Islet Amyloid Polypeptide Is a Target Antigen forDiabetogenic CD4+ T Cells

Thomas Delong, …Kathryn Haskins Diabetes 2011

Register 3 mimotope

12-14 wk vaccination

3-4 wkvaccination

Prevention of type 1 diabetes in mice by tolerogenic vaccination with a strongagonist insulin mimetope

Carolin Daniel,1,4 Benno Weigmann,3 Roderick Bronson,4and Harald von Boehmer1,2

JEM 2011

Stadinski et al Diabetogenic T cells recognize insulin bound to IAg7 inan unexpected, weakly binding registerPNAS 107:10978, 2010

All anti-B:9-23 only reacted with peptide in Register 3 with arginine in pocket 3

FIXING IN REGISTER 3 WITH DISULFIDE BONDS

Stadinski et al PNAS 2010

By mutating the MHC binding groove of NOD’s I-Ag7 insulin peptide B:9-23 is fixed in specific register 3, and this is low affinity register diabetogenic

T cell receptors “see” the peptide.

I-Ag7

Peptide

Peptide Presentation Model

AA Peptide Side Chains

ARG(22)

GLU

GLY

CYS

VAL

LEU

TYR

LEU

ALA(14)

Crystal Structure of I-Ag7

INSULIN PEPTIDE B:9-23 IN TWO REGISTERS (arrow 3)

Unique autoreactive T cells recognize insulin peptides generated within the islets of Langerhans in autoimmune diabetes James F Mohan,…& Emil R Unanue Nature Immunology March 2010

Rag-/- isletsOnly islets + type B

CD11c islets

InsulinomaType A

Insulin granules

Type B

TEETH

GROOVE

“TEETH”

T Cell Receptor Analogy

Conserved T Cell Receptor Alpha Chain Induces Insulin Autoantibodies

Kobayashi et al PNAS 105:10090-94 2008

Anti-B:9-23 TCR alpha Transgene No Transgene

Leader 1: MAL LVHFLPLLALLALWEPKPTQA

Leader 2: MALWMRFLPLLALLFLWESHPTQA

Human : MALWMRLLPLLALLALWGPDPAAA

10 20

B Chain 1: FVKQHLCGPHLVEALYLVCGERGFFYTPKS

B Chain 2: FVKQHLCGSHLVEALYLVCGERGFFYTPMS

Human : FVNQHLCGSHLVEALYLVCGERGFFYTPKT

25 30 40 50

C-Peptide 1: RREVEDPQVEQLELGGSPG…..DLQTLALEVARQ

C-Peptide 2: RREVEDPQVAQLELGGGPGAGDLQTLALEVAQQ

Human : RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQ

55 60 70 80

A Chain 1: KR GIVDQCCTSICSLYQLENYCN

A Chain 2: KR GIVDQCCTSICSLYQLENYCN

Human : KR GIVEQCCTSICSLYQLENYCN

88 100

Amino Acid Sequence of Mouse 1 and 2 and Human Insulin

B:9-23

0 10 20 30 40 50 600

20

40

60

80

100

ins1-, ins2-, Tg+ (n=25, P<0.01)

ins1+, ins2-, Tg+ (n=25)

Weeks of age

% D

iab

ete

s F

ree

Lack of progression to diabetes of NOD mice lacking both insulin native genes.

Ins1-, ins2-: n=Ins1+, ins2-: n=

2525

1014

2123

11

24 Life table update 5/19/05

Nakayama Nature 435:220,2005

Insulin is the primary antigen and precedes IGRP in hierarchy of autoantigens

Epitope and antigen spreading,expansion

Diabetes

Insulin specific T cells

Insultis

Krishnamurthy et al JCI:116:3258, 2006

Is there a primary antigen or immune response to multiple antigens required for autoimmunity?

Epitope and antigen spreading, expansion

Diabetes

T cells specific for one antigen

Diabetes

Expansion of T cells

T cells specific for multiple antigens

Insulitis Insulitis

OR

Krishnamurthy et al JCI:116:3258, 2006

Multiple Antigens are Targeted in Type 1 Diabetes

CPE

Hsp70

IGRP

Jun-B

SOX13

DNA TopII

S100

GAD67

Hsp90

Peripherin

GLUT2

GAD65

Glima38

HIP/PAP

Insulin

IAPP

Sulphatide

IA-2

Hsp60

Imogen38

IA-2

ICA69

GM2-1GT3

DMK

Nucleus

Synaptic-likeMicrovesicles

EndoplasmicReticulum

Cytosol

GolgiNetwork

SecretoryGranules

Mito

ch

on

dria

GD3

CD38

ZnT8

Chg A

Hsp10

RegRegII

Importin b

PDX1

Ab AbT T

H M

x

xx

x

x

x

x

xx

xx

xx

xx

x

x

x

xxx

xx

xx

x

xxxxxx

xx

x

x

xx

xxx

x

xxx

x

xxxxxxxx

xxxx

x

x

xxxx

Figure modified from Tissue Antigens 2003, 62:359Information from Expert Rev Clin Immunol 2010, 6:939

