Type 1 diabetes

Anti-insulin: health

• No cure; therapy is insulin for life;

physiologic glycaemic control never
achieved

Anti-insulin: disease

•Excess morbidity and mortality

•Incidence increasing by ~5% every 5

years; costs ~£1 billion of UK NHS budget

Peakman
 Type 1 diabetes is T cell mediated
•Infiltrating CD4+, CD8+ T cells
•Anti-T cell therapies are effective
•Islet cell autoantibodies  disease

CD4
T-cell
CD8
T-cell
CD4 TCR
Treg
Epitope

HLA II
HLA I

APC
Islet
Peakman autoantigen
 The Immune System

Innate Acquired (Adaptive)

• Rapid Microbial Defense • Long-lived Microbial
Defense
• Adaptive Immune System
Activation • Neoplasm surveillance
• Autoimmunity,
Transplantation
Rejection & Atopy

BDC
 The Innate Immune System
• Antimicrobial Peptides (e.g., Defensins, Cathelicidins)
• Phagocytes (Macrophages, Neutrophils, Monocytes,
Dendritic Cells)
• Pattern Recognition Receptors
• Alternative Complement System
• NK Cells
• B1B Cells*

* Aspects of both the innate and adaptive immune system

BDC
Selected Pattern Recognition
Receptors: Toll-like Receptors
TLR: Selected Role in Immunity: Localization:
Ligands:
TLR1 PGN, Zymosan, Antifungal & Dendritic Cells,
TLR2 Lipoproteins Antibacterial Macrophages,
TLR6 T Cells,
TLR4 LPS Antibacterial B Cells,
Epithelium
TLR5 Flagellin
TLR11 ?
TLR9 CpG Antibacterial &
Antiviral
TLR3 dsRNA Antiviral
TLR7 ssRNA
TLR8 ssRNA
TLR10 ? ?
Selected Pattern Recognition
Receptors: Other Families
Receptor Selected Ligands Role in Immunity Localization
NOD Pro teins
NOD1 PGN (Gm- ) Antibacterial Cytoplasmi c
NOD2 PGN (Gm + & -) Antibacterial Cytoplasmi c
CD14 LBP:LPS, PGN Antibacterial (wit h TLR4) Serum &
Phagocy te Cell
Surface
C-type Lectins
Macrophage Glycop roteins or Antibacterial, Antiviral, Macrophage,
Mannose Re ceptor Glyco li pids Antif unga l DCs
(MMR), DC-SIGN,
DEC-205
Surfactant A, D LPS, Opsoni zation o f Bacteria, Soluble in the
(Coll ectin Famil y) Lipop roteins, Vir us & Fung i; Cytokine Lungs
Oli gos accha rides Stimula tion; Apoptotic Cell
Clearance
MBP/MBL Mannose group s Complement Activation Serum
on bacterial (Antibacterial & Antivir al)
carbohyd rates
Scavenger
Receptors
SR-A, LPS, LTA, PGN Antibacterial; Apoptotic Macrophages ,
CD36 Cell Clearanc e Endoth elium
Systematic Mouse ligand Human ligand Receptor References
name (alias) (alias)
CCL1 TCA-3/I-309 I-309 CCR8
CCL2 JE/MCP-1 MCP-1 CCR2 Bertuzzi et al., 2004; Bradley et al., 1999; Cardozo et al., 2001; Cardozo et al., 2003;
Chen et al., 2001; Frigerio et al., 2002; Giarratana et al., 2004; Grewal et al., 1997;
Kutlu et al., 2003; Nomura et al., 2000; Schroppel et al., 2005; Yang et al., 2004
