Inflammatory & Immune Pharmacology Control of T-lymphocyte Clonal Expansion The Example of Organ Transplant Tolerance.

Inflammatory & Immune Inflammatory & Immune PharmacologyPharmacology

Control of T-lymphocyte Clonal ExpansionControl of T-lymphocyte Clonal Expansion

The Example of Organ Transplant ToleranceThe Example of Organ Transplant Tolerance

Why Inhibit T-Cell Clonal Expansion?Why Inhibit T-Cell Clonal Expansion?

• Transplant-specific Cytotoxic T-cells are Transplant-specific Cytotoxic T-cells are responsible for most organ transplant responsible for most organ transplant rejection eventsrejection events

• Can start drug just before foreign antigens are Can start drug just before foreign antigens are introduced (IE, just before the transplant)introduced (IE, just before the transplant)

T

Foreign Cell or ProteinForeign Cell or Protein

Antigen uptakeAntigen uptake

Antigen processingAntigen processing

Antigen presentationAntigen presentation

RECOGNITIONRECOGNITION

Ag-presenting cellAg-presenting cell

CLONALCLONALEXPANSIONEXPANSION

T

Foreign Cell or ProteinForeign Cell or Protein

Antigen uptakeAntigen uptake

Antigen processingAntigen processing

Antigen presentationAntigen presentation

RECOGNITIONRECOGNITION

Ag-presenting cellAg-presenting cell

CLONALCLONALEXPANSIONEXPANSION

II

IIII

IIIIII

APC

Receptor BindingReceptor Binding & Signaling& Signaling

Cell - cell signalingCell - cell signaling (IFN-(IFN-, IL-1, IL-1))

AutocrineAutocrinesignalingsignaling

(IL-2)(IL-2)

IV IV ProliferationProliferation

Steps Towards T-cell Clonal ExpansionSteps Towards T-cell Clonal Expansion

I. TCR Binding, I.C.SignalingI. TCR Binding, I.C.SignalingII. Cell-Cell SignalingII. Cell-Cell Signaling

APC

TT

Other interactions Other interactions that contributethat contributeto identificationto identificationand stabilityand stability

Signals for expression of Signals for expression of growth factors,growth factors, differentiation markers, differentiation markers, & other cytokines& other cytokines

IFN-IFN-IL-1IL-1

III. IL-2: Autocrine Growth & Differentiation FactorIII. IL-2: Autocrine Growth & Differentiation Factor

• IL-2 is released by IL-2 is released by the activated T cell the activated T cell•Acts through IL-2RActs through IL-2R

IL-2IL-2

IL-2RIL-2R

IL-2IL-2

MitogenesisMitogenesis

IV. IL-2 Driving Clonal Expansion IV. IL-2 Driving Clonal Expansion

CloneClone of identical T-cells, specific for a unique antigen of identical T-cells, specific for a unique antigen

Destroy Destroy transplanted tissuetransplanted tissue

T

T

TT T

T

T

TT T

TT

TT T

T

Drugs that Prevent IL-2 Driven Drugs that Prevent IL-2 Driven Clonal ExpansionClonal Expansion

• CyclosporinCyclosporin

• TacrolimusTacrolimus

• SirolimusSirolimus

• GlucocorticoidsGlucocorticoids

• Mycophenolic acidMycophenolic acid

• Anti-TCR antibody (anti-CD3) Anti-TCR antibody (anti-CD3)

Preventing IL-2-driven clonal expansionPreventing IL-2-driven clonal expansion

IL-2IL-2IL-2RIL-2R

IL-2IL-2

MitogenesisMitogenesis

Prevent TCR signalingPrevent TCR signalingfor IL-2 gene transcription:for IL-2 gene transcription:

cyclosporin & tacrolimuscyclosporin & tacrolimus glucocorticoidsglucocorticoids

Prevent mitogenic responsePrevent mitogenic responseto IL-2R stimulationto IL-2R stimulation

Sirolimus & MycophenolateSirolimus & Mycophenolate

Prevent activationPrevent activationof T-cell via TCRof T-cell via TCR

Anti-TCR AntibodyAnti-TCR Antibody

IL-2 mRNA DegradationIL-2 mRNA Degradation glucocorticoids glucocorticoids

Prevent TCR signalingPrevent TCR signalingfor IL-2 gene transcription:for IL-2 gene transcription:

cyclosporin & tacrolimuscyclosporin & tacrolimusglucocorticoidsglucocorticoids

Prevent mitogenic responsePrevent mitogenic responseto IL-2R stimulationto IL-2R stimulation