HUMAN MOUSE

Reported Antibody or T Cell

Dilorenzo

The Generation of Retrogenic Mice

Insertion: TCR alpha only oralpha_P2A_beta

Phoenix cells(293T cells transfected with pCL-Eco)

pMIG

pMIG

pMIG

SCID orCa-KOBM cells

SCID

3’LTR

5’LTR

IRE

S

GFP

Ψ

pMIG

MIG

CMV-env (Mo-MuLV)RSV-gag-pol (Mo-MuLV)

Anti-GAD TCR + or – B Lymphocytes TCR Retrogenic Induction High Levels GAD65 Autoantibodies

T Cell Islet Accumulation in Type 1 Diabetes is a Tightly Regulated, Cell-Autonomous Event Lennon et al

Immunity 31, 643-653, 2009

Nat Immunol. 2010 Mar;11(3):225-31. Epub 2010 Feb 7.Chromogranin A is an autoantigen in type 1 diabetes.

Stadinski, …Kappler, Haskins

Peptide WE14 bound to the NOD mouse major histocompatibility complex class II molecule I-A(g7) in an atypical manner, occupying only the carboxy-terminal half of the I-A(g7) peptide-binding groove.

T Cell Receptor Gene Segments

Chr. 14

Chr. 6

V

J

V

D

J

HLA Molecule Peptide

Antigen Presenting Cell

Antigen Recognition by CD8+ T Cells

T cellreceptor

Vb Va

Tcell

class IMHC

b2m

b cellT. DiLorenzo

V(D)J Rearrangement

1011 -1016 different possible

combinations.

Kuby Immunology

Child with IPEX syndrome

Awaiting Bone Marrow Transplant

9 Months of Age

IPEX: Immune Dysfunction, Polyendocrinopathy, Enteropathy, X-linked

• Scurfin gene (Foxp3/JM2)- Controls Regulatory T Cells!

• Approximately 80% of children with syndrome develop diabetes!

• Bone marrow transplant can reverse

“Typical” Insulitis of man differs from peri-insulitis NOD mouse: Bottazzo

In’t Veld and Atkinson, Diabetes (Submitted)

Pipeleers and Ling, Diabetes/Metabolism Reviews 8:209, 1992.Gepts et al, 1965, 1978; ,Doniach et al, 1973; Klöppel et al, 1984; Bottazzo et al, 1985; Foulis et al, 1986; Hänninen et al, 1992; Somoza et al, 1994; Lernmark et al, 1995; Shimada et al, 1999; Dotta et al, 2007; Uno et al, 2007; Butler et al, 2007, Gianani and Atkinson, 2010.

Duration of disease

≤1 week >1 week - ≤1 year

>1 year total

Onset childhood (0-14 yrs)

22/23 37/42 3/32 62/97

Onset young adult(15-39 yrs)

8/14 17/26 1/23 26/63

Meta-Analysis of Insulitis – T1D Patients Stratified to Age at Onset and Duration of Disease

Atkinson

b-cells

PancreaticLymph Node

T cells

ISLET

A MODEL

Ag

APC

t

tt

Mathis/Benoist

TRAV5D-04+TRAJ53 Restriction?

Insulin B:9-23: SHLVEALYLVCGERG?

Natural T Regulatory Cells Antigen Specific T Reg

IPEX Syndrome MAN:

foxP3 mutant DM in days of birth!

NOD anti-B9-23insulin TCR:

foxP3 mutant DM

?

MAN NOD MOUSE

IAg7DR3/DR4

Homann 2006,JCI

ENVIRONMENTAL FACTORS

Incidence type 1 DM Increasing 3%/year > 30 years!

