Formative Assessment

Question 1

How do DCs mature?

Answer 1

Maturation caused by microbial products/inflammatory cytokines/ damaged host cells
-> migrate to lymph nodes and present antigenic peptides to T cells

Question 2

How are the following identified?
Helper T cells
B cells
Macrophages
Endothelial cells
Lymph vessels

Answer 2

CD4: Helper T cells
B220: B cells
F4/80, CD169: macrophages
ERTR7: endothelial cells
Lyve-1: lymph vessels

Question 3

Describe the structure of a lymph node

Answer 3

• Subcapsular sinus- between capsule and cortex- afferent lymph draining
• Cortex- mainly B cells, B cell follicles (outer cortex)
• Paracortex- inner cortex- mainly T cells
• Medulla- blood vessels/macrophages, plasma cells- lymph flows out to efferent lymphatics
• IFA interfollicular area- afferent lymphatics, entry of DCs

Question 4

Outline DC subsets

Answer 4

• Can be classical (CDC, CDC11c+) or plasmocytoid (pDC)- looks more like plasma cell
• Different CDC subsets express different surface receptors (Eg CD103 in gut)
- CDC1 express XCR1+ and are cross-priming
- CDC2 express CD11b+ and are CD4 priming
• pDC- type I interferon production, antiviral function

Question 5

Outline cross-presentation

Answer 5

Exogenous proteins from the endosome enter the cytosol and are presented in MHC I via proteasome- allows uninfected DC to still present in MHC class I to CD8- allows recognition of viruses that could interfere with MHC I presentation

Question 6

Describe flow cytometry

Answer 6

• Monoclonal Antibodies against particular surface molecules are conjugated to fluorescent label/dye/fluorochrome
• Flow cytometer- measures intensity of fluorescence of each cell
• Using isotype controls distinguish positive and negative populations for each marker

Question 7

What do Forward scatter (FSC) and Side scatter (SSC) do?

Answer 7

Allow us to know the differentiation of different types of cells

Question 8

What do glow cytometry histograms do?

Answer 8

To determine whether two populations are different to each other

Question 9

How can we identify antigen specific cells using FC?

Answer 9

• Intracellular staining- activate cells with antigen to see which cells produce cytokines- these are antigen specific
• Tetramer/multimeter staining- peptide that is recognised by specific T cell is attached to fluorochrome

Question 10

How can Fc measure proliferation?

Answer 10

Cells lose fluorescence as they proliferate- by measuring fluorescence intensity we can determine level of proliferation

Question 11

How is Diptheria toxin used?

Answer 11

• Use a tissue specific enhancer/promoter with DT receptor (microinjection to fertilised eggs)
• Can knockout chosen cell population by injection of DT

Question 12

What are conditional gene knockouts?

Answer 12

• Technique to eliminate specific gene in certain tissue/cell type (inducible eg Tamoxifen)
• Control where and when gene is knocked out to avoid embryonic mortality, to elucidate role of gene
• eg CRE system

Question 13

Outline CRE-Lox recombination

Answer 13

Site specific recombination:
• LoxP sequences contain specific binding sites for Cre
• Specific DNA sequence is spliced using Cre recombinase
• Floxed mouse has LoxP gene either side of target gene
• Cell specific promoters used to control Cre-> in cells with active Cre recombinase the target gene function is disrupted

Eg.
• Infar1^flox/flox: LoxP (flox) sequences in INFR1 gene
• Itgax-CRE: Cre under control of CD11c promoter
-> Cre expressed in DCs expressing CD11c
-> INFR1 knocked out in DCs expressing CD11c