DIABETES

TYPE 1

DIABETES

TYPE 2

DIABETES

monogenic diabetes

idiopathic type 1

complex genetic disorders

pancreatic diseases

endocrinopathies

iatrogenic diabetes

autoantibodies

[DNA]

[insulitis]

world epidemysecond diabetes

epidemy

DNA

hormones

insulin secretion

pancreatic imaging

O

T

H

E

R

S absence of positive

diagnostic parameter

heterogeneity

T2D DIAGNOSIS

from physiology to disease

po

pu

lati

on

%

physiology

dia

gn

ostic

thre

sh

old

common

disease

multiple genes

multifactorial

chronicunknown cause

environment

genesmonogenic

diabetes

Psammomys obesus

NATURAL HISTORY OF TYPE 2 DIABETES

a highly chronic process

adapted from Weyer C et al. J Clin Invest 1999

insulin resistance + b cell compensation

DIABETES

insulin resistance + early b cell deficit

bcell f

un

cti

on

insulin sensitivity

normoglycemia

glucose intolerancediabetes

Diabetes Audit and Research Tayside study .

DIABETES

b CELL DEFECTS IN TYPE 2 DIABETES

N

O

R

M

A

L

glucose 500mg iv

+ 20mg/min 60min

Nesher & Cerasi Diabetes 2001

APOPTOSISOXYDATIVE STRESS

markers

nitrotyrosine

8-OH-2’-deoxyguanosine

Sakuraba et al, Diabetologia, 2002

nucleus staining

DNA damage DEDIFFERENCIATIONTalchai C et al. Cell 2012

D

I

A

B

E

T

E

S

reduced first phase insulin response

to i.v. glucose

FUNCTIONAL DEFECT GLUT1/2, Glucokinase

glucose oxydation

AMPK activation

insulin

MGAT4A glycosyl trase

foxo-1, PDX-1

TYPE 2

DIABETES

glucagon

insulin

DIABETES: GENES

T2D

λs ~ 3

DZ twin concordance ~ 20%

MZ twin concordance ~ 70%

Number of genes multiple

Dominant gene none

Proportion of heritability

explained< 10%

Predicted heritability > 50%

1

1.5

TYPE 2 DIABETES: MORE THAN 40 GENES

PT

PR

D

KC

NQ

1

TC

F7

L2

SR

R

DU

SP

9

CD

KN

2A

-2B

IRS

1

UB

E2

E2

FT

O

HH

EX

-ID

E

SL

C30

A8

CD

KA

L1

IGF

BP

2

TH

AD

A

PP

AR

G

CE

NT

D2

KC

NJ11

NO

TC

H2

C2C

D4A

/B

WF

S1

AD

CY

5

CH

CH

D9

CD

C12

3/C

AM

K1d

HM

GA

2

HN

F1

B

JA

ZF

1

RB

MS

1

TS

PA

N8

/LG

R5

MT

NR

1B

AD

AM

TS

9

ZB

ED

3

DU

SP

8

BC

L11

A

DG

KB

ZF

AN

D6

PR

OX

1

KL

F1

4

GC

K

TP

53

INP

1

HN

F1

A

GC

KR

PR

C1

PPARG, ENPP1,

ADIPOQ, SREBF1,

PPARGC1A, AHSG,

SHBG, IRS1, FTO

insulin resistance

KCNJ11, HNF1B

FOXO1, SLC30A8

HHEX, CDKAL1, WFS1

IGF2BP2, CDKN2A/B

SGK1, JAZF1, KCNQ1,

TCF7L2

ADAMTS9

CAPN10 CDC123/CAMK1D, THADA

TSPAN8/LGR5, MTNR1B

ISLET GENES

Schäfer et al. 2011

Travers ME et al. 2011* GWAS

2377 participants

255 T2D

[Framingham Offspring Study]

