Type 1A Diabetes(Immune Mediated)

Clinical Immunology SocietyGeorge S. Eisenbarth

Barbara Davis Center for Childhood Diabetes

Slides Chosen From Teaching Slides of:Type 1 Diabetes: Molecular, Cellular, Clinical

Immunology-www.barbaradaviscenter.org

Made possible through an unrestricted educational grant from KRONUS.

WWW.BARBARADAVISCENTER.ORG: Book: Immunology Type 1 Diabetes Teaching slides are Powerpoint slide sets that can be downloaded.1. Primer Immunology and Autoimmunity (Updated - 12/03)

Stephanie C. Eisenbarth2A. Cell Therapy of Diabetes (Updated - 3/02)Jan Nygaard Jensen and Jan Jensen2B. Proprotein Processing and Pancreatic Islet Function (Updated - 3/02)John Hutton, Tina Wasmeier, Rodabe Amaria, Nicholas Bright and John Creemers 2C. Stimulus-Secretion Coupling in the Pancreatic Beta-Cell (Updated - 3/02)Kirstine Juhl and John Hutton 3. Animal Models of Type 1 Diabetes: Genetics and Immunological Function (Updated - 8/02)Julie Lang and Donald Bellgrau4. The Role of T Cells in Beta Cell Damage in NOD Mice and Humans (Updated - 3/02)Katalin Kelemen5. Type 1 Diabetes Mellitus: An Inflammatory Disease Of The Islet (Updated - 12/03)Regine Bergholdt, Peter Heding, Karin Nielsen, Runa Nolsøe, Thomas Sparre, Joachim Størling,

2. Allan E. Karlsen, Jørn Nerup, Flemming Pociot and Thomas Mandrup-Poulsen. Steno Diabetes 3. Center, Gentofte, Denmark

6. The Immunobiology of Pancreatic Islet Transplantation (Updated - 11/01)Marilyne Coulombe and Ronald G. Gill7. Type I Diabetes Mellitus of Man: Genetic Susceptibility and Resistance (Updated - 4/02) A. Pugliese and G. S. Eisenbarth8. Autoimmune Polyendocrine Syndromes (Updated - 10/03)J.M. Barker and G. S. Eisenbarth9. Epidemiology of Type I Diabetes (Updated - 4/02)Marian Rewers, Jill Norris and Dana Dabelea10. Humoral Autoimmunity (Updated - 9/02) L. Yu and G.S. Eisenbarth11. Prediction of Type I Diabetes: The Natural History of the Prediabetic Period (Updated - 11/03)George S. Eisenbarth12. Clinical Trials for the Prevention of Type I Diabetes (Updated - 9/03)H. Peter Chase, Anthony R. Hayward & G. S. Eisenbarth

1986 NEJM “Stages” in Development of Type1Diabetes

Age (years)

Genetic Predisposition

Bet

a ce

ll m

ass

(?Precipitating Event)

Overtimmunologicabnormalities

Normal insulinrelease

Progressiveloss insulinrelease

Glucosenormal

Overtdiabetes

C-peptidepresent

NoC-peptide

Pea

k in

sulin

res

po

nse

to

intr

aven

ou

s g

luco

se

(1+

3 m

in)

imm

un

ore

acti

ve in

sulin

(μ

U/m

l)

0

50

100

150

200

250

300

350

'66 '67 '68 '69 '70 '71 '72 '73 '74 '75'66 '68 '70 '72 '74 '76 '78 '80 '82

ANTIBODY NEGATIVE

*

**

ANTIBODY POSITIVE

*

DM

Srikanta S. et al, New Engl J Med 308:322-325, 1983

Triplets Serial Intravenous Glucose Tolerance Tests

Stages Type IA Diabetes

•I Genetic Susceptibility• II Triggering• III Active Autoimmunity• IV Progressive Metabolic

Abnormalities• V Overt Diabetes• VI Insulin Dependence

Type 1A DiabetesType 1A Diabetes

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

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

• Oligogenic:Oligogenic: MHC+few major genesMHC+few major genes Genetic heterogeneity with Genetic heterogeneity with

different major non-MHC genes different major non-MHC genes for for different families (e.g. BB rat)different families (e.g. BB rat)

