonamers

21L L D K A V S N V I177-1866

22T T L I T N L S S V393-40245

22K A F L T Q L D E L245-25434

22G L E A L V P L A V2-1123

24R L L D L A Q E G L204-21312

28G L L D K A V S N V-1

1 2 3 4 5 6 7 8 9 10

Binding

scoreCYP2D6 sequence

HLA-A*0201

decamers

Tetramer

number

Peptide

number

21306-31457

HLA-A*0201 nonamers

21177-1866

22393-40245

22245-25434

222-1123

24204-21312

28176-1851

Amino acid position

Binding

scoreCYP2D6 sequence

HLA-A*0201

decamers

Tetramer

number

Peptide

number

21G M V T T S T T L306-31457

HLA-A*0201 nonamers

21L L D K A V S N V I177-1866

22T T L I T N L S S V393-40245

22K A F L T Q L D E L245-25434

22G L E A L V P L A V2-1123

24R L L D L A Q E G L204-21312

28G L L D K A V S N V-1

1 2 3 4 5 6 7 8 9 10

Binding

scoreCYP2D6 sequence

HLA-A*0201

decamers

Tetramer

number

Peptide

number

21306-31457

HLA-A*0201 nonamers

21177-1866

22393-40245

22245-25434

222-1123

24204-21312

28176-1851

Amino acid position

Binding

scoreCYP2D6 sequence

HLA-A*0201

decamers

Tetramer

number

Peptide

number

21G M V T T S T T L306-31457

HLA-A*0201 nonamers

21L L D K A V S N V I177-1866

22T T L I T N L S S V393-40245

22K A F L T Q L D E L245-25434

22G L E A L V P L A V2-1123

24R L L D L A Q E G L204-21312

28G L L D K A V S N V-1

1 2 3 4 5 6 7 8 9 10

Binding

scoreCYP2D6 sequence

HLA-A*0201

decamers

Tetramer

number

Peptide

number

21306-31457

HLA-A*0201 nonamers

21177-1866

22393-40245

22245-25434

222-1123

24204-21312

28176-1851

Amino acid position

Binding

scoreCYP2D6 sequence

HLA-A*0201

decamers

Tetramer

number

Peptide

number

21G M V T T S T T L306-31457

HLA-A*0201 nonamers

21L L D K A V S N V I177-1866

22T T L I T N L S S V393-40245

22K A F L T Q L D E L245-25434

22G L E A L V P L A V2-1123

24R L L D L A Q E G L204-21312

28G L L D K A V S N V-1

1 2 3 4 5 6 7 8 9 10

Binding

scoreCYP2D6 sequence

HLA-A*0201

decamers

Tetramer

number

Peptide

number

Cytotoxity IFN-g

21306-31457

HLA-A*0201 nonamers

21177-1866

22393-40245

22245-25434

222-1123

24204-21312

28176-1851

Amino acid position

Binding

scoreCYP2D6 sequence

HLA-A*0201

decamers

Tetramer

number

Peptide

number

21G M V T T S T T L306-31457

HLA-A*0201 nonamers

21L L D K A V S N V I177-1866

22T T L I T N L S S V393-40245

22K A F L T Q L D E L245-25434

22G L E A L V P L A V2-1123

24R L L D L A Q E G L204-21312

28G L L D K A V S N V-1

1 2 3 4 5 6 7 8 9 10

Binding

scoreCYP2D6 sequence

HLA-A*0201

decamers

Tetramer

number

Peptide

number

21306-31457

HLA-A*0201 nonamers

21177-1866

22393-40245

22245-25434

222-1123

24204-21312

28176-1851

Amino acid position

Binding

scoreCYP2D6 sequence

HLA-A*0201

decamers

Tetramer

number

Peptide

number

21G M V T T S T T L306-31457

HLA-A*0201 nonamers

21L L D K A V S N V I177-1866

22T T L I T N L S S V393-40245

22K A F L T Q L D E L245-25434

22G L E A L V P L A V2-1123

24R L L D L A Q E G L204-21312

28G L L D K A V S N V-1

1 2 3 4 5 6 7 8 9 10

Binding

scoreCYP2D6 sequence

HLA-A*0201

decamers

Tetramer

number

Peptide

number

21306-31457

HLA-A*0201 nonamers

21177-1866

22393-40245

22245-25434

222-1123

24204-21312

28176-1851

Amino acid position

Binding

scoreCYP2D6 sequence

HLA-A*0201

decamers

Tetramer

number

Peptide

number

21G M V T T S T T L306-31457

HLA-A*0201 nonamers

21L L D K A V S N V I177-1866

22T T L I T N L S S V393-40245

22K A F L T Q L D E L245-25434

22G L E A L V P L A V2-1123

24R L L D L A Q E G L204-21312

28G L L D K A V S N V-1

1 2 3 4 5 6 7 8 9 10

Binding

scoreCYP2D6 sequence

HLA-A*0201

decamers

Tetramer

number

Peptide

number

21306-31457

HLA-A*0201 nonamers

21177-1866

22393-40245

22245-25434

222-1123

24204-21312

28176-1851

Amino acid position

Binding

scoreCYP2D6 sequence

HLA-A*0201

decamers

Tetramer

number

Peptide

number

21G M V T T S T T L306-31457

HLA-A*0201 nonamers

21L L D K A V S N V I177-1866

22T T L I T N L S S V393-40245

22K A F L T Q L D E L245-25434

22G L E A L V P L A V2-1123

24R L L D L A Q E G L204-21312

28G L L D K A V S N V-1

1 2 3 4 5 6 7 8 9 10

Binding

scoreCYP2D6 sequence

HLA-A*0201

decamers

Tetramer

number

Peptide

number

Tetramers

Tetramer

labelled

with

phycoerythrin

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

p2 Tetramer p3 Tetramer p4 Tetramer p5 Tetramer p7 Tetramer HBVcore18-

27 Tetramer

%

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

p2 Tetramer p3 Tetramer p4 Tetramer p5 Tetramer p7 Tetramer HBVcore18-

27 Tetramer

%

% T

etr

am

er p

ositiv

e C

D8

T c

ells

Diagnosis

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

p2 Tetramer p3 Tetramer p4 Tetramer p5 Tetramer p7 Tetramer HBVcore18-

27 Tetramer

%

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

p2 Tetramer p3 Tetramer p4 Tetramer p5 Tetramer p7 Tetramer HBVcore18-

27 Tetramer

%

Remission

CYP2D6245-254 tetramer positive

CD8 T cells

M IFNγ

TNFα

IL1

CTL

IL2

IFNγ

NK

P

IL13 IL10

IL4 B

Th2

IL4 Tr

Co-

stimuli

Th0

Th1

Class I

Class II

Liver

cell

APC

IL12

Th17

IL6

TGFβ Th17

IL17

IL17

IL13 IL10

IL4

Th2

IL4 Tr

Co-

stimuli

Th0

Th1

APC

IL12

Th17

IL6

TGFβ IFNγ

IL2

IL17

Liberal R, et al. The impaired immune regulation of autoimmune hepatitis is linked to a defective Galectin-9/Tim-3 pathway. Hepatology 2012;56:677-686

Impairment of regulatory T cells

CD4 and CD8 autoepitopes defined

Expand regulatory T cells

What next?

Attempt new modes of treatment

Correlation with disease activity

T-regs

T-regs + T-regs T-regs T-regs

semi-mature DCs

First-in-human clinical trial

Thank you

Children’s Liver Disease

Foundation