Sirolimus & MycophenolateSirolimus & Mycophenolate

Prevent activationPrevent activationof T-cell via TCRof T-cell via TCR

Anti-CD3 AntibodyAnti-CD3 Antibody

Preventing IL-2-Driven T-Cell Clonal ExpansionPreventing IL-2-Driven T-Cell Clonal Expansion

IL-2 mRNA DegradationIL-2 mRNA Degradation glucocorticoids glucocorticoids

Prevent TCR signalingPrevent TCR signalingfor IL-2 gene transcription:for IL-2 gene transcription:

cyclosporin & tacrolimuscyclosporin & tacrolimusglucocorticoidsglucocorticoids

Preventing IL-2-Driven T-Cell Clonal ExpansionPreventing IL-2-Driven T-Cell Clonal Expansion

IL-2IL-2

IL-2RIL-2R

IL-2IL-2

MitogenesisMitogenesis

Prevent TCR signalingPrevent TCR signalingfor IL-2 gene transcription:for IL-2 gene transcription:

cyclosporin & tacrolimuscyclosporin & tacrolimusglucocorticoidsglucocorticoids

IL-2IL-2

TCRTCR

Receptor-associated tyrosineReceptor-associated tyrosinekinases (ZAP-70, lck & fyn)kinases (ZAP-70, lck & fyn)

Phospholipase C

Phospholipase C

phosphatidylphosphatidylinositolinositol

IPIP33

PKCPKC

DAGDAG

CaCa2+2+??

CalcineurinCalcineurinNF-ATNF-AT

PP

OctOct

PPNF-ATNF-AT

OctOct

NF-NF-B &B & Other TF’sOther TF’s

IL-2IL-2

Proximal IL-2 Promoter Transcription Factor BindingProximal IL-2 Promoter Transcription Factor Binding

NF-ATNF-AT

PP OctOct

PP

NF

-AT

NF

-AT

Oct

Oct

NF-NF-BB

NF-NF-BB

NF

-AT

NF

-AT

Oct

Oct

OA

PO

AP

AP

-1A

P-1

c-Fos

c-Fos

c-Jun

c-Jun

CalcineurinCalcineurin

Cyclosporin

NNCHCH33

OOHH

Cyclosporin

Fungal-derived, cyclic undecapeptideFungal-derived, cyclic undecapeptide

NNCHCH33

OOHH

Tacrolimus (FK506)

Fungal-derived macrolideFungal-derived macrolide

Cyclophilin & Immunophilins

• Intracellular peptidyl-prolyl topoisomerasesIntracellular peptidyl-prolyl topoisomerases

• Normal role is in post-translational folding of Normal role is in post-translational folding of proteinsproteins

• Targets of cyclosporin (cyclophilin) and Targets of cyclosporin (cyclophilin) and tacrolimus (FKBP = FK506-binding protein)tacrolimus (FKBP = FK506-binding protein)

• Ligand-bound, cyclophilin & FKBP acquire Ligand-bound, cyclophilin & FKBP acquire affinity for calcineurin affinity for calcineurin inhibit enzyme inhibit enzyme

IL-2IL-2

TCRTCR

Receptor-associated tyrosineReceptor-associated tyrosinekinases (ZAP-70, lck & fyn)kinases (ZAP-70, lck & fyn)

Phospholipase C

Phospholipase C

phosphatidylphosphatidylinositolinositol

IPIP33

PKCPKC

DAGDAG

CaCa2+2+??

