Lec 34- Immunosuppressive therapy Flashcards
1
Q
Clinical uses for immunosuppressant
A
- Suppress rejection of organ transplants
- Suppress graft vs host disease in bone marrow transplants -Autoimmune diseases (For example Myasthenia Gravis)
2
Q
Therapeutic approach to minimise early rejection
A
Induction immunosuppression (first 2 week post-transplant)
-Depletion of activated T cells e.g. anti-CD25 (basiliximab)
+PLUS cyclosporin or tacrolimus PLUS azathioprine or mycophenolate mofetile
- CD25 is a component of the IL-2 receptor which is present on activated T cells and is required for T cell proliferation
- So we are selectively knocking out T cell compartment and not the whole lot
- Ciclosporin and tacrolimus are calcinuerin inhibitors
3
Q
Later Therapeutic approach
A
- Standard initial therapy- to prevent allograft rejection= combination of
1) calcineurin inhibitor (ciclosporin or tacrolimus)
2) Anti-proliferative (azathioprine)
3) corticosteroids when needed - Maintenance therapy: lower dose of the above. 90% of patients survive 1 year after transplantation so patients need chronic immunosuppression
- Short courses of aggressive immunotherapy required to address acute rejection
4
Q
TCR
A
- T cells use transmembrane dimeric proteins called T cell receptors (TCR); Typically 1 TCR protein sequence per T cell (Most common TCR is alpha and beta, with gamma, delta less common)
- TCRs bind a specific combination of Ag fragment nestled into the cleft of of a glycoprotein encoded by genes in the major histocompatibility complex (Typically, MHC I and II)
5
Q
TCR signalling
A
- Fyn or Lck phosphorylates tyrosine residues on the CD3epsilon and ITAMs, allowing ZAP-70 to bind
- Lck activates ZAP-70 which in turn phosphorylates LAT and SLP-76, SLP-76 binds to activates phospholipase-C-gamma (PLC-g)
- PLC-g cleaves PIP2 and yield DAG and IP3 3 different pathways
1) DAG activates protein kinase C-0 –> Protein kinase C-0 activates a transcription factor NFkB
2) IP3 increases intracellular Ca2+ conc, activating a phosphatase, calcineurin –> calcineurin activates a transcription factor NFAT (Nuclear factor of activated T cells)
3) DAG activates RasGRP, which in turn activates a MAP kinase cascade -> Rat-Induced kinase cascade induces and activates Fos, a component of the AP-1 transcription factor 1,2,3, all cause transcription faction: NFkB: NFAT and AP-1 act to induce specific gene transcription, leading to cell proliferation and differentiation
6
Q
Mechanism of TCR activation: The calcineurin Pathway
A
- Activation of TCR (which aided by co-stimulator molecules also present in the plasma membrane APC) causes;
- A rise in intracellular Ca2+ which
- Activates calcineurin, a phosphatase which removes phosphate from NF-AT (Nuclear Factor of Activated T cells) -Dephosphorylated NF-AT enters the nucleus and with accessory transcription factors e.g. AP-1 binds to the promoters of some 1000 genes expressed in activated T cells
7
Q
IL-2 is the primary driver of T cell proliferation
A
- MAPK activates AP-1, Ca2+ activates NFAT
- PKC activates NF-kB (Oct1 is always bound to the promoter -NFAT allows production of IL-2
8
Q
TCR activation
A
- Increased IL-2 expression
- T cell proliferation
- Activation of cellular immune response
- Thes T cell responses can contribute to autoimmunity and to tissue rejection in transplant patients
9
Q
Immunophillins
A
- Cyclosporin A, Tacrolimus and sirolimus all work by binding to immunophillins (High-affinity receptor proteins in the cytoplasm that combine with immunosuppressant drugs leading to rotamase inhibition and, in T cells, thus to interruption of cell activation.)
