The Generation of Retrogenic Mice

1
The Generation of Retrogenic Mice
Insertion TCR alpha only or alpha_P2A_beta
SCID
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
2
Anti-GAD TCR or B Lymphocytes
TCR Retrogenic Induction High Levels GAD65
Autoantibodies
3
T Cell Islet Accumulation in Type 1 Diabetes is a
Tightly Regulated, Cell-Autonomous Event Lennon
et al Immunity 31, 643-653, 2009
4
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
Rag-/- islets
Only islets type B
Type B
CD11c islets
Insulin granules
5
Conserved T Cell Receptor Alpha Chain Induces
Insulin Autoantibodies Kobayashi et al PNAS
10510090-94 2008
Anti-B9-23 TCR alpha Transgene
No Transgene
6
Nat Immunol. 2010 Mar11(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.
7
T Cell Receptor Gene Segments
Antigen Presenting Cell
HLA Molecule
Peptide
V? J?
V? D? J?
Chr. 14
Chr. 6
8
Antigen Recognition by CD8 T Cells
T. DiLorenzo
9
V(D)J Rearrangement
1011-1016 different possible combinations.
Kuby Immunology
10
Peptide Presentation Model
AA Peptide Side Chains
Peptide
I-Ag7
11
GROOVE
TEETH
TEETH
T Cell Receptor Analogy
12
Child with IPEX syndrome Awaiting Bone Marrow
Transplant
9 Months of Age
13
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

14
Typical Insulitis of man differs from
peri-insulitis NOD mouse Bottazzo
15
(No Transcript)
16
(No Transcript)
17
A MODEL
b-cells
APC
ISLET
Ag
T cells
Pancreatic Lymph Node
Mathis/Benoist
18
MAN
NOD MOUSE
TRAV5D-04TRAJ53 Restriction?
?
Insulin B9-23 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
19
ENVIRONMENTAL FACTORS
Incidence type 1 DM Increasing 3/year gt
30 years!
Protective Factor Decreasing -Hygiene Hypothesis
Bach NEJM 347911, 2002 Triggering
Factors -Congenital Rubella-Rubenstein Diabetes
311088, 1982 -Kilham Rat Virus (BB-DR
rat)-Zipris J. Immunol 174131, 2005 -Poly-IC
Induction Interferon Alpha-Devendra Diabetes 54
2005 -Dietary Factors-Scott Ann Rev Nutr 26175,
2006
20
Natural T Regulatory Cells
Antigen Specific T Reg
T Cell Receptor
21
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
B9-23
22
Lack of progression to diabetes of NOD mice
lacking both insulin native genes.
25 25
10 14
21 23
1 1
2 4
Ins1-, ins2- n Ins1, ins2- n
Life table update 5/19/05
Nakayama Nature 435220,2005
23
Insulin is the primary antigen and precedes IGRP
in hierarchy of autoantigens
Krishnamurthy et al JCI1163258, 2006
24
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 JCI1163258, 2006
25
Natural peptides selected by diabetogenic DQ8 and
murine I-Ag7 molecules show common sequence
homology Suri et al JCI 1152268, 2005Structure
of Human insulin peptide DQ8, Lee et al Nature
Immunology 6501, 2001
Crystal DQ8B9-23 S H L V E A L Y L V
C G E R G
Wiley Nat Immunol
P1
P4
P9
P6
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
26
NOD Mouse anti-islet T Cell Clones/transgenics/ret
rogenics
27
Hafler DRB10401 A1-15
Peakman DRB10401 C19-A3
Peakman A2 CD8 PPI15-24
Gottlieb B9-23 DQ8
Durinovic-Bello DRB10401 CD4 73-90
28
Expanded T cells from pancreatic lymph nodes of
type 1 diabetic subjects recognize an insulin
epitopeKent et al, Nature 435224, 2005

• Pancreatic LN Single cell cloning - PHA
• 2/3 Patients Clonal expansion duration diabetes
  29 and 15 years
• Vbeta29-103J2-3(50)/Valpha8-302
  J4401(25)Vbeta5-101 J2-3(52)/Valpha3901
  J3301(26)
• DRB10401 RestrictedInsulin A1-15
• Caveat High concentration to stimulate 100uM

BDC
29
The insulin A-chain epitope recognized by human T
cells is posttranslationally modifiedMannering
et al, JEM 2021191-1197, 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

30
Ins2 deficiency augments spontaneous
HLA-A0201-restricted T cell responses to Insulin
Jarchum and DiLorenzo J Immunol 2010 vol 184
Humanized HLA-A2 Mice lacking Insulin 2!
Ins B10-18
CD8 Elispot
31
Thymus-specific deletion of insulin induces
autoimmune diabetes.Fan et al EMBO Journal
282812-2824 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 B9-23 though mice
  do not have I-Ag7.
• MYSTERIES Need for Ins1-/- Lack protection
  H-2b Presentation B9-23 by H-2b.

32
The frequency and immunodominance of
Islet-specific CD8 T-cell responses change after
Type 1 Diabetes Diagnosis and Treatment
Martinuzzi et al Diabetes 571312-1320, 2008
7-16 months post onset most ELSIPOT (HLA-A2)
responses decrease
PREPROINSULIN
PROINSULIN
New Responses
33
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 ProducedNRPKYNKANWF
  L NRP-V7 KYNKANVFL
• Higher Avidity Peptide/Tetramer V7
• 30 Intra-islet post 9 weeksgt.5 in Blood
  Predicts Diabetes NOD Mice

34
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
35
CTLs are targeted to kill ß cells in patientswith
type 1 diabetes through recognition ofa
glucose-regulated preproinsulin epitope Ania
Skowera,. and Mark Peakman JCI 2008118
3268-3271.
Interferon gamma HLA-A2 ELISPOT peripheral
blood Patients (SIgt3) DM vs Controls
36
Screening of Peptide Fractions by 51Cr Release
Cytotoxicity Assay Method Used to Discover IGRP
APC
T. DiLorenzo
37
Antigens for CD8 T Cells in Type 1 Diabetes
Patients
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
DiLorenzo
GAD, glutamic acid decarboxylase IAPP, islet
amyloid polypeptide
38
Antigens for Islet-infiltrating CD8 T Cells in
NOD Mice
Antigen Position MHC T cells How identified
DMK 138-146 H-2Db AI4 islet Positional scanning libraries
IGRP 206-214 H-2Kd 8.3 islet MHC 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 kinase GAD, glutamic
acid decarboxylase IGRP, islet-specific
glucose-6-phosphatase catalytic subunit-related
protein
DiLorenzo
39
Toma et al. Recognition of a subregion of human
proinsulin by class I-restricted T cells in type
1 diabetic patients PNAS 10210581, 2005

• Selected 8-11mer peptides of proinsulin
• PBMCs of 29/32 (90) recentlong term diabetics
  responded IFNgamma in ELISPOT assay versus
  minimal response of normals
• Significant Peptides often overlapped insulin B
  chain peptide B9-23(Proins 33-47) Proins
  34-42(B10-18) Proins 41-50(B17-26)Proins
  42-51Prins44-51 but also B chain-C-peptide
  (49-57)
• Multiple class I alleles, including A1 and B8

40
(No Transcript)