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The Generation of Retrogenic Mice. MIG. Insertion: TCR alpha only or alpha_P2A_beta. Ψ. 5’LTR. SCID. pMIG. IRES. GFP. 3’LTR. Phoenix cells (293T cells transfected with pCL-Eco). CMV-env (Mo-MuLV) RSV-gag-pol (Mo-MuLV). SCID or C a -KO BM cells. pMIG. pMIG. pMIG.

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The Generation of Retrogenic Mice

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The generation of retrogenic mice l.jpg

The Generation of Retrogenic Mice

MIG

Insertion:

TCR alpha only or

alpha_P2A_beta

Ψ

5’LTR

SCID

pMIG

IRES

GFP

3’LTR

Phoenix cells

(293T cells transfected with pCL-Eco)

CMV-env (Mo-MuLV)

RSV-gag-pol (Mo-MuLV)

SCID or

Ca-KO

BM cells

pMIG

pMIG

pMIG


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Anti-GAD TCR + or – B Lymphocytes

TCR Retrogenic Induction High Levels GAD65Autoantibodies


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T Cell Islet Accumulation in Type 1 Diabetes is a Tightly Regulated, Cell-Autonomous Event Lennon et al Immunity 31, 643-653, 2009


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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

InsulinomaType A

Only islets + type B

Rag-/- islets

Type B

CD11c islets

Insulin granules


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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


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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 l.jpg

T Cell Receptor Gene Segments

Antigen Presenting Cell

HLA Molecule

Peptide

V

J

V

D

J

Chr. 14

Chr. 6


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T

cell

Vb

Va

T cell

receptor

class I

MHC

b2m

b cell

Antigen Recognition by CD8+ T Cells

T. DiLorenzo


V d j rearrangement l.jpg

V(D)J Rearrangement

1011-1016 different possible combinations.

Kuby Immunology


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Peptide Presentation Model

AA Peptide Side Chains

Peptide

I-Ag7


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GROOVE

“TEETH”

TEETH

T Cell Receptor Analogy


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Child with IPEX syndrome

Awaiting Bone Marrow Transplant

9 Months of Age


Ipex immune dysfunction polyendocrinopathy enteropathy x linked l.jpg

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


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


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t

t

t

A MODEL

b-cells

APC

ISLET

Ag

T cells

Pancreatic

Lymph

Node

Mathis/Benoist


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MAN

NOD MOUSE

TRAV5D-04+TRAJ53 Restriction?

?

Insulin B:9-23: SHLVEALYLVCGERG?

IAg7

DR3/DR4

Homann 2006,JCI

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


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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


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Natural T Regulatory Cells

Antigen Specific T Reg

T Cell Receptor


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Amino Acid Sequence of Mouse 1 and 2 and Human Insulin

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

B:9-23


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

25

25

21

23

10

14

2

4

1

1

Ins1-, ins2-: n=

Ins1+, ins2-: n=

Life table update 5/19/05

Nakayama Nature 435:220,2005


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Insulin specific T cells

Insultis

Epitope and antigen spreading,

expansion

Diabetes

Insulin is the primary antigen and precedes IGRP in

hierarchy of autoantigens

Krishnamurthy et al JCI:116:3258, 2006


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Is there a primary antigen or immune response to

multiple antigens required for autoimmunity?

T cells specific for

multiple antigens

T cells specific for

one antigen

Insulitis

Insulitis

OR

Epitope and antigen

spreading, expansion

Expansion of T cells

Diabetes

Diabetes

Krishnamurthy et al JCI:116:3258, 2006


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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

Wiley Nat Immunol

P1

P4

P6

P9

Preferred AA in Bound Peptides

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

12% 20% 30,11% 45%

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

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

% amino acid at position


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


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Hafler

DRB1*0401 A1-15

Peakman

DRB1*0401 C19-A3

Peakman

A2 CD8 PPI:15-24

Gottlieb

B:9-23 DQ8

Durinovic-Bello

DRB1*0401 CD4 73-90


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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


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


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Ins2 deficiency augments spontaneous HLA-A*0201-restricted T cell responses to Insulin Jarchum and DiLorenzo J Immunol 2010 vol 184

Humanized HLA-A2 Mice lacking Insulin 2!

Ins B:10-18

CD8 Elispot


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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.


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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

PREPROINSULIN

PROINSULIN

New Responses


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“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


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% NRP-V7 tetramer+ CD8+ cells

% NRP-V7 tetramer+ CD8+ cells

Age (weeks)

Age (weeks)

Tan et al., JCI 1/2003

Female NOD Mice Peripheral Blood

Kd

NRP-V7 Peptide (KYNKANVFL)

Tetramer Analysis

Avidin

Kd

Kd

Kd

Diabetes

No Diabetes


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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


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CD8+ CTL

CTL

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

APC

T. DiLorenzo


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Antigens for CD8+ T Cells in Type 1 Diabetes Patients

DiLorenzo

GAD, glutamic acid decarboxylase; IAPP, islet amyloid polypeptide


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

DMK, dystrophia myotonica kinase

GAD, glutamic acid decarboxylase

IGRP, islet-specific glucose-6-phosphatase catalytic subunit-related protein

DiLorenzo


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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


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T cell epitopes on the diabetes autoantigen Phogrin (IA-2beta) are conserved

among different species

C terminus

TM

PTP

N terminus

Peptide 2: Amino acids 643-658

Peptide 7: Amino acids 762-777

GADPSADATEAYQEL (rat)

GADPSADATEAYQEL (mouse)

GGDPGADATAAYQEL (human)

KNRSLAVLTYDHSRI (rat)

KNRSLAVLTYDHSRI (mouse)

KNRSLAVLTYDHSRV (human)


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