T cell activation and signaling

Question 1

What are the three Th cell activation signals?

Answer 1

1. TCR + Ag/MHC
2. costimulation and survival signals
3. IL-2 + IL-2R -> proliferation; other cytokines -> differentiation

Question 2

What is an issue with TCR-MHC binding?

Answer 2

It quickly dissociates

Question 3

How can the TCR-Ag-MHC complex stay together long enough to generate a signal?

Answer 3

accessory/adhesion molecules

Question 4

What adhesion/accessory molecules are used by a CD4+ T cell and pAPC to hold the TCR-Ag-MHC complex together? Describe if each interaction is adhesive or costimulatory

Answer 4

• CD4 binds to MHC-II (adhesion)
  -CD28 on a T cell binds to CD80/86 on APC (costimulatory and adhesive)
• LFA-1 on a T cell binds to ICAM-1 on APC (adhesion)

Question 5

Why are CD4 and CD8 co-receptors important?

Answer 5

• must bind to MHC
• required for TCR signal

Question 6

What regions of MHC does CD8 and CD4 bind?

Answer 6

CD8: MHC-I a3 domain
CD4: MHC-II B2 domain

Question 7

What types of proteins are involved in TCR and BCR signaling? What does each type of protein do?

Answer 7

1. tyrosine as well as serine/threonine kinases
   - add a phosphate group
2. protein phosphotases
   - desphosphorylate proteins
3. lipid kinases and lipases
   - phosphorylate (kinases) or cleave (lipases) lipids to generate second messengers (e.g DAG and IP3)
4. G proteins
   - cycle between active (GTP-bound) and inactive (GDP-bound) states
5. TFs

Question 8

What T cell receptors have no intrinsic kinase activity and how do they compensate?

Answer 8

TCR and CD3

compensate by having ITAMs on their cytoplasmic tails (CD3)

Question 9

How do ITAMs work?

Answer 9

contain two tyrosine residues that can each be phosphorylated by kinases

Question 10

How does tyrosine phosphorylation of a protein modify its function?

Answer 10

1. regulate a protein’s enzymatic activity
2. regulate interactions between proteins
   - Src homology 2 (SH2) and protein tyrosine binding (PTB) domains bind phosphorylated tyrosines
3. regulate a protein’s subcellular localization

Question 11

What is the structure of Src family tyr kinases (SFNs), what is the function of each domain? Name an example of an SFN

Answer 11

1. lipid modifications (anchor to membrane)
2. UNIQ (localization)
3. SH3 (binds proline-rich regions of proteins)
4. SH2 (binds tyrosline phosphorylated sequences)
5. KINASE (kinase domain)
6. R (regulatory domain)

• Lck in T cells

Question 12

How are Src kinases regulated?

Answer 12

• closed/inactivate = SH2 binds to regulatory domain (bites its tail)
• primed/inactive = release of the regulatory domain from SH2 via dephosphorylation of the regulatory domain
• open/active = autotransphorsphorylation of the kinase domain

Question 13

Describe the steps of TCR signaling cascade

Answer 13

1. TCR binds to MHC-Ag
2. CD4/8 binds to MHC
3. CD4 brings along a kinase, Lck
   - Lck has a phosphorylated ITAM
4. CD45 dephosphorylates Lck
5. Lck phosphorylates ITAMs (P-ITAMs) on CD3
6. ZAP70 is attracted to the P-ITAMS
   - ZAP70 contains SH2 domains
7. ZAP70 phosphorylated by Lck
   - ZAP70 is activated
8. P-ZAP70 phosphorylates LAT an adaptor protein
9. PLCgI is attracted to P-LAT
10. PLCgI is phosphorylated by P-ZAP70
11. P-PLCgI metabolizes PIP2 into DAG and IP3
    - IP3 opens ER and Ca2+ channels –> Ca2+ enters cytoplasm
12. Ca2+ and DAG facilitate activation of TFs

Question 14

What are lipid rafts? What’s found in them (T cells)?

Answer 14

• rigid regions of the p.m rich in cholesterol and sphingolipids which are distinct from the surronding membrane
• site where GPI-anchored and lipid modified signaling proteins are found (e..g LAT and CD4 + inactivated Lck)

Question 15

What proteins are found in lipid rafts in inactive B cells and activated B cells?

Answer 15

inactive:
- Lyn (a Src kinase)

active:
- BCR + Ag
- Lyn
- Iga and Igb

Question 16

Describe costimulation in T cells. What does it do, when does it act?

Answer 16

• CD28 binds to CD80/86 on APC “costimulates” the T cell
• costimulation –> increase T cell response in conjugtion with TCR
• increases IL-2 and IL-2R ts
• cannot act unless TCR is engaged

Question 17

What signal is missing in a 3 signals for T cell activation model to create anergy? How does this occur, what are the consequences?

Answer 17

• no signal 2 (costimulation) –> anergy
• occurs when a non-APC presents self Ag on MHC-II (non-APCs do not carry CD80/86)
• anergic cells cannot reactivate even with signal 1 + 2

Question 18

What is the effect of CD28 signaling?

Answer 18

improves the IL-2R:
- moderate affinity (gamma + beta subunits) –> high affinity (gamma + beta + alpha subunits)

Question 19

Describe the “if”, “then” and “but we know” of issues of how naive CD8+ T cells are activated to become CTLs?

Answer 19

if:
- the TCR on Tc cells can only recognize endogenous Ag presented on MHC-I
- naive T cells can only be activated when there’s a second signal provided by APCs

then:
- therefore, Tc cells should only be activated when APCs are infected

but we know:
- Tc cells are activated in the absence of APC infection, how does this happen?

Question 20

How are naive CD8+ T cells activated?

Answer 20

1. licensing of DCs by Th1 cells:
   - CD40 on APC interacts with CD40L on an activated Th cell + microbial products –> TLR activation
2. cross-presentation of Ag.s
   - DC presents exogenous Ag on MHC-I + provides costim molecules + releases IL-12 –> signal 1, 2 and 3 –> proliferation and differentiation

Question 21

Describe the three signals required for naive CD8+ T cells to be activated

Answer 21

1. signal from TCR after recognition of Ag + MHC-I on a licensed APC
2. constimulatory signal (CD28 binds to CD80/86)
3. activation of its high affinity IL-2R by IL-2 released from Th1 or Th17

Question 22

For the following defects, name the impaired function and the disease it causes: ZAP70 and LFA-1

Answer 22

ZAP70:
- impaired function = defective signal from TCR
- disease = SCID

LFA-1:
- impaired function = defective T-APC adhesion
- disease = leukocyte-adhesion defect (LAD)