- Immunophillins are peptide-prolyl cis-trans isomerases (They have 2 different conformations, they hold a bond in a certain way)
- Ciclosporin A and tacrolimus inhibit calcineurin sirolimus (Rapamycin) inhibits mTOR (mammalian target of rapamycin)
10
Q
Mechanism of cyclosporin A (CsA)
A
- Cyclic peptides of 11 amino acids (The bonus of a cyclic peptide means its conformation is fixed and structure is strong) -Binds to an immunophillin called cyclophilin (CyP/CpN) as a cytoplasmic receptors -Complex of CsA:CyP inactivates calcineurin which is required for T cell signalling
- Inhibit IL-2 transcription which inhibits the clonal expansion of T cells (CD4 and 8) and reduces T cell effector functions
11
Q
Structure of the ternary complex
A
- CypA: cyclophillin
- CsA: Cyclosporin A
- CnA/CnB: 2 domains of calcineurin
12
Q
Tacrolimus (FK506)
A
- Macrolide antibiotic
- Binds to the immunophillin FK Binding Protein (FKBP) as cytoplasmic receptors
- More potent than cyclosporin A at inhibiting calcineurin
- Greater absorption and greater solubility than cyclosporin A
- Inital choice between cyclosporin and tacrolimus based on side effect profiles for individuals
- Used as rescue therapy for acute kidney rejection in cyclosporin-resistant cases
- FK506 binds to FKBP inhibiting calciuneurin (inhibit NFAT etc)
13
Q
Anti-proliferative: Azathioprine
A
- Undergoes metabolism to release 6- mercaptopurine (A purine analogue)
- Inhibit DNA and RNA synthesis
- Inhibits clonal proliferation of T cells
- Low selectivity
- Complications include myelosuppression (bone marrow suppression, by inhibiting proliferation we won’t get any of the WBC’s or RBC’s) and hepatotoxicity
14
Q
Mycophenolate mofetil
A
- More selective than azathioprine
- Inhibits purine biosynthesis- specific for T cell and B cell proliferation
- Used in cases of calcineurin inhibitor intolerance
- Acute rejection is 50% lower using MM versus azathioprine
- No evidence yet of efficacy in long-term graft survival
- Useful in treatment of acute cell rejection- reversing refractory rejection
15
Q
Sirolimus (Rapamycin)
A
- Novel, potent immunosuppressive
- Binds to FKBP family (FKBP12), thus can be used with cyclosporin (Different pathway)
- The sirolimus-FKBP complex inhibits mTOR and does not inhibit calcineurin
- Inhibits IL-2R signalling events (where as calcineurin prevents production of IL-2)
- Prevents cell cycle entry so prevents clonal expansion
- Used in cases of calcineurin inhibitor tolerance
16
Q
Corticosteriod
A
- Decreased T cells and macrophage cytokines (including IL-2)
- Decrease APC function
- Decrease MHC Ag expression consequently
- Decreased T helper cell proliferation
- Administered during acute rejection
17
Q
Management of rejection
A
-Suspected rejection- methylprednisolone -If steroids fail, anti-lymphocyte treatment
+Antithymocyte globulin (Thymoglobuline)
+Muromonab- CD3 depletion
18
Q
Rabbit anti-thymocyte globulin
A
-Mostly works by depleting T cells but also modulates cell surface receptor -take human T cell, stick it in rabbit get anti-serum (with Abs) and then neutralise T cells
19
Q
Belatacept
A
- Quick take on a CTLA4-FC fusion -chimeic (2 different proteins fused together)
- It has an Fc region -Has a soluble recombinant CTLA-4 part
- CD8 T cells have CD28 an activator when binding to APC (co-stimulator and CTLA-4 this is an inhibitor for T cell)
- By providing soluble CTLA-4 so it can bind to all of the Ags presented on MHC by APC meaning CD28 cannot bind and create positive stimulus to initiate CD8 T cell
20
Q
Mechanism of kidney graft damage
A
- Alloantigen (Ag that differs between members of the same species)
- Th1 binds with IL-2 which induces Tc cell, this then binds to kidney cell expressing alloantigen causing direct cell lysis -Th cell also produce IFN-g, this acts on endothelial cells to produce PDGF and PAF (platelet activating factor) (This works in an autocrine way, i.e. this will activate the cell that released it):
+This will cause the production of endothelia causing vasoconstriction-> schema and so graft cell death
+IL-8 will also be released which will initiate inflammation +Platelet aggregation will also occur not only will this cause thrombosis and necrosis. It also activates smooth muscle cell proliferation which leads to fibrosis and replacement of functional tissue with non-functional scar tissue
21
Q
Calcineurin pathway TCR activation- NF-AT
A
- Phosphorylation on serine and threonine residues keeps NFAT in the cytoplasm of unstimulated cells -ca2+ entry activates the serine phosphatase calcineurin which in turn dephosphorelates NFAT (Calmodulin when Ca2+ binds takes phosphate group off) , allowing it to enter the nucleus
- Dephosphorylated NFAT enters the nucleus and activates gene transcription
- Depletion of Ca2+ stores by IP3 results in the activation of CRAC channels (Calcium Release Activated Calcium Channels) which leads to NFAT activation
22
Q
T cell targets
A
1) drug target interferes with calcineurin, NFAT pathway (this activates IL-2)
2) when IL-2 is produced acts autocrine by binding to cell receptors and creates mTOR pathway
3) Antiproliferation
23
Q
Effects of mycophenolate mofetil and azathioprine
A
- MMF inhibits IMP dehydrogenase which stops production of inosine MP and adenosine MP (building blocks for DNA)
- Azathioprine inhibits bother imp-dehydrogenase and adenosine dehydrogenase
24
Q
mTOR inhibitors sirolimus mechanism
A
- Growth factor (GF- IL-2) binds to IL-2 receptor
- Causes production of AKT-TSC-Rheb and this forms 2 different complex
1) mTOR 2
2) mTORC1 (this is effected by drug targets) = cell proliferation, cell survival, tumour growth, angiogenesis
25
Q
The future
A
- All the drugs currently are non-specific (so inhibit T cells not just for the transplanted tissue but for bacteria, virus, fungi etc)
- Claim that we can use mesenchymal stem cells that are both anti-inflammatory (cytokines) and immunoprivileged:
- Known as mesoblast
26
Q
A