Protective Factor Decreasing

-Hygiene Hypothesis – Bach NEJM 347:911, 2002

Triggering Factors

-Congenital Rubella-Rubenstein Diabetes 31:1088, 1982

-Kilham Rat Virus (BB-DR rat)-Zipris J. Immunol 174:131, 2005

-Poly-IC Induction Interferon Alpha-Devendra Diabetes 54 2005

-Dietary Factors-Scott Ann Rev Nutr 26:175, 2006

T Cell Receptor

Natural T Regulatory Cells

Antigen Specific T Reg

Natural peptides selected by diabetogenic DQ8 and murine I-Ag7 molecules show common sequence

homology Suri et al JCI 115:2268, 2005Structure of Human insulin peptide DQ8, Lee et al

Nature Immunology 6:501, 2001

Crystal DQ8;B:9-23: S H L V E A L Y L V C G E R G

P1 P4 P9

I-Ag7 v,e,q I,L A,s D,E

12% 20% 30,11% 45%

P6

DQ8 E,d A,S A,V,s E,D

27,17% 19% 20% 60,25%

Preferred AA in Bound Peptides

Wiley Nat Immunol

% amino acid at position

Clone CD4/CD8 Source Antigen TCRα/β Tetramer Transgenic Retrogenic Comment AuthorBDC2.5 CD4 Spleen Chromagranin 7D-6/2 yes yes yes 10.1 same mimotope HaskinsBDC10.1 CD4 Islet Chromagranin 17/20 yes yes DM+++ retrogenic HaskinsBDC5.10.3 CD4 Islet Chromagranin /2BDC5.2.9 CD4 Amyloid(IAPP 1-20) 2/19BDC6.9 CD4 Islet ? IAPP 5D-4*01/2 yes yes Strain specific, chr 6 HaskinsNY4.1 CD4 Islet 5D-4*04/16 yes yes DM transgenic Santamaria

Phogrin-13 CD4 Immun LN IA2β 640-659 13-2/3 yes insulitis retrogenic HuttonPhogrin-18 CD4 Immun LN IA2β 755-777 10/16 yes insulitis retrogenic Hutton10.23 CD4 Immun LN IA2 676-688 13-4/4 yes Hutton

2H6 CD4 Panc LN insulin B:12-25 21/31 yes TGFβ protection Zekzer12-4.1 CD4 Islet insulin B:12-22 5D-4*04/1 yes yes DM retrogenic Wegmann12-4.4 CD4 Islet insulin B:12-22 5D-4*04/5 yes DM retrogenic Wegmann 8-1.1 CD4 Immun LN insulin B:12-22 5D-4*04/15 yes yes DM retrogenic Wegmann12-1.19 CD4 Islet insulin B:9-23 13-1/19 yes DM retrogenic Wegmann12-2.35 CD4 Islet insulin B:9-18 7-4/13-1 yes DM retrogenic Wegmann6-4.3 CD4 Islet insulin B:9-23 5D-4*04/1 Wegmann12-2.4 CD4 Islet insulin B:9-16 WegmannPCR1-10 CD4 anti 8-1.1 isletinsulin B:9-23 5D-4*04/16 yes Same alpha 8-1.1 NakayamaAS91 CD4 Islet insulin B12-20 5-1/ yes UnanueAS150 CD4 Islet insulin B13-21 UnanueI.29 CD4 Islet insulin 10/1 yes Unanue

6C5 CD4 islet 9D-4/19 young NOD Fathman

5A CD4 Immun Spl GAD ZekzerB16.3 CD4 spleen GAD65 pp286–300 6-2/5 yes suppress DM McDevitt

BW5147 CD4 spleen GAD65 pp206–220 3/13-2 suppress DM McDevitt

PA15.14B CD4 spleen GAD pp206–220 6D-6/13-3 yes no DM retrogenic VignalliIA4 CD4 GAD pp217–236 6-6/2 yes no DM retrogenic Tisch

PA18.10F10CD4 GAD pp510–524 7-4/4 yes no DM retrogenic VignalliPA18.9H7 CD4 GAD pp524–538 21/15 yes no DM retrogenic Vignalli530.45.19 CD4 GAD pp530–543 (5D-4/2) yes no DM retrogenic SercarzPA17.9G7 CD4 Immun GAD pp284–300 4.5/10 yes no DM, encephalitis Vignalli4B5 CD4 GAD pp217-236:290-309 2.4/6 no DM, encephalitis TischPA19.9G11 CD4 Immun GAD pp221-237:284-300 17.1/5.2 no DM, encephalitis Vignalli

