• One Critical Protein• One Critical Peptide• One Critical Register• ? Germline Encoded Critical TCR SegmentTHUS: Targeting Trimolecular Complex Possible for Prevention

FOR A GIVEN SPECIES(?)/MHC/ORGAN SPECIFIC AUTOIMMUNE DISEASE

HYPOTHESIS

THE NOD EXAMPLE

TCR rearrangement

Vα

Jα

Vβ

JβDβ

T Cell Receptor Gene Segments

Chr. 14

Chr. 6

V

J

V

D J

HLA Molecule Peptide

Antigen Presenting Cell

Antigen

APC

Endocytosis

T Cell Receptor

Peptide

MHC II

CD4+ T cell

T cell Recognition of Antigen on an APC

Trimolecular Complex

The Trimolecular Complex NOD MOUSE

MHC PEPTIDE

INS B:9-23

TCR

Liu et al Diabetes 2012

Interfering with formation of the trimolecular recognition complex

Zhang et al, J. Diabetes 2011

Deletion of T cells expressing specific TCR alpha or

TCR beta genes

TCR

B:9-23

MHC

TCRB:9-23

MHC

Antibody binds to MHC/peptidecomplex and blocks it

from interacting with TCR

B:9-23

MHC

Small molecule prevents frombinding/dislodges the B:9-23

peptide from MHC

TCR

Michels et al, J. Immunol 2011

*

“Stages” in Development of Type 1A Diabetes

Age (years)

Genetic Predisposition

Bet

a ce

ll m

ass

(?Precipitating Event)

Overtimmunologicabnormalities

Normal insulinrelease

Progressiveloss insulinrelease

Glucosenormal

Overtdiabetes

C-peptidepresent Minimal

C-peptide

Eisenbarth, 2012

nPOD 6052-02 Tail: 12 yo 1 year diabetes-Lobular Pseudoatrophic Islets

Insulin and Ki67 Staining

Glucagon/anti-CD3 Staining

Type 1 diabetes

• No cure; therapy is insulin for life; physiologic glycaemic control never achieved

• Incidence increasing by ~5% every 5 years; costs ~£1 billion of UK NHS budget

• Excess morbidity and mortality

Anti-insulin: health

Anti-insulin: disease

Peakman

CD8 T-cell

HLA I

• Infiltrating CD4+, CD8+ T cells• Anti-T cell therapies are effective• Islet cell autoantibodies disease

CD4 T-cell

HLA II

TCR

Epitope

CD4Treg

Type 1 diabetes is T cell mediated

Isletautoantigen

APC

Peakman

The Immune System

Innate• Rapid Microbial

Defense• Adaptive

Immune System Activation

Acquired (Adaptive)• Long-lived

Microbial Defense

• 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 ReceptorsTLR: Selected

Ligands:Role in Immunity: Localization:

TLR1 PGN, Zymosan, Lipoproteins

Antifungal & Antibacterial

Dendritic Cells,Macrophages,T Cells,B Cells,Epithelium

TLR2TLR6TLR4 LPS AntibacterialTLR5 FlagellinTLR11 ?TLR9 CpG Antibacterial &

AntiviralTLR3 dsRNA AntiviralTLR7 ssRNATLR8 ssRNATLR10 ? ?

Selected Pattern Recognition Receptors: Other Families

Receptor Selected Ligands Role in Immunity LocalizationNOD ProteinsNOD1 PGN (Gm-) Antibacterial CytoplasmicNOD2 PGN (Gm + & -) Antibacterial CytoplasmicCD14 LBP:LPS, PGN Antibacterial (with TLR4) Serum &

Phagocyte CellSurface

C-type LectinsMacrophageMannose Receptor(MMR), DC-SIGN,DEC-205

Glycoproteins orGlycolipids

Antibacterial, Antiviral,Antifungal

Macrophage,DCs

Surfactant A, D(Collectin Family)

LPS,Lipoproteins,Oligosaccharides

Opsonization of Bacteria,Virus & Fungi; CytokineStimulation; Apoptotic CellClearance

Soluble in theLungs

MBP/MBL Mannose groupson bacterialcarbohydrates

Complement Activation(Antibacterial & Antiviral)

