Cytotoxic T cells

Question 1

What is the markers on cytotoxic T cell ?

Answer 1

Mostly involves CD8+ cells but some CD4+ T cells – most information on CD8+ T cells (Tc)

Question 2

how are CD4+ T cells activated ?

Answer 2

CD4+ T cells activated in peripheral lymphoid organs by antigen presenting cell - usually a dendritic cell – that is expressing the specific antigen for the T cell receptor . This leads to clonal expansion of the specific T cell and production of memory cells .

Question 3

what are MHC class II and MHC class I molecules restricted by ?

Answer 3

MHC class II molecules are restricted with CD4+ T cells while MHC class I molecules are restricted with CD8+ T cells.

Question 4

can cytotoxic T cells directly kill a pthogen and why >

Answer 4

Tc kill other cells and once it killed a cell it can go on to another target cell – cannot directly kill pathogens. This is due to MHC restriction

Question 5

do they tend to hit the same trget twice ?

Answer 5

nope

Question 6

what’s the difference in timespan of killing for in vivo and in vitro ?

Answer 6

In vitro study showed it took 45 mins for a target cell to completely disintegrate by apoptosis - in vivo studies suggest its much quicker

Question 7

activation stages for cytotoxic T cell ?

Answer 7

Recognition of specific Ag using T cell receptor by APC

2. Environmental cues important

• cytokines upregulate activity e.g. IL-2, IL-12, IL-15,
• Cytotoxic CD8+ T cells have their activity upregulated by a range of cytokines e.g. IL-2, IL-12, IL-15, IL-18, IL-23, IL-27 – leading to more cells producing their effectors molecules/increase in amount or effector molecule produced/cell
• So innate responses can provide the environment for developing specific immune response
• In comparison to NK cells , Tc killing involves granules which are induced AFTER stimulation or direct death signal

Question 8

on a stidy comparing the activity of cytotoxic T cell against influenza A , what did the activity depend on ?

Answer 8

Studies comparing the activity of cytotoxic T cells against influenza A virus showed that activity

i) depends on the epitope recognised by T cell (i.e. specificity)
ii) reaction depends on the person used to isolate the cells CD8+ T cells , this involves MHC molecules

Question 9

what did the low affinty Fc receptors on self specific CD8+ T cells find?

Answer 9

low affinity Fc receptor on self-specific CD8+ T cell found they acted as a receptor for antibody-mediated cytotoxicity. Antibody acts as an opsonin.

Question 10

they also found that receptor such as NKG2 , CD244 and CD94 where upregulated what did this reveal ?

Answer 10

Upregulated receptors for other molecules – NKG2D, CD244, CD94 these could have a role in stimulation and killing by Tc.

Question 11

what did upregulatiion of receptors IL-12 and IL-18 reveal ?

Answer 11

receptors for IL-12 and IL-18 up regulated on effector and memory cells. Therefore, products of innate immune cells influence specific response

Question 12

soluble effectors released from CD8+?

Answer 12

cytotoxins such as perforin and granzymes , mediators were IFN gamma and TNF alpha

Question 13

membrane bound effectors for CD8+ T cells ?

Answer 13

Fas ligand

Question 14

Th1 soluble effectors ?

Answer 14

IL-2 , IL-3 , TNF alpha , IFN gamma and GM-CSF

Question 15

membrane bound effectors for Th1 >

Answer 15

FasL on activation , antigen specific and TNF beta

Question 16

TH2 Soluble effectors /

Answer 16

IL-3 , 4 , 5,6, 10 , 13 GM-CSF

Question 17

membrane bound effectors Th2 ?

Answer 17

FasL low

Question 18

how come cytoxic T cells are serial killers ?

Answer 18

need conjugation ( close contact) between cytotoxic T lymphoctye (CTL) and target cell – called an immunological synapse.

Can kill infected cell but not a pathogen directly since it will NOT express MHC molecules)

kill the cell target directly by granules/perforin induce apoptosis in target cell

Kill a cell indirectly by cytokines they produce which are directly toxic (TNF-a) – or induce toxic reactions (IFN-gamma) which induces phagocytosis.

Question 19

what does the production of TNF and IFN beta have on the target cell >

Answer 19

Produced TNF and IFN gamma which can act on TNF receptor and IFN receptor on target cell.

Question 20

what occurs at the immunological synapse ?

Answer 20

If there’s an interaction between CTL and target through the receptors then at the immunological synapse granzymes are released which enter the target cell and induce apoptosis

Question 21

death signal ?

Answer 21

Another way that apoptosis is initiated in the target cell is through the death signal that is induced via the Fas-L ligand of the Tc and the CD95 of the target cell and caspases are activated and apoptosis is initiated.

Question 22

differences between cytotoxic T cells and Nk cells ?

Answer 22

Know that NK are innate – they have no immunological memory

Know that activation is different for the two cell types – NK cells - balance between activation/inhibition important – Tc cells recognition via T cell receptor

Know that Tc produce granules on stimulation whereas NK constitutively have granules present

Question 23

Perforin role ?