CCL3 MIP-1 MIP-1  CCR1 & 5 Bradley et al., 1999; Cameron et al., 2000; Giarratana et al., 2004; Lohmann et al., 2002
CCL4 MIP-1 MIP-1  CCR5 Bradley et al., 1999; Cameron et al., 2000; Lohmann et al., 2002)
CCL5 RANTES RANTES CCR1, 3 & 5 Bradley et al., 1999; Carvalho-Pinto et al., 2004; Frigerio et al., 2002; Weber et al., 2006
CCL6 C10 unknown CCR1
CCL7 MARC/MCP-3 MCP-3 CCR1, 2 & 3 Bradley et al., 1999; Matos et al., 2004
CCL8 MCP-2 MCP-2 CCR3 & 5
CCL9/10 MIP-1 unknown CCR1
CCL11 Eotaxin-1 Eotaxin-1 CCR3
CCL12 MCP-5 unknown CCR2 Bradley et al., 1999
CCL13 unknown MCP-4 CCR2 & 3
CCL14 unknown HCC-1 CCR1 & 5
CCL15 unknown HCC-2/MIP-1d CCR1 & 3
CCL16 unknown HCC-4 CCR1 & 2
CCL17 TARC TARC CCR4 Giarratana et al., 2004; Kim et al., 2002
CCL18 unknown PARC unknown
CCL19 ELC MIP-3b/ELC CCR7 Bouma et al., 2005a; Bouma et al., 2005b
CCL20 MIP-3/LARC MIP-3a/LARC CCR6 Cardozo et al., 2003
CCL21 SLC/6Ckine SLC/6Ckine CCR7 Bouma et al., 2005b; Giarratana et al., 2004
CCL22 MDC MDC CCR4 Giarratana et al., 2004; Kim et al., 2002
CCL23 unknown MIPIF-1/MIP-3 CCR1
CCL24 Eotaxin-2 Eotaxin-2 CCR3
CCL25 TECK TECK CCR9
CCL26 unknown Eotaxin-3 CCR3
CCL27 CTACK CTACK CCR10
CCL28 MEC MEC CCR3 & 10
CXCL1 KC GRO CXCR2 Cardozo et al., 2001; Matos et al., 2004
CXCL2 MIP-2 GRO CXCR2
CXCL3 unknown GRO  CXCR2
CXCL4 PF4 PF4 CXCR3B
CXCL5 LIX ENA-78 CXCR2 Matos et al., 2004
CXCL6 unknown GCP-2 CXCR1 & 2
CXCL7 TCK-1 NAP-2 CXCR2
CXC8 unknown IL-8 CXCR1 & 2
CXCL9 MIG MIG CXCR3 Christen et al., 2003; Frigerio et al., 2002; Matos et al., 2004
CXCL10 IP-10 IP-10 CXCR3 Baker et al., 2003a; Baker et al., 2003b; Bradley et al., 1999; Cardozo et al., 2001;
Cardozo et al., 2003; Christen et al., 2004; Christen et al., 2003; Ejrnaes et al., 2005;
Frigerio et al., 2002; Giarratana et al., 2004; Morimoto et al., 2004; Nicoletti et al., 2002;
Rhode et al., 2005; Shimada et al., 2001
CXCL11 I-TAC I-TAC CXCR3 Cardozo et al., 2003
CXCL12 SDF-1/PBSF SDF-1/ CXCR4 Dubois-Laforgue et al., 2001; Kawasaki et al., 2004; Kayali et al., 2003
CXCL13 BLC BLC/BCA-1 CXCR5
CXCL14 BRAK BRAK unknown
CXCL15 Lungkine unknown unknown
CXCL16 SR-PSOX SR-PSOX CXCR6
XCL1 lymphotactin SCM-1/ATAC XCR1 Bradley et al., 1999; Weber et al., 2006
XCL2 unknown SCM-1b XCR1
CX3CL1 fractalkine fractalkine CX3CR1 Cardozo et al., 2001
Table 1. T1D, chemokines and their receptors (modified after Lut et al., 2006).
Chemokines with a putative role in T1D pathogenesis are identified by gray background shading.
 The Adaptive Immune System

• Cell-mediated Immunity (Cytotoxicity)

• T cells
• CD4+ (Th1 & Th2)
• CD8+ (Tc1 & Tc2)

• Humoral Immunity (Antibody production)

