Lecture 8 - Molecular events in T cell activation

Question 1

Cell signalling: what is it and what components are involved in it?

Answer 1

Turning an extracellular signal
into a cellular response

Ligand, receptor, intracellular signals, target (often nucelus)

Question 2

Protein phosphorylation: what is the process and what enzymes are involved?

Answer 2

Protein becomes (de)phosphorylated and becomes either activated or inactivated

• Kinase - serine/threonine kinases or tyrosines
• Phosphatases

Question 3

Scaffold proteins: what are they and what are some examples?

Answer 3

Large proteins that get phosphorylated and recruit and phosphorylate other proteins

ITAMs, LAT, SLP76, SH2, etc

Question 4

Are only proteins phosphorylated?

Answer 4

No, membrane lipids can too - PI3K, PIP2, etc

Question 5

Signal amplification: what is it and what examples are there?

Answer 5

Growing a signal larger

• Kinase cascades - Raf/MAPK cascade
• Release of secondary messengers (IP3/Ca²⁺)

Question 6

Intracellular signals: how do they modulate gene expression?

Answer 6

Regulation of transcription factors:
* Assembly of TF subunits
* Phosphorylation status of TFs
* Location of TF subunits

Question 7

CD4+ T-cell activation: what does it require?

Answer 7

• Signal 1 - antigen recognition by CD4+ T-cell receptor (presented by MHCII)
• Signal 2 - co-stimulatory molecule interaction (e.g. CD28-CD80/86)
• Signal 3 - cytokine signal to promote differentiation of T cells

Question 8

Signal 1 and 2 for T-cell activation: are there other components that increase their interaction strength?

Answer 8

Yes - adhesion receptors (e.g. LFA1-ICAM1 interaction)

Question 9

TcR complex: what is it and what is it composed of?

Answer 9

The name given to the entire complex of both the TCR recognition site along with the CD3 molecules associated which are required to transmit signals

• α and β chains of the TcR recognition area
• CD3 complex composed of:
• Heterodimer of ε and δ chains
• Heterodimer of ε and γ chains
• Homodimer of ζ chains

Question 10

CD3: how do they transmit signals?

Answer 10

They contain ITAM motifs in cytoplasmic domains which are required for signalling

Question 11

ITAMs: what are they, where are they found, what do they do, how is this affected by its structure, and what are they phosphorylated by?

Answer 11

Immunoreceptor tyrosine-based activation motifs (ITAMs)

Many different immunoreceptors - B-cell receptor complex, NK cell receptors, Fc receptors

Creates binding sites for additional signalling proteins (scaffold proteins) - 6-9 amino acids between tyrosine residues, good for allowing SH2 binding

Lck = Src-family kinase

Question 12

How many ITAMs does CD3 have in total?

Answer 12

• Two on each heterodimer, with one given to each chain
• Six on the homodimer, with three given to each chain

Question 13

ZAP-70: what is it, what does it do, how is it phosphorylated, why can it be phosphorylated, and what enhances its signalling?

Answer 13

Zeta-chain-associated protein kinase 70

Phosphorylates several different proteins to trigger signalling - ie LAT (linker of activated t-cells) and SLP-76

• Recruited to phosphorylated ITAMs via its SH2 domains
• Activated via phosphorylation by Lck

It has two SH2 domains which can recruit to the scaffolding of the ITAMs

Signalling proposed to be enhanced by TcR complex clustering

Question 14

SMACs: what are they, what do they do, what is their structure, and how do they do their function?

Answer 14

Supramolecular activating complexes - the name given to the massive complex made when TcRs bind to MHC complexes and cause phosphorylation of ITAMs

Result in the phosphorylation of ITAMs

Three layers:
* Central SMAC (cSMAC) - in the centre, this is where signal 1/2 molecules are
* Peripheral SMAC (pSMAC) - molecules involved in adhesion, (LFA1, CD2, etc) and some more signal 2 molecules (CD4, LCK, etc)
* Distal SMAC (dSMAC) - CD43/44/45

CD45 is involved in the dephosphorylation of ITAMs so the size exclusion SMACs do (because has a large extracellular domain) results in ITAMs being able to be phosphorylated without being turned off

Question 15

LAT/SLP-76: how do they continue the signal produced at the TcR?

Answer 15

• Adaptor protein Gads brings them together
• Gads/LAT/SLP-76 complex recruits PLC-γ using PIP3
• Once attached to PIP3, the kinase Itk activates PLC-γ
• PLC-γ cleaves PIP2 into IP3 and DAG
• IP3 triggers Ca²⁺-mediated signalling events by opening ER calcium stores
• DAG recruits signalling proteins to the membrane (PKC-θ and RasGRP (ER))
• PKC-θ

Question 16

PIP3: where does it come from in the TcR-MHC signalling stream and as well as its role in forming IP3/DAG and PLC-γ attachment, what else may it do?

Answer 16

Signal 2 (costimulatory molecules) results in the recruitment of PI3K which phosphorylates PIP2 into PIP3

Recruit kinases PDK (role in NF-κB activation and phosphorylation of protein kinase C-θ) and Akt (role in cell survival and proliferation)

Question 17

PLC-γ: what is it, what does it do, and what is it activated by?

Answer 17

Phospholipase C gamma

Cleaves PIP2 into IP3 and DAG

Itk once bound to PIP3 by the Gads/LAT/SLP-76 complex

Question 18

B7: what is it also known as?

Answer 18

CD80 or CD86

Question 19

Ca²⁺ signalling induced by the TcR: how does it work?

Answer 19

• IP3 opens up Ca²⁺ stores in the ER
• STIM1 aggregation (mechanism of aggregation is not clear) along with the released Ca²⁺ causes the opening of more Ca²⁺
• Creating a massive amount of intracellular Ca²⁺, resulting in

Question 20

Note: important to realise that pathways mentioned here are not the only signals triggered by the TcR

Answer 20

read tb for ER

IP3/Ca²⁺ cause NFAT activation
DAG/RasGRP cause AP-1 activation

Question 21

TcR signalling downstream pathways

Answer 21

• Calcineurin pathway
• NF-kB pathway
• AP-1 pathway
• MAPK pathway

Question 22

Calcineurin pathway:

Answer 22

• Phosphorylated NFAT - inactive
• Calmodulin binds to calcium (produced from IP3) - undergoes a conformational change, allowing calcineurin to bind
• Calcineurin dephosphorylates NFAT, allowing it to translocate to the nucleus and affect gene expression

Question 23

NF-kB pathway

Answer 23

• PLC-θ causes DAG production
• DAG causes PKC-θ recruitment to the membrane
• CARMA1 (scaffold protein) gets phosphorylated and forms a multimolecular complex
• CARMA1 causes Bcl-10 and MALT-1 recruitment
• Bcl-10 and MALT-1 cause TRAF-6 recruitment
• TRAF-6 makes a K63 ubiquitin tag which results in TAB1/2 recruitment
• TAB1/2 adaptor proteins allow TAK1 binding
• TAK1 can act on the IKK complex, deactivating it and stopping the NF-kB inhibition, resulting in NF-kB production

Question 24

IKK complex: what is it, what is its structure, what does it do, and how does it interact with the NF-kB pathway?

Answer 24

Inhibitor of kB kinase complex

• α and β subunits
• γ subunit (NEMO) - the subunit that allows TAK1 interaction

Regulates the activity of the inhibitor of kB

• Once ubiquitinated by TRAF6 and phosphorylated on its β subunit by TAK1, the β subunit can dissociate and cause the breakdown of the inhibitor of kB, causing NF-kB to be able to affect gene transcription

Question 25

Final image in this lecture

Answer 25

ER