CalcineurinCalcineurinNF-ATNF-AT

PP

OctOct

PPNF-ATNF-AT

OctOct

NF-NF-B &B & Other TF’sOther TF’s

IL-2IL-2

Action of Cyclosporin or TacrolimusAction of Cyclosporin or Tacrolimus

NF-ATNF-AT

PP OctOct

PP

NF

-AT

NF

-AT

Oct

Oct

NF-NF-BB

NF

-AT

NF

-AT

Oct

Oct

OA

PO

AP

AP

-1A

P-1

CalcineurinCalcineurin

CycloCyclo CyclophilinCyclophilin

CycloCyclo

IL-2IL-2

Action of Cyclosporin or TacrolimusAction of Cyclosporin or Tacrolimus

NF-ATNF-AT

PP OctOct

PP

NF

-AT

NF

-AT

Oct

Oct

NF-NF-BB

NF

-AT

NF

-AT

Oct

Oct

OA

PO

AP

AP

-1A

P-1

CalcineurinCalcineurin

CycloCyclo CyclophilinCyclophilin

CycloCyclo

CycloCyclo

IL-2IL-2

Action of Cyclosporin or TacrolimusAction of Cyclosporin or Tacrolimus

NF-ATNF-AT

PP OctOct

PP

NF

-AT

NF

-AT

Oct

Oct

NF-NF-BB

NF

-AT

NF

-AT

Oct

Oct

OA

PO

AP

AP

-1A

P-1

CalcineurinCalcineurin

CycloCyclo CyclophilinCyclophilin

CycloCyclo

CycloCyclo

GlucocorticoidsGlucocorticoids

• Suppression of transcriptionSuppression of transcription

• Reduced IL-2 mRNA stabilityReduced IL-2 mRNA stability

-116 -110 -106 -99 -97 -88| | | | | |

Ets c-Jun/ATF-2 NF-kBC/EBP

ACCGCT T CCT CCAGAT GAGCT CATGGGT T T CT CCACCAAGGAAGT T T T CCGCT GG

NF-NF-B/GR InteractionB/GR Interaction

Rel-A

NF-NF-BB I-I-BB

Rel

-A

NF

-N

F-

BB

GRNLSNLS

GCGC

GRNLSNLS

GC

GRNLSNLS

GC

GRNLSNLS

GC

GRNLSNLS

GC

GRNLSNLS

GC

I-I-BB Induction Induction

Rel-A

NF-NF-BB I-I-BB

GRNLSNLS

GC

GRNLSNLS

GC

I-I-BB

I-I-BB

Rel-A

NF-NF-BB

c-Ju

nPP

c-F

osc-

Fos

Physical Interaction Between AP-1 & GRPhysical Interaction Between AP-1 & GR

AP-1

-65 -59

| |

5' - G C T G G T T G A A T G A T T C T T T C

c-Ju

nPP

c-F

osc-

Fos

GRNLSNLS

GCGC

GRNLSNLS

GC

GRNLSNLS

GC

GRNLSNLS

GC

GRNLSNLS

GC

GRNLSNLS

GC

IL-2IL-2

NF-ATNF-AT

PP OctOct

PP

NF

-AT

NF

-AT

Oct

Oct

NF-NF-BB

NF

-AT

NF

-AT

Oct

Oct

OA

PO

AP

AP

-1A

P-1

CalcineurinCalcineurin

Glucocorticoids & IL-2 ExpressionGlucocorticoids & IL-2 Expression1) IL-2 Gene Transcription1) IL-2 Gene Transcription

GRNLSNLS

GC

GRNLSNLS

GC

NF

-N

F-

BB

c-Fos

c-Fos

c-Jun

c-Jun

Preventing IL-2-Driven T-Cell Clonal ExpansionPreventing IL-2-Driven T-Cell Clonal Expansion

IL-2 mRNA DegradationIL-2 mRNA Degradation glucocorticoids glucocorticoids

Coding Region 3’ UTRPromoter

mRNA SynthesismRNA Synthesis

Coding

3’UTR

AAAAAAAAA

UAUUUAU

Poly-A

AU-rich regionAU-rich region

Glucocorticoids & IL-2 ExpressionGlucocorticoids & IL-2 Expression2) IL-2 mRNA Stability2) IL-2 mRNA Stability

IL-2IL-2

AcceleratedAcceleratedDegradationDegradation

Prevent mitogenic responsePrevent mitogenic responseto IL-2R stimulationto IL-2R stimulation

Sirolimus & MycophenolateSirolimus & Mycophenolate

Preventing IL-2-Driven T-Cell Clonal ExpansionPreventing IL-2-Driven T-Cell Clonal Expansion

Preventing IL-2-driven clonal expansionPreventing IL-2-driven clonal expansion

IL-2IL-2

IL-2RIL-2R

IL-2IL-2

MitogenesisMitogenesis

Prevent mitogenic responsePrevent mitogenic responseto IL-2R stimulationto IL-2R stimulation

Sirolimus (Rapamycin)Sirolimus (Rapamycin)

NNCHCH33

OOHH

Fungal-derived macrolideFungal-derived macrolide

Sirolimus (Rapamycin)

Sirolimus (Rapamycin)Sirolimus (Rapamycin)

• Structurally related to cyclosporine & Structurally related to cyclosporine & tacrolimustacrolimus

• Also binds an immunophilin “Mammalian Also binds an immunophilin “Mammalian Target of Rapamycin” (mTOR)Target of Rapamycin” (mTOR)