Sex

Family history

Age

BMI

Fasting plasma glucose

Systolic blood pressure

HDL-Cholesterol

Fasting triglycerides

Meiggs JB et al. N Engl J Med 2008

Sex-adjusted

reclassification by genotype score 4.1%

11.9% < 50

0.47 > 50

sex-adjusted OR

for T2D 1.12

GENETIC DIVERSITY

TYPE 1 DIABETES MELLITUS

• insulitis

Gepts W 1965, Diabetes

Gianani R 2010, Diabetologia

b cell selectivity

an autoimmune disease

• islet cell autoantibodies

Bottazzo & Doniach 1974, Lancet

anti-GAD

anti-IA2

anti-insulin

anti-ZnT8

others

aAc threshold Specificity Sensitivity

IAA ≥50 nU/ml 99% 64%

GAD/IA-21 ≥4 UI/ml 98% 96%

GAD ≥ 5 UI/ml 98% 92%

IA-2 ≥ 7.5 UI/ml 100% 64%

ZnT8 ≥15 UI/ml 99% 68%

TYPE 1 DIABETES: A CHRONIC DISEASE

ß c

ell m

ass

diabetes

hyperglycemia

Bingley et al. Diabetologia 2006

patientduration of

diabetes

diabetes

recurrenceInsulitis ICA

1 22 years 60 days CD8+ T cells -

2 17 years 44 days CD8+ T cells -

3 27 years 92 days CD8+ T cells +

THE EFFECTOR versus REGULATORY BALANCE

Regulation

• Regulatory T cells (IL-10, TGF-β)

• Regulatory B cells (IL-10)

• NK T cells

• Regulatory dendritic cells (IDO)

Immunity

• Pathogenic CD4 & CD8 T cells

• Pathogenic B cells

• Activating dendritic cells

(iNOS, IL-12)

Innate immunity

• IFNγ

• Class I

• CXCL10

• neutrophils

incidence: > 65/105/year in Finland

3-4%/year,

earlier age at onset, milder HLA susceptibility

0

20

40

60

1921 50s 1972 1992

inc

ide

nc

e [

10

5/y

ea

r]

VIRUSES

congenital rubella

coxsackie virus B

varicella-zoster

mumps

cytomegalovirus

Epstein-Barr

OTHER

SUN EXPOSURE

vitamin D

NUTRITIONAL

FACTORS

cow milk proteins

absence of a unique environmental factorgenesenvironment

monogenic

T1DAIRE

FoxP3

SIRT1

congenital

rubella

fulminant

type 1 ?