BDC

Human Leukocyte Antigen

human MHC

cell-surface proteins

important in self vs. nonself distinction

present peptide antigens to T cells

CLASS I: A,B,C CLASS II: DR,DQ,DP

HLAJ. Noble

The Major Histocompatibility Complex

Human

Mouse

DP DQ DR B C A

K I-A I-E D L

Chromosome 6

Chromosome 17

Class II Class III Class I

Class II Class III Class IClass I

Complement Proteins

Cytokines Class I-like genesand pseduogenes

Antigen Processing Genes

TERMINOLOGY

DRB1*02

DQB1*0302DRB1*0401

DRB1*0401

DRB1*0301

DQB1*0302

DRB1*0401

DQB1*02(DQ2)

Allele:

Haplotype:

Genotype

J. Noble

DR4

DR3

DR4

DR4

DQ2

DQ8

DQ8

DQB1*0402

Asp57

Leu56

-chain

-chain

BDC BDC

0

1

2

3

4

5

Od

ds r

ati

o

0

20

40

60

80

Tra

ns

mis

sio

n f

req

ue

ncy

(%)

******

**

* *

*p< 0.05 vs. control haplotype

High risk

ProtectiveModerate risk

461 389 40 51 182 82 99 20 121 55 124 27 135 34

HBDI Families: Odds Ratio

HBDI Families: Transmission from Heterozygous Parents

BDC

Insulin Gene (INS)

Class I VNTR26-63 repeats

21 alleles

Predisposing

IDDM2

Insulin Gene (INS)

Class III VNTR140-200 repeats

15 alleles

IDDM2

Protective

The IDDM2 Locus

VNTR = Variable Number of Tandem Repeats

Inherited Susceptibility Loci

LOCUS CHROMOSOME CANDIDATE GENES or MICROSATELLITES

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

OTHERS

Autoimmune Polyendocrine Syndromes• APS-II (Autoimm Polyendocrine)

• APS-I (AIRE mutation)

• XPID: (Scurfy Mutation)

• Anti-insulin Receptor Abs + “Lupus”

• Hirata (Anti-insulin Autoantibodies)

• POEMS (Plasmacytoma,..)

• Thymic Tumors + Autoimmunity

• Congenital Rubella + DM +Thyroid

APS-SyndromesBetterle et al. Endocrine Reviews 23:327-364Neufeld and Blizzard: 1980, Pinchera, in Symposium

Autoimmune Endocrine Aspects of Endocrine Disorders

• APS-I:>=2 of Candidiasis, Hypopara,Addison’s

• APS-II:Addison’s + Autoimmune Thyroid and/or Type 1 Diabetes

• APS-III: Thyroid Autoimmune + other autoimmune [not above]

• APS-IV: Two or more organ-specific autoimmune, not I,II, or III.

General Paradigm

• Identify Genetic Susceptibility

• Detect Initial Autoantibodies

• Monitor Metabolic Decompensation

• Treat Overt Disease Prior to

Morbidity/Mortality

• Basic/Clinical Research to Allow Prevention

Associated Autoimmune IllnessesCeliac Disease: Diarrhea, weight loss, growth

failure, abdominal pain, osteoprorosis, anemia

Hyperthyroid: Hypothyroid:

Weight loss, feeling warm, anxiety, bulging eyes Weight gain, feeling cold

Pernicious Anemia: Anemia, movement problems

Addison’s Disease: Darkening of skin, loss of weight, dizziness, nausea

Ovarian Failure: Premature menopause, hot flashes, infertility

Myasthenia Gravis: Muscle weakness, double vision

Diabetes Mellitus: Increased urination, thirst, appetite, weight loss, coma

Comparison APS-I and APS-II APS-I APS-II

• Onset Infancy• Siblings

AIRE gene mutated• Not HLA Associated• Immunodeficiency

AsplenismMucocutaneous Candidiasis

• 18% Type 1 DM

• Older Onset• Multiple Generations• DR3/4 Associated• No Defined

Immunodeficiency• 20% Type 1 DM

BDC

APS-I• Autoimmune Polyendocrine Syndrome Type

1• Autosomal Recessive mutations AIRE

(Autoimmune Regulator) gene• Mucocutaneous Candidiasis/Addison’s

Disease/Hypoparathyroidism• 18% Type 1 Diabetes• “Transcription Factor” in Thymus