Answer 23

Form pores for granules to enter target cell

• Granules taken up in endosomes and perforin allows release into cytosol to induce apoptosis?
• Perforin essential for granule mediate cytotoxicity (KO knock out gene sequence for perforin studies) and ability to control tumours and infections lost in KO studies.
• lots of different granzymes e.g. humans have four – B, A, K and M and mice have 10
• B and A most abundant in humans and B major constituent

Question 24

overview of granzymes B ?

Answer 24

Overview of Granzyme B suggests that perforin forms a pore which allows granzyme to enter the target cell. The granzyme then has various effects on the signalling pathways within the target cell , it activates caspase 3 and 7 which activates a caspase cascade and results in substrate proteolysis and cell death.

The granzyme can have more than one effect within a cell , it can also act on BID in a golgi and this activates cells which are pro apoptotic and cause an apoptosome to form which results in apoptosis.

Multiple pathways are available for apoptosis , so that if a pathogen develops the ability to block a pathway there are other ways to induce apoptosis.

Question 25

Granzymes A ?

Answer 25

Slower inducer of cell death( apoptosis) compared to granzyme B

damages DNA in target cell by causing single-strand nicks which then results in apoptosis - moves to nucleus and activates a DNAse (NM23-H1)

Need TREX1 (3’-5’ endonuclease) an enzyme who’s activation prevents DNA annealing and repairing

Activates oxidative stress in cell - increases reactive oxygen-species (ROS) - damaging .

Granzyme A enter the nucleus of the target cell and has an effect on TREX1 to prevent repair of damaged DNA. Also, on NM23H1 which causes nicks on single strand of DNA. It also effects the potential of the golgi and causes oxidative stress.

Question 26

Granzyme B ?

Answer 26

2Kda serine protease - aspase – enzyme which cleaves after aspartic acid residues

• Could enter cells in perforin independent way which uses mannose-6-phosphate receptor
• Knockout studies using mice shown granzyme B deficient CTL are less effective at killing and required for caspase activity
• Direct killing - can act on caspases - e.g. directly on caspase 3, 6, 7, 8, 9, 10 and via caspase 3 on caspase 2, 6, and 9 - stimulates apoptosis in target cell
• Indirect killing - triggers mitochondrial permeabilization - release of inter-membrance spacer proteins e.g. cytochrome C = forms a complex (apoptosome - APAF-1 and caspase 9) - causes cell death.

Question 27

Granzyme H ?

Answer 27

Granzyme H - induces a caspase and BID-independent cell death via chromosomal condensation and nuclear fragmentation.

Question 28

Granzyme C?

Answer 28

induces mitochondrial depolarization and cytochrome c release , similar to granzyme B.

Question 29

Granzyme K ?

Answer 29

induces ROS and may kill like granzyme A

Question 30

explanation of direct death signal ?

Answer 30

formation of a death inducing signalling complex (DISC) which activates caspases directly

• Fas ligand (FasL) present on activated cytotoxic cell binds to Fas (CD95) on cell membrane of target cell this gives a death signal and the cell dies by apoptosis - activates caspase cascade – at least 12 types of caspases
• CD28 – mediates in protection against Fas activated cell death as it inhibits intracellular signalling pathway.
• Or direct killing using TNF alpha related apoptosis-inducing ligand – TRAIL
• Cancer cells can express FasL can cause apoptosis of T cells which express Fas , this is a protective mechanism by cancer cells to vade cell death.

Question 31

final step of cell killing ?

Answer 31

When target cell dies by apoptosis it is removed by phagocytes or undergoes secondary necrosis and cell membrane lyses

Question 32

Why can T cells no kill without MHC restriction ?

Answer 32

Must be primed – cytokines, growth factors, mitogen or antigen

APC needs to present the antigen to the T cells

Close contact required to release granulysin – insert into microbial membrane – osmotic lysis

This procces may need perforin

Mediates killing bacteria, protozoa, fungi of pathogens by indirect method of killing.

Question 33

why are Tc not killed by their products ?

Answer 33

a membrane associated form of cathepsin B which is found in granules of Tc it can cleave perforin and protect the activated Tc.

• inside granules pH maintained low (pH6) and keeps perforin in its inactive state – only activated in neutral pH i.e. when granules released from the cell. Also surrounded by proteoglycans (serglycin and calreticulin) inside the granule
• Serpin PI-9 - acts as a pseudo-substrate for granzyme B - forms irreversible complex which inhibits its function
• CD28 enhances T cells survival in vitro - perhaps by increasing glucose consumption

Question 34

are mice relevant to humans ?

Answer 34

Genetic deficiency in perforin

Familial hemophagocytic lymphohistiocytosis is a lack of perforin which is rare, fatal autosomal recessive immune disorder

• Causes an uncontrolled activation of T cells and macrophages over production of inflammatory cytokines
• curable with bone marrow transplant
• little/no perforin produced – linked to three gene loci and the Tc and NK activity reduced/absent.

Question 35

Car T cells ?

Answer 35

chimeric antigen receptor (CAR T) cell therapy , This allows us to make new therapies – by expanding cancer-specific T cells and introducing the patient’s own primed T cells back into the individual or make new anticancer vaccines.