Serum

ScavengerReceptorsSR-A,CD36

LPS, LTA, PGN Antibacterial; ApoptoticCell Clearance

Macrophages,Endothelium

Systematic Mouse ligand Human ligand Receptor Referencesname (alias) (alias)

CCL1 TCA-3/I-309 I-309 CCR8CCL2 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., 2002CCL4 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., 2006CCL6 C10 unknown CCR1CCL7 MARC/MCP-3 MCP-3 CCR1, 2 & 3 Bradley et al., 1999; Matos et al., 2004CCL8 MCP-2 MCP-2 CCR3 & 5CCL9/10 MIP-1 unknown CCR1CCL11 Eotaxin-1 Eotaxin-1 CCR3CCL12 MCP-5 unknown CCR2 Bradley et al., 1999CCL13 unknown MCP-4 CCR2 & 3CCL14 unknown HCC-1 CCR1 & 5CCL15 unknown HCC-2/MIP-1d CCR1 & 3CCL16 unknown HCC-4 CCR1 & 2CCL17 TARC TARC CCR4 Giarratana et al., 2004; Kim et al., 2002CCL18 unknown PARC unknownCCL19 ELC MIP-3b/ELC CCR7 Bouma et al., 2005a; Bouma et al., 2005bCCL20 MIP-3/LARC MIP-3a/LARC CCR6 Cardozo et al., 2003CCL21 SLC/6Ckine SLC/6Ckine CCR7 Bouma et al., 2005b; Giarratana et al., 2004CCL22 MDC MDC CCR4 Giarratana et al., 2004; Kim et al., 2002CCL23 unknown MIPIF-1/MIP-3 CCR1CCL24 Eotaxin-2 Eotaxin-2 CCR3CCL25 TECK TECK CCR9CCL26 unknown Eotaxin-3 CCR3CCL27 CTACK CTACK CCR10CCL28 MEC MEC CCR3 & 10CXCL1 KC GRO CXCR2 Cardozo et al., 2001; Matos et al., 2004CXCL2 MIP-2 GRO CXCR2CXCL3 unknown GRO CXCR2CXCL4 PF4 PF4 CXCR3BCXCL5 LIX ENA-78 CXCR2 Matos et al., 2004CXCL6 unknown GCP-2CXCL7 TCK-1 NAP-2 CXCR2CXC8 unknown IL-8CXCL9 MIG MIG CXCR3 Christen et al., 2003; Frigerio et al., 2002; Matos et al., 2004CXCL10 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., 2003CXCL12 SDF-1/PBSF SDF-1/ CXCR4 Dubois-Laforgue et al., 2001; Kawasaki et al., 2004; Kayali et al., 2003CXCL13 BLC BLC/BCA-1 CXCR5CXCL14 BRAK BRAK unknownCXCL15 Lungkine unknown unknownCXCL16 SR-PSOX SR-PSOX CXCR6XCL1 lymphotactin SCM-1/ATAC XCR1 Bradley et al., 1999; Weber et al., 2006XCL2 unknown SCM-1b XCR1CX3CL1 fractalkine fractalkine CX3CR1 Cardozo et al., 2001

CXCR1 & 2

CXCR1 & 2

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• IL-12 induces differentiation• Cytokine Production:

Interferon-Interleukin-2

• Intracellular Pathogens • Macrophage Activation• Delayed Type

Hypersensitivity

Th2• IL-4 induces

differentiation• Cytokine Production:

Interleukin-4Interleukin-5Interleukin-13

• Extracellular Pathogens• B Cell activation & IgE• Eosinophil responses• Immediate Type

Hypersensitivity BDC

T cell signaling molecules and autoimmunity

(Mustelin T, et al. Mol Immunol. 2004)

Human T1D loci (Ref1)

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

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

2 light chains ( or )2 heavy chains (5 isotypes: IgG, M, A, D, E)2 Binding sites (Divalent)Secreted into circulationBinds Soluble Antigen

