Type 1 insulin-dependent Type 1 insulin-dependent autoimmune diabetes. autoimmune diabetes.

Ciriaco A. PiccirilloCanada Research Chair

Department of Microbiology & Immunology

McGill University Health Center

Ciro.piccirillo@mcgill.ca

Diabetes Classification

• Type 1– Immune Mediated– Insulin deficient, autoantibodies

• Type 2– No Autoantibodies and treated without insulin

• Other Specific forms of Diabetes– Gestational Diabetes

• Monogenic:Monogenic: Single gene defect.Single gene defect. APS-I: AIRE autosomal recessive APS-I: AIRE autosomal recessive IPEX: Scurfy Gene X-linked IPEX: Scurfy Gene X-linked

• Polygenic:Polygenic: Summation of small effects of multiple genes creating diabetes susceptibility Summation of small effects of multiple genes creating diabetes susceptibility – e.g. NOD mousee.g. NOD mouse

The Non-Obese Diabetic (NOD) mouse.Model of spontaneous Type 1 insulin-dependent diabetes (T1D).

-islet Insulin

Normal

Type 1Autoimmune

diabetes

pLN

pancreas

Abnormal peripheral T cell tolerance:

Self-reactive T cell

Checkpoints1. Peri-insulitis (Th2)

2.Insulitis (Th1)

Cellularity heterogeneous

Salivary glandsThyroidNuclear antigensTestesOvaries

Multi-organ Autoimmunity

Cumulative incidence of T1D:

80% in females,

30% in males

(at 30 weeks)

Activated TH1 CD4+ T Cell

CD4+ Cell(TH2 )

CD4+ Cell

(TH0 )

DR3, DR4,,DQ8/insulin peptide

CD2

Macrophage/dendritic cell

Fc R

IFN-

IL-12 CD40L

CD40

, TCR

IL-1, TNF, LT, NO, PGE-2

B Cell?anti-insulin, GAD ab anti-Mog

IL-4

Immunopathophysiology of Diabetes

?Antibody mediated injury

Dendritic cell/APC

CD40L

IL-4CD40L

CD8+ CTL

FasLperforin

cell death islet cells

LOSS OF FIRST PHASE LOSS OF FIRST PHASE INSULIN RESPONSE INSULIN RESPONSE

TIMETIME

Stages in Development of Type 1 DiabetesStages in Development of Type 1 Diabetes B

ET

A C

EL

L M

AS

SB

ET

A C

EL

L M

AS

S

DIABETES“PRE”-

DIABETES

GENETICPREDISPOSITION

INSULITISBETA CELL INJURY

NEWLY DIAGNOSED DIABETES

MULTIPLE ANTIBODY POSITIVEMULTIPLE ANTIBODY POSITIVE

GENETICALLY AT RISK

(?Precipitating Event)Progressiveloss insulinrelease

Glucosenormal

Overt immunologicabnormalities

Normal insulin release

Autoantibodies/Autoreactive B Cells Contribute to NOD Diabetes

• Immunoglobulin knockout prevention NOD DMSerreze et al, J. Immunol 1998, 161:3912-3918

• I-Ag7 on B cells needed for NOD diabetes.Noorchashm et al, J. Immunol 1999, 163, 743-750

• Anti-Insulin VH125 Heavy Chain Increases diabetes in NOD mice.Hulbert et al, J. Immunol, 2001, 167: 5535-5538

• Transplacental autoantibodies accelerate NOD diabetes.Greeley et al, Nature Immunol.

Progression to Diabetes increases with number of Autoantibodies(GAD, ICA512, Insulin)

T1D in NOD mice is T cell dependentT1D in NOD mice is T cell dependent

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

8-9 wks 13 wks 18 wks

NO

D S

CID

NO

D S

CID

NO

DN

OD

0%

11%

70%

*Diabetic T cell transfer into normal or immunodeficient NOD*T cell depletion studies*Combined contributions of CD4+ and CD8+ T cells:

- Lessons from knockouts*CD4+ and CD8+ T cell clones can induce T1D alone.

