Antigen Receptor Signaling and T-cell Activation - Kane Flashcards
What is Lck and what is its function?
Lck is a Src tyrosine kinase closely associated with the co-receptors of T-cells and plays a role in the phosphorylation cascades involved in TCR signaling.
TCR signaling: Step 1
TCR binds to MHC-peptide and co-receptor brings Lck to close proximity of ITAMs
TCR signaling: Step 2
Lck phosphorylates ITAMs in zeta chains and CD3 chains of the TCR.
TCR signaling: Step 3
Zap70 is recruited to ITAM phosphorylated tyrosines and Zap70 gets phosphorylated by Lck.
TCR signaling: Step 4
Zap70 phosphorylates LAT and PLCy1, and PLCy1 associates with LAT
TCR signaling: Step 5
PLCy1 cleaves phosphoinositol substrates and creates second messengers such as DAG and IP3
TCR signaling: Step 6
Second messengers activate txn factors such as NF-AT
What is an SH2 domain and what does it do?
SH2 domains are specific regions of proteins that can mediate direct binding of phosphorylated tyrosine substrates. For example, SH2 domains Zap70 can bind to z-chains phosphorylated by Lck.
What is an SH3 domain?
SH3 domains are specific regions of proteins that recognize poly-proline residues.
Describe the PLCy1 pathway
Upon activation of PLCy1, it cleaves PIP to generate 2 second messengers, DAG and IP3.
What does IP3 do?
IP3 is a second messenger that associates with the ER and causes an efflux of intracellular Ca. Efflux of Ca, causes activation of calcineurin which dephosphorylates NF-AT (txn factor).
What do the drugs cyclophilin and FKBP do?
These drugs inhibit calcineurin preventing activation of NF-AT. This shuts down T-cell activation despite upstream signaling.
Describe co-stimulation of T-cells?
CD28 on T-cells interact with B7 receptors on APCs to stimulate activation. Absence of this signal can lead to a state of anergy.
How is co-stimulation regulated? Describe how this is druggable.
CTLA on T-cells regulate stimulation by B7 on APCs by competitively inhibiting CD28. Drugs such as CTLA-Ig can competitively inhibit B7 to prevent co-stimulation of T-cells.
How can you enhance T-cell responses via drugs involved in the co-stimulation pathway?
You can target CTLA to inhibit it so that it cannot dampen co-stimulation of T-cells via CD28 and B7 interactions.
What is LFA1?
Adhesion molecule constitutively expressed on T-cells that interact with ICAMs on APCs to stabilize TCR interaction with MHC-peptide.
What cytokine is critical for T-cell activation and proliferation?
IL-2. This cytokine is upregulated in activated T-cells and can act in an auto or paracrine fashion to stimulate T-cell proliferation.
How do CD4+ T-cells help other immune cells?
They bind to and secrete cytokines to enhance effector functions of phagocytes. They can also stimulate B-cells to secrete antibodies.
How is the T-cell response resolved (down-regulated)
Apoptosis of T-cells following the acute phase of the response.
What are the features of lymphocyte memory?
Higher precursor frequency (decreases proliferation time). Shorter activation time.
What is T-cell exhaustion?
During a chronic infection or cancer where repeated stimulation of T-cells impedes the development of memory T-cells.
Which proteins direct T-cell circulation?
L-Selectin interacting with modified sugars on epithelial cells or lymph node venules. Naiive and activated T-cells express different adhesion molecules.
What do chemokines do?
Small molecules that cause chemotaxis to direct immune cells or promote inflammation of tissues to promote infiltration of immune cells to tissues.
What are some methods to track phosphorylation in cells?
P32 orthophosphate - radioactive or Phospho-specific antibodies (4G10 –> tyrosine phos.).
Western blotting vs. Flow cytometry
Western blots have a disadvantage as its a lot of steps and lots of samples but better visualization of band sizes. Flow uses small number of cells and visualization of sub-pops but more complicated controls.
Visualizing activation of transcription factors?
Transcriptional reporters
How can you measure T-cell proliferation?
Monitoring DNA replication in stimulated T-cells with labeled nucleotides.
