Lecture 15; T cell activation and effector subsets Flashcards
Describe the antigen binding site on the tcR;
The hypervariable region can be;
- a/b
or
- g/d
TcR’s
What do the TcR recognise?
MHC molecules only
Describe the CD3 complex signalling proteins;
Four seperate chains that can be Phosphorylated
g,d,epsilom, zeta
What is the t cell antigen receptor complex?
CD4
CD3
TcR
CD3
What are CD4 and CD8 and what do they both have in common for signalling?
Co receptors of TcR
They both have Src Kinases called Lck
Bound non-covalently to their IC domains
During exogeous activation, what second signal must t cells recieve?
T cells must recieve a second signal via the CD28
CD28 binds CD80,86 on APC
This is absolutely essential for Tcell activation
What are important T cell adhesion molecules?
LFA1-ICAM-1
CD2-LFA3
What are the key protein kinases for T cell intracellular signalling;
Protein Tyrosine Kinases;
- Lck
- ZAP70
What is a tyrosine phosphotase involved in the regulation of t cell?
CD45 (tyrosine phosphotase)
What down regulates the t cell response?
CTLA-4 can outcompete CD28 preventing t cell signalling
Also results in RAS upregulation and apoptosis
what is a SMAC?
Supramolecular Activation Clusters
Molecules that are colocated to the membrane during T cell activation
What is in a SMAC?
- TcR + CD3 (antigen recognition)
- CD3 g,d,e,z (signalling)
- CD4 or CD8 (coreceptor)
- Costimulatory molecule (CD28) (Stim) CTLA-4 (inhib)
- Kinases (Lck, ZAP70)
- Adhesion molecules (LFA1-ICAM-1 & CD2-LFA3)
What can cause dysregulation of smac?
CTLA-4 or SHP
What is the function of SMACs?
- Increase proximal activity or internal kinases to TcR
Describe SMAC formation;
Microclusters are formed first then congeal into one large cluster.
Describe CD3 gamma;
IG like membrane domains with short cytoplasmic tail
Non-covalently associates with a/b (TCR)
Contains 1 ITAM
Each CD3 is similar
CD3g> CD3d>CD3e>CD3z
Describe CD3 delta;
IG like membrane domains with short cytoplasmic tail
Ive charge in transmembrane domain
Contains 1 ITAM
(same as CD3 e)
Describe CD3 e;
IG like membrane domains with short cytoplasmic tail
Ive charge in transmembrane domain
Contains 1 ITAM
Same as CD3 d
(structure will be different)
Describe CD3 z;
- 9 AA EC domain
- Homodimer
- 2x3 ITAMS
- Phosphorylated by Lck and is associated with CD4/8
What is the CD3/TcR complex?
A dimer
How is CD3 and TcR unusual?
They all have charge AA residues in their TM domains, thus form complexes of dimers
Where is Lck found?
- Lck belongs to the Src family of PTK
- Lck is bound non-covalently to CD4/CD8 cytoplasmic tail
- Phosphorylates CD3 ζ-chain ITAM
What regulates Lck?
- Regulation:
- Csk phosphorylates negatively regulate
- CD45 dephosphorylates to activate
What are ITAMs?
Immunoreceptor Tyrosine-based Activation Motif
What do ITAMs do?
IC domain of TCR
- Binds Src family kinases, e.g. Syk, ZAP-70 - - Recruits signalling proteins that have tandem SH2 domains
What are ITIM?
Immunoreceptor Tyrosine-based Inhibition Motif Inhibitory receptors
What do ITIMS do?
- Binds SHP-1 and SHP-2 phosphatases via SH2 domain.
- Binds SHIP – ionosotide phosphatase targets IP3
- Dephosphorylates kinases associated with ITAMs
- CTLA-4 on T cells – directly opposes CD28
What are 1-3 of the earliest events in t cell activation?
- CD45 is a phosphatase that primes lck by removing phosphate
- CD4/CD8 bring Src kinase (lck) proximal to the CD3 ζ-chain
- At least 2 ITAMs phosphorylated on ζ−ζ-chain.