Clones/Lines CD4/CD8 Source Antigen TCR Tetramer Transgenic Comment Author

BDC2.5 CD4 NOD Spleen Unknown V 4, V1 Yes Yes Haskins

BDC6.9 CD4 NOD Spleen Unknown V 4, V13.1 Yes Chromosome 6 Haskins

BDC6-4.3 CD4 NOD Islet InsulinB:9-23

V13.3-J53 Wegmann

BDC12-2.4 CD4 NOD Islet InsulinB:9-23

V13.3-J53 Wegmann

2H6 CD4 PeripancreaticLymph Node

InsulinB:12-25

V14 TGF betaProtective

Zekzer

5A CD4 ImmunizedSplenocytes

GAD524-543

Zekzer

4.1 CD4 Unknown Yes Yes Santamaria

Phogrin-15 CD4 ImmunizedLymph Node

PhogrinPeptide 2

V 8, V10 Kelemen

Phogrin -12 CD4 ImmunizedLymph Node

PhogrinPeptide 7

V 5,V11.3 Kelemen

G9C8 CD8 NOD Islet InsulinB:15-23

Yes Wong

8.3 CD8 NOD Islet NRP-A7mimotope

V17-J42 Yes Yes PerforinindependentFas mediated

Santamaria

A/4 CD8 NOD Islet V8-J52 Yes Serreze

Lines CD4CD4CD8

GAD: 530-543GAD: 524-538GAD: 546-554

V4

V12

“Driver”Protective

QuinnQuinnQuinn

Autoantigens: Lessons from diabetogenic T cells.

Diverse T cell response - epitope spreading/cascade?

Checkpoints in T1D development

Checkpoint 1 Insulitis (peri)

-Starts at weaning: immunological changes related to food uptake and changes in the intestinal flora-Increased homing of T cells : expression of addressins MadCam and PNAd on pancreatic blood vessel epithelium-3-4 weeks of age, non-destructive-Th2 dominated

Checkpoint 2 Beta cell loss & diabetes- T cells gain more aggressive effector mechanisms: Th1/Th2 balance, expression of Fas Ligand on CTLs, direct cytotoxicity.-Loss of protective mechanisms:

-Protective cytokines, Regulatory T cells-Amplification : Epitope spreading-10-12 weeks of age, destructive-Th1 dominated

Diabetogenic MHC I-Ag7• The unusual H-2g7 MHC haplotype of NOD mice:

Kd, I-Ag7,I-Enull, Db: Idd1 on chromosome 17.

• I-Ag7 and some HLA-DQB alleles: encode serine, alanine, or valine at position 57 and mediate T1D susceptibility

• Aspartic acid at position 57 is associated with resistance.

• Mutations to Aspartic acid reduce disease incidence but does not reduce insulitis.

• Homozygosity is required for disease:– Possible requirement for a threshold of MHC-peptide complexes for tolerance

induction.– T1D incidence increases with HLA haplotype combinations (DR2/3)

DQB1*0402Asp57

Leu56

-chain

-chain

Defective Central ToleranceDiabetogenic MHC I-Ag7

1. I-Ag7 haplotype is poor peptide binder.

2. Failure to efficiently negatively select autoreactive T cells

3. Failure to positively select Treg cells.

Abnormal peripheral T cell Tolerance in NOD mice.

• Hyporesponsive T cell responses:– TCR induced proliferation and cytokine production (IL-2 / IL-4).– Deficient PKC/Ras/MAPK pathway

– Weak MLR response

• Deficient frequency of NK-T cells:– IL-4 producing cells

– Th1/Th2 balance

• Aberrant regulatory T cell network.

T cell immunoregulation in the NODEvidence

• Delay between insulitis onset and diabetes• Prediabetic T cells prevent adoptive transfer of

disease into NOD.scid mice.• Thymectomy• Cyclophosphamide• Treg cells are numerous and heterogeneous

Balance of effector and regulatory Balance of effector and regulatory mechanisms determines peripheral tolerancemechanisms determines peripheral tolerance

Type 1 insulin dependent autoimmune diabetes

-islet Ags

nTreg

CD4+Foxp3+

Functional deficiency in CD4+CD25+ Treg cells in autoimmunity ?

nTreg

Self-reactiveTeff cell

Autoimmune disease:

Organ-specific - T1D, MS/EAE, Sjogren’s, ThyroiditisSystemic - SLE, APS, RA

CD4+CD25+

-islet Insulin

Normal

T1D

Age

T1D

Aberrant activation of effector T cells?

HealthT1D

Health

The Non-Obese Diabetic (NOD) mouse.

Model of spontaneous type 1 insulin-dependent diabetes (T1D).

Abnormal T cell tolerance to Abnormal T cell tolerance to -islet antigens-islet antigens

CD4+CD25+ nTreg cells in T1D.

•Functional deficiency in CD4+CD25+ Treg cells:– NOD mice succumb to T1D more rapidly in their absence– Delayed administration blocks disease.– NOD mice deficient for B7.1/2, CD40, and CD28 molecules have a more aggressive disease course.

•CD4+CD25+ Treg cells are absent

• Faulty “signals” in NOD mice?•Development, activation requirement, survival or function?

