9. Lymphocyte signalling 1 Flashcards
Why is learning about T cell signalling important?
- Most signalling pathways are not restricted to T cells.
- It is a big part of drug development as they are developed against individual molecules to change a function, and it is important to understand how the interactions of the molecules affect each other.
What is the basic function of a receptor?
- A receptor takes extracellular information and uses it to cause a biochemical change that can alter cell behaviour.
- The binding to a receptor will trigger some sort of conformational change.
- This change triggers a biochemical change through enzymes. eg phosphorylation of tyrosine.
- this can be done with 1 molecule like EGFR or 10 like TCR.
What do the protein chains in the TCR contain?
- Signalling motifs that allow the signal from the a/ß chains to be transmitted across the plasma membrane.
- As antigen recognition and signal transduction are on separate proteins, there needs to be something that holds them together.
- The a/ß chains contain positively charged residues in the transmembrane domain.
- The other chains contain negatively charged residues in the transmembrane domain.
- This does have a charge imbalance but this holds the complex together.
- Having charged residues in the membrane is a huge energy burden.
What protein chains make up a TCR?
- a/ß chains that are different in every TCR and bind the MHC complex for antigen recognition.
- CD3yε dimer. This is the same in every TCR.
- CD3δε dimer. This is the same in every TCR.
- Zeta dimer. This is the same in every TCR.
What other complex does the TCR work with?
- the MHC peptide complex
- The peptide and the MHC is recognised by the TCR.
- The invariant chains in the MHC bind the co-receptors associated with the TCR.
- CD4 co-receptor binds MHC2
- CD8 co-receptor binds MHC1
- Basic T cell activation needs antigen recognition and co-receptor binding.
What are the critical signalling motifs in the TCR/BCR?
- ITAMs: immunoreceptor tyrosine-based activation motif.
- These have 2 tyrosine residues that can be phosphorylated by kinases.
- It has a tyrosine and then 3 positions later the must be a large aliphatic amino acid. This is a leucine or isoleucine.
- This is repeated after about 10 amino acids.
How many ITAMs are in a TCR?
1.δ, y and e chains have 1 ITAM each
2. z chains have 3 ITAMs each
3. Totals 10 ITAMs and 20 tyrosine residues
Where is the enzymatic activity in the TCR?
- None of the protein chains in the TCR have enzymatic domains.
- It needs to recruit enzymes to the complex
What enzymes do the TCR recruit for initial signalling?
Tyrosine kinases Lck and ZAP-70 provide the enzyme activity needed for the TCR to generate a signal.
What is Lck?
- A Src family kinase
- It is always associated with co-receptors CD4 and CD8.
- If an MHC-peptide binds the TCR, Lck. is bought into the complex.
- Lck then phosphorylates the ITAMs in the TCR.
What are Src family kinases?
- Originally found in viruses
- They are a large family of kinases
- Include Src, Lck, Csk, Fyn and Lyn
Src family kinase domains: Lipid tail
- Lipid modification of the protein
- Helps it localised to the plasma membrane and stay there for longer.
- Very common in proteins working close to the membrane.
Src family kinase domains: Unique domain
Along with the lipid tail the unique src domain control the interaction with CD4 and CD8.
Src family kinase domains: SH2 domain
- A protein recruitment domain.
- It binds to phosphorylated tyrosines
Src family kinase domains: SH3 domains
- A protein recognition domain
- IT binds to a proline X X proline motif
Src family kinase domains: kinase domain
- The enzymatic domain with some protein protein interaction domains.
- A tyrosine residue in the kinase domain is an activating tyrosine that can activate itself through autophosphorylation.
- A c-terminal tyrosine residue that is inhibitory when it is phosphorylated.
What are the 2 main functions of signalling?
- Causing a change in localisation
- Enzymatic activity
What happens if a signal is always on?
Oncogenic transformation
What needs to happen when a signal turns on?
A method of turning it off is upregulated
How are Src family kinases inhibited/turned off?
- Through proline X X proline motif in the linker-SH3 region that the SH3 can bind.
- C-terminal tyrosine being phosphorylated.
- Through a physical blockade that is often intramolecular.
- This is a different domain in the same protein that binds the functional domain to prevent it eg the SH2 binding the C-terminal tyrosine
What is different about the regulation of Lck in T cells?
- Evidence suggests Lck has similar activity levels in inactive and active T cells.
- The key element of the regulation of Lck and other Src kinases is the localisation and the bringing of Lck to the TCR and plasma membrane.
- CD4/8 is heavily involved in this.
How do different phosphorylation patterns change the activity of Lck?
- If the Inhibitory tyrosine is phosphorylated by another src family kinases called Csk the activity is inhibited.
- If the stimulatory tyrosine is phosphorylated by Lck itself, its activity is activated.
- Both can be dephosphorylated by the transmembrane phosphatase CD45.
What does Lck’s change in location allow it to do?
Allows the Lck to phosphorylate the ITAMs on the TCR and recruit ZAP-70.
What is ZAP-70?
- A kinase that carries out the enzymatic activity needed to activate the TCR.
- Binds to ITAMs to become active.
How does ZAP-70 autoinhibit itself?
The SH2 linker binds to the kinase domain to prevent its function. This happens when the SH2 is not bound to a phosphorylated tyrosine.
