Insertion: TCR alpha only oralpha_P2A_beta

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

pMIG

pMIG

pMIG

SCID orC-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

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

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

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.

Chr. 14

Chr. 6

V

J

V

D

J

Peptide

Antigen Presenting Cell

Antigen Recognition by CD8+ T Cells

T cellreceptor

V V

Tcell

class IMHC

2m

cellT. DiLorenzo

V(D)J Rearrangement

1011 -1016 different possible

combinations.

Kuby Immunology

I-Ag7I-Ag7

PeptidePeptide

AA Peptide Side ChainsAA Peptide Side ChainsAA Peptide Side ChainsAA Peptide Side Chains

TEETH

GROOVE

“TEETH”

T Cell Receptor Analogy

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

-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

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

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

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 Produced

NRP=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; isletPositional scanning

libraries

IGRP 206-214 H-2Kd 8.3; isletMHC purification and

mass 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