• Does not inhibit calcineurinDoes not inhibit calcineurin

• Does not inhibit IL-2 or IL-2RDoes not inhibit IL-2 or IL-2R

• Inhibits Inhibits mitogenic responsemitogenic response to IL-2 to IL-2

GG00

GG11

GG22

SS

MM

GG0 0 G G11:CDK:CDK22/cyclin E complex inhibited by /cyclin E complex inhibited by

p27-CDK-inhibitorp27-CDK-inhibitor

Late GLate G11: p27-CDK-inhibitor degradation: p27-CDK-inhibitor degradation

GG1 1 S Transition S Transition

Cyclin ECDKCDK22 p27p27

Cyclin ECDKCDK22

GG00

GG11

GG22

SS

MM

GG0 0 G G11:CDK:CDK22/cyclin E complex inhibited by /cyclin E complex inhibited by

p27-CDK-inhibitorp27-CDK-inhibitor

Late GLate G11: p27-CDK-inhibitor degradation: p27-CDK-inhibitor degradation

GG1 1 S Transition S Transition

Cyclin ECDKCDK22 p27p27

mTORmTORSirSir

Cyclin ECDKCDK22 p27p27

SirolimusSirolimus

mTORmTOR

AAAAAA

PP

p70s6 Kinase

60 40

synthesis labile synthesis labile proteinsproteins

SirolimusSirolimus SirSir

mTORmTORSirSir

mTORmTOR

AAAAAA

PP

p70s6 KinasemTORmTOR

SirSir

60

40

synthesis ofsynthesis of labile proteinslabile proteins

SirolimusSirolimus

PreventsPrevents p27-CDK-inhibitorp27-CDK-inhibitor

SirSir

mTORmTORSirSir

mTORmTOR

AAAAAA

PP

p70s6 KinasemTORmTOR

SirSir

CDKCDK22/cyclin E/cyclin E

activityactivityGG11/S arrest/S arrest

60

40

synthesis ofsynthesis of labile proteinslabile proteins

Mycophenolic AcidMycophenolic Acid

• Fungal derivedFungal derived• Inhibitor of de-novo guanosine monophosphate Inhibitor of de-novo guanosine monophosphate

synthesissynthesis• B and T cells depend on de-novo synthesis:B and T cells depend on de-novo synthesis:

-lack salvage pathway for guanine recovery-lack salvage pathway for guanine recovery• Other cells have salvage pathwayOther cells have salvage pathway• Specific inhibition of DNA synthesis, RNA synthesis Specific inhibition of DNA synthesis, RNA synthesis

& other GTP or cGMP requiring pathways& other GTP or cGMP requiring pathways

COOH

OCH3

CH3

OH

O

CH3O

HClOCH3

CH3

OH

O

CH3OO

O N O

Mycophenolate mofetil Mycophenolate mofetil

(prodrug)(prodrug)

esteraseesterase

MycophenolateMycophenolate

Ribose-5P + ATPRibose-5P + ATP

5-phosphoribosyl-1-pyrophosphate (PRPP)5-phosphoribosyl-1-pyrophosphate (PRPP)

Guanine Guanine Guanosine MP Guanosine MP Inosine MP Inosine MP Adenosine MP Adenosine MP

Salvage PathwaySalvage Pathway(deficient in (deficient in T & B cells)T & B cells)

IMP dehydrogenaseIMP dehydrogenase

MycophenolateMycophenolate

Prevent activationPrevent activationof T-cell via TCRof T-cell via TCR

Anti-CD3 AntibodyAnti-CD3 Antibody

Preventing IL-2-Driven T-Cell Clonal ExpansionPreventing IL-2-Driven T-Cell Clonal Expansion

Preventing IL-2-driven clonal expansionPreventing IL-2-driven clonal expansion

IL-2IL-2

IL-2RIL-2R

IL-2IL-2

MitogenesisMitogenesis

Prevent Antigen/APC activationPrevent Antigen/APC activationof T-cell via TCRof T-cell via TCR

Anti-CD3 AntibodyAnti-CD3 Antibody

TCR Binding, I.C.Signaling & Cell-Cell SignalingTCR Binding, I.C.Signaling & Cell-Cell Signaling

APC

TT

Other interactions Other interactions that contributethat contributeto identificationto identificationand stabilityand stability

Signals for expression of Signals for expression of differentiation markers, differentiation markers, growth factors & growth factors & other cytokinesother cytokines

IFN-IFN-IL-1IL-1

CD3 is a component of the TCR CD3 is a component of the TCR It contributes to signaling It contributes to signaling

APC

TT

CD3CD3

IFN-IFN-IL-1IL-1

IL-2IL-2

TT

CD3CD3

IFN-IFN-

IL-2IL-2

Anti-CD3Anti-CD3 AntibodyAntibody

IL-2IL-2

Anti-CD3Anti-CD3

MitogenesisMitogenesis

SirolimusSirolimus

MycophenolateMycophenolate

IL-2IL-2

Summary:Summary: Preventing IL-2-Driven T-Cell Clonal ExpansionPreventing IL-2-Driven T-Cell Clonal Expansion

CyclosporinCyclosporinTacrolimusTacrolimusGlucocorticoidsGlucocorticoids

GlucocorticoidsGlucocorticoids