BDC

XPID: X-linked polyendocrinopathy, immune dysfunction and diarrhea

• Other NamesIPEX: Immunodysregulation, Polyendocrinopathy, Enteropathy, X-linkedXLAAD: X-Linked Autoimmunity Allergic Dysregulation

• Foxp3 Gene Mutation

• Loss of Regulatory T LymphocytesBone Marrow Transplant with Chimera “Cures” Scurfy Mouse and Man

BDC

Mutations for XPID Syndrome Scurfy/Foxp3/JM2 Gene

Fork Head HomologyZn Zip

X

X

Scurfy

D

ORF

XLAAD-100

XLAAD-200

Zn = Zinc-finger domain, Zip = Zip Motif

ORF = Predicted Open Reading Frame

Modified from Review by Patel, JCI, 2000

Major DR/DQ Associations• Type 1 Diabetes

DR3: DRB1*0301/DQA1*0501/DQB1*0201DR4: DRB1*0401/DQA1*0301/DQb1*0302

• Celiac DiseaseThe same as Type 1 DM plusDR5/DR7 = DQA1*0501/DQB1*0201 in trans

• Addison’s DiseaseThe same as Type 1 DM but DRB1*0404 preference (Yu, JCEM 84:328,1999)

BDC

Known Initiators

DISEASE INITIATOR ASSOCIATIONCeliac Gliadin/wheat

glutenPredominant

InsulinAutoImmune

SH-Drugsmethimizole

Predominant

Type 1 DM Cong Rubella RareThyroiditis Iodine “Common”Graves’ Anti-CD52 RareMyasthenia Penicillamine Rare

Mediator/Autoantigen(s)

Graves’ Antibody TSH ReceptorMyasthenia Antibody ACh ReceptorInsulin Auto Antibody InsulinCeliac ? TransglutaminaseType 1 DM T Cell Insulin/GAD/

ICA512Addison’s T Cell 21-OHThyroiditis T Cell Thyroglobulin

Peroxidase

Celiac Disease

• Intestinal Autoimmune Disorder

• Anti-Transglutaminase (EMA)

• 1/200 General Population U.S./Europe1/20 Patients with Type 1 DM1/6 Patients Type 1 DM who are DR3/DR3

• Gliadin Induction

• Hypothesis: transglutaminase+gliadin

Prevalence of TGA by HLA-DR amongst patients with type 1 DM, relatives of DM patients and

general population

0%

5%

10%

15%

20%

25%

DR3+ DR3-

IDDMRelativesPopulation

Prevalence

HLA-DR

BDC

Stages Type IA Diabetes

• I Genetic Susceptibility

•II Triggering• III Active Autoimmunity• IV Progressive Metabolic

Abnormalities• V Overt Diabetes• VI Insulin Dependence

Environment

•Congenital Rubella•Controversy re Enteroviruses/

other virus•Controversy re bovine milk•Hygiene Hypothesis•2 JAMA papers re early cereal

Ziegler, JAMA 2003: 290:721

0

5

10

15

20

25

30

0 2 4 6 8

Age (years)

Isle

t au

toim

mu

nit

y, %

<=3 mo.

>6 mo.

>3 to6 mo.

DR3/4 DQ8: Norris JAMA 290:1713

0

5

10

15

20

25

0 2 4 6 8

Age (years)

Isle

t A

uto

imm

un

ity,

%

<=3 mo.

4 o 6 mo.

>=7 mo.

BabyDiab and DAISY

Age introduction gluten (Ziegler) or cereal (Norris) greatly increases development of anti-islet autoantibodies in infants followed from birth.

