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Cellular and Molecular Immunology > 13. Lymphocyte signalling 5: Activation in time and space > Flashcards

13. Lymphocyte signalling 5: Activation in time and space Flashcards

1
Q

What is the key challenge in design drugs?

A
  1. Finding a target that is more important for the disease process then it is for functioning body.
  2. Ideally this will be an element of disease biology that is distinct from healthy biology.
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2
Q

How can cytokine secretion be blocked with treatment?

A
  1. Cytokines secretion is driven by co stimulation
  2. A blocking antibody for CD80/86 can prevent this co stimulation.
  3. This selectively reduces the cytokines secreted from T cells.
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3
Q

How is the 4-1BB signalling motif used in drugs?

A
  1. It is a signalling motif from the TNF receptors that helps drive Th1 signalling.
  2. Used to stimulate T cells to attack cancer cells.
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4
Q

What can treatments targeting signalling mechanisms be?

A

Blocking or stimulating

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5
Q

How is calcineurin targeted by drugs?

A
  1. FK506
  2. This blocks the function of calcineurin.
  3. This prevents T cell activation.
  4. This is not used very often but it is used after solid organ transplantation as immunosuppression.
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6
Q

What can drugs that target T cell proliferation also treat?

A

Cancer

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7
Q

Why are kinases good drug targets?

A

They have an active site which is an obvious target to prevent function.

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8
Q

What do PI3-kinase inhibitors do?

A
  1. prevents the conversion of PIP2 to PIP3
  2. PIP3 signals lots of proliferative signalling like Akt and mTORC.
  3. Inhibitors shuts off this catabolic and proliferative signalling.
  4. These are used to treat cancers with PI3-kinase mutations that make it constitutively active.
  5. This includes metastatic breast cancer.
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9
Q

What do BTK inhibitors do?

A
  1. This is the B cell analogue of ITK.
  2. B cell lymphomas often rely on BTK mutations and excessive activation.
  3. BTK is the starting point of proliferative signalling as it recruits and activates PLC.
  4. They can also target other tyrosine kinases.
  5. They treat B cell cancers like chronic lymphoid leukaemia or marginal zone lymphoma.
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10
Q

What do Ras inhibitors do?

A
  1. Ras is one of the most common mutations in cancers, as it is a key driver of proliferation.
  2. The key mutation in KRas is a G12C mutation which makes up 70% of Ras mutations.
  3. This locks Ras in the active signalling state so the MAPK signalling pathway is always active.
  4. A new drug recognises specifically this mutations and covalently inactivates Ras.
  5. However most patients don’t respond well.
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11
Q

Why is Ras a bad drug target?

A
  1. It is a really small molecule
  2. Has no enzymatic activity, so no obvious target
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12
Q

What are better targets in the Ras signalling pathway?

A

Downstream kinases like MEK

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13
Q

What do MEK inhibitors do?

A
  1. They are used more often then Ras inhibitors but only for tumours carrying a specific set of mutations.
  2. Also block the activation of the MAPK pathway.
  3. Mostly used in melanoma and in combination with other treatments.
  4. Resistance is a problem
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14
Q

Why Ras pathway and other proliferation inhibitors lead to resistance?

A
  1. There are multiple pathways for proliferation in the cell.
  2. Tumours heavily rely on 1 pathway that they have mutated.
  3. When this pathway is blocked by treatments/inhibitors, it creates pressure on the other pathways.
  4. This leads to mutations in other signalling pathways and increasing proliferation in the cancer cells.
  5. This means that treatments stop working and creates cancer cell variants.
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15
Q

What do Akt inhibitors do?

A
  1. They are used for metastatic breast cancer.
  2. Used for mutations in the PI3-kinase pathway
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16
Q

What are the key challenges in designing drugs for signalling?

A
  1. The target has to be critical for the function of the diseased cell.
  2. The drug has to allow continued function of other cells in the body.
  3. Redundant pathways that are normal safe guarding mechanisms for the cell make for rapid drug resistance.
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17
Q

Where do activated T cell sit?

A

In the secondary lymphoid organs

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18
Q

What needs to happen to activate a T cell?

A
  1. The specific T cell needs to find the APC presenting the corresponding antigen.
  2. This means the proximity of the cells is important.
  3. T and B cells need to be activated at the same time to create an effective immune response
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19
Q

What is important to create an effective immune response?

A

When and where signalling is activated.

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20
Q

How can space and time in T cell activation be observed?

A
  1. Tissue staining
  2. Time-lapse microscopy
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21
Q

How can space and time in T cell activation be manipulated?

A
  1. It is difficult as these things are hard to change/measure.
  2. You need to observe different time points or locations to see changes in function to prove or disprove a hypothesis.
  3. Not as easy as a knock out.
  4. T cells can make good examples as they move around and activate quickly.
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22
Q

What needs to be remembered when testing T cell activation considering space and time?

A
  1. Signalling is dynamic
  2. Signalling is controlled by ligand engagement and the receptors need to be where the cellular interface is.
  3. This can make it difficult to test
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23
Q

What are the challenges in naive T cell activation in space and time?

