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

Why Inhibit T-Cell Clonal Expansion? Transplant-specific Cytotoxic T-cells are responsible for most organ transplant rejection eventsTransplant-specific Cytotoxic T-cells are responsible for most organ transplant rejection events Can start drug just before foreign antigens are introduced (IE, just before the transplant)Can start drug just before foreign antigens are introduced (IE, just before the transplant)

T Foreign Cell or Protein Antigen uptake Antigen processing Antigen presentation RECOGNITION Ag-presenting cell CLONALEXPANSION

T Foreign Cell or Protein Antigen uptake Antigen processing Antigen presentation RECOGNITION Ag-presenting cell CLONALEXPANSION

I II III APC Receptor Binding & Signaling & Signaling Cell - cell signaling (IFN- , IL-1  ) (IFN- , IL-1  ) Autocrinesignaling(IL-2) IV Proliferation Steps Towards T-cell Clonal Expansion

I. TCR Binding, I.C.Signaling II. Cell-Cell Signaling APC T Other interactions that contribute to identification and stability Signals for expression of growth factors, differentiation markers, differentiation markers, & other cytokines IFN-  IFN-  IL-1 

III. IL-2: Autocrine Growth & Differentiation Factor IL-2 is released by the activated T cell IL-2 is released by the activated T cell Acts through IL-2RActs through IL-2R IL-2 IL-2R IL-2 Mitogenesis

IV. IL-2 Driving Clonal Expansion Clone of identical T-cells, specific for a unique antigen Clone of identical T-cells, specific for a unique antigen Destroy transplanted tissue Destroy transplanted tissue T T T T T T T T T T T T T T T T

Drugs that Prevent IL-2 Driven Clonal Expansion CyclosporinCyclosporin TacrolimusTacrolimus SirolimusSirolimus GlucocorticoidsGlucocorticoids Mycophenolic acidMycophenolic acid Anti-TCR antibody (anti-CD3)Anti-TCR antibody (anti-CD3)

Preventing IL-2-driven clonal expansion IL-2 IL-2R IL-2 Mitogenesis Prevent TCR signaling for IL-2 gene transcription: cyclosporin & tacrolimus glucocorticoids glucocorticoids Prevent mitogenic response to IL-2R stimulation Sirolimus & Mycophenolate Prevent activation of T-cell via TCR Anti-TCR Antibody  IL-2 mRNA Degradation glucocorticoids

Prevent TCR signaling for IL-2 gene transcription: cyclosporin & tacrolimus glucocorticoids Prevent mitogenic response to IL-2R stimulation Sirolimus & Mycophenolate Prevent activation of T-cell via TCR Anti-CD3 Antibody Preventing IL-2-Driven T-Cell Clonal Expansion  IL-2 mRNA Degradation glucocorticoids

Prevent TCR signaling for IL-2 gene transcription: cyclosporin & tacrolimus glucocorticoids Preventing IL-2-Driven T-Cell Clonal Expansion

IL-2 IL-2R IL-2 Mitogenesis Prevent TCR signaling for IL-2 gene transcription: cyclosporin & tacrolimus glucocorticoids

IL-2 TCR Receptor-associated tyrosine kinases (ZAP-70, lck & fyn) Phospholipase C  phosphatidylinositol IP 3 PKC DAG Ca 2+ ? Calcineurin NF-ATP OctP NF-AT Oct NF-  B & Other TF’s Other TF’s

IL-2 Proximal IL-2 Promoter Transcription Factor Binding NF-ATP OctP NF-AT Oct NF-  B NF-AT Oct OAP AP-1 c-Fos c-Jun Calcineurin

Cyclosporin

N CH 3 OH Cyclosporin Fungal-derived, cyclic undecapeptide

N CH 3 OH Tacrolimus (FK506) Fungal-derived macrolide

Cyclophilin & Immunophilins Intracellular peptidyl-prolyl topoisomerasesIntracellular peptidyl-prolyl topoisomerases Normal role is in post-translational folding of proteinsNormal role is in post-translational folding of proteins Targets of cyclosporin (cyclophilin) and tacrolimus (FKBP = FK506-binding protein)Targets of cyclosporin (cyclophilin) and tacrolimus (FKBP = FK506-binding protein) Ligand-bound, cyclophilin & FKBP acquire affinity for calcineurin  inhibit enzymeLigand-bound, cyclophilin & FKBP acquire affinity for calcineurin  inhibit enzyme

IL-2 TCR Receptor-associated tyrosine kinases (ZAP-70, lck & fyn) Phospholipase C  phosphatidylinositol IP 3 PKC DAG Ca 2+ ? Calcineurin NF-ATP OctP NF-AT Oct NF-  B & Other TF’s Other TF’s

IL-2 Action of Cyclosporin or Tacrolimus NF-ATP OctP NF-AT Oct NF-  B NF-AT Oct OAP AP-1 Calcineurin Cyclo Cyclophilin Cyclo

IL-2 Action of Cyclosporin or Tacrolimus NF-ATP OctP NF-AT Oct NF-  B NF-AT Oct OAP AP-1 Calcineurin Cyclo Cyclophilin Cyclo Cyclo

IL-2 Action of Cyclosporin or Tacrolimus NF-ATP OctP NF-AT Oct NF-  B NF-AT Oct OAP AP-1 Calcineurin Cyclo Cyclophilin Cyclo Cyclo