G9C8 CD8 Islet insulin B:15-23 yes Wong

8.3 CD8(Kd) Islet IGRP 206-214 17/ yes yes Perforin independent, Fas Santamaria

AI4 CD8(D6) DMK 138-146 DMK Widespread DiLorenzo

Clone CD4/CD8 Source Antigen TCRα/β Tetramer Transgenic Retrogenic Comment AuthorBDC2.5 CD4 Spleen Chromagranin 7D-6/2 yes yes yes 10.1 same mimotope HaskinsBDC10.1 CD4 Islet Chromagranin 17/20 yes yes DM+++ retrogenic HaskinsBDC5.10.3 CD4 Islet Chromagranin /2 HaskinsBDC5.2.9 CD4 Amyloid(IAPP 1-20) 2/19 HaskinsBDC6.9 CD4 Islet ? IAPP 5D-4*01/2 yes yes Strain specific, chr 6 HaskinsNY4.1 CD4 Islet 5D-4*04/16 yes yes DM transgenic Santamaria

Phogrin-13 CD4 Immun LN IA2β 640-659 13-2/3 yes insulitis retrogenic HuttonPhogrin-18 CD4 Immun LN IA2β 755-777 10/16 yes insulitis retrogenic Hutton10.23 CD4 Immun LN IA2 676-688 13-4/4 yes Hutton

2H6 CD4 Panc LN insulin B:12-25 21/31 yes TGFβ protection Zekzer12-4.1 CD4 Islet insulin B:12-22 5D-4*04/1 yes yes DM retrogenic Wegmann12-4.4 CD4 Islet insulin B:12-22 5D-4*04/5 yes DM retrogenic Wegmann 8-1.1 CD4 Immun LN insulin B:12-22 5D-4*04/15 yes yes DM retrogenic Wegmann12-1.19 CD4 Islet insulin B:9-23 13-1/19 yes DM retrogenic Wegmann12-2.35 CD4 Islet insulin B:9-18 7-4/13-1 yes DM retrogenic Wegmann6-4.3 CD4 Islet insulin B:9-23 5D-4*04/1 Wegmann12-2.4 CD4 Islet insulin B:9-16 WegmannPCR1-10 CD4 anti 8-1.1 isletinsulin B:9-23 5D-4*04/16 yes Same alpha 8-1.1 NakayamaAS91 CD4 Islet insulin B12-20 5-1/ yes UnanueAS150 CD4 Islet insulin B13-21 UnanueI.29 CD4 Islet insulin 10/1 yes Unanue

6C5 CD4 islet 9D-4/19 young NOD Fathman

5A CD4 Immun Spl GAD ZekzerB16.3 CD4 spleen GAD65 pp286–300 6-2/5 yes suppress DM McDevitt

BW5147 CD4 spleen GAD65 pp206–220 3/13-2 suppress DM McDevitt

PA15.14B CD4 spleen GAD pp206–220 6D-6/13-3 yes no DM retrogenic VignalliIA4 CD4 GAD pp217–236 6-6/2 yes no DM retrogenic Tisch

PA18.10F10CD4 GAD pp510–524 7-4/4 yes no DM retrogenic VignalliPA18.9H7 CD4 GAD pp524–538 21/15 yes no DM retrogenic Vignalli530.45.19 CD4 GAD pp530–543 (5D-4/2) yes no DM retrogenic SercarzPA17.9G7 CD4 Immun GAD pp284–300 4.5/10 yes no DM, encephalitis Vignalli4B5 CD4 GAD pp217-236:290-309 2.4/6 no DM, encephalitis TischPA19.9G11 CD4 Immun GAD pp221-237:284-300 17.1/5.2 no DM, encephalitis Vignalli

G9C8 CD8 Islet insulin B:15-23 yes Wong

8.3 CD8(Kd) Islet IGRP 206-214 17/ yes yes Perforin independent, Fas Santamaria

AI4 CD8(D6) DMK 138-146 DMK Widespread DiLorenzo

NOD T CELL CLONES

NOD Mouse anti-islet T Cell Clones/transgenics/retrogenics

Clone CD4/CD8 Source Antigen TCRα/β Tetramer TransgenicRetrogenicComment AuthorBDC2.5 CD4 Spleen 7D-6/2 yes yes yes 10.1 same mimotope HaskinsBDC10.1 CD4 Islet 17/20 yes yes DM+++ retrogenic HaskinsBDC6.9 CD4 Islet 5D-4*01/2 yes yes Strain specific, chr 6 HaskinsNY4.1 CD4 Islet 5D-4*04/ yes yes yes DM transgenic Santamaria

