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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