• B Cells

BDC
 Th1 and Th2 CD4+ T Cells
Th1 Th2
• IL-12 induces differentiation • IL-4 induces differentiation
• Cytokine Production: • Cytokine Production:
Interferon- Interleukin-4
Interleukin-2 Interleukin-5
• Intracellular Pathogens Interleukin-13
• Macrophage Activation • Extracellular Pathogens
• Delayed Type • B Cell activation & IgE
Hypersensitivity • Eosinophil responses
• Immediate Type
Hypersensitivity
BDC
 T cell signaling molecules and autoimmunity

Human T1D loci (Ref1)

MHC : λs ≈ 3
Insulin : odds 1.9
CTLA4 : odds 1.2
PTPN22 odds 1.7

Cblb : Komeda rat

(Yokoi N, Nat Genet, 2002)

Pten: Cre-loxP knock-out

(Suzuki A, Immunity, 2001)

Zap70: Sakaguchi mice

(Sakaguchi N, Nature, 2003)
(Mustelin T, et al. Mol Immunol. 2004)

Concannon P et al. Diabetes. 2005 Oct;54(10):2995-3001.

H. Ueda
 B and T Lymphocyte Antigen Receptors

VH VH V V
VL CH1 CH1 VL  e e 
z z C C
CL CL

CH2 CH2

Ig/Ig CH3 CH3 Ig/Ig

fyn lck
Zap 70
Blk, Fyn or Lyn

2 light chains ( or ) 2 Chains / (95%) or / (5%)

2 heavy chains (5 isotypes: IgG, M, A, D, E)
2 Binding sites (Divalent)
Secreted into circulation
Binds Soluble Antigen
1 Binding site (Monovalent)
Membrane Bound, Not Secreted
Binds Antigen Complexed with MHC
BDC
 J. Noble

HLA
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

 DQB1*0402

 -chain

Leu56
-chain
Asp57

BDC BDC
 Hahn, Wucherpfenning et al. Nature Immunology 6:490-496, 2005

Topology of Self-peptide/MHC Binding by Ob.1A12 TCR

Autoimmune (MBP Peptide+DR2) Anti-viral (HA Peptide+DR1)
Ob.1A12 HA1.7

Red: TCR
Yellow: TCR
Hahn, Wucherpfenning et al. Nature Immunology 6:490-496, 2005

Ob.1A12 HA1.7

Anti-viral
(HA
Peptide+D
R1)

Autoimmune Red: TCR

(MBP Yellow: TCR
Peptide+DR2)
 The Human Leukocyte Antigen Complex (6p21.31)

Class III
Class II (1.1 Mb) (0.7Mb) Class I (2.2Mb)

DP DQ DR B C A

Centromere Telomere

Frequent Class I-like genes

Recombination and pseudogenes
Complement
and Cytokines

Recombination Recombination
is Rare is Rare

BDC
HLA Class I and II Molecules Have a Distinct Structure and Function

•Binds 8-10mers •Binds 13-25mers

•Expressed on most •Expressed on APCs,
Nucleated cells Macs, B cells, activated
•Presents Cytosolic T cells
Proteins to CD8+ T cells •Presents Vesicular
Proteins to CD4+ T cells

1 2 1 1

2 3 2 2

Class I Class II BDC

 Cis and Trans- Class II Dimerization

DQA1 DQB1

Maternal 0501 0201 cis

DQA1*0501/DQB1*0201

0301 0302 cis

Paternal

trans
DQA1*0301/DQB1*0302

DQA1*0301/DQB1*0201 DQA1*0501/DQB1*0302
BDC
 HLA-Peptide: TCR
2 Helix NH3+
1 Helix

TCR
alpha

TCR
beta

BDC
COO-
“Tetramer” for T Cell Analysis

DQ PEPTIDE

DQ DQ

Avidin

DQ
BDC
HLA Class II tetramer ( DR0401-hGAD555-567)

streptavidin

Leucine
zippers spacers

MHC peptide

W.W.Kwok & G.T.Nepom,

Benaroya Research Institute at Virginia Mason
 T cell Recognition of Antigen on an APC
Antigen

