Lecture 6 - Innate Stimulation of DCs

Question 1

List some of the various classes of PRR

Answer 1

TLRs
• Plasma membrane associated
• Endosome associated

RLRs
• Cytosolic

NLRPs
• Cytosolic

Question 2

Describe the generalised signal transduction pathways triggered by PRR ligation

What are the outcomes of these pathways?

Answer 2

1. TLRs
   • Ligation of TLR by ligand
   • Adaptor molecule activation: MyD88 or TRIF
   • Adaptor molecules activates TF: either NFκB or IRF
   • Transcription factors turn on the transcription genes:
   - NFκB: IL-1, IL-6, TNF, CXCL8, co-stimulatory molecules → acute inflammation, stimulation of adaptive immunity
   - IRF: type I IFN → antiviral state

2. NLRs
 • Various molecules activate NLRP3:
- extracellular ATP
- crystals etc.
 • NLRP3 associates with Caspase-1 to form the inflammasome
 • Active caspase-1 on inflammasome cleaves cytokines to activate them:
Pro-IL-1β → IL-1β
Pro-IL-18 → IL-18
 • Induction of acute inflammation

Question 3

Describe DC mediated activation of T cells

Answer 3

Immunological synapse:

Signal 1
• MHC:peptide and TCRs

Signal 2
• Costimulation
• CD80/86 and CD28
• CD40 - CD40L

Signal 3
• Cytokines (IL-2)
• Depending on these cytokines, the CD4+ T cells are polarised down certain lineages

Question 4

Describe the process of ELISA

Answer 4

Used in the case to detect the secretion of cytokines

1. Wells coated with anti-cytokine abs (e.g. anti-IL6)
2. Supernatents of the DC cultures are added to the wells
3. Incubation
4. Wash away the supernatant
5. Cytokines remain bound to the abs
6. Second Ab against cytokine conjugated with biotin added (Sandwich)
7. Streptavidin-HRP added, which binds biotin
8. Addition of a substrate, which HRP converts into a compound with a colour
9. Comparison with a standard curve

Advantage of use of two Abs:
• Becomes more sensitive
• Amplifies the signal
• Allows for a faster test, less incubation time needed

Question 5

Describe the process of FACS

Answer 5

Used in this case to detect cell surface molecule expression

1. DCs cell suspension stained with various Abs tagged with fluorochromes:
 • CD11b
 • CD11c
 • MHC II
 • CD40
 • CD86

2. FACS analysis

Question 6

Describe the process of qPCR

Answer 6

Looking at mRNA expression of:
 • NLRP3
 • IL-6
 • pro-IL-1β
 • β-actin

1. mRNA extracted from various DCs
2. Preparation of cDNA through reverse transcriptase
3. cDNA used as template for qPCR amplification

Controls:
• cDNA template, because SYBR green binds all dsDNA
• β-actin: a housekeeping gene, gives an idea of ‘normal’ levels of mRNA

Question 7

What is SYBR green?

Answer 7

A fluorescent dye that binds dsDNA

Question 8

What are CD11b and CD11c?

Answer 8

Markers of DCs

Found on myeloid cells

Question 9

What is C(T)?

Answer 9

The ‘threshold’ cycle / ‘quantification’ cycle

This is the cycle number at which the fluorescence signal crosses the threshold level

Question 10

How is the ‘threshold level’ determined?

Answer 10

Through the use of a ‘no template’ control

Question 11

What does running the products of PCR on agarose gel do?

Answer 11

Separates the DNA on the basis of size

Question 12

How does gel electrophoresis?

Answer 12

Positive electrode at the opposite end to the wells

DNA is negatively charged, and will move through the gel towards the positive electrode

The distance the DNA product travels is inversely proportional to its size

This is then compared to a DNA molecular mass standard (‘ladder’)