Stages Type IA Diabetes

• I Genetic Susceptibility• II Triggering

•III Active Autoimmunity• IV Progressive Metabolic

Abnormalities• V Overt Diabetes• VI Insulin Dependence

Insulin Autoantibodies:A Chain L13

ReceptorBindingRegion

Experimental Autoimmune Diabetes

B:9-23 Peptide ----- Insulin Autoantibodies

B:9-23 Peptide + Poly-IC ------ Insulitis

B:9-23 Peptide + Poly-IC + B7.1 Islet -- Diabetes

Moriyama et al. PNAS 99: 5539-5544, 2002

B Chain 1: FVKQHLCGPHLVEALYLVCGERGFFYTPKS 2

B Chain 2: FVKQHLCGSHLVEALYLVCGERGFFYTPMS

Difference of Amino acid sequence Difference of Amino acid sequence between preproinsulin 1 and 2between preproinsulin 1 and 2

B:9-23

Leader 1: MALLYHFLPL LALLALWEPKPTQA 6

Leader 2: MALWMRFLPL LALLFLWESHPTQA

A Chain 1: GIVDQCCTSI CSLYQLENYC N 0A Chain 2: GIVDQCCTSI CSLYQLENYC N

C-Peptide 1: EVEDPQVEQLELGGSPGDLQTLALEVARQ 5

C-Peptide 2: EVEDPQVAQLELGGGPGAGDLQTLALEVAQQ

insulin 1 KO male

0 10 20 30 40 500

20

40

60

80

100

weeks of age

% o

f d

iab

ete

s f

ree

Insulin 1 KO female

0 10 20 30 40 500

20

40

60

80

100

weeks of age

% o

f d

iab

ete

s f

ree

Insulin 2 KO male

0 10 20 30 40 500

20

40

60

80

100

weeks of age

% o

f d

iab

ete

s f

ree

Insulin 2 KO female

0 10 20 30 40 500

20

40

60

80

100

weeks of age

% o

f d

iab

ete

s f

ree

PNAS 2003,18:10376

Diabetes Autoimmunity Study in the Young Sibling/offspring cohortGeneral population cohort

enrolled = 293 high risk 72

429 moderate risk 220

347 average - low risk 401

1,069 All 693

relatives 1,491 1,007

screened = 21,713

HLA-defined IDDM risk groupsDenver population, n=9,338

IDDM risk by age 20 HLA-DR DQB1 Frequency %

High 1:15 3/4 0201/0302 2.4 Moderate 4/x 0302/ 12.7 1:60-1:200 4/4 0302/ 3.0 3/3 0201/0201 1.4

Average 1:300 3/x 0201/ 12.5 3/4 0201/not 0302 1.0

Lower than 1:300 4/x, 4/4 /not 0302 6.6 others 60.4

DAISY 7/96

Autoantibodies

•Insulin

•Glutamic Acid Decarboxylase

•ICA512 (IA-2)

IAA assay

3. Add Protein A/G-Sepharose to reaction mix in a 96-well filtration plate

High Throughput Anti-Insulin Autoantibody Assay

Sera

2. Incubate 72 hours at 4 C

4. Incubate for 45 min at 4 C

5. Wash each well using the vacuum- operated 96-well plate washer

6. Count radioactivity with 96-well plate beta counter

Ag-Ab mixture

Vacuum

o

o

(125)Insulin

Beta Counter

Protein A/G Sepharose

1. mix (125)I-insulin and sera

1

10

100

1000

10000

5 10 15 20 25 30 35

Age (years)

An

ti-i

ns

uli

n a

uto

an

tib

od

ies

(n

U/m

l)

Insulin Autoantibodies Versus Age of Diabetes Onset

Diabetes Care 11:736-739, 1988

The Levels of mIAA in Prediabetic Children

0.0001

0.001

0.01

0.1

1

10

0 2 4Age (years)

Le

ve

l of m

IAA

(ind

ex

)

0.0001

0.001

0.01

0.1

1

10

0 2 4

Age (years)

Level o

f mIA

A (in

dex)