Endocytosis

CD4+
T cell
APC T Cell
Receptor

Peptide

MHC II
 T cell Activation by an Activated APC
IL-1
IL-6
IL-12
IL-12 Receptor

CD28
“Signal 3” B7 CD4+
T cell
LPS T Cell
“Signal 2” Receptor
TLR4

“Signal 1” Peptide Signal 1: Specificity

MHC II Signal 2: Activation

Signal 3: Differentiation
Antigen Presenting Cell (APC)
The 2-Signal Model of Lymphocyte Activation
Absence of Signal 2

APC
T Cell
TCR MHC

Tolerance
Clonal Anergy or Deletion

Signal 1 + Signal 2
CD28 B7

APC
T Cell TCR MHC

cytokines

Activation BDC
 APC and T cell Interactions

B7 (CD80/86) CTLA-4 Activation

B7 (CD80/86) CD28 Activation

MHC II TCR Recognition

CD4+
APC T Cell
CD58 (LFA-3) CD2 Adhesion

Activation CD40 CD40L

 Molecular Interactions of Helper T Cells and APC

CD4+ T Cell
CTLA-4 CD28 p56 lck CD3 CD2
 e z zh h

CD40L C C TCR
CD45 LFA-1
V V
VLA-1
peptide
B7 B7 LFA-3
CD4 MHC II ICAM-1
Collagen

CD80/CD86 CD40 APC/ B cell

L. Chess 2002
 T cell activation is regulated by signals derived from the TCR
/CD3/CD4 complex and the CD40L and CD28/CTLA-4 co-stimulatory
molecules
CD4+ T Cell

Co-stimulatory signals Antigen specific TCR signals

(- ) / [+]
lck CD3
  e z z hh

C C
CD28/ V V
, TCR
CTLA-
CD40L
4 Peptide antigen

CD40 CD4 MHC class II

signal
CD80 (B7.1)/ [+]
CD86 (B7.2)
Antigen Presenting Cell (APC)
L. Chess 2002
 TCR

CD4 CD45

TCR signaling CD28

Zap70 Lck Lck

Fyn
Tec Shc PTK
PLC1
Grb2
PIP2
SOS
(PMA)
(ION) IP3 + DAG Ras

Ca++ PKC

calcineurin MAPK

NFB

NFAT activation

Fathman
 T cell activation induces expression
of functional T cell surface molecules

Induction and
Activated activation of B cells Late Activated
MHC/peptid APCs
CD4+ T cell CD4+ T cell
e CD40L
TCR TCR

TCR
CD25
Resting
APC CD4+ T cell (+)
Qa-1/V
(-) VLA-1
Collagen
TCR
(anti-Qa-1/V) Migration of
sites of
inflammation
Activated Regulatory
CD8+ T cell CD8+ T cell
Down-regulation of
Activated CD4+ T Cells
L. Chess 2002
 Immunological tolerance

• Definition:
– specific immune unresponsiveness to an antigen that
is induced by exposure of lymphocytes to that antigen
(tolerogen vs immunogen)
• Significance:
– All individuals should be tolerant of their own
antigens (self-tolerance); breakdown -->autoimmunity
– The induction of tolerance could be exploited to treat
autoimmune diseases
– Mechanisms of tolerance must first be understood

Fathman
Mechanisms of unresponsiveness to self antigens

• Central tolerance
– Immature self-reactive T lymphocytes that recognize self antigens
in the thymus undergo negative selection (deletion)
• Peripheral tolerance
– Mature self-reactive T lymphocytes that escape central tolerance
and recognize self antigens in peripheral tissues can be inactivated
(anergy), killed (deletion) or regulated (suppressed)
• “Clonal ignorance”
– Mature self-reactive lymphocytes do not respond to self antigens
in non-inflamed settings

Fathman
The Control of Activated CD4+ T Cells by Regulatory T cells

NKT cells/
CD4+CD25+ cells
CD4+CD25- cells

Apoptosis

(- ) TH1 CD4+ cells

peptide/APC IL-12/
IFN-
(- )
IL-10 IFN-
IL-4
(- )
Activated CD4 T cells
Resting CD4 T cells
(- ) TH2 CD4+ cells

