Lecture 15; T cell activation and effector subsets

Question 1

Describe the antigen binding site on the tcR;

Answer 1

The hypervariable region can be;

• a/b

or

• g/d

TcR’s

Question 2

What do the TcR recognise?

Answer 2

MHC molecules only

Question 3

Describe the CD3 complex signalling proteins;

Answer 3

Four seperate chains that can be Phosphorylated

g,d,epsilom, zeta

Question 4

What is the t cell antigen receptor complex?

Answer 4

CD4

CD3
TcR
CD3

Question 5

What are CD4 and CD8 and what do they both have in common for signalling?

Answer 5

Co receptors of TcR

They both have Src Kinases called Lck

Bound non-covalently to their IC domains

Question 6

During exogeous activation, what second signal must t cells recieve?

Answer 6

T cells must recieve a second signal via the CD28

CD28 binds CD80,86 on APC

This is absolutely essential for Tcell activation

Question 7

What are important T cell adhesion molecules?

Answer 7

LFA1-ICAM-1

CD2-LFA3

Question 8

What are the key protein kinases for T cell intracellular signalling;

Answer 8

Protein Tyrosine Kinases;

• Lck
• ZAP70

Question 9

What is a tyrosine phosphotase involved in the regulation of t cell?

Answer 9

CD45 (tyrosine phosphotase)

Question 10

What down regulates the t cell response?

Answer 10

CTLA-4 can outcompete CD28 preventing t cell signalling

Also results in RAS upregulation and apoptosis

Question 11

what is a SMAC?

Answer 11

Supramolecular Activation Clusters

Molecules that are colocated to the membrane during T cell activation

Question 12

What is in a SMAC?

Answer 12

• 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)

Question 13

What can cause dysregulation of smac?

Answer 13

CTLA-4 or SHP

Question 14

What is the function of SMACs?

Answer 14

• Increase proximal activity or internal kinases to TcR

Question 15

Describe SMAC formation;

Answer 15

Microclusters are formed first then congeal into one large cluster.

Question 16

Describe CD3 gamma;

Answer 16

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

Question 17

Describe CD3 delta;

Answer 17

IG like membrane domains with short cytoplasmic tail

Ive charge in transmembrane domain

Contains 1 ITAM

(same as CD3 e)

Question 18

Describe CD3 e;

Answer 18

IG like membrane domains with short cytoplasmic tail

Ive charge in transmembrane domain

Contains 1 ITAM

Same as CD3 d

(structure will be different)

Question 19

Describe CD3 z;

Answer 19

• 9 AA EC domain
• Homodimer
• 2x3 ITAMS
• Phosphorylated by Lck and is associated with CD4/8

Question 20

What is the CD3/TcR complex?

Answer 20

A dimer

Question 21

How is CD3 and TcR unusual?

Answer 21

They all have charge AA residues in their TM domains, thus form complexes of dimers

Question 22

Where is Lck found?

Answer 22

• Lck belongs to the Src family of PTK
• Lck is bound non-covalently to CD4/CD8 cytoplasmic tail
• Phosphorylates CD3 ζ-chain ITAM

Question 23

What regulates Lck?

Answer 23

• Regulation:
• Csk phosphorylates negatively regulate
• CD45 dephosphorylates to activate

Question 24

What are ITAMs?

Answer 24

Immunoreceptor Tyrosine-based Activation Motif

Question 25

What do ITAMs do?

Answer 25

IC domain of TCR - Binds Src family kinases, e.g. Syk, ZAP-70 - - Recruits signalling proteins that have tandem SH2 domains

Question 26

What are ITIM?

Answer 26

Immunoreceptor Tyrosine-based Inhibition Motif Inhibitory receptors

Question 27

What do ITIMS do?

Answer 27

* 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

Question 28

What are 1-3 of the earliest events in t cell activation?

Answer 28

1. CD45 is a phosphatase that primes lck by removing phosphate 2. CD4/CD8 bring Src kinase (lck) proximal to the CD3 ζ-chain 3. At least 2 ITAMs phosphorylated on ζ−ζ-chain.