Phogrin-13 CD4 Immun LN IA2β 640-659 13-2/3 yes insulitis retrogenic HuttonPhogrin-18 CD4 Immun LN IA2β 755-777 10/16 yes insulitis retrogenic Hutton10.23 CD4 IA2 676-688 13-4/4 yes Hutton

2H6 CD4 Panc LN insulin B:12-25 21/31 yes TGFβ protection ZekzerBDC12-4.1 CD4 Islet insulin B:12-22 5D-4*04/1 yes yes DM retrogenic WegmannBDC12-4.4 CD4 Islet insulin B:12-22 5D-4*04/5 yes DM retrogenic WegmannBDC 8-.1.1 CD4 Immun LN insulin B:12-22 5D-4*04/15 yes DM retrogenic WegmannBDC12-1.19 CD4 Islet insulin B:9-23 13-1/19 DM retrogenic WegmannBDC12-2.35 CD4 Islet insulin B:9-18 7-4/13-1 DM retrogenic WegmannBDC6-4.3 CD4 Islet insulin B:9-23 5D-4*04/1 WegmannBDC12-2.4 CD4 Islet insulin B:9-16 Wegmann6C5 CD4 islet 9D-4 young NOD Fathman

5A CD4 Immun Spl GAD ZekzerB16.3 CD4 spleen GAD65 pp286–300 6-2/5 yes suppress DM McDevittBW5147 CD4 spleen GAD65 pp206–220 3/13-2 suppress DM McDevittPA15.14B CD4 spleen GAD pp206–220 6D-6/13-3 yes no effect retrogenic VignalliIA4 CD4 GAD pp217–236 6-6/2 yes no effect retrogenic TischPA17.9G7 CD4 GAD pp284–300 6-2/4 yes no effect retrogenic VignalliPA18.10F10 CD4 GAD pp510–524 7-4/4 yes no effect retrogenic VignalliPA18.9H7 CD4 GAD pp524–538 21/15 yes no effect retrogenic Vignalli530.45.19 CD4 GAD pp530–543 (5D-4/2) yes no effect retrogenic Sercarz

G9C8 CD8 Islet insulin B:15-23 yes Wong

8.3 CD8 Islet IGRP 206-214 yes yes Perforin independent, Fas Santamaria

Peakman

A2 CD8 PPI:15-24

Durinovic-Bello

DRB1*0401 CD4 73-90

Gottlieb

B:9-23 DQ8

Peakman

DRB1*0401 C19-A3

Hafler

DRB1*0401 A1-15

Expanded T cells from pancreatic lymph nodes of type 1 diabetic subjects recognize an insulin epitopeKent et al, Nature 435:224, 2005

• Pancreatic LN: Single cell cloning - PHA• 2/3 Patients Clonal expansion

duration diabetes 29 and 15 years• Vbeta29-1*03J2-3(50%)/Valpha8-3*02 J44*01(25%)

Vbeta5-1*01 J2-3(52%)/Valpha39*01 J33*01(26%)

• DRB1*0401 RestrictedInsulin A1-15

•Caveat: High concentration to stimulate 100uM

BDC

The insulin A-chain epitope recognized by human T cells is posttranslationally modified

Mannering et al, JEM 202:1191-1197, 2005• CD4 T cells cloned with CFSE method from peripheral

blood of one patient with diabetes and one child with insulin autoantibodies

• Subset of the clones reacted with insulin A-chain 1-13 epitope

• T Cells DR restricted and only reacted with peptide with vicinal disulfide bond between adjacent cysteines A6 and A7

• No reactivity with this peptide of clones from two normal controls with DR4

• Oral report by Sally Kent that their A1-15 peptide reactive T cells from pancreatic lymph node do not require vicinal disulfide cross-link

Ins2 deficiency augments spontaneous HLA-A*0201-restricted T cell responses to Insulin Jarchum and

DiLorenzo J Immunol 2010 vol 184

Ins B:10-18

Humanized HLA-A2 Mice lacking Insulin 2!

CD8 Elispot

Thymus-specific deletion of insulin induces autoimmune diabetes.

Fan et al EMBO Journal 28:2812-2824 2009

• Ins1-/- mice (only) with cre mediated thymic mTEC deletion of ins2 develop autoimmune diabetes by age 3 weeks.

• Diabetes develops in H-2b mice!• ELISPOT Responses to Insulin B:9-23

though mice do not have I-Ag7.• MYSTERIES: Need for Ins1-/-; Lack

protection H-2b; Presentation B:9-23 by H-2b.