Glucocorticoids Suppression of transcriptionSuppression of transcription Reduced IL-2 mRNA stabilityReduced IL-2 mRNA stability

NF-  B/GR Interaction Rel-A NF-  B I-  B Rel-A NF-  B GR NLS GC GR NLS GC GR NLS GC GR NLS GC GR NLS GC GR NLS GC

I-  B  Induction Rel-A NF-  B I-  B  GR NLS GC GR NLS GC I-  B  Rel-A NF-  B

c-Jun P c-Fos Physical Interaction Between AP-1 & GR AP-1 c-Jun P c-Fos GR NLS GC GR NLS GC GR NLS GC GR NLS GC GR NLS GC GR NLS GC

IL-2 NF-ATP OctP NF-AT Oct NF-  B NF-AT Oct OAP AP-1 Calcineurin Glucocorticoids & IL-2 Expression 1) IL-2 Gene Transcription GR NLS GC GR NLS GC NF-  B c-Fos c-Jun

Preventing IL-2-Driven T-Cell Clonal Expansion  IL-2 mRNA Degradation glucocorticoids

Coding Region3’ UTR Promoter mRNA Synthesis Coding 3’UTR AAAAAAAAA UAUUUAU Poly-A AU-rich region Glucocorticoids & IL-2 Expression 2) IL-2 mRNA Stability IL-2 AcceleratedDegradation

Prevent mitogenic response to IL-2R stimulation Sirolimus & Mycophenolate Preventing IL-2-Driven T-Cell Clonal Expansion

Preventing IL-2-driven clonal expansion IL-2 IL-2R IL-2 Mitogenesis Prevent mitogenic response to IL-2R stimulation Sirolimus (Rapamycin)

N CH 3 OH Fungal-derived macrolide Sirolimus (Rapamycin)

Structurally related to cyclosporine & tacrolimusStructurally related to cyclosporine & tacrolimus Also binds an immunophilin “Mammalian Target of Rapamycin” (mTOR)Also binds an immunophilin “Mammalian 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 mitogenic response to IL-2Inhibits mitogenic response to IL-2

G0G0G0G0 G1G1G1G1 G2G2G2G2 S M G 0  G 1 :CDK 2 /cyclin E complex inhibited by p27-CDK-inhibitor p27-CDK-inhibitor Late G 1 : p27-CDK-inhibitor degradation   G 1  S Transition G 1  S Transition Cyclin E CDK 2 p27 Cyclin E CDK 2

G0G0G0G0 G1G1G1G1 G2G2G2G2 S M G 0  G 1 :CDK 2 /cyclin E complex inhibited by p27-CDK-inhibitor p27-CDK-inhibitor Late G 1 : p27-CDK-inhibitor degradation   G 1  S Transition G 1  S Transition Cyclin E CDK 2 p27 mTORSir Cyclin E CDK 2 p27

Sirolimus mTOR AAAAAA P p70 s6 Kinase synthesis labile proteins

SirolimusSir mTORSirmTOR AAAAAA P p70 s6 Kinase mTORSir  synthesis of labile proteins labile proteins

Sirolimus Prevents  p27-CDK-inhibitor Sir mTORSirmTOR AAAAAA P p70 s6 Kinase mTORSir  CDK 2 /cyclin E activity G 1 /S arrest  synthesis of labile proteins labile proteins

Mycophenolic Acid Fungal derivedFungal derived Inhibitor of de-novo guanosine monophosphate synthesisInhibitor of de-novo guanosine monophosphate synthesis B and T cells depend on de-novo synthesis: -lack salvage pathway for guanine recoveryB and T cells depend on de-novo synthesis: -lack salvage pathway for guanine recovery Other cells have salvage pathwayOther cells have salvage pathway Specific inhibition of DNA synthesis, RNA synthesis & other GTP or cGMP requiring pathwaysSpecific inhibition of DNA synthesis, RNA synthesis & other GTP or cGMP requiring pathways

COOH OCH 3 CH 3 OH O CH 3 O  HCl OCH 3 CH 3 OH O CH 3 O O O NO Mycophenolate mofetil (prodrug) esterase Mycophenolate

Ribose-5P + ATP  5-phosphoribosyl-1-pyrophosphate (PRPP) 5-phosphoribosyl-1-pyrophosphate (PRPP)  Guanine  Guanosine MP  Inosine MP  Adenosine MP Salvage Pathway (deficient in T & B cells) IMP dehydrogenase Mycophenolate

Prevent activation of T-cell via TCR Anti-CD3 Antibody Preventing IL-2-Driven T-Cell Clonal Expansion

Preventing IL-2-driven clonal expansion IL-2 IL-2RIL-2Mitogenesis Prevent Antigen/APC activation of T-cell via TCR Anti-CD3 Antibody

TCR Binding, I.C.Signaling & Cell-Cell Signaling APC T Other interactions that contribute to identification and stability Signals for expression of differentiation markers, growth factors & other cytokines IFN-  IFN-  IL-1 

CD3 is a component of the TCR It contributes to signaling APC T CD3 IFN-  IFN-  IL-1  IL-2

T CD3 IFN-  IFN-  IL-2 Anti-CD3 Antibody

IL-2 Anti-CD3 Mitogenesis Sirolimus MycophenolateIL-2Summary: Preventing IL-2-Driven T-Cell Clonal Expansion CyclosporinTacrolimusGlucocorticoids Glucocorticoids