DM

DM

DM

DM

DM

Yu et al. PNAS: 97:1701-1706, 2,000 BDC

DAISY AUTOANTIBODIES:Initial Test <Age 1

0 1 2 3 40

20

40

60

80

100

Percent with Persistent Autoantibodies (GAA/IAA/ICA512)

3/4SOC

3/4NEC

not 3/4SOC

not 3/4NEC

3/4 SOC: 15 9 5 43/4 NEC: 151 110 67 18-3/4 SOC: 69 56 39 16 3-3/4 NEC: 492 300 208 110

p<.0001

12/27/97

0

20

40

60

80

100

0 2.5 5 7.5 10 12.5 15

3 Abs2 Abs1 Ab

Progression to Diabetes vs Number of Autoantibodies(GAD, ICA512, Insulin)Percent not Diabetic

Years of Follow-up

3 Ab n = 41 17 8 1 2 Abs n = 44 27 15 4 2 11 Abs n = 93 23 14 10 6 4

Stages Type IA Diabetes

• I Genetic Susceptibility• II Triggering• III Active Autoimmunity

•IV Progressive Metabolic Abnormalities

• V Overt Diabetes• VI Insulin Dependence

We can now predict type 1 diabetes.

We cannot now prevent type 1

diabetes.

What are we missing?

Assay for Pathogenic T

cells.

? TETRAMER

? ELISPOT

MHC peptide

HLA Class II tetramer (DR0401-hGAD555-567)

Leucinezippers spacers

streptavidin

W.W.Kwok & G.T.Nepom, BenaroyaResearch Institute at Virginia Mason

Female NOD Mice Peripheral Blood

Avidin

Kd

NRP-V7 Peptide (KYNKANVFL)

Kd

Kd

Kd

Tetramer Analysis

0

0.2

0.4

0.6

0.8

1

1.2

5 10 14 18 21 24 27 30

Age (weeks)% t

etra

mer

+ C

D8+

cel

ls

Diabetes

0

0.2

0.4

0.6

0.8

1

1.2

5 9 12 15 18 21 24 27 30

Age (weeks)% t

etra

mer

+ C

D8+

cel

ls

No Diabetes

Trudeau,Santamaria,Tan: JCI 2003

IGRP-2nd Beta Cell Specific Ag

Multiple Trials New Onset Planned/

Underway•Anti-CD3 Monoclonal•Anti-IL2 Receptor + MMF•Altered Peptide Ligand B:9-23

insulin•HSP 60, p277 Peptide (LADA

Pts)•GAD65 (LADA patients)

Changes from Study Entry to 12 Months in the Total C-Peptide Response to Mixed-Meal Tolerance Testing

Herold K. et al., N Engl J Med 2002; 346:1692-8.

To

tal

Are

a u

nd

er t

he

C-P

epti

de

Res

po

nse

Cu

rve

(nm

ol/

l/4

hr)

Monoclonal-Antibody Group

To

tal

Are

a u

nd

er t

he

C-P

epti

de

Res

po

nse

Cu

rve

(nm

ol/

l/4

hr)

Control Group

Large NIH Prevention Initiatives

• Immune Tolerance Network

• DPT-1 ===> TrialNet

• Autoimmunity Centers Excellence

• Autoimmunity Prevention Centers

Rewers-BDC

IDS Guidelines for Intervention TrialsIDS Guidelines for Intervention TrialsGreenbaum and Harrison:Diabetes 52:1059, 2003Greenbaum and Harrison:Diabetes 52:1059, 2003

• Diagnosis ADA criteria• Document: age,sex,pubertal, family history,glucose,

bicarb,ketoacidosis, weight loss, symptoms,HbA1c,islet autoab, insulin Rx, HLA

• Phase I >=18• GAD, IA-2, IAA(<2 wks), and if DM ICA C-peptide>=.2 nmol/L, early =

<12 weeks from diagnosis• >=2 year trials• Randomize, blind, mask, safety review, tight control, and continue

insulin• 2 hr. AUC C-Peptide with meal tolerance test, no AM insulin except

pump basal, fasting glucose 4-11.1 mmol/l• Measure islet autoAb other immune with HLA