The frequency and immunodominance of Islet-specific CD8+ T-cell responses change after Type 1 Diabetes

Diagnosis and Treatment Martinuzzi et al Diabetes 57:1312-1320, 2008

7-16 months post onset most ELSIPOT (HLA-A2) responses decrease

New Responses

PROINSULIN

PREPROINSULIN

“Santamria” NRP(IGRP206-214): Class I (Kd)Recognized

Peptide• CD8 T cell from NOD islets (e.g. clone

8.3)• Conserved Valpha/ nDn/ J alpha

(Va17,Ja42)Vbeta not conserved but contributes

• Accelerated diabetes TCR Transgenic• Mimotope Defined-Kd Tetramer

ProducedNRP=KYNKANWFL; NRP-V7: KYNKANVFL

• Higher Avidity Peptide/Tetramer V7• 30% Intra-islet post 9 weeks

>.5% in Blood Predicts Diabetes NOD Mice

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)% N

RP

-V7

te

tra

me

r+ C

D8

+ c

ell

s

Diabetes

0

0.2

0.4

0.6

0.8

1

1.2

5 9 12 15 18 21 24 27 30

Age (weeks)% N

RP

-V7

te

tra

me

r+ C

D8

+ c

ell

s

No Diabetes

Tan et al., JCI 1/2003

CTLs are targeted to kill β cells in patientswith type 1 diabetes through recognition ofa glucose-regulated preproinsulin epitope Ania Skowera,…. and Mark Peakman JCI 2008:118 3268-3271.

Interferon gamma HLA-A2 ELISPOT peripheral blood

Patients+ (SI>3)

DM vs Controls

CD8+ CTL

CTL

Screening of Peptide Fractions by 51Cr Release Cytotoxicity Assay Method Used to Discover IGRP

APC

T. DiLorenzo

GAD, glutamic acid decarboxylase; IAPP, islet amyloid polypeptide

Antigen Position MHC Why examined

GAD65 114-123 A2 MHC binding

IAPP 5-13 A2 MHC binding

Insulin B10-18 A2 Proteasome cleavage

B14-22 A3, A11 Proteasome cleavage

B15-23 A24 MHC binding

B15-24 A24 Proteasome cleavage

B17-26 A1, A3, A11 Proteasome cleavage

B18-27 A1, A2, B8, B18 Proteasome cleavage

B20-27 A1, B8 Proteasome cleavage

B21-29 A3 Proteasome cleavage

B25-C1 B8 Proteasome cleavage

B27-C5 B8 Proteasome cleavage

Antigens for CD8+ T Cells in Type 1 Diabetes Patients

DiLorenzo

Antigen Position MHC T cells How identified

DMK 138-146 H-2Db AI4; islet Positional scanning libraries

IGRP 206-214 H-2Kd 8.3; isletMHC purification andmass spectrometry

21-29 H-2Kd Islet MHC binding

225-233 H-2Db Islet MHC binding

241-249 H-2Db Islet MHC binding

324-332 H-2Kd Islet MHC binding

Insulin 1/2 B15-23 H-2Kd G9C8; islet Expression cloning

DMK, dystrophia myotonica kinaseGAD, glutamic acid decarboxylaseIGRP, islet-specific glucose-6-phosphatase catalytic subunit-related protein

Antigens for Islet-infiltrating CD8+ T Cells in NOD Mice

DiLorenzo

Toma et al. Recognition of a subregion of human proinsulin by class I-restricted T cells in type 1

diabetic patients PNAS 102:10581, 2005

• Selected 8-11mer peptides of proinsulin• PBMCs of 29/32 (90%) recent+long term

diabetics responded IFNgamma in ELISPOT assay versus minimal response of normals

• Significant Peptides often overlapped insulin B chain peptide B:9-23(=Proins 33-47); Proins 34-42(B10-18); Proins 41-50(B17-26);Proins 42-51;Prins44-51; but also B chain-C-peptide (49-57)

• Multiple class I alleles, including A1 and B8

Peptide 2: Amino acids 643-658Peptide 7: Amino acids 762-777

C terminus

PTP

GADPSADATEAYQEL (rat) GADPSADATEAYQEL (mouse) GGDPGADATAAYQEL (human)

KNRSLAVLTYDHSRI (rat)KNRSLAVLTYDHSRI (mouse)KNRSLAVLTYDHSRV (human)

TM

N terminus

T cell epitopes on the diabetes autoantigen Phogrin (IA-2beta) are conservedamong different species