common forms

numerous

numerous

TYPE 1 DIABETES MELLITUS

immune genesvirus-response genes

β-genes

λs 15

sibling incidence 5-6%

child of diabetic mother 2-3%

child of diabetic father 6%

ls 15

dizygotic twin

concordance0-13%

monozygotic twin

concordance21-70%

offspringmultiple

autoantibody detection

age 9 m 2 y 5 y

n 985 554 110

from mother 4.4% 11.6% 15.5%

from father 4.2% 9.7% 9.7%

total 4.4% 11.1% 14.6%

RISK OF DIABETES IN NEWBORNS

FROM A DIABETIC PARENT

Ziegler A. et al. Diabetes1999

no autoantibody

one autoantibody

su

rviv

al w

ith

ou

t d

iab

ete

s %

0

20

40

60

80

100

0 8 years42 6

> one autoantibody

TTT

T

TTT

T

TYPE 1 DIABETES

isletcirculating

naive T cells

TTT

TT

TT

T

draining

lymph node

Larger et al. J Exp Med 1996

Gagneurault et al. J Exp Med 2002

Turley et al. J Exp Med 2003

MONOCLONAL TREATMENTS OF T1D

Keymeulen B et al. 2005, N Engl J Med

anti-CD3

Ludvigsson et al, N Engl J Med, 2008

GAD-alum

n = 35

n = 34

n = 40

n = 40

4-doses n = 111

2-doses n = 108

Placebo n = 115

Ludvigsson et al, N Engl J Med, 2012Sherry N et al. Lancet 2011

anti-CD3 n = 417

placebo n = 99

all patients

primary composite outcomeInsulin < 0.5 U/kg& HbA1c < 6.5% 14d fd 19.8%

14d ld 13.7%

6d fd 20.8%

placebo 20.4%children

Allegheny County

childhood-onset (< 18) T1D registry

diagnosed 1965-79

Secrest et al. Diabetes 2010

acute

renal

cardio

vascular

infections

cancer

n = 1075

age Controls

mortality rate

/10 3 person-yrs

Standardized

Mortality Rate

♂ 0-4 1.4880 2.0

5-9 0.1511 3.7

19-14 0.2218 4.6

15-19 0.7166 3.3

20-24 0.8752 2.3

25-29 0.9381 4.5

30-34 1.1388 6.3

35-39 1.4270 3.3

♀ 0-4 1.2640 0.0

5-9 0.1550 0.0

19-14 0.1289 4.6

15-19 0.2802 5.8

20-24 0.2927 3.2

25-29 0.3477 4.6

30-34 0.5285 5.1

35-39 0.7086 2.7

3.9

4.0

Skrivarhaug et al. Diabetologia 2006 [Norway]

T1D MORTALITY

excess mortality compared to

the general population

intermediate disease compared to

high risk autoimmune diseases

normal life expectancy

In absence of nephropathy

T CD4TNF-a, IFN-g…

b cell

TYPE 1 DIABETES

FROM T CELLS TO DIABETES PREVENTION

Insulin

new preclinical

models

Towards trials

APC

MHC-resticted

peptides

β-cell proteasesproteasome

class IER

T CD8

IMMUNE IMAGE OF β-CELLSautoantigen peptides insertion in class I MHC molecules

Toma et al, PNAS2005; Toma et al. Diabetes 2009; Luce Diabetes 2011

CD8+ T CELL RESPONSE TO A*0201-RESTRICTED

PREPROINSULIN PEPTIDES [ELISPOTassay]

$ Val42, L14, L16, A23 & A24 were identified as C-terminal residues

generated by proteasome digestion in vitro.

2-11

6-14

15-24

30-39

33-42

34-42

42-51

101-109

PDHase 208-16

Nef 83-91

MATA2 58-66

hu

man

pre

pro

insu

lin

candidate peptide C-termini

proteasome digestion $

anchoring residues

A*0201: C,L,V in 2

in vitro binding to

soluble A*0201

T1D patients

P22P01

basal

P04

16.1 8.9 19.2

Insulin

B18-27

14.4

controlpositive

168.978.3

+++ +++

145.5

+++

33.3 161.1

++++

peptide CD8+ IFNγ response

long

standing

recent

onset

total

34-42* 2/4 4/10 6/14

6-14* 7/13 1/4 8/17

6-16* 5/15 2/4 7/19

14-23* 7/13 0/4 7/17

15-24* 7/13 1/4 8/17

class I preproinsulin peptide

A2.1 2-11

A2.1 6-14

A2.1 15-24

A2.1 30-39

A2.1 33-42

A2.1 34-42

A2.1 42-51

A2.1 101-109

controls PDHase 2008-216 MATA2 58-66 Nef 83-91

b cells

TCD8+

TCD8+

TETRAMER RECOGNITION OF BLOOD CD8+ T CELLS

class I

peptide

biotin

streptavidin

100

101

102

103

104

100

101

102

103

104

Dt1A5 TTM A2 PPIh_Dt1A5 TTM A2 33-42.fcsÉCD3+ CD8+

PerCP-A: CD8 PerCP-A

PE

-A:

TT

M P

E-A

0.810 0.79

99.20

100

101

102

103

104

100

101

102

103

104

Dt1A5 TTM A2 PPIh_Dt1A5 TTM A2 6-14.fcsÉCD3+ CD8+

PerCP-A: CD8 PerCP-A

PE

-A:

TT

M P

E-A

0.0370 0.037

1000

100

101

102

103

104

100

101

102

103

104

25-01-08_D124D 6-14.fcsÉCD3+ CD8+

PerCP-A: CD8 PerCP-A

PE

-A:

TT

M P

E-A

0.60 0.54

99.60

Sandrine Luce

CD8+ T cell expansions in peripheral blood

Recent-onset: leader sequence peptides

TETRAMER RECOGNITION OF BLOOD CD8+ T CELLS

A2.1-restricted peptides

Luce et al. Diabetes 2011

2-11

6-14

15-24

30-39

33-42

34-42

42-51

101-109

PDHase 208-16

Nef 83-91

MATA2 58-66

hu

man

pre

pro

insu

lin

Long-standing: B chain peptides

single cell PCR Insulin-specific CD8: central memory

CMV-specific: effector memrory

Control CD8: 33% IL-10

mouse

MHC HLAIAg7

HLADQ8HLADQ8

insulin/A2.1/DQ8 mice

YES

Insulin

A2.1

DQ8 genes

OUF

A2/DQ8 Tg Human insulin

x

NEW PRECLINICAL T1D MODELS

♂ ♂ ♀ ♀

YES

YESCol

Col

Intraperitoneal glucose tolerance test

AU

C m

g.h

/dL

beta cell mass

mg

0

10

20

30

40

E/T 1 10 30

Live-dead cell-mediated cytotoxicity assay

MatA2, recall MatA2

MatA2, recall MatA2, anti-A2PBS, recall MatA2

PBS, recall MatA2, anti-A2PBS

BrDU proliferation assay

NEW PRECLINICAL T1D MODELS

CD8+ IFNγ Elispot assay

MATA2MATA

+anti-A2

0

SPLENOCYTES C57B6CD4+CD25+FoxP3+

YESCD4+CD25+FoxP3+

CD3+CD4+CD8+

DIABETES IN RIP-B7 TRANSGENIC YES MICE

B7.2

CD28

CD4

MHC

TCR

T cell

Luce et al. unpublished

Age (weeks)

dia

be

tes

(%

)

% t

ota

l is

lets

non diabetic diabetic

CD4

CD

8

CD11b

CD

11c

CD19

TC

Rβ

dapi anti-glucagon anti-CD3

RIP-B7 YES

TLR3

TRIF

DIABETES IN RIP-B7 TRANSGENIC YES MICE

CD8+ TMr+ expansion assay

CD4+ T cell proliferation

FROM PALLIATIVE TREATMENT TO PREVENTION OF TYPE 1 DIABETES

T CD4

APC

T CD8

NKTNK

T reg

B

damaged

islet of Langerhans

AUTHORS FROM RESEARCH TO CLINIC

YES mice

insulin

Luce S et al. Diabetes 2011

“We always overestimate the change that will occur in the next

two years and underestimate the change that will occur in the

next ten. Don’t let yourself be lulled into inaction.” Bill Gates

Carlier et al. PloSOne 2012

Immunotherapy

EU FP7 exalt project

HLApeptideautoantigen

T

M1IFNg+

Th1

M1

M1

M1

M1

Th17

CD8+

CytokinesIL1βTNFa

IFNgIL6MCP-1

FFA

insulin

resistance

insulin

resistance

insulin

resistance

insulinsecretion

A T2D/T1D CONNECTION ?

CRP, SAA

islet inflammation

β cell debris β cell

dysfuntion

T2Dsystemicinflammation

T1D?

local inflammation

T2D β-cell genesT1D

β-cell& immune genes

TLR4, TLR2TLR3, TLR7, TLR9, RIG-1

Département Hospitalo-Universitaire AUToimmune and HORmonal diseaseS

Uth rs

DHU

DIABETES

ADRENAL DISEASES

VASCULITIDES

Andrea Toma

Claire Briet

Isabelle Tardivel

Chantal Bécourt

Sandrine Luce

Etienne Larger

Ute Rogner

Roberto Mallone

François Lemonnier

Agnès Lehuen

MINISTRY OF RESEARCH (ANR)

NOVO/JDRF/EFSD joint program

« Contradiction is not a sign of falsity, nor the lack of

contradiction a sign of truth. »

Blaise Pascal

« Ni la contradiction n'est marque

de fausseté, ni l'incontradiction

n'est marque de vérité. »