Immune Cell Signalling

Question 1

How many signals are required for effective T-cell activation?

Answer 1

• Three signals are needed for effective T cell activation
• Presence of each signal is vital
• Order is also important

Question 2

No Signal 1 (aka no antigen)

Answer 2

-Strong systemic cytokine (e.g. high-dose IL-2 treatment)
pre-exposure induces CD4 anergy – lose antigen
responsiveness

Question 3

No Signal 2 (aka no co-stimulation/ no PRR signals)

Answer 3

– CD4 and CD8 anergy
– Note: Treg induction requires co-stimulation

Question 4

No Signal 3

Answer 4

– In the absence of cytokines CD8 T cells proliferate but fail
to gain effector function
– Determines whether stimulation results in tolerance (Treg)
or effector functions (Th1, Th2, etc.)

Question 5

Describe TCR signalling

Answer 5

*The activation of the TCR and CD28 signaling pathway leads to intracellular signaling events which regulate IL-2 secretion and T cell survival

*Several Transcription factors/signaling pathways activated

Question 6

What is autocrine IL-2 essential for?

Answer 6

Essential for clonal expression

Question 7

How many key signaling pathways are there? What do they regulate?

Answer 7

*Four Key signaling pathways regulate IL-2 expression in T cells (as well as other effector functions)

Question 8

Name the 4 key signaling pathways

Answer 8

*NF-kB (nuclear factor -kB) pathway
*NFAT = nuclear factor of activated T cells
*AP1 = activator protein 1, a heterodimer of Jun and Fos Proteins
*PI3k (phosphoinositide 3-kinase) and Akt pro-survival pathway

Question 9

What do Kinases and Phosphatases do?

Answer 9

*Kinases add phosphate groups - phosphorylation
*Phosphatases remove phosphate groups - Dephosphorylation
*Enable signal amplification

Question 10

ITAMS

Answer 10

• ITAMs (immunoreceptor tyrosine based activation motif) in TCR complex
• 2 YXXL/I motifs are separated by about 6-9 aa within each ITAM
• Y=tyrosine, L=Leucine, I=isoleucine, X=any aa.

Question 11

How does phosphorylation of ITAMs by Lck occur?

Answer 11

• Antigen recognition by the T-cell receptor and its co-receptors leads to phosphorylation of ITAMs by Src-family kinases
• Lck, a Src family tyrosine kinase is associated with the cytoplasmic tails of CD4 and CD8.
• Following TCR binding to its ligand and CD4/CD8 to invariant regions of MHC molecules, Lck is brought into close proximity to the ITAMs of CD3
  and z chains of the TCR complex. Leads to activation of Lck which then phosphorylates the ITAMs.

Question 12

What do Phosphorylated ITAMs recruit?

Answer 12

• Phosphorylated ITAMs recruit and activate the tyrosine kinase
  ZAP-70, which phosphorylates scaffold proteins such as LAT
  (linker for the activation of T cells).
• ZAP-70 (!-associated protein of 70KDa) is a tyrosine kinase
  with 2 SH2-domains

Question 13

What does phosphorylated LAT provide?

Answer 13

Phosphorylated LAT provides docking sites for other proteins including
adapter proteins Grb2 and PLC-!1 (phospholipase C gamma-1), a key
enzyme in T cell activation.

Question 14

What does adaptor protein Grb2 recruit?

Answer 14

Once Grb-2 is attached to LAT Grb-2 recruits Sos (GTP/GDP exchange
factor for Ras). Leads to MAPK activation.

Question 15

Steps in TCR signaling - part 1

Answer 15

1. Antigen-recognition forms lipid raft
2. Lck associated with CD4/CD8 phosphorylates CD3z ITAMs
3. ZAP-70 is binds to p-ITAMs and gets phosphorylated
4. p-ZAP-70 phosphorylates LAT
5. PLC-ɣ and Grb-2/SOS

Question 16

What does the activation of PLC-y require?

Answer 16

• The activation of PLC-y requires a co-stimulatory signal (provided by activation of CD28 pathway)

Question 17

How is PIP3 created?

Answer 17

PIP2 (phosphatidyl inositol bisphosphate) can be phosphorylated by PI3K
(phosphatidyl inositol-3-kinase) to create PIP3 during cellular
activation.

Question 18

PIP3

Answer 18

PIP3 creates binding sites for proteins with PH domains at the
internal face of the plasma membrane

Question 19

What does PLC-y generate?

Answer 19

• Activated PLC-! generates the second messengers diacylglycerol (DAG) and inositol trisphosphate (IP3)
• IP3 is released into the cytoplasm where it interacts with receptors on the ER inducing the release of stored Ca2+ ions into the cytoplasm

Question 20

What induces a conformational change in Calmodulin?

Answer 20

• Ca2+ binding to the calcium binding protein Calmodulin induces a conformational change in Calmodulin which allows it to bind to
  Calcineurin and activate it.

Question 21

What is Calcineurin?

Answer 21

*Calcineurin is a phosphatase, an important
target of calcineurin is NFAT.
*The transcription factor NFAT (nuclear factor of activated T cells)
is phosphorylated at given serine/threonine residues which keep it in an inactive state.

Question 22

Active Calcineurin

Answer 22

*Active Calcineurin dephosphorylates NFAT exposing the Nuclear localization signal (NLS) of NFAT allowing its movement into the nucleus to regulate the transcription of genes including IL-2.

Question 23

How does DAG/PKC activate NFkB?

Answer 23

• DAG generated by PLCy1 recruits the serine/threonine kinase PKC
• PKC then activates the IKK complex (IKKa/b/NEMO)
• Degradation of IkB allows NF-kB to move into the nucleus and regulates the
  transcription of genes including IL-2

Question 24

AP-1

Answer 24

• AP-1 is a heterodimer of c-Jun and Fos proteins – TF involved in IL-2 transcription
• c-Jun requires activation (phosphorylation) via JNK (c-Jun N terminal kinase) (part of MAPK pathway)
• Fos is transcribed in response to activation of the TF Elk-1 (part of MAPK pathway)