A
  1. The T cell needs to find the right APC, and to do this, the T cells move through the lymph nodes.
  2. Once activated, something needs to keep the T cell in the lymph node.
  3. The T cell then needs to proliferate.
  4. This all needs to be coordinated and this is done by linking signalling events to ensure it happens at the right time.
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24
Q

How can T cell activity in the lymph node be imaged?

A
  1. Using two-photon microscopy.
  2. This allows imaging of intact lymph nodes, and you can see what’s going on with the T cells in the lymph node.
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25
Q

What was initially observed in lymph nodes when using two-photon microscopy?

A
  1. Mouse models had dendritic cells injected and left for a day so they can drain to the draining lymph nodes.
  2. Then inject T cell that can recognise the MHC presented on the Dendritic cells into the lymph node.
  3. The T cells are very motile and always searching for the right dendritic cell.
  4. The T cells should bind to the Dendritic cells, but they seemed to ignore them and not bind.
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26
Q

Why were the T cells not binding to the Dendritic cells?

A
  1. They thought it was due to differences between in vitro and in vivo experiments.
  2. But it was actually due to the point in time
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27
Q

What was observed at a later time point in lymph nodes when using two-photon microscopy?

A
  1. The T cells were binding to the Dendritic cells.
  2. They were binding for quite a long time.
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28
Q

What is the problem using imaging in experiment?

A

You need to be able to quantify what you see somehow

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29
Q

What was seen when measuring the contact times for the initial time point in the lymph node?

A
  1. At 2-4 hours after injection, the contact times were short
  2. Minutes
30
Q

What was seen when measuring the contact times for the later time point in the lymph node?

A
  1. At 8-12 hours, the contact times are over an hour.
31
Q

Why was there an observed difference in how quickly T-cells and dendritic cells bind in vitro and in vivo?

A
  1. In vitro in perfect conditions they bind really quickly
  2. In vivo, the conditions are different, and binding takes longer
  3. This was due to the affinity of antigen
32
Q

How readily do low-affinity peptides on APC bind T cells?

A
  1. Not very much
  2. after 24 hours 2 TCR were in contact with a dendritic cell.
  3. This is the limiting concentrations needed for T cell activation
33
Q

How readily do high-affinity peptides on APC bind T cells?

A
  1. They bind very readily to TCRs.
  2. After 24 hours 156 T cells were in contact with a dendritic cell.
  3. With the same concentration as a low affinity peptide there is significantly more binding.
34
Q

What type of responses do stronger stimuli produce in T cells?

A
  1. Contact time is longer
  2. Contact occurs quicker
  3. Signalling happens quicker
35
Q

What is inversely proportional to stimulus strength?

A

Time

36
Q

How do weaker stimuli cause T cell activation?

A
  1. Time compensates for signal strength
  2. The cell takes more time making the decision to trigger signalling due to the weakness of the signalling.
  3. But you can still get the same level of response.
37
Q

What happens when you increase the stimulus strength in T cell activation?

A

You decrease the activation time of the T cell.

38
Q

Why can’t dendritic cells keep T cells in the lymph node?

A

They only bind transiently.

39
Q

What keeps T and B cells in the lymph nodes after activation?

A
  1. All T and B cells express the sphingosine 1-phosphate receptor (S1P1).
  2. This detects and binds sphingosine 1-phosphate circulating in the blood.
  3. This interaction is required for the T and B cells to leave the lymph node and enter circulation.
  4. During activation S1P1 is down regulated keeping the cells in the lymph node.
40
Q

What is sphingosine 1-phosphate?

A
  1. A lipid
  2. It is present in serum
  3. It circulates in the blood, mostly bound to albumin.
41
Q

What is sphingosine 1-phosphate receptor?

A
  1. It is highly expressed on all lymphocytes.
  2. It controls the exit of T and B cells from the lymph node.
42
Q

How was the sphingosine 1-phosphate receptor discovered?

A
  1. Using the immunosuppressive drug FTY720.
  2. It is an S1P1 antagonist so blocks its function.
  3. This caused all the lymphocytes to be retained by the lymph node.
  4. used to treat highly active MS
43
Q

What is the time scale of S1P1 expression?

A
  1. It is highly expressed on naive T cells to allow them to circulate.
  2. After activation it is down regulated so the cell cannot leave the lymph node.
  3. S1P1 was regained after about 5 days and the active T cells can leave the lymph node to fight infection
44
Q

How is S1P1 down regulated in T cell activation?

A
  1. CD69 is upregulated in T cell activation
  2. CD69 goes to the cell surface binds to S1P1 and internalises it.
  3. This causes S1P1 to be degraded
  4. The cell then cannot leave the lymph node.
45
Q

When is CD69 upregulated?

A

At the same time as the peak contact time from 6-12 hours.

46
Q

What does a committed T cell during activation also commit to?

A

Staying in the lymph node

47
Q

What does the T cell need to do once it is activated?

A

Proliferate

48
Q

What is required for T cell proliferation?