Regulatory immunity
CD4/CD8 interactions
CD8 or CD4 CD8 or CD4 suppressor
suppressor effector precursor
L. Chess 2002
 Regulatory T Cell Subsets
Regulatory T cell Murine Markers Proposed Mechanisms of Inhib ition
Suppressor Cell CD8+ Recogn ition of Qa-1:peptide on ac tivated CD4+ T
cells  indu ction of cytotoxicity
Natural Treg CD4+, CD25+ Cell-contact dependen t but no t antigen- specifi c;
CTLA-4+, Ligation of B7 on effector cells; IL-2
GITR+, Foxp3+ sequestration; CTLA-4 interaction w ith IDO 
(intracellula r) tolerogen ic DCs; IL-10 & TGF-beta produc tion
Adaptive Treg CD4+, CD25 -, Cell-contact dependen t but no t antigen- specifi c
Foxp3- inhibition
Tr1 CD4+, CD45Rb lo Cell-contact independen t; IL-10 & IL-4 secretion
Th3 CD4+, CD45Rb lo Cell-contact independen t; TGF-beta secretion
Invariant NKT cell Invar ia nt TCR CD1d:glyco lipid complex recogn iti on; IL-10
(V14-J281), secretion
CD4+, CD8-,
NK1.1+
 Regulatory T Cells in Autoimmunity
CD4+CD45RBlo TGF, IL10 Colitis by CD45RBhi
Th3 TGF,+/- EAE, glomeruloneph, MG,
IL10,IL4 clone DM
TR1 IL10, normal IBD by CD45RBhi ,
TGF,no IL4 Respond IL-15
CD4+CD25+ CTLA4 Thymectomy autoimmune
(? =TR1) Constituitive CTLA4
Other CD4+ IL2, IFN MBP EAE model
CD8+ Oral tolerance intestine
CD4-CD8-TCR Fas Skin Allograft
Gamma/Delta Gut, Nasal Insulin
NKT IL4,IL10,IFN, CD1 Activated
TGF

BDC Roncarolo et al. Curr Opinion Immunol 2000

 XPID: X-linked Polyendocrinopathy,
Immune dysfunction and Diarrhea
Foxp3 Gene Essential CD4-CD25 T Reg
• XLAAD: Autoimmunity Allergic Dysregulation
• Defect in scurfin protein (gene = Foxp3/JM2)
or “scurfy mouse”
• Immunopathogenesis relates to a deficiency of
T regulatory cells
-Scurfy x Nude: No Autoimmunity
-CD4+ T cells into Nude: Disease
-Bone Marrow into irradiated: No Disease
-Mixed Chimera: No Disease
BDC
Requirements for the development
of an autoimmune disease

Nature Immunology (9): 759-761 (2001) Fathman

 Immunopathophysiology of Diabetes

Dendritic cell/ Activated

CD2
APC CD4+ TH1 CD4+ T
Cell Cell
(TH0 )
DR3, IL-12 CD40L
,, TCR IFN-
DR4,,DQ8/insulin
peptide CD40
IL-4
Macrophage/dendritic cell

CD4+ Cell Fc R
FasL
(TH2 ) perforin
CD40L
CD8+ CTL IL-1, TNF, LT, NO, PGE-2
IL-4
CD40L

?anti-insulin,
B Cell GAD ab anti-
Mog
 cell death
?Antibody mediated injury  islet cells
L. Chess 2002
 Induction of CD4+ TH1 mediated
autoimmunity:
A paradigm for the pathogenesis of
rheumatoid arthritis, multiple sclerosis and
type I diabetes (1) expansion of CD4+,
autoreactive TH1 cells
specific for autoantigens
MHC/self-peptide CD4
(2) migration and
CD4 MHC/V infiltration of these self
reactive CD4+ TH1 cells
TCR Vx
into tissues and induction
TCR Vx
of inflammation and
CD4+ Vx T cell APC autoimmunity
Activated autoreactive CD4+
TCR Vx TH1 cell (3) induction of regulatory
cells which control the
growth and activation of
the pathogenic
autoreactive repertoire of
CD4+ T cells
L. Chess 2002
1984:Subset Participants Immunology in Diabetes Rome