What are 4-6 of the earliest events in T cell activation?
- Syk/ZAP70 protein kinase binds phosphorylated ζ-chain via its SH2 domain. 5.Phosphorylation of many adaptor proteins including LAT and SLP-76.
- Phospholipase C-γ docks onto phosphorylated LAT-SLP-76.
What are 7-9 of the earliest events in T cell activation?
- PLC-γ cleaves PIP2 to IP3 and DAG.
- DAG activates PKC-θ and RasGRP-Ras pathway
- IP3 mediates an increase in Ca2+ and activation of the Ser Thr phosphatase calcineurin
What happens once a t cell is activated?
- Actin reorganisation (cell enlarges)
- Cell adhesion molecule upregulation
- Gene expression and t cell regulation
What genes are turned on in t cell activation?
- NFκB (Transcription factor)
- NF-AT(turns on IL-2 gene)
- AP-1 (binds IL-2 promoter)
Nuclear translocation of NF-AT, N F-κB and AP-1 to induce specific gene transcription, proliferation and differentiation
What turns on NFkB?
- DAG activates PKC-θ
* PKC-θ activates NF-κB
What activates NF-AT?
- IP3 increases Ca2+
- Ca2+ binds Calmodulin
- Activates Calcineurin phosphatase
- Calcineurin de-phosphorylates NF-ATc
What activates AP-1?
- Guanine exchange factors (GEF) activate Ras
- Ras activates MAP kinase cascade
- MAP kinase activates c-Fos component of AP-1
What is IL2 to T cells?
Key growth factor
What pathways do cd28 activate?
PKC
RAS-GRP
Thus IL2 = growth and expansion of t cells
What does CTLA4 do signalling pathways exactly?
- Dephosphorylates TcR
- Exclusion or endocytosis of CD28 (cant activate PKC)
What are the different t cells subsets (cd4) that can arise from activation and proliferation?
- IFN g cytokine = TH1 synthesis
- IL 4 = TH2
- TGF-b and IL6 = TH17
What causses different t cells subsets to arise?
The types of PRR that are activated during infection
Determines cytokine release
What do TH1 do?
Clearance of intracellular pathogens
immunopathology
Autoimmune
Basically activates CD8
What does TH2 do?
- Clearance of EC pathogens
- Allergy
- Atopy
i.e IL 6 release!
Whats the role of TH17?
- il17 production
- Clearance of pathogens
- Tissue inflammation
- immunopathology, Autoimmunity.
What is another t cell that can be produced under certain circumstances?
iTreg
If food or self is presented then this mechanism prevents self reaction
TNF-b
How can t cells be manipulated?
• Therapeutic purposes: Organ transplantation – Cyclosporin – FK506 • Manipulation by pathogens – Bacterial and viral superantigens • A clinical trial gone wrong – CD28 superantagonist
What is alloreactivity?
How t cells respond to foreign MHC and to what degree.
What does cyclosporin do?
Inhibits the translocation of Nuclear Factor for activated T cells (NF-AT )
(inhibits t cells)
What does FK-506 (Tacrolimus) do?
Also inhibits the translocation of Nuclear Factor for activated T cells (NF-AT )
What is the Mechanism of action of cyclosporin & tacrolimus?
Both target calcinueron and prevent NF-AC from being phosphorylated and activating IL2
What produces super antigens?
• Superantigens are produced by bacteria, mycoplasma and viruses,
e.g.:
– staphylococcal enterotoxins (SEs): food poisoning
– Toxic shock syndrome toxin-1 (TSST-1): toxic shock syndrome
What do superantigens do?
- Recognised by T cells without processing into peptides
* Can stimulate 2-20% of all T cells = cytokine storm
What did CD28 superagonist do?
TGN1412 acted like a superantigen and nearly killed people
What do PRR do in t cell activation?
• PRR (danger) signalling provides context for T cell effector activation