2 Chains / (95%) or / (5%)

1 Binding site (Monovalent)Membrane Bound, Not SecretedBinds Antigen Complexed with MHC

Zap 70

fyn lck

ez

V V

C C

CH1

CH2

CH3

CH2

CH3

CH1

CL CL

VL VL

VH VH

e

z

Ig/Ig Ig/Ig

Blk, Fyn or Lyn

B and T Lymphocyte Antigen Receptors

BDC

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

HLAJ. Noble

DQB1*0402

Asp57

Leu56

-chain

-chain

BDC BDC

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

HA1.7Ob.1A12

Red: TCRYellow: TCR

Autoimmune (MBP Peptide+DR2) Anti-viral (HA Peptide+DR1)

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

Ob.1A12 HA1.7

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

Autoimmune (MBP Peptide+DR2)

Anti-viral (HA Peptide+DR1)

Red: TCRYellow: TCR

The Human Leukocyte Antigen Complex (6p21.31)

DP DQ DR B C A

Class II (1.1 Mb)Class III

Class I (2.2Mb)

Complement and Cytokines

Class I-like genesand pseudogenes

Frequent Recombination

Recombinationis Rare

TelomereCentromere

Recombinationis Rare

(0.7Mb)

BDC

32

1 2 1

2

1

2

• Binds 8-10mers• Expressed on most Nucleated cells• Presents Cytosolic Proteins to CD8+ T cells

• Binds 13-25mers• Expressed on APCs, Macs, B cells, activated T cells• Presents Vesicular Proteins to CD4+ T cells

Class I Class II

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

BDC

0501 0201

0301 0302

cis

cis

DQA1 DQB1

trans

DQA1*0301/DQB1*0201 DQA1*0501/DQB1*0302

DQA1*0501/DQB1*0201

DQA1*0301/DQB1*0302

Maternal

Paternal

Cis and Trans- Class II Dimerization

BDC

HLA-Peptide: TCR

CDR1

CDR2CDR3

CDR3CDR2

CDR1

NH3+

COO-

TCR

alpha

TCR

beta

2 Helix 1 Helix

BDC

AvidinDQDQ

DQ

“Tetramer” for T Cell Analysis

DQ PEPTIDE

BDC

MHC peptide

HLA Class II tetramer (DR0401-hGAD555-567)

Leucinezippers spacers

streptavidin

W.W.Kwok & G.T.Nepom, BenaroyaResearch Institute at Virginia Mason

Antigen

APC

Endocytosis

T Cell Receptor

Peptide

MHC II

CD4+ T cell

T cell Recognition of Antigen on an APC

CD4+ T cell

T Cell Receptor

Peptide

LPS

TLR4

Antigen Presenting Cell (APC)

MHC II

“Signal 1”

“Signal 2”

“Signal 3”

IL-1

IL-6

IL-12

CD28B7

IL-12 Receptor

Signal 1: Specificity

Signal 2: Activation

Signal 3: Differentiation

T cell Activation by an Activated APC

Absence of Signal 2

Activation

Clonal Anergy or Deletion

TCR MHC

APC

Tolerance

T Cell

TCR MHC

APC

Signal 1 + Signal 2

B7

T Cell

cytokines

CD28

The 2-Signal Model of Lymphocyte Activation

BDC

CD2

APC MHC II

B7 (CD80/86)

APC and T cell Interactions

CTLA-4

CD40L

CD28

B7 (CD80/86)

TCR

CD40

CD58 (LFA-3)

CD4+ T Cell

Activation

Activation

Recognition

Adhesion

Activation

e

V

CCV

z zp56 lck

peptide

CD3CD28

CD40

LFA-3

LFA-1

ICAM-1MHC IIB7

CD40L

CD4

CD45TCR

CD2

CD4+ T Cell

APC/ B cell

h h

Molecular Interactions of Helper T Cells and APC

B7

CTLA-4

VLA-1

Collagen

CD80/CD86

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

e z zCD3

MHC class II

, TCR

h h

Peptide antigen

CD4+ T Cell

Antigen Presenting Cell (APC)

V V

C C

CD4

Antigen specific TCR signals

CD40

CD40L

signal

CD28/CTLA-4

CD80 (B7.1)/CD86 (B7.2)

lck

Co-stimulatory signals

(- ) / [+]

[+]

L. Chess 2002

PIP2

IP3 + DAG

PLC1

Ca++

NFAT activation

calcineurin

TCR

CD4 CD45CD28

MAPK

Shc

Ras

SOS

Grb2

PKC

NFB

TCR signaling

(ION)(PMA)