A

IL-2

49
Q

What do T cells start to make and secrete once they are activated?

A

IL-2

50
Q

How does IL-2 act on T cells?

A
  1. autocrine
    (2. sometimes systemically)
51
Q

When does IL-2 production start?

A
  1. 6-12 hours after activation
  2. This is the same time as the maximum contact and the upregulation of CD69.
  3. This drives T cell proliferation in the lymph node.
52
Q

What needs to happen to the T cell to receive the IL-2 signalling?

A

They need to express the IL-2 receptor.

53
Q

What protein chains make up the IL-2 receptor and when are they expressed?

A
  1. Gamma chain which is always expressed.
  2. Beta chain which is always expressed.
  3. Alpha chain (CD25) which is expressed when the T cell is activated.
54
Q

What kind of receptor does the IL-2R gamma and beta chain make?

A

A low affinity IL-2 receptor that doesn’t respond very well to IL-2.

55
Q

What kind of receptor does the IL-2R alpha, gamma and beta chain make?

A
  1. This forms that active IL-2R heterotrimer.
  2. This creates a good high affinity receptor that responds to IL-2 signalling.
56
Q

What is the difference in IL-2 receptors on Treg cells?

A
  1. All 3 receptor chains are always expressed so the active receptor is always expressed.
  2. They can always respond and bind to IL-2 all the time.
57
Q

What happens if you knock out CD25 in the IL-2 receptor?

A
  1. You create autoimmunity.
  2. This is because all the Treg cells cannot function without IL-2, so all the immune regulation is lost.
  3. The other T cell can activate using the low affinity receptor to create the autoimmune effect.
58
Q

When is CD25 expressed?

A

During the same 6-12 hours that CD69 is unregulated, S1P1 is downregulated, and IL-2 is unregulated.

59
Q

What are the tasks T cells need to do at the same time to activate effectively?

A
  1. Find and see the antigen on the Dendritic cell.
  2. Stay in the lymph node using S1P1 and CD69.
  3. Making IL-2 and proliferation.
  4. Respond to IL-2 by expressing CD25
60
Q

What is critical for T cell activation?

A

The synchronisation of antigen recognition, staying in the lymph node, making IL-2 and making CD25.

61
Q

What happens to T cells in the tumour microenvironment?

A

They become exhausted, so they don’t clear the tumour cells.

62
Q

What could be used to overcome T cell exhaustion?

A

IL-2

63
Q

What are the problem with trying to use IL-2 as a drug/treatment?

A
  1. Space - There are more Treg cells in the TME than in any other place in the body. These absorb all the IL-2 and prevent it from reaching the effector T cells.
  2. Space - IL-2 high doses cause T cell exhaustion and leukopenia and over-activation of Treg.
  3. Time - exposure to IL-2 for too long causes activation induced cell death
64
Q

What synthetic receptors are used in IL-2 based therapies?

A
  1. CAR receptor to activate the T cell
  2. SynNotch to trigger transcription of IL-2
65
Q

What is the CAR receptor?

A
  1. A synthetic receptor with a antibody fragment for antigen recognition.
  2. Targets a specific receptor on cancer cells
  3. Uses CD3 zeta chain motif and 4-1BB costimulatory domain to activate the T cells
  4. Needs more stimulation from IL-2
66
Q

What is the synNotch receptor?

A
  1. a synthetic notch receptor
  2. It recognises a tumour marker.
  3. You can put in whatever transcription factor as the cytoplasmic domain.
  4. This can be engineered to bind to the IL-2 promoter and activate transcription
67
Q

What is a notch receptor?

A
  1. When a ligand binds there are 2 proteolytic cleavage sites that cleave off the cytoplasmic domain.
  2. This cytoplasmic domain is a transcription factor.
  3. Once cleaved it can enter the nucleus and activate transcription
68
Q

How does the CAR and synNotch receptors work together in cancer treatment?

A
  1. Engineer them both onto T cells
  2. CAR receptor recognises the tumour cells
  3. The synNotch receptor activates as well and makes IL-2 locally. The IL-2 is not soaked up the Tregs.
  4. This means the Engineered T cell can proliferate in the body.
  5. This makes T cells that can detect and kill cancer cells and proliferate using IL-2.
69
Q

Why won’t IL-2 work when administered separately to the engineered T cells?

A
  1. Providing it systemically means it will all get soaked up by Tregs.
  2. Forcing constitutive expression of IL-2 causes the T cells to die from exhaustion.
  3. Driving IL-2 expression through transcription factors also causes exhaustion.
70
Q

When is the only time engineered T cells and IL-2 treatment works?

A
  1. When the T cell that is in contact with the tumour and makes its own IL-2 effectively.
  2. The time and space of the IL-2 production is critical for effective cancer treatment.
71
Q

What are the main principles of activation of T cells?

A
  1. T cell activation operates a different time scales from seconds to days.
  2. Time scales vary depending on the physiological state of T cells.
  3. Functionally linked biological processes are dynamically coordinated.
  4. T cell engineering is only effective when time and space are considered.

Cellular and Molecular Immunology (17 decks)

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