Adoptive transfer NOD model of T1D.

DiabetogenicTeff CD4+CD25-

ProtectivenTreg CD4+CD25+

Diabetes?

NOD.TCR -/-

Wild-Type

BDC2.5 Islet-specific

V4+CD4+ TCR Tg

NOD

Are there functional deficiencies in CD4+ nTreg cells in NOD?

0

20

40

60

80

100

120

0 5 10 15 20 25 30

Days post-transfer

Dia

bet

es i

nci

den

ce (

%)

WholeDep++++++

BDC2.5Teff Treg

+ -+ +- +

0

20

40

60

80

100

0 5 10 15 20 25 30

;

Thymus

Peripheral

Days post-transfer

Dia

bet

es

inci

de

nc

e (%

)D

iab

ete

s in

cid

en

ce

(%)

BDC2.5Teff Treg

+ -+ +

Functional CD4+CD25+ nTreg cells in NOD mice.

Age-dependent loss in nTreg cells?

Roland Tisch JEM 2005

Immunomonitoring of nTreg cells in health and disease.

nTreg X,Y,ZnTreg

Peripheral CD4+ T cells expressing CD25

Activated nTregActivated effectorsAnergized effectors

Induced Treg

HealthPre-clinical or

symptomatic disease

Immune activation

Normal Peri-insulitis Insulitis/T1D

Neonatal Adult

NOD BDC2.5

Are there quantitative differences in the cellular frequency ofCD4+ nTreg cells in NOD mice?

PancreaticLN

Non-draining

LN

CFSE

BDC2.5

BDC2.5 + Treg

PancreaticLN

Non-drainingLN

CD4+CD25+ nTreg cells do not affect the activation or proliferation

of diabetogenic T cells.

CD69

Resistance to T1D correlates with an increased infiltrate of CD4+ Foxp3+ nTreg cells

in pancreatic environments.

- nTreg function+ nTreg cells

- nTreg cells

Use of nTreg cells for the cure of T1D.

Primary Established

Tarbell et al. JEM 2004

Genetic determinants of nTreg cell development in

NOD mice?

Foxp3 dependent development of nTreg cells.

Deficiency of Foxp3+ nTreg cells promotes T1D.

Primary cause or consequence?

Inherited Susceptibility Loci: Both MHC and non-MHC genes are required.

LOCUS CHROMOSOME CANDIDATE GENES

IDDM1 6p21 HLA-DQ\DR

IDDM2 11p15 INS VNTR

IDDM3 15q26 D15s107

IDDM4 11q13 MDU1, ZFM1, RT6, FADD/MORT1, LRP5

IDDM5 6q24-27 ESR, MnSOD

IDDM6 18q12-q21 D18s487, D18s64, JK (Kidd locus)

IDDM7 2q31 D2s152, IL-1, NEUROD, GALNT3

IDDM8 6q25-27 D6s264, D6s446, D6s281

IDDM9 3q21-25 D3s1303

IDDM10 10p11-q11 D10s193, D10s208, D10s588

IDDM11 14q24.3-q31 D14s67

IDDM12 2q33 CTLA-4, CD28

IDDM13 2q34 D2s137, D2s164, IGFBP2, IGFBP5

IDDM14 ? NCBI # 3413

IDDM15 6q21 D6s283, D6s434, D6s1580

IDDM16 ? NCBI # 3415

IDDM17 10q25 D10s1750-D10s1773

Insulin Gene (INS)

Class I VNTR26-63 repeats

Predisposing

IDDM2

Insulin Gene (INS)

Class III VNTR140-200 repeats

IDDM2

Protective

The IDDM2 Locus

VNTR = Variable Number of Tandem Repeats • VNTR stimulates INS steady-state transcription in ß-cells

• VNTR length inversely correlates with INS mRNA levels in ß-cells in vivo

• Class III VNTR alleles = LOWER (~30%) INS transcription than predisposing class I VNTR alleles

• Class III VNTR alleles = Higher thymic INS transcription than predisposing alleles

Low incidence of T1D in Low incidence of T1D in

Idd3Idd3 recombinant congenic NOD mice recombinant congenic NOD mice

ChrChr

22

33

44

55

66

77

88

99

1100

11

1122 11

33 1144 11

551166

1177 11

88 1199XX

1111

20%20% Wicker LS et al. J Exp Med Wicker LS et al. J Exp Med 19941994Lyons PA et al. Genome Res Lyons PA et al. Genome Res 20002000

B6B6

..B6-B6-Idd3Idd3

NODNOD

1%1%

B6B6

NOD.NOD.B6-chr3B6-chr3

80%80%