PTK

Zap70 LckLck

TecFyn

Fathman

MHC/peptide

TCR

Qa-1/V

Activated CD4+ T cell

RestingCD4+ T cell

Down-regulation of Activated CD4+ T Cells

T cell activation induces expression of functional T cell surface molecules

Regulatory CD8+ T cell

TCR (anti-Qa-1/V)

Activated CD8+ T cell

(+) (-)

TCR

APC

TCR

Late Activated CD4+ T cell

VLA-1Collagen

CD40L

Migration of sites of

inflammation

Induction and activation of B cells

APCs

CD25

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

TH1 CD4+ cells

TH2 CD4+ cells

CD8 or CD4 suppressor precursor

CD8 or CD4 suppressor effector

Activated CD4 T cells

peptide/APC

Regulatory immunityCD4/CD8 interactions

(- )

(- )

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

Apoptosis

Resting CD4 T cells

IL-12/IFN-

IL-4

NKT cells/ CD4+CD25+ cellsCD4+CD25- cells

(- )

(- )

IL-10 IFN-

L. Chess 2002

Regulatory T Cell Subsets

Regulatory T cell Murine Markers Proposed Mechanisms of InhibitionSuppressor Cell CD8+ Recognition of Qa-1:peptide on activated CD4+ T

cells induction of cytotoxicityNatural Treg CD4+, CD25+

CTLA-4+,GITR+, Foxp3+(intracellular)

Cell-contact dependent but not antigen-specific;Ligation of B7 on effector cells; IL-2sequestration; CTLA-4 interaction with IDO tolerogenic DCs; IL-10 & TGF-beta production

Adaptive Treg CD4+, CD25-,Foxp3-

Cell-contact dependent but not antigen-specificinhibition

Tr1 CD4+, CD45Rblo Cell-contact independent; IL-10 & IL-4 secretionTh3 CD4+, CD45Rblo Cell-contact independent; TGF-beta secretionInvariant NKT cell Invariant TCR

(V14-J281),CD4+, CD8-,NK1.1+

CD1d:glycolipid complex recognition; IL-10secretion

Regulatory T Cells in AutoimmunityCD4+CD45RBlo TGF, IL10 Colitis by CD45RBhi

Th3 TGF,+/-IL10,IL4

EAE, glomeruloneph, MG,clone DM

TR1 IL10, normalTGF,no IL4

IBD by CD45RBhi ,Respond IL-15

CD4+CD25+(? =TR1)

CTLA4 Thymectomy autoimmuneConstituitive CTLA4

Other CD4+ IL2, IFN MBP EAE modelCD8+ Oral tolerance intestineCD4-CD8-TCR Fas Skin AllograftGamma/Delta Gut, Nasal InsulinNKT IL4,IL10,IFN,

TGFCD1 Activated

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

Activated TH1 CD4+ T Cell

CD4+ Cell(TH2 )

CD4+ Cell

(TH0 )DR3, DR4,,DQ8/insulin peptide

CD2

Macrophage/dendritic cell

Fc R

IFN- IL-12 CD40L

CD40,, TCR

IL-1, TNF, LT, NO, PGE-2

B Cell ?anti-insulin, GAD ab anti-Mog

IL-4

Immunopathophysiology of Diabetes

?Antibody mediated injury

Dendritic cell/APC

CD40L

IL-4CD40L

CD8+ CTL

FasLperforin

cell death islet cells

L. Chess 2002

MHC/self-peptide CD4

MHC/VCD4

CD4+ Vx T cell APCActivated autoreactive CD4+

TCR Vx TH1 cell

TCR Vx TCR Vx

(1) expansion of CD4+, autoreactive TH1 cells specific for autoantigens(2) migration and infiltration of these self reactive CD4+ TH1 cells into tissues and induction of inflammation and autoimmunity(3) induction of regulatory cells which control the growth and activation of the pathogenic autoreactive repertoire of CD4+ T cells

Induction of CD4+ TH1 mediated autoimmunity:

A paradigm for the pathogenesis of rheumatoid arthritis, multiple sclerosis and

type I diabetes

L. Chess 2002

1984:Subset Participants Immunology in Diabetes Rome