Question 29

What are 4-6 of the earliest events in T cell activation?

Answer 29

4. Syk/ZAP70 protein kinase binds phosphorylated ζ-chain via its SH2 domain. 5.Phosphorylation of many adaptor proteins including LAT and SLP-76. 6. Phospholipase C-γ docks onto phosphorylated LAT-SLP-76.

Question 30

What are 7-9 of the earliest events in T cell activation?

Answer 30

7. PLC-γ cleaves PIP2 to IP3 and DAG. 8. DAG activates PKC-θ and RasGRP-Ras pathway 9. IP3 mediates an increase in Ca2+ and activation of the Ser Thr phosphatase calcineurin

Question 31

What happens once a t cell is activated?

Answer 31

- Actin reorganisation (cell enlarges) - Cell adhesion molecule upregulation - Gene expression and t cell regulation

Question 32

What genes are turned on in t cell activation?

Answer 32

- 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

Question 33

What turns on NFkB?

Answer 33

* DAG activates PKC-θ | * PKC-θ activates NF-κB

Question 34

What activates NF-AT?

Answer 34

* IP3 increases Ca2+ * Ca2+ binds Calmodulin * Activates Calcineurin phosphatase * Calcineurin de-phosphorylates NF-ATc

Question 35

What activates AP-1?

Answer 35

* Guanine exchange factors (GEF) activate Ras * Ras activates MAP kinase cascade * MAP kinase activates c-Fos component of AP-1

Question 36

What is IL2 to T cells?

Answer 36

Key growth factor

Question 37

What pathways do cd28 activate?

Answer 37

PKC RAS-GRP Thus IL2 = growth and expansion of t cells

Question 38

What does CTLA4 do signalling pathways exactly?

Answer 38

- Dephosphorylates TcR | - Exclusion or endocytosis of CD28 (cant activate PKC)

Question 39

What are the different t cells subsets (cd4) that can arise from activation and proliferation?

Answer 39

- IFN g cytokine = TH1 synthesis - IL 4 = TH2 - TGF-b and IL6 = TH17

Question 40

What causses different t cells subsets to arise?

Answer 40

The types of PRR that are activated during infection Determines cytokine release

Question 41

What do TH1 do?

Answer 41

Clearance of intracellular pathogens immunopathology Autoimmune Basically activates CD8

Question 42

What does TH2 do?

Answer 42

- Clearance of EC pathogens - Allergy - Atopy i.e IL 6 release!

Question 43

Whats the role of TH17?

Answer 43

- il17 production - Clearance of pathogens - Tissue inflammation - immunopathology, Autoimmunity.

Question 44

What is another t cell that can be produced under certain circumstances?

Answer 44

iTreg If food or self is presented then this mechanism prevents self reaction TNF-b

Question 45

How can t cells be manipulated?

Answer 45

``` • Therapeutic purposes: Organ transplantation – Cyclosporin – FK506 • Manipulation by pathogens – Bacterial and viral superantigens • A clinical trial gone wrong – CD28 superantagonist ```

Question 46

What is alloreactivity?

Answer 46

How t cells respond to foreign MHC and to what degree.

Question 47

What does cyclosporin do?

Answer 47

Inhibits the translocation of Nuclear Factor for activated T cells (NF-AT ) (inhibits t cells)

Question 48

What does FK-506 (Tacrolimus) do?

Answer 48

Also inhibits the translocation of Nuclear Factor for activated T cells (NF-AT )

Question 49

What is the Mechanism of action of cyclosporin & tacrolimus?

Answer 49

Both target calcinueron and prevent NF-AC from being phosphorylated and activating IL2

Question 50

What produces super antigens?

Answer 50

• 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

Question 51

What do superantigens do?

Answer 51

* Recognised by T cells without processing into peptides | * Can stimulate 2-20% of all T cells = cytokine storm

Question 52

What did CD28 superagonist do?

Answer 52

TGN1412 acted like a superantigen and nearly killed people

Question 53

What do PRR do in t cell activation?

Answer 53

• PRR (danger) signalling provides context for T cell effector activation