How is ZAP-70 activated?
- A ITAM in the TCR has been phosphorylated by Lck.
- The SH2 domain in ZAP-70 can bind the phosphorylated tyrosines.
- The phosphorylated tyrosines on the ITAMs are a constant distance apart and the SH2 bind and are held in place.
- This releases the kinase domain, and the enzyme is now active.
What is the general rule of enzymes activated by signalling?
Binding to the recruitment domains allows the enzymatic activity to take place
EGFR activation mechanism
- EGF binds and trigger dimerisation
- Dimerisation allows kinase cross activation
- Recognition of a high affinity ligand
TCR Activation mechanism
- Peptide-MHC binding to TCR causes a conformational change, probably through physical force.
- This allows Lck recruitment, ITAM phosphorylation, ZAP70 recruitment and activation.
- Recognition of a spectrum of low-affinity ligands and selecting the right one
Why is TCR binding tricky for a T cell?
- The T cell needs to decide on a ligand
- It is a needle in a haystack situation for the T cells to find the one specific MHC it can bind.
- Affinity for the correct MHC is low, and for the incorrect, it is even lower, so it is hard to distinguish.
What is the function of the CD28 receptor?
Costimulation which is the 2nd signal needed for T cell activation.
What does CD28 bind?
CD80 or CD86 on the APC
Where is CD28 expressed?
On a T cell
What are the members of the CD28 family of receptor?
- CD28
- ICOS
- CTLA4
- PD1
What is the function of ICOS?
- Costimulatory receptor
- Similar signalling properties to CD28
- important for signalling the development of T follicular helper cells.
What is the function of Tfh cells?
Help B cells do class switching and affinity maturation
what is the function of CTLA4?
- It is an antagonist of CD28 so it is an inhibitor of CD28.
- Often a drug target
What is the function of PD1?
- Expressed on a lot of cells
- It is a T cell activation marker that is upregulated as soon as T cells are activated.
- Further upregulated in T cell in persistent immune responses like viruses and cancer.
- Good drug target
- Involved in the suppression of the persistent immune responses.
Different signalling motifs: ITAM YXXL/I
- tyrosine and leucine/isoleucine motif.
- Immunoreceptor tyrosine-based activation motif
- Recruits Syk family kinases
Different signalling motifs: ITAM YXXM
- Tyrosine and methionine motif
- Immunoreceptor tyrosine-based activation motif
- Recruits lipid kinases like PI3
- In CD28 and ICOS ITAMs
Different signalling motifs: ITIMs
- Immunoreceptor tyrosine-based inhibitory motif.
- Recruits phosphatases
Different signalling motifs: ITSMs
- Immunoreceptor tyrosine-based switch motif
- Can be activating or inhibitory.
- Recruit phosphatases or kinases
- Needs a small adaptor protein to recruit a kinase
What receptor shares ligand with CD28?
- CTLA4
- They both bind with CD80/86
Where is CD80/86 expressed?
On an APC
Where is CD28 and CTLA4 expressed?
on T cells
What are the methods CTLA4 uses to inhibit T cells?
- Ligand competition
- Ligand depletion
How does CTLA4 compete with CD28?
- CTLA4 has a higher binding affinity for CD86 then CD28 does.
- It can also bind as a dimer.
- If there is enough CLTA4 on the surface of a T cell CD28 will never bind CD80/86.
How does CTLA4 inhibit T cells in the lymph node?
- CTLA4 is constitutively expressed on Treg cells which mostly live in the lymph node.
- CTLA4 blocks CD80/86 and prevents it binding to CD28.
- This means activation is inefficient or doesn’t happen,
- CLTA4 can also be shed and act as a soluble inhibitor.
How does CTLA4 cause ligand depletion?
- Through transendocytosis
- If a receptor engages a ligand it always wants to internalise it but mostly it doesn’t happen.
- However, CTLA4 binds CD86 very tightly and can pull it off the APC and internalise it.
What is another minor function of CLTA4?
inhibitory signalling
How does PD1 carry out its inhibitory function?
- It has a ITSM that can recruit and tyrosine phosphatase.
- This is called SHP2.
- It counters the kinase activity that is happening from Lck and ZAP70.
Where is PD1 expressed?
- Mostly talked about in immune cells.
- Expressed on lots of other cells.
- Inhibitory mechanism is different when more complex signalling/cells are involved.
How can CTLA4 be turned into a medication?
- CD28 is responsible for activation of T cell in some autoimmunity.
- If you dimerise CTLA4 using an Fc antibody domain this creates a competitive inhibitor for CD28.
- This CLTA4 antibody binds to CD86 and prevents the T cell from being activated.
What are some examples treatments by blocking inhibitory receptors?
- Anti-PD1 drugs like Nivolumab.
- Anti-PD-L1 drugs like Atezolizumab
- Anti-CTLA4 drugs like Ipilimumab
What are the main principles to remember in signalling?
- Receptors come in families.
- Family members can be activating (CD28) or inhibitory (CTLA4).
- Receptors contain protein interaction motifs like ITAM/ITIM.
- Receptor signalling moves downstream through protein recruitment like Lck.
- Signalling intermediates are often autoinhibited.
